Note: Descriptions are shown in the official language in which they were submitted.
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/08486
NEUROTROPHIC FACTOR RECEPTORS
Field of the Invention
s
The present invention relates to receptors for neurotrophic factors. In
~ particular, the invention relates to receptors for glial cell line-derived
neurotrophic
factor (GDNF) and neurturin and provides nucleic acid and amino acid sequences
encoding the receptors. The present invention also relates to therapeutic
techniques
to for the treatment of neurotrophic factors-responsive conditions.
2. Background of the Invention
Glial Cell line-Derived Neurotrophic Factor
15 Glial cell line-derived neurotrophic factor (GDNF) was initially isolated
and
cloned from rat B49 cells as a potent neurotrophic factor that enhances
survival of
midbrain dopaminergic neurons (Lin et al., Science, 260, 1130-1132, 1993).
Recent
studies have indicated that this molecule exhibits a variety of other
biological
activities, having effects on several types of neurons from both the central
and
2o peripheral nervous systems. In the central nervous system (CNS), GDNF has
been
shown to prevent the axotomy-induced death of mammalian facial and spinal cord
motor neurons (Li et al., Proceedings Of The National Academy Of Sciences,
U.S.A., 92, 9771-9775, 1995; Oppenheim et al., Nature, 373, 344-346, 1995; Yan
et
al., Nature, 373, 341-344, 1995; Henderson et al., Science, 266, 1062-1064,
1994;
25 Zurn et al., Neuroreport, 6, 113-118, 1994), and to rescue developing avian
motor
neurons from natural programmed cell death (Oppenheim et al., 1995 supra).
Local
administration of GDNF has been shown to protect nigral dopaminergic neurons
from axotomy-induced (Kearns and Gash, Brain Research, 672, 104-111, 1995;
Beck
et al., Nature, 373, 339-341, 1995) or neurotoxin-induced degeneration (Sauer
et al.,
3o Proceedings Of The National Academy Of Sciences U.S.A., 92, 8935-8939,
1995;
Tomac et al., Nature, 373, 335-339, 1995). In addition, local administration
of
GDNF has been shown to induce sprouting from dopaminergic neurons, increase
levels of dopamine, noradrenaline, and serotonin, and improve motor behavior
(Tomac et al., 1995 supra).
35 More recently, GDNF has been reported to be a potential trophic factor for
brain noradrenergic neurons and Purkinje cells. Grafting of fibroblasts
ectopically
expressing GDNF prevented 6-hydroxydopamine-induced degeneration and
CA 02291608 1999-11-26
WO 98/54213 PCTIUS98108486
2
promoted the phenotype of adult noradrenergic neurons in vivo {Arenas et al.,
Neuron, 15, 1465-1473, 1995}, while exogeneously applied GDNF effectively
promoted survival and morphological differentiation of embryonic Purkinje
cells in
vitro (Mount et al., Proceedings Of The National Academy Of Sciences U.S.A.,
92,
9092-9096, 1995). In the peripheral nervous system, GDNF has been shown to
promote the survival of neurons in nodose, ciliary, and sympathetic ganglia,
as well
as small populations of embryonic sensory neurons in dorsal root ganglia {DRG)
and
trigeminal ganglia (Trupp et aL, Journal Of Cell Biology, 130, 137-148, 1995;
Ebendal et al., Journal Of Neuroscience Research, 40, 276-284, I 995;
Oppenheim et
al., 1995 supra; Yan et al., 1995 supra; Henderson et al., 1994 supra). GDNF
has
also been reported to enhance the expression of vasoactive intestinal peptide
and
preprotachykinin-A mRNA in cultured superior cervical ganglion {SCG) neurons,
and thus effects the phenotype of SCG neurons and induces bundle-like
sprouting
(Trupp et al., 1995 supra).
Expression of GDNF has been observed in a number of different cell types
and structures of the nervous system. In the CNS, GDNF mRNA expression has
been observed by reverse transcriptase polymerise chain reaction (RT-PCR) in
both
developing and adult rat striatum, the major target of nigral dopaminergic
innervation, and widely in other regions, including hippocampus, cortex,
thalamus,
septum, cerebellum, spinal cord, and medulla oblongata (Arenas et al., supra
1995;
Poulsen et al., Neuron, 13, 1245-1252, 1994; Springer et al., Experimental
Neurology, 127, 167-170, 1994; Stroemberg et al., Experimental Neurology, 124,
401-412, 1993; Schaar et al., Experimental Neurology, 124, 368-371, 1993). In
human, GDNF transcripts have also been detected in striatum, with highest
level in
the caudate and lower levels in the putamen. Detectable levels are also found
in
hippocampus, cortex, and spinal cord, but not in cerebellum (Schaar et al.,
Experimental Neurology, 130, 387-393, 1994; Springer et al., 1994 supra). In
the
periphery, GDNF mRNA expression has been reported in DRG and SCG of
postnatal day 1 rats, sciatic nerve, and primary cultures of neonatal Schwann
cells
(Trupp et al., 1995 supra; Hoffer et al., Neuroscience Letters, 182, 107-111,
1994;
Henderson et al., 1994 supra; Springer et al., 1994 supra). In addition,
recent studies
have shown that GDNF transcripts are also widely expressed in peripheral non-
neuronal organs, including postnatal testis and kidney, embryonic whisker pad,
stomach, and skin. Expression can be detected at lower levels in embryonic
muscle,
adrenal gland and limb bud, and in postnatal lung, liver and ovary (Trupp et
al., 1995
supra; Henderson et al., 1994 supra). So far, however, the biological
significance of
the non-neuronal expression of GDNF is not clear.
CA 02291608 2003-O1-29 --
WO 98/54213 PCT/US98/08486
A neurotrophic factor refered to as "neurturin" is described in Nature
384(5):467-470, 1996. Detailed descriptions of the preparation and
characterization
of GDNF protein products may be found in U.S. Patent No. 6,362,319
(also see WO 93/06116, Publication No. EP 610 254). Additional GDNF protein
products are described in U.S. Patent No. 6,184,200. As used herein, the term
to "GDNF protein product" includes biologically active synthetic or
recombinant
GDNF proteins and analogs, as well as chemically modified derivatives thereof.
GDNF analogs include deletion variants such as tnuicated GDNF proteins, as
well as
insertion and substitution variants of GDNF. Also included are GDNF proteins
that
are substantially homologous to the human GDNF protein.
IS
~DNF Theranv
GDNF therapy is helpful in the treatment of nerve damage caused by
conditions that compromise the survival and/or proper function of one or more
types
of nerve cells. Such nerve damage may occur from a wide variety of different
2o causes. Nerve damage may occur to one or more types of nerve cells by: ( 1
)
physical injury, which causes the degeneration of the axonal processes and/or
nerve
cell bodies near the site of injury; (2) temporary or permanent cessation of
blood
flow to parts of the nervous system, as in stroke; (3) intentional or
accidental
exposure to neurotoxins, such as chemotherapeutic agents (e.g., cisplatinum)
for the
25 treatment of cancer or dideoxycytidine (ddC) for the treatment of AIDS; (4)
chronic
metabolic diseases; such as diabetes or renal dysfunction; or (5)
neurodegenerative
diseases such as Parkinson's disease, Alzheimer's disease, and amyotrophic
lateral
sclerosis (ALS), which result from the degeneration of specific neuronal
populations.
Several studies indicate that GDNF therapy is particularly helpful in the
3o treatment of neurodegenerative conditions such as the degeneration of the
dopaminergic neurons of the substantia nigra in Parltinson's disease. The only
current treatments for Parkinson's disease are palliative, aiming at
increasing
dopamine levels in the striatum. The expected impact of GDNF therapy is not
simply to produce an increase in the dopaminergic neurotransmission at the
35 dopaminergic nerve terminals in the striatum (which will result in a relief
~of the
- ~ symptoms), but also to slow down, or even stogy the progression of the
degenerative
processes and to repair the damaged nigrostriatal pathway and restore its
function.
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/08486
4
GDNF may alsa be used in treating other forms of damage to or improper
function
of dopaminergic nerve cells in human patients. Such damage or malfunction may
occur in schizophrenia and other forms of psychosis. The only current
treatments for
such conditions are symptomatic and require drugs which act upon dopamine
receptors or dopamine uptake sites, consistent with the view that the improper
functioning of the dopaminergic neurons which innervate these receptor-bearing
neuronal populations may be involved in the disease process.
Receptors
A number of receptors which mediate binding and response to protein factors
have been characterized and molecularly cloned, including receptors for
insulin,
platelet derived growth factor, epidermal growth factor and its relatives, the
fibroblast growth factors, various interleukins, hematopoietic growth factors
and
ciliary neurotrophic factor (U.S. 5,426,177). Study results indicate that some
receptors can bind to multiple (related) growth factors, while in other cases
the same
factor can bind and activate multiple (related) receptors (e.g., Lupu et al.,
Science,
249:1552-1555, 1990; Dionne et al., EMBO J., 9:2685-2692, 1990; Miki et al.,
Science, 251:72-75, 1991 }. Most receptors can broadly be characterized as
having
an extracellular portion or domain responsible for specifically binding a
protein
factor, a transmembrane domain which spans the cell membrane, and an
intracellular
domain that is often involved in initiating signal transduction upon binding
of the
protein factor to the receptor's extracellular portion. Although many
receptors are
comprised of a single polypeptide chain, other receptors apparently require
two or
more separate subunits in order to bind to their protein factor with high-
affinity and
to allow functional response following binding (e.g., Hempstead et al.,
Science,
243:373-375, 1989; Hibi et al., Cell, 63:1149-1157, 1990).
The extracellular and intracellular portions of a given receptor may share
common structural motifs with the corresponding regions of other receptors,
suggesting evolutionary and functional relationships between different
receptors.
These relationships can often be quite distant and may simply reflect the
repeated use
of certain general domain structures. For example, a variety of different
receptors
that bind unrelated factors make use of "immunoglobulin" domains in their
extracellular portions, while other receptors utilize "cytokine receptor"
domains in
their factor-binding regions (e.g., Akira et al., The FASEB J., 4:2860-2867,
1990).
A large number of receptors with distinct extracellular binding domains (which
thus
bind different factors) contain related intracytoplasmic domains encoding
tyrosine-
specific protein kinases that are activated in response to factor binding
(e.g., Ullrich
CA 02291608 1999-11-26
WO 98/54213 PCT/US98I08486
and Schlessinger, Cell, 61:203-212, 1990). The mechanisms by which factor-
binding "activates" the signal transduction process is poorly understood, even
in the
case of receptor tyrosine kinases. For other receptors, in which the
intracellular
domain encodes a domain of unknown function or in which the binding component
associates with a second protein of unknown function (e.g., Hibi et al., Cell,
63:1149-1157, 1990), activation of signal transduction is not well
characterized.
The mode of action of GDNF in vivo is not clearly elucidated in the art, in
part due to the absence of information on a receptor for GDNF. Two groups have
independently found that striatum injected [ 1251]_labeled GDNF can be
retrogradely
1 o transported by dopaminergic neurons in the substantia nigra (Tomac et al.,
Proceedings Of The National Academy Of Sciences Of The United States Of
America. 92, 8274-8278, 1995; Yan et al., 1995 supra). Retrograde transport of
[ 1251~_GDNF by spinal cord motor neurons, DRG sensory neurons and neurons in
the B layer of retina ganglia was also been observed. These retrograde
transport
phenomena can all be specifically inhibited by 100-fold or higher
concentrations of
unlabeled GDNF, suggesting a saturable, receptor-mediated transport process.
In
vitro, recombinant GDNF has been shown to enhance the survival and promote
dopamine uptake of cultured dopaminergic neurons at very low concentrations.
The
observed half maximal effective concentration (ECSp) of GDNF on these neurons
is
0.2 to 1.6 pM (Lin et al., 1993 supra). GDNF has also been shown to support
the
survival of dissociated motor neurons at low concentrations. The reported ECSp
of
GDNF on motor neurons, in a 5 to 10 fM range, is even lower than that on
dopaminergic neurons (Henderson et al., 1994 supra).
Taken together, these observations indicate that receptors) for GDNF
expressed in these cells have very high ligand binding affinities. Similar to
members
of the TGF-13 family, the widely diversified tissue distribution and varied
biological
function of GDNF on different populations of cells suggest that different
types of
receptors) for GDNF or receptor complexes may exist. Saturation steady-state
and
competitive binding of [1251]_GDNF to E10 chick sympathetic neurons has shown
that these neurons express GDNF binding sites differing from those observed in
dopaminergic and motor neurons. The half maximal saturation concentration and
the
half maximal inhibition concentration of GDNF an these binding sites is in the
range
of 1 to 5 nM {Trupp et al., 1995 supra). Similarly, the ECSp of GDNF in
supporting
the survival of sympathetic neurons from P 1 rat SCG has also been reported to
be in
the nanomolar range (Trupp et al., 1995 supra).
To better understand the mechanism by which GDNF activates signal
transduction to exert its affects on cells, it would be beneficial to identify
the
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/084~v
6
receptors) which mediate binding and response to this protein factor. It would
also
be beneficial for GDNF therapy to identify and make possible the production of
accessory molecules which provide for or enhance GDNF signal transduction.
Moreover, the identification of a protein receptor for GDNF would provide
powerful
applications in diagnostic uses, for.example, as an aid in determining if
individuals
would benefit from GDNF protein therapy. Furthermore, the protein receptor for
GDNF could be a key component in an assay for identifying additional molecules
which bind to the receptor and result in desired biological activity.
to
SUMMARY OF THE INVENTION
The present invention provides nucleic acid sequences which encode
neurotrophic factor receptor proteins having amino acid sequences as depicted
in the
15 Figures as well as biologically equivalent analogs. The neurotrophic factor
receptor
protein and protein products of the present invention are designated herein as
filial
cell line-derived neurotrophic factor receptor (GDNFR) protein and protein
products.
Particular receptor proteins refered to herein include GDNFR-a, and filial
cell line-
derived neurotrophic factor receptor-a-related receptor proteins 2 and 3 {GRR2
and
20 GRR3). The novel proteins are functionally characterized by the ability to
bind
GDNF and/or neurturin specifically, and to act as part of a molecular complex
which
mediates or enhances the signal transduction affects of GDNF and/or neurlurin.
GDNFR protein products are typically provided as a soluble receptor protein
and in a
substantially purified form.
25 In one aspect, the present invention provides for the production of GDNFR
protein products by recombinant genetic engineering techniques. In an
alternative
embodiment, the GDNFR proteins are synthesized by chemical techniques, or a
combination of the recombinant and chemical techniques.
In another aspect of the present invention, the GDNFR proteins may be made
3o in glycosylated or non-glycosylated forms. Derivatives of GDNFR protein
typically
involve attaching the GDNFR protein to a water soluble polymer. For example,
the
GDNFR protein may be conjugated to one or more polyethylene glycol molecules
to
decrease the precipitation of the GDNFR protein product in an aqueous
environment.
Yet another aspect of the present invention includes the various
35 polynucleotides encoding GDNFR proteins. These nucleic acid sequences are
used
in the expression of GDNFR in a eukaryotic or prokaryotic host cell, wherein
the
expression product or a derivative thereof is characterized by the ability to
bind to
CA 02291608 1999-11-26
WO 98/54213 PCT/US98108486
GDNF and thereby form a complex capable of mediating GDNF activity, such as
increasing dopamine uptake by dopaminergic cells. The polynucleotides may also
be used in cell therapy or gene therapy applications. Suitable nucleic acid
sequences
include those specifically depicted in the Figures as well as degenerate
sequences,
naturally occurring allelic variations and modified sequences based on the
present
invention.
Exemplary nucleic acid sequences include sequences encoding a
neurotrophic factor receptor protein comprising an amino acid sequence as
depicted
in the Figures capable of complexing with glial cell line-derived neurotrophic
factor
(GDNF) and/or neurturin and mediating cell response to GDNF and/or neurturin,
and
biologically equivalent analogs thereof. Such sequences include: (a} a
sequence set
forth in Figure 1 (SEQ ID NO. 1 ) comprising nucleotides encoding Metl through
Ser465 or Figure 3 (SEQ ID NO. 3) comprising nucleotides encoding Metl through
Ser'16g encoding a neurotrophic factor receptor (GDNFR-a} capable of
complexing
with glial cell line-derived ncurotrophic factor (GDNF) and mediating cell
response
to GDNF, as well as GRR2 and GRR3; (b) a nucleic acid sequence which ( 1 )
hybridizes to a complementary sequence of (a) and (2) encodes an amino acid
sequence with GDNFR activity; and (c) a nucleic acid sequence which but for
the
degeneracy of the genetic code would hybridize to a complementary sequence of
(a)
2o and (2) encodes an amino acid sequence with GDNFR activity. Also disclosed
herein are vectors such nucleic acid sequences wherein the sequences typically
are
operatively linked to one or more operational elements capable of effecting
the
amplification or expression of the nucleic acid sequence. Host cells
containing such
vectors are also contemplated. Typically, the host cell is selected from
mammalian
cells and bacterial cells, such as a COS-7 cell or E. coli, respectively.
A further aspect of the present invention involves vectors containing the
polynucleotides encoding GDNFR proteins operatively linked to amplification
and/or expression control sequences. Both prokaryotic and eukaryotic host
cells may
be stably transformed or transfected with such vectors to express GDNFR
proteins.
3o The present invention further includes the recombinant production of a
GDNFR
protein wherein such transformed or transfected host cells are grown in a
suitable
nutrient medium, and the GDNFR protein expressed by the cells is, optionally,
isolated from the host cells and/or the nutrient medium. The present invention
further includes the use of polynucleotides encoding GDNFR protein and vectors
containing such polynucleotides in gene therapy or cell therapy.
The host cell may also be selected for its suitability to human implantation,
wherein the implanted cell expresses and secretes a neurotrophic factor
receptor of
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/08486
the present invention. The host cell also may be enclosed in a semipermeable
membrane suitable for human implantation. The host cell may be transformed or
transfected ex vivo. An exemplary device for treating nerve damage involves:
(a) a
semipermeable membrane suitable for implantation; and (b) cells encapsulated
within the membrane, wherein the cells express and secrete a neurotrophic
factor
receptor as disclosed herein. The membrane is selected from a material that is
permeable to the neurotrophic factor receptor protein but impermeable to
materials
detrimental to the encapsulated cells.
Methods for the recombinant production of a neurotrophic factor receptor of
to the present invention are also disclosed. An exemplary method involves: (a)
culturing a host cell containing a nucleic acid sequence encoding a GDNFR
protein
of the present invention, such as an amino acid sequence depicted in the
Figures
capable of complexing with glial cell line-derived neurotrophic factor andlor
neurturin and mediating cell response to GDNF and/or neurturin, or
biologically
15 equivalent analogs thereof; (b) maintaining said host cell under conditions
suitable
for the expression of said neurotrophic factor receptor by said host cell; and
(c)
optionally, isolating said neurotrophic factor receptor expressed by said host
cell.
The host cell may be a prokaryotic cell or a eukaryotic cell. If bacterial
expression is
involved, the method may further include the step of refolding the
neurotrophic
2o factor receptor.
The present invention includes an isolated and purified protein comprising an
amino acid sequence as depicted in the Figures capable of complexing with
glial cell
line-derived neurotrophic factor and/or neurturin and mediating cell response
to
GDNF and/or neurturin, and biologically equivalent analogs thereof. Exemplary
25 analogs include, but are not limited to, proteins comprising the amino acid
sequence
Serl g through Pro446~ Asp25 t~.ough Leu44~ and Cys29 through Cys442 as
depicted in Figure 2 (SEQ ID N0:2) as well as proteins comprising the amino
acid
sequence Metl ~ through Pro44g and Cys29 through Cys443 as depicted in Figure
4
(SEQ ID N0:4). The proteins of the present invention may be glycosylated or
non-
3o glycosylated and may be produced by recombinant technology or chemical
synthesis. The present invention further includes nucleic acid sequences
encoding a
receptor protein comprising such amino acid sequences.
Also disclosed herein are pharmaceutical compositions comprising a GDNFR
protein of the present invention in combination with a pharmaceutically
acceptable
35 carrier. A variety of other formulation materials may be used to facilitate
manufacture, storage, handling, delivery and/or efficacy.
Another aspect of the present invention includes the therapeutic use of
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/08486
9
GDNFR genes and proteins. For example, a circulating or soluble GDNFR protein
product may be used alone or in conjunction with GDNF andlor neurturin in
treating
disease of or injury to the nervous system by enhancing the activity of
transmembrane signaling of GDNF and/or neurturin. Thus, the proteins and
pharmaceutical compositions of the present invention may be used in treating
improperly functioning dopaminergic nerve cells, Parkinson's disease,
Alzheimer's
disease and amyotrophic lateral sclerosis. Alternatively, a recombinant GDNFR
gene may be inserted in the cells of tissues which would benefit from
increased
sensitivity to GDNF or neurturin, such as motor neurons in patients suffering
from
1 o amyotrophic lateral sclerosis. In yet another embodiment, GDNFR may be
used to
block GDNF or neurturin activity in cases where the GDNF or neurturin activity
is
thought to be detrimental. The GDNFR protein may be used to verify that
observed
effects of GDNF or neurturin are due to the GDNFR protein.
In another aspect of the invention, GDNFR probes may be used to identify
cells and tissues which are responsive to GDNF or neurlurin in normal or
diseased
states. Alternatively, the probes may be used to detect an aberrancy of GDNFR
protein expression in a patient suffering from a GDNF- or neurturin-related
disorder.
In a further aspect of the invention, GDNFR probes, including nucleic acid as
well as antibody probes, may be used to identify GDNFR-related molecules. For
2o example, the present invention provides for such molecules which form a
complex
with GDNFR protein and thereby participate in GDNFR protein function. As
another example, the present invention provides for receptor molecules which
are
homologous or cross-reactive antigenically, but not identical to GDNFR-a, GRR2
or
GRR3, including consensus sequence molecules as depicted in the Figures.
The present invention also provides for the development of both binding and
functional assays for GDNF or neurturin based on the receptor. For example,
assay
systems for detecting GDNF activity may involve cells which express high
levels of
GDNFR-a, and which are therefore extremely sensitive to even very low
concentrations of GDNF or GDNF-like molecules. Similar assays may involve
3o neurturin and GRR2. In yet another embodiment, soluble GDNFR may be used to
bind or detect the presence of GDNF or GDNF-like molecules.
In addition, the present invention provides for experimental model systems
for studying the physiological role of GDNF or neurturin. Such systems include
assays involving anti-GDNFR antibodies or oligonucleotide probes as well as
animal
models, such as transgenic animals which express high levels of GDNFR and
therefore are hypersensitive to GDNF and/or neurturin or animals derived using
embryonic stem cell technology in which the endogenous GDNFR genes were
CA 02291608 1999-11-26
WO 98/54213 PCT/US98I0848(
deleted from the genome. An anti-GDNFR antibody will binds a peptide portion
of
the neurotrophic factor receptor proteins. Antibodies include monoclonal and
polyclonal antibodies. Alternatively, immunological tags for which antibodies
already exist may be attached to the GDNFR protein to aid in detection. Such
tags
5 include but are not limited to Flag (IBI/Eastman Kodak) and myc sequences.
Other
tag sequences such as polyhistidine have also been used for detection and
purification on metal chelating columns.
Yet another aspect of the present invention involves the use of GDNFRs to
identify ligands which activate receptors as described in the following
detailed
1o description and examples. Proteins as well as small molecule neurotrophic
factor
mimetics may be identified and studied following the binding studies described
herein.
Additional aspects and advantages of the invention will be apparent to those
skilled in the art upon consideration of the following description, which
details the
practice of the present invention.
BRIEF DESCRIPTION OF THE FIGURES
2o Figure 1 depicts a nucleic acid sequence (SEQ ID NO:1 ) encoding human
filial cell line-derived neurotrophic factor receptor (GDNFR-a}. The amino
acid
sequence of the full length GDNFR protein is encoded by nucleic acids 540 to
1934.
Figure 2 depicts the amino acid sequence (SEQ ID N0:2) of the full length
human GDNFR-a protein.
Figure 3 depicts a nucleic acid sequence (SEQ ID N0:3) encoding rat
GDNFR-a. The amino acid sequence of the full length GDNFR-a protein is encoded
by nucleic acids 302 to 1705.
Figure 4 depicts the amino acid sequence (SEQ ID N0:4) of the full length
rat GDNFR-a protein
Figure 5 depicts the alignment and comparison of portions of GDNFR-a
cDNA sequences produced in various clones as well as the consensus sequence
for
human GDNFR-a.
CA 02291608 2003-O1-29 -
WO 98/54213 PCTIUS98/08486
11
Figure 6 depicts the identification of Neuro-2A derived cell lines expressing
GDNFR-a.
Figure 7A and 7B depict the results of the equilibrium binding of
~ 1251~GDNF to cells expressing GDNFR-a.
Figure 8 depicts the results of the chemical cross-linking of [ 125I]GDNF to
GDNFR-a and Ret Expressed in cells expressing GDNFR-a.
' Figure 9 depicts the results of the induction of c-Ret autophosphorylation
by
GDNF in cells expressing GDNFR-a.
Figure 10 depicts the results of the induction of c-Ret autophosphorylation by
GDNF and soluble GDNFR-a.
Figure 11 depicts the results of the blocking of c-Ret autophosphorylation by
a Ret-Fc fission protein.
Figure 12 depicts the results of the induction of c-Ret autophosphorylation by
GDNF in motor neurons.
Figure 13 depicts a model for GDNF signaling mediated by GDNFR-a and
Ret.
Figure 14 depicts a nucleic acid sequence (SEQ ID N0:35) encoding human
glial cell line-derived neurotrophic factor receptor-a-related protein 2
(GRR2). The
amino acid sequence of the full length GRR2 protein is encoded by nucleic
acids
1585 to 2989.
3o Figure 15 depicts a nucleic acid sequence (SEQ ID X10:37) encoding human
glial cell line-derived neurotrophic factor receptor-a-related protein 3
(GRR3).
Figure 16 depicts a nucleic acid sequence (SEQ ID N0:39) encoding rat glial
ceU line-derived neurotrophic factor receptor-a-related protein 2 (rat GRR2),
..
Figure 17 depicts a nucleic acid sequence (SEQ ID N0:41 ) encoding rat glial
cell line-derived neurotrophic factor receptor-a-related protein 3 (rat GRR3).
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/08486
12
Figure 18 depicts the alignment and comparison of various human, rat and
mouse GDNFR amino acid sequences.
Figure I 9 depicts the alignment and comparison of human, rat and mouse
GDNFR-a, GRR2 AND GRR3 amino acid sequences and an exemplary consensus
GDNFR sequence.
Figure 20 depicts the alignment and comparison of human and rat GDNFR-a
and GRR2 peptide sequences.
Figure 21 (Panels A and B} depicts the binding of neurturin and GDNF to
LA-N-% and NGR-38 cells. LA-N-5 (Panel A) and NGR-38 (Panel B) cells were
incubated with 50 pM of either ['zSI~NTN or ['ZSI]GDNF in the absence (light
gray
bars) or presence of unlabeled GDNF (dark gray bars) or neurturin (black bars)
at
4°C for two hours.
Figure 22 depicts the results of the chemical cross-linking of neurturin and
GDNF to GDNFR-a and GRR2.
Figure 23 depicts the results of neurturin induced ret autophosphorylation in
NGR-38 cells.
Figure 24 depicts the results of neurturin induced ret autophosphorylation in
LA-N-5 cells.
Figure 25 (Panels A and B) depicts the results of neurturin and GDNF
induced MAP kinase activation in LA-N-5 and NGR-38 cells.
3d Figure 26 depicts the amino acid sequences of GDNFR-a, GRR2 and GRR3
are aligned and a consensus sequence is shown above the three receptor
sequences.
Upper case letters in the consensus sequence indicate amino acids that are
conserve
in all three receptors, lower case letters indicate that two of the three
receptors sl-:~
that amino acid, and dots indicate all three receptors have a different amino
ac~ ~
that position. Predicted signal peptide sequences are underlined in GDNFR-~<
GRR3; no signal peptide is predicted for GRR2. The hydrophobic C-terming
regions of all three putative receptors are underlined. Potential N-glycosy~a
CA 02291608 1999-11-26
WO 98154213 PCT/US98/08486
13
are shown in boldface and sites conserved between two receptors are outlined
by
boxes.
DETAILED DESCRIPTION OF THE INVENTION
Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic
factor which exhibits a broad spectrum of biological activities on a variety
of cell
types from both the central and peripheral nervous systems. It is a
glycosylated,
l0 disulfide-linked dimer which is distantly related {less than 20% homology)
to the
transforming growth factor-13 (TGF-f3) superfamily. GDNF's ability to enhance
the
survival of dopaminergic neurons and other neuron populations demonstrates its
therapeutic potential for the treatment of Parkinson's disease as well as
other forms
of nerve damage or malfunction.
t5 The described biological activities of the neurturin neurotrophic factor
(Nature 384(5):467-470, 1996) include promoting the survival of nodose ganglia
sensory neurons and a small population of dorsal root ganglia sensory neurons,
in
addition to superior cervical ganglion sympathetic neurons. The activity
suggests the
possibility of a common or similar signaling pathway. In addition, the
biological
2o activities of neurturin may extend to motor neurons and dopaminergic
neurons.
Thus, neurturin may be useful in the treatment of diseases for which the use
of
GDNF may be indicated.
The present invention is based upon the discovery of a high affinity receptor
first found on the surface of cultured retinal cells from postnatal rats.
These
25 receptors possess an estimated GDNF binding affinity comparable to that of
the
receptors found in dopaminergic and motor neurons; midbrain dopaminergic
neurons
(Lin et al., 1993 supra; Sauer et al., 1995 supra; Kearns and Gash, 1995
supra; Beck
et al., 1995 supra; Tomac et al., 1995a supra), facial and spinal cord motor
neurons
(Li et al., 1995 supra; Oppenheim et al., 1995 supra; Yan et al., 1995 supra;
Zurn et
3o al., 1994 supra; Henderson et al., 1994 supra). The receptor molecule has
been
named GDNF receptor-alpha (GDNFR-a) since it is the first known component of a
receptor system for GDNF. The present invention also provides the first
description
of the expression cloning and characterization of GDNFR-a protein. Cells
modified
to express the recombinant receptor bind GDNF with high affinity. Additional
35 receptor proteins include glial cell line-derived neurotrophic factor
receptor-a related
receptor proteins 2 and 3 (GRR2 and GRR3).
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/0848b
14
Using a dopamine uptake assay and [1251]_GDNF binding on cultured cells,
high affinity receptors to GDNF were detected on the surface of rat
photoreceptor
cells. As further described in the Examples, the study of photoreceptor cells
lead to
the isolation of a cDNA clone by expression cloning for GDNFR-a. The nucleic
acid sequence for GDNFR-a encodes a protein of 468 amino acids with 31
cysteine
residues and three potential N-glycosylation sites. Next, a nucleic acid
sequence
from the rat cDNA clone was used to isolate its human homolog which was found
to
be nearly identical to the rat receptor at the amino acid level. The human
GDNFR-a
cDNA sequence encodes a protein of 465 amino acids with the positions of all
1o cysteine residues and potential N-glycosylation sites conserved relative to
the rat
receptor. This high degree of primary sequence conservation indicated an
important
role for this receptor in the biological function of GDNF.
As discussed above, many receptors have three main domains: an
extracellular or cell surface domain responsible for specifically binding a
protein
I5 factor; a transmembrane domain which spans the cell's membrane; and an
intracellular or cytoplasmic domain that is typically involved in initiating
signal
transduction when a protein factor binds to the extracellular domain. It was
determined, however, that GDNFR-a is unrelated in sequence or structural
characteristics to any known protein (such as the consensus sequences found in
2o either receptor kinases or cytokine receptors), lacks a cytoplasmic domain,
lacks the
C-terminal charged residues characteristic of a transmembrane domain and is
anchored to the cell membrane by glycosyl-phosphatidylinositol (GPI) linkage,
as
described in greater detail below. Although the absence of an intracellular
catalytic
domain precluded a direct role in transmembrane signaling, the high binding
affinity
25 and strong evolutionary sequence conservation further suggested that this
receptor
was important for GDNF function.
Because GDNFR-a lacks a cytoplasmic domain, it was thought that this
receptor must act in conjunction with one or more accessory molecules which
play a
role in transmembrane signaling. It was then discovered that transgenic mice
which
3o lack the gene for GDNF die and have no kidneys. Transgenic mice which lack
the
gene for c-ret proto-oncogene (Schuchardt, et al., Nature, 367, 380-383, 1994)
were
found to have a similar phenotype. The c-ret proto-oncogene encodes a receptor
tyrosine kinase (RTK) whose normal function had not yet been determined. All
RTKs have a similar topology: they possess an extracellular ligand-binding
domain,
35 a transmembrane domain and a cytopiasmic segment containing the catalytic
protein-
tyrosine kinase domain. Binding of a ligand leads to the activation of the
kinase
domain and phosphorylation of specific substrates in the cell that mediate
CA 02291608 2003-O1-29
WO 98/54213 PCT/US98/08486
intracellular signaling. The present invention involves the discovery that a
soluble
form of GDNFR-a may be used to mediate the binding of GDNF to the c-retproto-
oncogene and thereby elicit a cellular response to GDNF as well as modify its
cell-
type specificity. a
5 Similar species, called "receptor alpha" components, provide ligand binding
specificity but do not have the capacity to transduce signal on their own.
Such
components are found in the ciliary neurotrophic factor (CNTF) and interleukin-
6
(IL,-6) receptor systems. Like GDNFR-a, and in contrast to IL-6 receptor, CNTF
receptor binds its ligand with high affinity, has a hydrophobic C-terminus, no
to cytoplasmic domain, and is anchored to the cell membrane by GPI linkage
(Davis et
al., 1991 ). In order to mediate signal transduction, CNTF binds first to CNTF
receptor, creating a complex which is able to bind gp130. This inactive
complex
then binds to L1F receptor to form the active signaling complex (Davis, et
al.,
Science, 260, 1805-1807, 1993). As with the present invention, CNTF receptor
(the
15 ligand specifzc binding component) must be present for signaling to occur
but it need
not be membrane bound (Economides et al., Science, 270, 1351-1353,,1995).
As further described below, the GDNFR protein may be anchored to a~.~cell.
surface, or it may be provided in a soluble form. In either case, the GDNFR
protein
forms a ligand complex with GDNF and/or neurturin, and the ligand complex
binds
to cell surface receptor to effectuate intracellular signaling. Thus, a
soluble form of
GDNFR-protein may be used to potentiate the action of a neurotrophic factor
that
binds thereto andlor modify its cell-type specificity.
The GDNFR proteins are unrelated to previously known receptors. There are
no apparent matches in the GenBank and Washington University-Merck databases
for related sequences. An expressed sequence tag (EST) found in the Washington
University-Merck EST database shows 75% homology to a small portion of the
coding region of GDNFR-a (approximately 340 nucleotides of the 521 nucleotides
of sequence generated from the 5' end of the clone). This clone (GenBank
accession
#H12981) was isolated from an oligo-dT primed human infant brain library and
3o cloned directionally into the Lafmid BA vector (Hillier, L. et al,
unpublished data).
The 3' end of the #H 12981 clone has been sequenced, but it exhibits no
homology to
any part of GDNFR-a. The appearance of homologybetween this #H12981 clone
and GDNFR-a over a short region, which homology then disappears, suggests that
the #H12981 clone represents an unspliced transcript, or cloning artifact
rather than a
bona fide cDNA transcript.
Thus, the present invention enables the cloning of a GDNFR protein by
providing a method for selecting target cells which express GDNFR protein. By
* trademark
CA 02291608 1999-11-26
WO 98!54213 PCT/US98/08486
16
providyng a means of enriching for GDNFR protein-encoding sequences, the
present
invention further provides for the purification of GDNFR protein and the
direct
cloning of GDNFR-encoding DNA. The present description of the GDNFR nucleic
acid and amino acid sequences provides the information needed to reproduce
these
entities as well as a variety of GDNFR analogs. With this information, GDNFR
protein products may be isolated or generated by any means known to those
skilled
in the art. A variety of means for the recombinant or synthetic production of
GDNFR protein are disclosed.
As used herein, the term "GDNFR protein product" includes biologically
1o active purified natural, synthetic or recombinant GDNFR-a, GRR2 and GRR3
(jointly referred to as glial cell line derived neurotrophic factor receptors,
GDNFR,
GDNFR protein), GDNFR analogs (i.e., GDNFR homologs and variants involving
insertion, substitution and deletion variations, such as based on the
consensus
sequences depicted in the Figures), and chemically modified derivatives
thereof.
15 GDNFR analogs are substantially homologous to the GDNFR amino acid
sequences
set forth in the Figures.
The term "biologically active", as used herein, means that the GDNFR
protein product demonstrates high affinity binding to GDNF and/or neurturin
and
mediates or enhances GDNF-induced or neurturin-induced signal transduction.
20 Using the present disclosure, it is well within the ability of those of
ordinary skill in
the art to determine whether a GDNFR protein analog has substantially the same
biological activity as the GDNFR protein products set forth in the Figures.
The term "substantially homologous" amino acid sequence, as used herein,
refers to an amino acid sequence sharing a degree of "similarity" or homology
to the
25 GDNFR amino acid sequences set forth in the Figures such that the
homologous
sequence has a biological activity or function similar to that described for
these
GDNFR amino acid sequences. It will be appreciated by those skilled in the
art, that
a relatively large number of individual or grouped amino acid residues can be
changed, positionally exchanged (e.g.s, reverse ordered or reordered) or
deleted
3o entirely in an amino acid sequence without affecting the three dimensional
configuration or activity of the molecule. Such modifications are well within
the
ability of one skilled in the art following the present disclosure. The
identification
and means of providing such modified sequences are described in greater detail
below. It is preferable that the degree of homology of a substantially
homologous
35 protein (peptide) is equal to or in excess of 70% (i.e., a range of from
70% to 100%
homology). Thus, a preferable "substantially homologous" GDNFR amino acid
sequence may have a degree of homology greater than or equal to ?0% of the
amino
CA 02291608 2003-O1-29
a j
WO 98/54213 PCT/US98/08486
17
acid sequences set forth for GDNFR-a, GRR2, GRR3 and consensus sequences
thereof as depicted in the Figures. More preferably the degree of homology may
be
equal to or in excess of 80% .or 85%. Even more preferably it is equal to or
in excess
of 90%, or most preferably it is equal to or in excess of 95%.
s The percentage of homology as described herein is calculated as the
percentage of amino acid residues found in one protein sequence which align
with
identical or similar amino acid residues in the second protein sequence. Thus,
in the
case of GDNFR protein homology, the degree of sequence homology may be
determined by optimally aligning the amino acid residues of the comparison
1o molecule to those of a reference GDNFR polypeptide, such as depicted in the
Figures or those encoded by the nucleic acid sequences depicted in the
Figures, to
maximize matches of residues between the two sequences. It will be appreciated
by
those skilled in the art that such alignment may include appropriate
conservative
residue substitutions and will disregard truncations and internal deletions or
1 s insertions of the comparison sequence by introducing gaps as required;
see, for
example Dayhoff; Atlas of Protein Sequence and Structure Vol. 5, wherein an ~
,
average of three or four gaps in a length of 100 amino acids may be introduced
to .
assist in alignment .(p. 124, National Biochemical Research Foundation,
Washington,
D.C., 1972). Once so
2o aligned, the percentage is determined by the number of aligned residues in
the
comparison polypeptide divided by the total number of residues in the
comparison
polypeptide. It is further contemplated that the GDNFR protein sequences of
the
present invention may be used to form a portion of a fusion protein or
chimeric
protein which has, at least in part, GDNFR protein activity. The alignment and
25 homology of such a protein would be determined using that portion of the
fusion
protein or chimeric protein which is related to GDNFR protein activity.
The sources of such substantially homologous GDNFR proteins include.the
GDNFR proteins of other mammals (such as depicted in the Figures) which are
expected to have a high degree of homology to the human GDNFR protein. For
3o example, 'the degree of homology between the rat and human GDNFR-a proteins
disclosed herein is about 93%. Substantially homologous GDNFR proteins may be
isolated from such mammals by virtue of cross-reactivity with antibodies to
the
GDNFR amino acid sequences depicted in the Figures. Alternatively, they may be
expressed by nucleic acid sequences which are isolated thmugh hybridization
with
35 the gene or with segments of the gene encoding the GDNFR proteins or which
hybridize to a complementary sequence of the nucleic acid sequences
illustrated in
the Figures. Suitable hybridization conditions are described in further detail
below.
CA 02291608 1999-11-26
WO 98154213 PCT/US98108486
18
The novel GDNFR protein products are typically isolated and purified to
form GDNFR protein products which are substantially free of unwanted
substances
that would detract from the use of the present polypeptides for an intended
purpose.
For example, preferred GDNFR protein products may be substantially free from
the
presence of other human (e.g., non-GDNFR) proteinaceous materials or
pathological
agents. Preferably, the GDNFR protein products are about 80% free of other
proteins which may be present due to the production technique used in the
manufacture of the GDNFR protein product. More preferably, the GDNFR protein
products are about 90% free of other proteins, particularly preferably, about
95% free
of other proteins, and most preferably about >98% free of other proteins. In
addition, the present invention furnishes the unique advantage of providing
polynucleotide sequences for the manufacture of homogeneous GDNFR proteins.
A variety of GDNFR variants are contemplated, including addition, deletion
and substitution variants. For example, a series of deletion variants may be
made by
is removing one or more amino acid residues from the amino and/or carboxy
termini of
the GDNFR protein. Using rules for the prediction of signal peptide cleavage
as
described by von Heijne (von Heijne, Nucleic Acids Research, 14, 4683-4690,
1986), the first amino acid residue of the GDNFR-a protein which might be
involved
in GDNF binding is SerlB, as depicted in the full length amino acid sequence
of
2o human GDNFR-a in Figure 2 (SEQ ID N0:2). Amino acid residues Metl through
SerlB are in the amino-terminal hydrophobic region that is likely to be part
of a
signal peptide sequence, and therefore, not be included in the mature form of
the
receptor protein. Similarly, the last amino acid residue of the GDNFR-a
protein
which is likely to be necessary for GDNF binding is Ser446. Amino acid
residues
25 Leu447 through Ser465 are in the carboxy-terminal hydrophobic region that
is
involved in the GPI linkage of the protein to the cell surface. Thus, it is
contemplated that any or all of the residues from Metl through SerlB and/or
Leu447
through Ser465 (as depicted in Figure 2 (SEQ ID N0:2) may be removed from the
protein without affecting GDNF binding to the GDNFR-a protein, thereby leaving
a
30 "core" sequence of Alal9 through Pro446, Using known analysis techniques,
it is
further contemplated that N-terminal truncations may include the removal of
one or
more amino acid residues up to and including G1y24. Thus, GDNFR-a truncation
analogs also may include the deletion of one or more amino acid residues from
either
or both termini such that an amino acid sequence of Asp25 through Pro446 or
35 Leu447 forms the basis for a core molecule. Additional GDNFR-a analogs are
contemplated as involving amino acid residues SerlB through Pro449 as depicted
in
the GDNFR-a amino acid sequence of Figure 4 (SEQ ID N0:4) , i.e., deleting one
or
CA 02291608 1999-11-26
WO 98/54213 PCT/US98108486
19
more amino acid residues from either or both termini involving the hydrophobic
regions depicted as amino acid residues Metl through Serl g and/or Pro44g
through
Ser468. Similar analogs may be designed using the amino acid sequences for
GRR2
and GRR3, as well as consensus sequences, as depicted in the Figures.
In addition, it is contemplated that one or more amino acid residues may be
removed from either or both of the amino and carboxy termini of the GDNFR
protein until the first and last cysteine residues in the full length sequence
are
reached. It is advantageous to retain the cysteine residues for the proper
intramolecular binding of the GDNFR protein. As depicted in the full length
amino
1o acid sequence of human GDNFR-a in Figure 2 (SEQ ID N0:2), any or all of
amino
acid residues from Metl to Asp2g may be removed from the amino terminal
without
removing the first cysteine residue which appears as Cys29. Similarly, any or
all of
amino acid residues from G1y443 to Ser465 may be removed from the carboxy
terminal without removing the last cysteine residue which appears as Cys442,
ether
15 GDNFR-a analogs may be made using amino acid residues Cys29 through Cys443
as depicted in the GDNFR-a amino acid sequence of Figure 4 {SEQ ID N0:4) ,
i.e.,
deleting all or part of the terminal regions depicted as amino acid residues
Metl
through Asp2g and/or Ser444 through Ser46g. Similar analogs may be designed
using the amino acid sequences for GRR2 and GRR3, as well as consensus
20 sequences, as depicted in the Figures.
It will be appreciated by those skilled in the art that, for the same reasons,
it
is contemplated that these identified amino acid residues may be replaced,
rather
than deleted, without affecting the function of the GDNFR protein.
Alternatively,
these identified amino acid residues may be modified by intra-residue
insertions or
2s terminal additions without affecting the function of the GDNFR protein. In
yet
another embodiment, a combination of one or more deletions, substitutions or
additions may be made.
The present GDNFR proteins or nucleic acids may be used for methods of
3o treatment, or for methods of manufacturing medicaments for treatment. Such
treatment includes conditions characterized by excessive production of GDNF or
neurturin, wherein the present GDNFRs, particularly in soluble form, may be
used to
complex to and therefore inactivate such excessive GDNF or neurturin. This
treatment may be accomplished by preparing a soluble receptor (e.g., use of
the
3s GDNF or neurturin binding domain) or by preparation of a population of
cells
containing such GDNFR, and transplanting such cells into the individual in
need
thereof. The present GDNFR protein products may also be used for treatment of
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/08486
those having defective GDNF and/or neurturin receptors. For example, one may
treat an individual having defective GDNFRs by preparation and delivery of a
soluble receptor, or by preparation of a population of cells containing such
non-
defective GDNFR and transplanting such cells into an individual. Or, an
individual
may have an inadequate number of GDNF or neurturin receptors, and cells
containing such receptors may be transplanted in order to increase the number
of
GDNF or neurturin receptors available to an individual. Such compositions may
be
used in conjunction with the delivery of GDNF or neurturin. It is also
contemplated
GDNFR protein products may be used in the treatment of conditions responsive
to
I o the activation of the c-ret receptor tyrosine kinase.
In yet another aspect of the present invention, a further advantage to the
novel compositions is the use of GDNFR to stabilize GDNF protein or neurturin
pharmaceutical compositions. In another aspect of the present invention, a
GDNFR
may be used to screen compounds for antagonist activity.
15 Other aspects and advantages of the present invention will be apparent to
those skilled in the art. For example, additional uses include new assay
systems,
transgenic animals and antibody production.
Studv Models
2o The present invention provides for assay systems in which GDNF or
neurturin activity or activities similar to GDNF or neurturin activity
resulting from
exposure to a peptide or non-peptide compound may be detected by measuring an
elicited physiological response in a cell or cell line which expresses the
GDNFR
molecules of the present invention. A physiological response may comprise any
of
the biological effects of GDNF or neuriurin, including but not limited to,
dopamine
uptake, extension of neurites, increased cell survival or growth, as well as
the
transcriptional activation of certain nucleic acid sequences (e.g.
promoter/enhancer
elements as well as structural genes), GDNF-related processing, translation,
or
phosphorylation, and the induction of secondary processes in response to
processes
3o directly or indirectly induced by GDNF, to name but a few.
For example, a model system may be created which may be used to study the
effects of excess GDNF activity. In such a system, the response of a cell to
GDNF
may be increased by engineering an increased number of suitable GDNFRs on the
cells of the model system relative to cells which have not been so modified. A
system may also be developed to selectively provide an increased number of
such
GDNFRs on cells which normally express GDNFRs. In order to ensure expression
of GDNFR, the GDNFR gene may be placed under the control of a suitable
promoter
CA 02291608 2003-O1-29
21
sequence. It may be desirable to put the GDNFR gene under the control of a
constitutive and/or tissue specific promoter (including but not limited to the
CNS
neuron specific enolase, neurofilament, and tyrosine hydroxylase promoter), an
inducible promoter (such as the metallothionein promoter), the UV activated
promoter in the human immunodeficiency virus long terminal repeat (Valeri et
al.,
1988, Nature 333:78-81), or the CMV promoter (as contained in pCMX, infra) or
a
developmentally regulated promoter.
By increasing the number of cellular GDNFRs, the response to endogenous
GDNF may be increased. If the model system contains little or no GDNF, GDNF
to may be added to the system. It may also be desirable to add additional GDNF
to the
model system in order to evaluate the effects of excess GDNF activity. Over
expressing GDNF (or secreted GDNF) may be one method for studying the effects
of
elevated levels of GDNF on cells already expressing GDNFR.
GDNFR Thera~es
In another aspect, certain conditions may benefit from an increase in GDNF
and/or neurturin responsiveness. It may, therefore, be beneficial to increase
the
number or binding affinity of GDNFRs in patients suffering from conditions
responsive to GDNF and/or neurturin therapy. This could be achieved through
gene
2o therapy, whereby selective expression of recombinant GDNFR in appropriate
cells is
achieved, for example, by using GDNFR genes controlled by tissue specific or
inducible promoters or by producing localized infection with replication
defective
viruses carrying a recombinant GDNFR gene.
It is envisioned that conditions which will benefit from GDNFR or combined
GDNF or neurturin/GDNFR delivery include, but are not limited to, motor neuron
disorders including amyotrophic lateral sclerosis, neurological disorders
associated
with diabetes, Parkinson's disease, Alzheimer's disease, arid Huntington's
chorea.
Additional indications for the use of GDNFR or combined GDNF or
neurturin/GDNFR delivery are described above and further include the treatment
of:
glaucoma or other diseases and conditions involving retinal ganglion cell
degeneration; sensory neuropathy caused by injury to, insults to, or
degeneration of,
sensory neurons; pathological conditions, such as inherited retinal
degenerations and
age, disease or injury-related retinopathies, in which photoreceptor
degeneration
occurs and is responsible for vision loss; and injury or degeneration of inner
ear
sensory cells, such as hair cells and auditory neurons for preventing and/or
treating
hearing loss due to variety of causes.
CA 02291608 1999-11-26
WO 98/54213 PCTIUS98/08486
22
Trans~;enic Animals
In yet another aspect, a recombinant GDNFR gene may be used to inactivate
or "knock out" the endogenous gene (e.g., by homologous recombination) and
thereby create a GDNFR deficient cell, tissue, or animal. For example, a
recombinant GDNFR-a gene may be engineered to contain an insertional mutation
which inactivates GDNFR-a. Such a construct, under the control of a suitable
promoter, may be introduced into a cell, such as an embryonic stem cell, by
any
conventional technique including transfection, transduction, injection, etc.
Cells
containing the construct may then be selected, for example by 6418 resistance.
1o Cells which lack an intact GDNFR-a gene are then identified (e. g., by
Southern
blotting or Northern blotting or assay of expression). Cells lacking an intact
GDNFR-a gene may then be fused to early embryo cells to generate transgenic
animals deficient in GDNFR. A comparison of such an animal with an animal not
expressing endogenous GDNF would reveal that either the two phenotypes match
completely or that they do not, implying the presence of additional GDNF-like
factors or receptors. Such an animal may be used to define specific neuronal
populations, or other in vivo processes, normally dependent upon GDNF. Thus,
these populations or processes may be expected to be effected if the animal
did not
express GDNFR-«, and therefore, could not respond to GDNF. Similar constructs
2~ may be made and procedures followed for GRR2 and GRR3.
Diagnostic A~IJlications
According to the present invention, GDNFR probes may be used to identify
cells and tissues which are responsive to GDNF or neurturin in normal or
diseased
states. The present invention provides for methods for identifying cells which
are
responsive to GDNF or neurturin by detecting GDNFR expression in such cells.
GDNFR expression may be evidenced by transcription of GDNFR mRNA or
production of GDNFR protein. GDNFR expression may be detected using probes
which identify GDNFR nucleic acid or protein or by detecting "tag" sequences
3o artificially added to the GDNFR protein.
One variety of probe which may be used to detect GDNFR expression is a
nucleic acid probe, which may be used to detect GDNFR-encoding RNA by any
method known in the art, including, but not limited to, in situ hybridization,
Northern blot analysis, or PCR related techniques. Nucleic acid products of
the
invention may be labeled with detectable markers (such as radiolabels and non-
isotopic labels such as biotin) and employed in hybridization processes to
locate the
human GDNFR gene position and/or the position of any related gene family in a
CA 02291608 1999-11-26
WO 98154213 PCT/US98/08486
23
chromosomal map. They may also be used for identifying human GDNFR gene
disorders at the DNA level and used as gene markers for identifying
neighboring
genes and their disorders. Contemplated herein are kits containing such
labeled
materials.
Polypeptide products of the invention may be "labeled" by association with a
detectable marker substance or label (e.g., a radioactive isotope, a
fluorescent or
chemiluminescent chemical, an enzyme or other label available to one skilled
in the
art} to provide reagents useful in detection and quantification of GDNF or
neurturin
in solid tissue and fluid samples such as blood or urine. Such products may
also be
1o used in detecting cells and tissues which are responsive to GDNF or
neurturin in
normal or diseased states.
Another possible assay for detecting the presence of GDNF or neurturin in a
test sample or screening for the presence of a GDNF-like molecule involves
contacting the test sample with a GDNFR protein, suitable for binding GDNF or
neurturin, immobilized on a solid phase, thereby producing GDNFR-bound GDNF
or neurturin protein. The GDNFR-bound GDNF or neurturin may optionally be
contacted with a detection reagent, such as a labeled antibody specific for
GDNF or
neurturin, thereby forming a detectable product. Such assays may be developed
in
the form of assay devices for analyzing a test sample. In a basic form, such
devices
include a solid phase containing or coated with an appropriate GDNFR protein.
A
method for analyzing a test sample for the presence of GDNF-like protein may
involve contacting the sample to an assay reagent comprising GDNFR protein,
wherein said GDNFR protein reacts with the GDNF-like protein present in the
test
sample and produces a detectable reaction product indicative of the presence
of
GDNF.
The assay reagents provided herein may also be embodied as part of a kit or
article of manufacture. Contemplated is an article of manufacture comprising a
packaging material and one or more preparations of the presently provided
nucleic
acid or amino acid sequences. Such packaging material will comprise a label
indicating that the preparation is useful for detecting GDNF, neurturin, GDNFR
or
GDNFR defects in a biological sample. As such, the kit may optionally include
materials to carry out such testing, such as reagents useful for performing
protein
analysis, DNA or RNA hybridization analysis, or PCR analysis on blood, urine,
or
tissue samples.
Anti-GDNFR Antibody
According to the present invention, GDNFR protein, or fragments or
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/084<~:
24
derivatives thereof, may be used as an immunogen to generate anti-GDNFR
antibodies. To further improve the likelihood of producing an anti-GDNFR
immune
response, the amino acid sequence of GDNFR may be analyzed in order to
identify
portions of the molecule which may be associated with increased
immunogenicity.
s For example, the amino acid sequence may be subjected to computer analysis
to
identify surface epitopes which present computer-generated plots of
hydrophilicity,
surface probability, flexibility, antigenic index, amphiphilic helix,
amphiphilic sheet,
and secondary structure of GDNFR. Alternatively, the amino acid sequences of
GDNFR from different species could be compared, and relatively non-homologous
regions identified; these non-homologous regions would be more likely to be
immunogemc across various species.
Also comprehended are polypeptide fragments duplicating only a part of the
continuous amino acid sequence or secondary conformations within GDNFR, which
fragments may possess one activity of mammalian GDNFR (e.g., immunological
t5 activity) and not others (e.g., GDNF protein binding activity). Thus, the
production
of antibodies can include the production of anti-peptide antibodies. The
following
exemplary peptides were synthesized using GDNFR sequences:
Table 1
2o GDNFR-a Peptides
SJP-6 H2N-QSCSTKYRTL-cooHhuman GDNFR-a, AA 40-49 (SEQ ID N0:25)
SJP-7 H2N-CKRGMKKEKN-COOHhuman GDNFR-a, AA 89-98 (SEQ ID N0:26)
SJP-8 H2N-LLEDSPYEPV-COOHhuman GDNFR-a, AA 115-124 (SEQ ID
N0:27)
SJP-9 H2N-CSYEERERPN-COOHrat GDNFR-a, AA 233-242 (SEQ ID N0:28)
SJP-10 H2N-PAPPVQTTTATTTT-COOH rat GDNFR-a, AA 356-369 (SEQ ID N0:29)
Peptides SJP-6, 7, and 8 are identical in rat and human GDNFR-a. Peptides SJP-
9
and 10 are derived from the rat sequence and are each one amino acid different
from
25 human. Both polyclonal and monoclonal antibodies may be made by methods
known in the art using these peptides or other portions of GDNFR.
Monoclonal antibodies directed against GDNFR may be prepared by any
known technique which provides for the production of antibody molecules by
continuous cell lines in culture. For example, the hybridoma technique
originally
3o developed by Kohler and Milstein to produce monoclonal antibodies (Nature,
256:495-497, 1975), as well as the trioma technique, the human B-cell
hybridoma
technique (Kozbor et al., Immunology Today 4:72, 1983), the EBV-hybridoma
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/08486
technique (Cole et al., in "Monoclonal Antibodies and Cancer Therapy," Alan R.
Liss, Inc. pp. 77-96, 1985), and the like, may be used.
Human monoclonal antibodies or chimeric human-mouse {or other species)
monoclonal antibodies also may be prepared for therapeutic use and may be made
by
any of numerous techniques known in the art (e.g., Teng et al., Proc. Natl.
Acad. Sci.
U.S.A., 80:7308-7312, 1983; Kozbor et al., Immunology Today, 4:72-79, 1983;
Olsson et al., Meth. Enzymol., 92:3-I6, 1982). Chimeric antibody molecules may
be
prepared containing a mouse antigen-binding domain with human constant regions
(Morrison et al., Proc. Natl. Acad. Sci. U.S.A., 81:6851, 1984; Takeda et al.,
Nature,
10 314:452, 1985).
Various procedures known in the art also may be used for the production of
polycional antibodies. For the production of antibody, various host animals
including, but not limited to, rabbits, mice, rats, etc., can be immunized by
injection
with GDNFR protein, or a fragment or derivative thereof. Various adjuvants may
be
z5 used to increase the immunological response, depending on the host species
selected.
Useful adjuvants include, but are not limited to, Freund's (complete and
incomplete),
mineral gels such as aluminum hydroxide, surface active substances such as
lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole
limpet
hemocyanins, dinitrophenol, and human adjuvants such as BCG (Bacille Calmette-
2o Guerin) and Corynebacterium parvum.
A molecular clone of an antibody to a GDNFR epitope also may be prepared
by known techniques. Recombinant DNA methodology (see e.g., Maniatis et al.,
Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y., 1982) may be used to construct nucleic acid sequences
which
25 encode a monoclonal antibody molecule, or antigen binding region thereof.
Antibody molecules may be purified by known techniques, e.g.,
immunoabsorption or immunoaffinity chromatography, chromatographic methods
such as high performance liquid chromatography, or a combination thereof, etc.
The
present invention provides for antibody molecules as well as fragments of such
3o antibody molecules. Antibody fragments which contain the idiotype of the
molecule
can be generated by known techniques. For example, such fragments include but
are
not limited to: the F(ab')2 fragment which can be produced by pepsin digestion
of
the antibody molecule; the Fab' fragments which can be generated by reducing
the
disulfide bridges of the F(ab')2 fragment, and the Fab fragments which can be
generated by treating the antibody molecule with papain and a reducing agent.
Such selective binding molecules may themselves be alternatives to GDNFR
protein, and may be formulated as a pharmaceutical composition.
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/08486
26
Recombinant Expression of GDNFR Protein
The present invention provides various polynucleotides encoding GDNFR
proteins. The expression product or a derivative thereof is characterized by
the
ability to bind to GDNF or neurturin.so that further interactions with
signaling
molecules can occur, thereby providing or enhancing GDNF or neurturin activity
such as increasing dopamine uptake by dopaminergic cells. The polynucleotides
may also be used in cell therapy or gene therapy applications.
According to the present invention, novel GDNFR protein and DNA
l0 sequences coding for all or part of such receptors are provided. Novel
nucleic acid
sequences of the invention are useful in securing expression in procaryotic or
eucaryotic host cells of polypeptide products having at least a part of the
primary
structural conformation and one or more of the biological properties of
recombinant
human GDNFR. The nucleic acids may be purified and isolated, so that the
desired
15 coding region is useful to produce the present polypeptides. Alternatively,
the
nucleic acid sequence may be used for diagnostic purposes, as described more
fully
below. Exemplary DNA sequences of the present invention comprise nucleic acid
sequences encoding the GDNFR-a amino acid sequences depicted in Figures 2 and
4
and set forth in SEQ. ID NOs:2 and 4. In addition, DNA sequences disclosed by
the
2o present invention include: (a) the GDNFR DNA sequences depicted in the
Figures
(and complementary strands); (b) a DNA sequence which hybridizes (under
hybridization conditions disclosed in the cDNA library screening section
below, or
equivalent conditions or more stringent conditions) to the DNA sequence in
subpart
(a) or to fragments thereof; and (c) a DNA sequence which, but for the
degeneracy of
25 the genetic code, would hybridize to the DNA sequence in subpart (a).
Specifically
comprehended in parts (b) and (c) are genomic DNA sequences encoding allelic
variant forms of human GDNFR and/or encoding GDNFR from other mammalian
species, and manufactured DNA sequences encoding GDNFR, fragments of
GDNFR, and analogs of GDNFR which DNA sequences may incorporate codons
30 facilitating transcription and translation of messenger RNA in microbial
hosts. Such
manufactured sequences may readily be constructed according to the methods
known
in the art as well as the methods described herein.
Recombinant expression techniques, conducted in accordance with the
descriptions set forth herein or other known methods, may be used to produce
these
35 polynucleotides and express the various GDNFR proteins. For example, by
inserting
a nucleic acid sequence which encodes a GDNFR protein into an appropriate
vector,
one skilled in the art can readily produce large quantities of the desired
nucleotide
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/08486
27
sequence. The sequences can then be used to generate detection probes or
amplification primers. Alternatively, a polynucleotide encoding a GDNFR
protein
can be inserted into an expression vector. By introducing the expression
vector into
an appropriate host, the desired GDNFR protein may be produced in large
amounts.
As further described herein, there are numerous host/vector systems available
for the propagation of nucleic acid sequences and/or the production of GDNFR
proteins. These include, but are not limited to, plasmid, viral and
insertional vectors,
and prokaryotic and eukaryotic hosts. One skilled in the art can adapt a
host/vector
system which is capable of propagating or expressing heterologous DNA to
produce
to or express the sequences of the present invention.
By means of such recombinant techniques, the GDNFR proteins of the
present invention are readily produced in commercial quantities with greater
purity.
Furthermore, it will be appreciated by those skilled in the art that, in view
of the
present disclosure, the novel nucleic acid sequences include degenerate
nucleic acid
sequences encoding the GDNFR proteins specifically set forth in the Figures,
sequences encoding variants of GDNFR proteins, and those nucleic acid
sequences
which hybridize, preferably under stringent hybridization conditions, to
complements of these nucleic acid sequences (see, Maniatis et. al., Molecular
Cloning (A Laboratory Manual); Cold Spring Harbor Laboratory, pages 387 to
389,
2o 1982.) Exemplary stringent hybridization conditions are hybridization in 4
x SSC at
62-67°C, followed by washing in 0.1 x SSC at 62-67°C for
approximately an hour.
Alternatively, exemplary stringent hybridization conditions are hybridization
in 45-
55% formamide, 4 x SSC at 40-45°C. DNA sequences which hybridize to the
complementary sequences for GDNFR protein under relaxed hybridization
conditions and which encode a GDNFR protein of the present invention are also
included herein. Examples of such relaxed stringency hybridization conditions
are 4
x SSC at 45-55°C or hybridization with 30-40% formamide at 40-
45°C.
Preparation of Polynucleotides Encodin~GDNFR
3o Based upon the disclosure of the present invention, a nucleic acid sequence
encoding a full length GDNFR protein or a fragment thereof may readily be
prepared
or obtained by a variety of means, including, without limitation, chemical
synthesis,
cDNA or genomic library screening, expression library screening, and/or PCR
amplification of cDNA. These methods and others useful for preparing nucleic
acid
sequences are known in the art and are set forth, for example, by Sambrook et
al.
(Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY, 1989), by Ausubel et al., eds (Current Protocols in
CA 02291608 1999-11-26
WO 98/54213 PCTIUS98/08486
28
Molecular Biology, Current Protocols Press, 1994), and by Berger and Kimmel
(Methods in Enzymology: Guide to Molecular Cloning Techniques, vol. 152,
Academic Press, Inc., San Diego, CA, 1987). Preferred nucleic acid sequences
encoding GDNFR are mammalian sequences.
Chemical synthesis of a nucleic acid sequence which encodes a GDNFR
protein can also be accomplished using methods known in the art, such as those
set
forth by Engels et al. (Angew. Chem. Intl. Ed., 28:716-734, 1989). These
methods
include, inter alia, the phosphotriester, phosphoramidite and H-phosphonate
methods
of nucleic acid sequence synthesis. A preferred method for such chemical
synthesis
is polymer-supported synthesis using standard phosphoramidite chemistry.
Typically, the DNA encoding the desired polypeptide will be several hundred
base
pairs (bp) or nucleotides in length. Nucleic acid sequences larger than about
100
nucleotides can be synthesized as several fragments using these methods. The
fragments can then be ligated together to form a sequence for the expression
of a full
length GDNFR protein or a portion thereof.
Alternatively, a suitable nucleic acid sequence may be obtained by screening
an appropriate cDNA library (i.e., a library prepared from one or more tissue
source{s) believed to express the protein) or a genomic Library (a library
prepared
from total genomic DNA). The source of the cDNA library is typically a tissue
that
is believed to express GDNFR in reasonable quantities. Typically, the source
of the
genomic library is any tissue or tissues from a mammalian species believed to
harbor
a gene encoding GDNFR. The library can be screened for the presence of the
GDNFR cDNA/gene using one or more nucleic acid probes (such as
oligonucleotides, cDNA or genomic DNA fragments based upon the presently
disclosed sequences) that will hybridize selectively with GDNFR cDNA(s) or
genes) present in the library. The probes typically used for such library
screening
usually encode a small region of the GDNFR nucleic acid sequence from the same
or
a similar species as the species from which the library was prepared.
Alternatively,
the probes may be degenerate, as discussed herein.
Library screening is typically accomplished by annealing the oligonucleotide
probe or cDNA to the clones in the library under conditions of stringency that
prevent non-specific binding but permit binding (hybridization) of those
clones that
have a significant level of homology with the probe or primer. Typical
hybridization
and washing stringency conditions depend in part on the size (i.e., number of
nucleotides in length) of the cDNA or oligonucleotide probe, and whether the
probe
is degenerate. The probability of obtaining a clones) is also considered in
designing
the hybridization solution (e.g., whether a cDNA or genomic library is being
CA 02291608 1999-11-26
WO 98/54213 PCT/US98108486
29
screened; if it is a cDNA library, the probability that the cDNA of interest
is present
at a high level).
Where DNA fragments (such as cDNAs) are used as probes, typical
hybridization conditions include those as set forth in Ausubel et al., eds.,
supra.
After hybridization, the blot containing the library is washed at a suitable
stringency,
depending on several factors such as probe size, expected homologry of probe
to
clone, type of library being screened, number of clones being screened, and
the like.
Examples of stringent washing solutions (which are usually low in ionic
strength and
are used at relatively high temperatures) are as follows. One such stringent
wash is
to 0.015 M NaCI, 0.005 M NaCitrate and 0.1% SDS at 55-65°C. Another
such
stringent buffer is 1 mM Na2EDTA, 40 mM NaHP04, pH 7.2, and 1 % SDS at about
40-50°C. One other stringent wash is 0.2 X SSC and 0.1% SDS at about 50-
65°C.
There are also exemplary protocols for stringent washing conditions where
oligonucleotide probes are used to screen cDNA or genomic libraries. For
example,
a first protocol uses 6 X SSC with 0.05 percent sodium pyrophosphate at a
temperature of between about 35 and 62°C, depending on the length of
the probe.
For example, 14 base probes are washed at 35-40°C, 17 base probes at 45-
50°C, 20
base probes at 52-57°C, and 23 base probes at 57-63°C. The
temperature can be
increased 2-3°C where the background non-specific binding appears high.
A second
2o protocol uses tetramethylammonium chloride (TMAC) for washing. One such
stringent washing solution is 3 M TMAC, 50 mM Tris-HCI, pH 8.0, and 0.2% SDS.
Another suitable method for obtaining a nucleic acid sequence encoding a
GDNFR protein is by polymerase chain reaction (PCR). In this method,
poly(A)+RNA or total RNA is extracted from a tissue that expresses GDNFR. A
cDNA is then prepared from the RNA using the enzyme reverse transcriptase
(i.e.,
RT-PCR). Two primers, typically complementary to two separate regions of the
GDNFR cDNA (oligonucleotides), are then added to the cDNA along with a
polyrnerase such as Taq polymerase, and the polymerase amplifies the cDNA
region
between the two primers.
3o Where the method of choice for preparing the nucleic acid sequence encoding
the desired GDNFR protein requires the use of oligonucleotide primers or
probes
(e.g., PCR, cDNA or genomic library screening), the oligonucleotide sequences
selected as probes or primers should be of adequate length and sufficiently
unambiguous so as to minimize the amount of non-specific binding that will
occur
during library screening or PCR amplification. The actual sequence of the
probes or
primers is usually based on conserved or highly homologous sequences or
regions
from the same or a similar gene from another organism, such as the rat nucleic
acid
CA 02291608 1999-11-26
WO 98154213 PCT/US98/~8486
sequence involved in the present invention. Optionally, the probes or primers
can be
fully or partially degenerate, i.e., contain a mixture of probes/primers, all
encoding
the same amino acid sequence, but using different codons to do so. An
alternative to
preparing degenerate probes is to place an inosine in some or ail of those
codon
5 positions that vary by species. The oligonucleotide probes or primers may be
prepared by chemical synthesis methods for DNA as described above.
GDNFR proteins based on these nucleic acid sequences encoding GDNFR,
as well as mutant or variant sequences thereof, are also contemplated as
within the
scope of the present invention. Mutant or variant sequences include those
sequences
to containing one or more nucleotide substitutions, deletions, and/or
insertions as
compared to the wild type sequence and that results in the expression of amino
acid
sequence variations as compared to the wild type amino acid sequence. In some
cases, naturally occurring GDNFR amino acid mutants or variants may exist, due
to
the existence of natural allelic variation. GDNFR proteins based on such
naturally
15 occurnng mutants or variants are also within the scope of the present
invention.
Preparation of synthetic mutant sequences is also well known in the art, and
is
described for example in Wells et al. (Gene, 34:315, 1985) and in Sambrook et
al.,
supra.
In some cases, it may be desirable to prepare nucleic acid and/or amino acid
2o variants of naturally occurring GDNFR. Nucleic acid variants (wherein one
or more
nucleotides are designed to differ from the wild-type or naturally occurring
GDNFR)
may be produced using site directed mutagenesis or PCR amplification where the
primers) have the desired point mutations (see Sambrook et al., supra, and
Ausubel
et al., supra, for descriptions of mutagenesis techniques). Chemical synthesis
using
25 methods described by Engels et al., supra, may also be used to prepare such
variants.
Other methods known to the skilled artisan may be used as well. Preferred
nucleic
acid variants are those containing nucleotide substitutions accounting for
codon
preference in the host cell that is to be used to recombinantly produce GDNFR.
Other preferred variants are those encoding conservative amino acid changes
(e.g.,
30 wherein the charge or polarity of the naturally occurring amino acid side
chain is not
altered substantially by substitution with a different amino acid) as compared
to wild
type, and/or those designed to either generate a novel glycosylation andlor
phosphorylation sites) on GDNFR, or those designed to delete an existing
glycosylation and/or phosphorylation sites) on GDNFR. Yet other preferred
variants are those encoding a GDNFR based upon a GDNFR consensus sequence as
depicted in the Figures.
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/0848G
31
Vectors
The cDNA or genomic DNA encoding the desired GDNFR protein is
inserted into a vector for further cloning (amplification of the DNA) or for
expression. Suitable vectors are commercially available, or the vector may be
specially constructed. Possible vectors include, but are not limited to,
cosmids,
plasmids or modified viruses, but the vector system must be compatible with
the
selected host cell. Such vectors include, but are not limited to,
bacteriophages such
as lambda derivatives, or plasmids such as pBR322, pUC, or Bluescript~ plasmid
derivatives (Stratagene, La Jolla CA). The recombinant molecules can be
introduced
o into host cells via transformation, transfection, infection,
electroporation, or other
known techniques.
For example, the GDNFR-encoding nucleic acid sequence is inserted into a
cloning vector which is used to transform, transfect, or infect appropriate
host cells
so that many copies of the nucleic acid sequence are generated. This can be
accomplished by ligating a DNA fragment into a cloning vector which has
complementary cohesive termini. If the complementary restriction sites used to
fragment the DNA are not present in the cloning vector, the ends of the DNA
molecules may be enzymatically modified. It also may prove advantageous to
incorporate restriction endonuclease cleavage sites into the oligonucleotide
primers
2o used in polymerase chain reaction to facilitate insertion of the resulting
nucleic acid
sequence into vectors. Alternatively, any site desired may be produced by
ligating
nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may
comprise specific chemically synthesized oligonucleotides encoding restriction
endonuclease recognition sequences. In an alternative method, the cleaved
vector
and GDNFR-encoding nucleic acid sequence may be modified by homopolymeric
tailing. In specific embodiments, transformation of host cells with
recombinant
DNA molecules that incorporate an isolated GDNFR gene, cDNA, or synthesized
DNA sequence enables generation of multiple copies of the gene. Thus, the
GDNFR-encoding nucleic acid sequence may be obtained in large quantities by
growing transformants, isolating the recombinant DNA molecules from the
transformants and, when necessary, retrieving the inserted gene from the
isolated
recombinant DNA.
The selection or construction of the appropriate vector will depend on 1 )
whether it is to be used for DNA amplification or for DNA expression, 2) the
size of
the DNA to be inserted into the vector, and 3) the host cell (e.g., mammalian,
insect,
yeast, fungal, plant or bacterial cells) to be transformed with the vector.
Each vector
contains various components depending on its function (amplification of DNA or
CA 02291608 1999-11-26
WO 98/54~i3 PCT/US98/08486
32
expression of DNA) and its compatibility with the intended host cell. For DNA
expression, the vector components may include, but are not limited to, one or
more
of the following: a signal sequence, an origin of replication, one or more
selection or
marker genes, enhancer elements, promoters, a transcription termination
sequence,
and the like. These components may be obtained from natural sources or
synthesized by known procedures. The vectors of the present invention involve
a
nucleic acid sequence which encodes the GDNFR protein of interest operatively
linked to one or more amplification, expression control, regulatory or similar
operational elements capable of directing, controlling or otherwise effecting
the
1o amplification or expression of the GDNFR-encoding nucleic acid sequence in
the
selected host cell.
Expression vectors containing GDNFR nucleic acid sequence inserts can be
identified by three general approaches: (a) DNA-DNA hybridization; (b) the
presence or absence of "marker" gene functions, and (c) the expression of
inserted
sequences. In the first approach, the presence of a foreign nucleic acid
sequence
inserted in an expression vector can be detected by DNA-DNA hybridization
using
probes comprising sequences that are homologous to an inserted GDNFR-encoding
nucleic acid sequence. In the second approach, the recombinant vector/host
system
can be identified and selected based upon the presence or absence of certain
"marker" gene functions (e.g., thymidine kinase activity, resistance to
antibiotics,
transformation phenotype, occlusion body formation in baculovirus, etc.)
caused by
the insertion of a foreign nucleic acid sequence into the vector. For example,
if a
GDNFR-encoding nucleic acid sequence is inserted within the marker gene
sequence
of the vector, recombinants containing the GDNFR insert can be identified by
the
absence of the marker gene function. In the third approach, recombinant
expression
vectors can be identified by detecting the foreign protein product expressed
by the
recombinant nucleic acid sequence. Such assays can be based on the physical or
functional properties of the expressed GDNFR protein product, for example, by
binding of the GDNFR-a protein to GDNF or to an antibody which directly
3o recognizes GDNFR-a.
Signal Sequence
The signal sequence may be a component of the vector, or it may be a part of
GDNFR DNA that is inserted into the vector. The native GDNFR DNA encodes a
signal sequence at the amino terminus of the protein that is cleaved during
post-
translational processing of the protein to form the mature GDNFR protein.
Included
within the scope of this invention are GDNFR polynucleotides with the native
signal
CA 02291608 1999-11-26
WO 98/54213 PCTILIS98/08486
33
sequence as well as GDNFR polynucleotides wherein the native signal sequence
is
deleted and replaced with a heterologous signal sequence. The heterologous
signal
sequence selected should be one that is recognized and processed, i.e.,
cleaved by a
signal peptidase, by the host cell. For prokaryotic host cells that do not
recognize
and process the native GDNFR signal sequence, the signal sequence is
substituted by
a prokaryotic signal sequence selected, for example, from the group of the
alkaline
phosphatase, penicillinase, or heat-stable enterotoxin II leaders. For yeast
secretion,
the native GDNFR signal sequence may be substituted by the yeast invertase,
alpha
factor, or acid phosphatase leaders. In mammalian cell expression the native
signal
1o sequence is satisfactory, although other mammalian signal sequences may be
suitable.
Origin of Replication
Expression and cloning vectors generally include a nucleic acid sequence that
enables the vector to replicate in one or more selected host cells. In cloning
vectors,
this sequence is typically one that enables the vector to replicate
independently of the
host chromosomal DNA, and includes origins of replication or autonomously
replicating sequences. Such sequences are well known for a variety of
bacteria,
yeasts, and viruses. The origin of replication from the plasmid pBR322 is
suitable
2o for most Gram-negative bacteria and various origins (e.g., SV40, polyoma,
adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells.
Generally, the origin of replication component is not needed for mammalian
expression vectors (for example, the SV40 origin is often used only because it
contains the early promoter).
Selection Gene
The expression and cloning vectors may contain a selection gene. This gene
encodes a "marker" protein necessary for the survival or growth of the
transformed
host cells when grown in a selective culture medium. Host cells that were not
3o transformed with the vector will not contain the selection gene, and
therefore, they
will not survive in the culture medium. Typical selection genes encode
proteins that
(a) confer resistance to antibiotics or other toxins, e.g., ampicillin,
neomycin,
methotrexate, or tetracycline; (b) complement auxotrophic deficiencies; or (c)
supply
critical nutrients not available from the culture medium.
Other selection genes may be used to amplify the gene which will be
expressed. Amplification is the process wherein genes which are in greater
demand
for the production of a protein critical for growth are reiterated in tandem
within the
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/08486
34
chromosomes of successive generations of recombinant cells. Examples of
suitable
selectable markers for mammalian cells include dihydrofolate reductase (DHFR)
and
thymidine kinase. The mammalian cell transformants are placed under selection
pressure which only the transformants are uniquely adapted to survive by
virtue of
the marker present in the vector. Selection pressure is imposed by culturing
the
transformed cells under conditions in which the concentration of selection
agent in
the medium is successively changed, thereby leading to amplification of both
the
selection gene and the DNA that encodes GDNFR. As a result, increased
quantities
of GDNFR are synthesized from the amplified DNA.
For example, cells transformed with the DHFR selection gene are first
identified by culturing all of the transformants in a culture medium that
contains
methotrexate, a competitive antagonist of DHFR. An appropriate host cell when
wild-type DHFR is used is the Chinese hamster ovary cell line deficient in
DHFR
activity (see, for example, Urlaub and Chasin, Proc. Natl. Acad. Sci., U.S.A.,
77(7):
421b-4220, 1980). The transformed cells are then exposed to increased levels
of
methotrexate. This leads to the synthesis of multiple copies of the DHFR gene,
and,
concomitantly, multiple copies of other DNA present in the expression vector,
such
as the DNA encoding a GDNFR protein.
2o Promoter
The expression and cloning vectors of the present invention will typically
contain a promoter that is recognized by the host organism and operably linked
to the
nucleic acid sequence encoding the GDNFR protein. Promoters are untranslated
sequences located upstream (5') to the start codon of a structural gene
(generally
within about 100 to 1000 bp) that control the transcription and translation of
a
particular nucleic acid sequence, such as that encoding GDNFR. Promoters are
conventionally grouped into one of two classes, inducible promoters and
constitutive
promoters. Inducible promoters initiate increased levels of transcription from
DNA
under their control in response to some change in culture conditions, such as
the
presence or absence of a nutrient or a change in temperature. A large number
of
promoters, recognized by a variety of potential host cells, are well known.
These
promoters are operably linked to the DNA encoding GDNFR by removing the
promoter from the source DNA by restriction enzyme digestion and inserting the
desired promoter sequence into the vector. The native GDNFR promoter sequence
may be used to direct amplification and/or expression of GDNFR DNA. A
heterologous promoter is preferred, however, if it permits greater
transcription and
higher yields of the expressed protein as compared to the native promoter, and
if it is
CA 02291608 2003-O1-29
W O 98/54213
~ PCT/US98I08486
compatible with the host cell system that has been selected for use.
Promoters suitable for use with prokaryotic hosts include the beta-lactamase
and lactose promoter systems; alkaline phosphatase, a tryptophan (trp)
promoter:.:
system; and hybrid promoters such as the tac promoter. Other known bacterial
promoters are also suitable. Their nucleotide sequences have been published,
thereby enabling one skilled in the art to ligate them to the desired DNA
sequence(s),
using linkers or adaptors as needed to supply any required restriction sites.
Suitable promoting sequences for use with yeast hosts are also well known in
the art. Yeast enhancers are advantageously used with yeast promoters.
Suitable
promoters for use with mammalian host cells are well known and include those
obtained from the genomes of viruses such as polyoma virus, fowlpox virus,
adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma
virus,
cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian
Virus 40..
(SV40). Other suitable mammalian promoters include heterologous mammalian
15 promoters, e.g., heat-shock promoters and the actin promoter. A promoter
for
possible use in the production of GDNFR proteins in CHO cells is SRa (see
Takebe
et al., Mol. Cell. Biol., 8(1): 466-472, 1988). A suitable expression vector
is
pDSRa2. The pDSRa2 plasmid.constructs containing the appropriate GDNFR
cDNA may be prepared substantially in accordance with the process described in
the
20 U,S. Patent No. 5,714,465 (also see, Publication No. EP 398,753 and WO
90/14363
(1990)).
Additional promoters which may be of interest in controlling GDNFR
25 expression include, but are not limited to: the SV40 early promoter region
(Bernoist
and Chambon, Nature, 290:304-310, 1981); the CMV promoter; the promoter
contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et
al.,
Cell, 22:787-797, 1980); the herpes thymidine kinase promoter (Wagner et al.,
Proc.
Natl. Acad. Sci. U.S.A., 78:144-1445, 1981 ); the regulatory sequences of the
3o metallothionine gene (Brinster et al., Nature, 296:39-42, 1982);
prokaryotic
expression vectors such as the beta -lactamase promoter (Villa Kamaroff, et
al.,
Proc. Natl. Acad. Sci. U.S.A., 75:3727-3731, 1978); or the tac promoter
(DeBoer, et
al., Proc. Natl. Acad. Sci. U.S.A., 80:21-25, 1983). Also of interest are the
following
animal transcriptional control regions, which exhibit tissue specificity and
have been
35 utilized in transgenic animals: the elastase I gene control region which is
active in
pancreatic acinar cells (Swift et al., Cell, 38:639-646, 1984; Ornitz et al.,
Cold
Spring Harbor Symp. Quart. Biol. 50:399-409, 1986; MacDonald, Hepatology,
CA 02291608 1999-11-26
WO 98/54213 PCT/US98108486
36
7:425-515, 1987); the insulin gene control region which is active in
pancreatic beta
cells (Hanahan, Nature, 315:115-122, 1985); the immunoglobulin gene control
region which is active in lymphoid cells (Grosschedl et al., Cell, 38:647-658,
1984;
Adames et al., Nature, 318:533-538, 1985; Alexander et al., Mol. Cell. Biol.,
7:1436-
1444, I 987); the mouse mammary tumor virus control region which is active in
testicular, breast, lymphoid and mast cells (Leder et al., Cell, 45:485-495,
1986),
albumin gene control region which is active in liver (Pinkert et ai., Genes
and Devel.,
i :268-276, 1987); the alpha-fetoprotein gene control region which is active
in liver
(Krumlauf et al., Mol. Cell. Biol., 5:1639-1648, 1985; Hammer et al., Science,
to 235:53-58, 1987); the alpha 1-antitrypsin gene control region which is
active in the
liver (Kelsey et al., Genes and Devel., 1:161-171, 1987); the beta-globin gene
control
region which is active in myeloid cells (Mogram et al., Nature, 315:338-340,
1985;
Kollias et al., Cell, 46:89-94, 1986); the myelin basic protein gene control
region
which is active in oligodendrocyte cells in the brain (Readhead et al., Cell,
48:703-
712, 1987); the myosin light chain-2 gene control region which is active in
skeletal
muscle {Sani, Nature, 314:283-286, 1985); and the gonadotropic releasing
hormone
gene control region which is active in the hypothalamus (Mason et al.,
Science,
234:1372-1378, 1986).
Enhancer Element
An enhancer sequence may be inserted into the vector to increase the
transcription of a DNA sequence encoding a GDNFR protein of the present
invention by higher eukaryotes. Enhancers are cis-acting elements of DNA,
usually
about 10-300 by in length, that act on the promoter to increase its
transcription.
Enhancers are relatively orientation and position independent. They have been
found 5' and 3' to the transcription unit. Several enhancer sequences
available from
mammalian genes are known (e.g., globin, elastase, albumin, alpha-feto-protein
and
insulin). Typically, however, an enhancer from a virus will be used. The SV40
enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer,
and
3o adenovirus enhancers are exemplary enhancing elements for the activation of
eukaryotic promoters. While an enhancer may be spliced into the vector at a
position
5' or 3' to GDNFR DNA, it is typically located at a site 5' from the promoter.
Transcription Termination
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant,
animal, human, or nucleated cells from other multicellular organisms} will
also
contain sequences necessary for terminating transcription and stabilizing the
mRNA.
CA 02291608 1999-11-26
WO 98154213 PCTlUS98108486
37
Such sequences are commonly available from the 5' and occasionally 3'
untranslated
regions of eukaryotic DNAs or cDNAs. These regions contain nucleotide segments
transcribed as polyadenylated fragments in the untranslated portion of the
mRNA
encoding GDNFR.
The construction of suitable vectors containing one or more of the above-
listed components together with the desired GDNFR-encoding sequence is
accomplished by standard ligation techniques. Isolated plasmids or DNA
fragments
are cleaved, tailored, and religated in the desired order to generate the
plasmids
to required. To confirm that the correct sequences have been constructed, the
ligation
mixtures may be used to transform E. coli, and successful transformants may be
selected by known techniques, such as ampicillin or tetracycline resistance as
described above. Plasmids from the transformants may then be prepared,
analyzed
by restriction endonuclease digestion, and/or sequenced to confirm the
presence of
15 the desired construct.
Vectors that provide for the transient expression of DNA encoding GDNFR
in mammalian cells may also be used. In general, transient expression involves
the
use of an expression vector that is able to replicate efficiently in a host
cell, such that
the host cell accumulates many copies of the expression vector and, in turn,
2o synthesizes high levels of the desired protein encoded by the expression
vector.
Transient expression systems, comprising a suitable expression vector and a
host
cell, allow for the convenient positive identification of proteins encoded by
cloned
DNAs, as well as for the rapid screening of such proteins for desired
biological or
physiological properties. Thus, transient expression systems are particularly
useful
25 in identifying variants of the protein.
Selection and Transformation of Host Cells
Host cells (e.g., bacterial, mammalian, insect, yeast, or plant cells)
transformed with nucleic acid sequences for use in expressing a recombinant
3o GDNFR protein are also provided by the present invention. The transformed
host
cell is cultured under appropriate conditions permitting the expression of the
nucleic
acid sequence. The selection of suitable host cells and methods for
transformation,
culture, amplification, screening and product production and purification are
well
known in the art. See for example, Gething and Sambrook, Nature, 293: 620-625
35 (1981), or alternatively, Kaufman et al., Mol. Cell. Biol., 5 (7): 1750-
1759 (1985) or
Howley et al., U.S. Pat. No. 4,419,446. Additional exemplary materials and
methods
are discussed herein. The transformed host cell is cultured in a suitable
medium, and
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/08486
38
the expressed GDNFR protein is then optionally recovered, isolated and
purified
from the culture medium (or from the cell, if expressed intracellularly) by an
appropriate means known to those skilled in the art.
Different host cells have characteristic and specific mechanisms for the
translational and post-translational processing and modification (e.g.,
glycosylation,
cleavage) of proteins. Appropriate cell lines or host systems can be chosen to
ensure
the desired modification and processing of the foreign protein expressed. For
example, expression in a bacterial system can be used to produce an
unglycosylated
core protein product. Expression in yeast may be used to produce a
glycosylated
to product. Expression in mammalian cells can be used to ensure "native"
glycosylation of the heterologous GDNFR protein. Furthermore, different
vector/host expression systems may effect processing reactions such as
proteolytic
cleavages to different extents.
Suitable host cells for cloning or expressing the vectors disclosed herein are
i5 prokaryote, yeast, or higher eukaryote cells. Eukaryotic microbes such as
filamentous fungi or yeast may be suitable hosts for the expression of GDNFR
proteins. Saccharomyces cerevisiae, or common baker's yeast, is the most
commonly used among lower eukaryotic host microorganisms, but a number of
other
genera, species, and strains are well known and commonly available.
2o Host cells to be used for the expression of glycosylated GDNFR protein are
also derived from multicellular organisms. Such host cells are capable of
complex
processing and glycosylation activities. In principle, any higher eukaryotic
cell
culture might be used, whether such culture involves vertebrate or
invertebrate cells,
including plant and insect cells. The propagation of vertebrate cells in
culture (tissue
25 culture) is a well known procedure. Examples of useful mammalian host cell
lines
include, but are not limited to, monkey kidney CV 1 Iine transformed by SV40
(COS7), human embryonic kidney line (293 or 293 cells subcloned for growth in
suspension culture), baby hamster kidney cells, and Chinese hamster ovary
cells.
Other suitable mammalian cell lines include but are not limited to, HeLa,
mouse
3o L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK
hamster cell lines.
Suitable host cells also include prokaryotic cells. Prokaryotic host cells
include, but are not limited to, bacterial cells, such as Gram-negative or
Gram-
positive organisms, for example, E. coli, Bacilli such as B. subtilis,
Pseudomonas
35 species such as P. aeruginosa, Salmonella typhimurium, or Serratia
marcescans. For
example, the various strains of E. coli (e.g., HB 101, DHSa, DH10, and MC 1061
) are
well-known as host cells in the field of biotechnology. Various strains of
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/08486
39
Streptomyces spp. and the like may also be employed. Presently preferred host
cells
for producing GDNFR proteins are bacterial cells (e.g., Escherichia coli) and
mammalian cells (such as Chinese hamster ovary cells, COS cells, etc.)
The host cells are transfected and preferably transformed with the above-
s described expression or cloning vectors and cultured in a conventional
nutrient
medium. The medium may be modified as appropriate for inducing promoters,
selecting transformants, or amplifying the genes encoding the desired
sequences.
Transfection and transformation are performed using standard techniques which
are
well known to those skilled in the art and which are selected as appropriate
to the
1o host cell involved. For example, for mammalian cells without cell walls,
the calcium
phosphate precipitation method may be used. Electroporation, micro injection
and
other known techniques may also be used.
Culturing the Host Cells
15 Transformed cells used to produce GDNFR proteins of the present invention
are cultured in suitable media. The media may be supplemented as necessary
with
hormones andlor other growth factors (such as insulin, transfernn, or
epidermal
growth factor), salts (such as sodium chloride, calcium, magnesium, and
phosphate),
buffers (such as HEPES), nucleosides (such as adenosine and thymidine),
antibiotics
20 (such as gentamicin), trace elements (defined as inorganic compounds
usually
present at final concentrations in the micromolar range), and glucose or other
energy
source. Other supplements may also be included, at appropriate concentrations,
as
will be appreciated by those skilled in the art. Suitable culture conditions,
such as
temperature, pH, and the like, are also well known to those skilled in the art
for use
2s with the selected host cells.
Once the GDNFR protein is produced, it may be isolated and purified by
standard methods including chromatography (e.g., ion exchange, affinity, and
sizing
column chromatography), centrifugation, differential solubility, or by any
other
standard technique for the purification of proteins. For example, GDNFR-a
protein
3o may be isolated by binding to an affinity column comprising GDNF or anti-
GDNFR-
a antibody bound to a stationary support. Similarly, GRR2 protein may be
isolated
by binding to an affinity column comprising neurturin or anti-GRR2 antibody
bound
to a stationary support.
3s Homologous Recombination
It is further envisioned that GDNFR proteins may be produced by
homologous recombination, or with recombinant production methods utilizing
CA 02291608 2003-O1-29 _
WO 98/54213 ~ j PCT/US98/08486
. ~ 40
contml elements introduced into cells already containing DNA encoding GDNFR.
For example, homologous recombination methods may be used to modify a cell
that
contains a normally transcriptionally silent GDNFR gene or under expressed
gene
and thereby produce a cell which expresses GDNFR. Homologous recombination is
s a technique originally developed for targeting genes to induce or correct
mutations in
transcriptionally active genes (Kucherlapati, Prog, in Nucl. Acid Res. and
Mol. Biol.,
36:301, 1989). The basic technique was developed as a method for introducing
specific mutations into specific regions of the mammalian genome (Thomas et
al.,
Cell, 44:419-428, 1986; Thomas and Capecchi, Cell, 51:503-512, 1987;
Doetschman
1o et al., Proc. Natl. Acad. Sci., 85:8583-8587, 1988) or to correct specific
mutations
within defective genes (Doetschman et al., Nature, 330:576-578, 1987).
Exemplary
homologous recombination techniques are described in U.S. 5,272,071
(EP 91 90 3051, EP Publication No. 505 500; PCTlCTS90/07642, International
Publication No. WO 91/09955) .
1s
Through homologous recombination, the DNA sequence to be inserted into
the genome can be directed to a specific region of the gene of interest by
attaching:it
to targeting DNA. The targeting DNA is DNA that is complementary (homologous)
to a region of the genornic DNA. Small pieces of targeting DNA that are
2o complementary to a specific region of the genome are put in contact with
the
parental strand during the DNA replication process. It is a general property
of DNA
that has been inserted into a cell to hybridize, and therefore, recombine with
other
pieces of endogenous DNA through shared homologous regions. If this
complementary strand is attached to an oligonucleotide that contains a
mutation or a
25 different sequence of DNA, it too is incorporated into the newly
synthesized strand
as a result of the recombination. As a result of the proo&eading function, it
is
possible for the new sequcnce of DNA to serve as the template. Thus, the
transferred
DNA is incorporated into the genome.
If the sequence of a particular gene is known, such as the nucleic acid
30 sequence, the pre-pm sequence or expression control sequence of GDNFR
presented
herein, a piece of DNA that is complementary to a selected region of the gene
can be
synthesized or otherwise obtained, such as by appropriate restriction of the
native
DNA at specific recognition sites bounding the region of interest. This piece
serues
as a targeting sequence upon insertion into the cell and will hybridize to its
35 homologous region within the genome. If this hybridization occurs during
DNA
replication, this piece of DNA, and any additional sequence attached thereto,
will act
as an Okazaki fragment and will be backstitched into the newly synthesized
daughter
--~ CA 02291608 2003-O1-29
WO 98/54213
PCT/US98/08486
41
strand of DNA.
Attached to these pieces of targeting DNA are regions of DNA which may
interact with the expression of a GDNFR protein. For example, a
promoter/enharicer
element, a suppresser, or an exogenous transcription modulatory element is
inserted
s in the genome of the intended host. cell in proximity and orientation
sufficient to
influence the transcription of DNA encoding the desired GDNFR protein. The
control element does not encode GDNFR, but instead controls a portion of the
DNA
present in the host cell genome. Thus, the expression of GDNFR proteins may be
achieved not by transfection of DNA that encodes the GDNFR gene itself, but
rather
1 o by the use of targeting DNA (containing regions of homology with the
endogenous
gene of interest) coupled with DNA regulatory segments that provide the
endogenous gene sequence with recognizable signals for transcription of a
GDNFR
protein.
15 A. GDNFR varian4,t~
As discussed above, the terms "GDNFR analogs" as used herein include
polypeptides in which amino acids have been deleted from ("deletion
variants"),
inserted into ("addition variants"), or substituted for ~"substitution
variants") residues
within the amino acid sequence of naturally-occurring GDNFR polypeptides
2o including those depicted in the Figures. Such variants are prepared by
introducing
appropriate nucleotide chaages into the DNA encoding the polypeptide or by in
vitro
chemical synthesis of the desired polypeptide. It will be appreciated by those
skilled
in the art that many combinations of deletions, insertions, and substitutions
can be
made to an amino acid sequence such as mature human GDNFR provided that the
25 final molecule possesses GDNFR activity.
Based upon~the present description of particular GDNFR-a, GRR2 and
GRR3 amino acid sequences from multiple species, as well as the consensus
sequences derived therefrom, one can readily design and manufacture a variety
of
nucleic acid sequences suitable for use in the recombinant (e.g., microbial)
3o expression of polypeptides having primary conformations which differ from
those
depicted in the Figures in terms of the identity or location of one or more
residues.
Mutagenesis techniques for the replacement, insefion or deletion of one or
more
selected amino acid residues encoded by the nucleic acid sequences depicted in
Figures 2, and 4 are well lmown to one skilled in the art (e.g., U.S. Pat. No.
35 4,518,584). There are
two principal variables in the construction of substitution variants: the
location of
the mutation site and the nature of the mutation. In designing GDNFR
substitution
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/08486
42
variants, the selection of the mutation site and nature of the mutation will
depend on
the GDNFR characteristics) to be modified. The sites for mutation can be
modified
individually or in series, e.g., by ( 1 ) substituting first with conservative
amino acid
modifications and then with more radical selections depending upon the results
achieved, (2) deleting the target amino acid residue, or (3) inserting amino
acid
residues adjacent to the located site. Conservative changes in from 1 to 30
contiguous amino acids are preferred. N-terminal and C-terminal deletion GDNFR
protein variants may also be generated by proteolytic enzymes.
For GDNFR deletion variants, deletions generally range from about 1 to 30
1 o contiguous residues, more usually from about 1 to 10 contiguous residues,
and
typically from about 1 to 5 contiguous residues. N-terminal, C-terminal and
internal
intrasequence deletions are contemplated. Deletions may be introduced into
regions
of the molecule which have low homology with non-human GDNFR to modify the
activity of GDNFR. Deletions in areas of substantial homology with non-human
15 GDNFR sequences will be more likely to significantly modify GDNFR
biological
activity. The number of consecutive deletions typically will be selected so as
to
preserve the tertiary structure of the GDNFR protein product in the affected
domain,
e.g., cysteine crosslinking. Non-limiting examples of deletion variants
include
truncated GDNFR protein products lacking N-terminal or C-terminal amino acid
2o residues. For example, one may prepare a soluble receptor by elimination of
the
peptide region involved in a glycosyl-phosphatidylinositol (GPI) anchorage of
GDNFR receptor to the cytoplasrnic membrane.
For GDNFR addition variants, amino acid sequence additions typically
include N-andlor C-terminal fusions or terminal additions ranging in length
from one
25 residue to polypeptides containing a hundred or more residues, as well as
internal or
medial additions of single or multiple amino acid residues. Polypeptides of
the
invention may also include an initial methionine amino acid residue (at
position -1
with respect to the first amino acid residue of the desired polypeptide).
Internal
additions may range generally from about 1 to 10 contiguous residues, more
3~ typically from about 1 to S residues, and usually from about 1 to 3 amino
acid
residues. Examples of N-terminal addition variants include GDNFR with the
inclusion of a heterologous N-terminal signal sequence to the N-terminus of
GDNFR
to facilitate the secretion of mature GDNFR from recombinant host cells and
thereby
facilitate harvesting or bioavailability. Such signal sequences generally will
be
35 obtained from, and thus be homologous to, the intended host cell species.
Additions
may also include amino acid sequences derived from the sequence of other
neurotrophic factors. For example, it is contemplated that a fusion protein of
GDNF
CA 02291608 1999-11-26
WO 98154213 PCT/US9810848G
43
and GDNFR-a, or neurturin and GRR2, may be produced, with or without a linking
sequence, thereby forming a single molecule therapeutic entity.
GDNFR substitution variants have one or more amino acid residues of the
GDNFR amino acid sequence removed and a different residues) inserted in its
place. Such substitution variants include allelic variants, which are
characterized by
naturally-occurring nucleotide sequence changes in the species population that
may
or may not result in an amino acid change. As with the other variant forms,
substitution variants may involve the replacement of single or contiguous
amino acid
residues at one or more different locations.
Specific mutations of the GDNFR amino acid sequence may involve
modifications to a glycosylation site (e.g., serine, threonine, or
asparagine). The
absence of glycosylation or only partial glycosylation results from amino acid
substitution or deletion at any asparagine-linked glycosylation recognition
site or at
any site of the molecule that is modified by addition of an O-linked
carbohydrate.
An asparagine-linked glycosylation recognition site comprises a tripeptide
sequence
which is specifically recognized by appropriate cellular glycosylation
enzymes.
These tripeptide sequences are either Asn-Xaa-Thr or Asn-Xaa-Ser, where Xaa
can
be any amino acid other than Pro. A variety of amino acid substitutions or
deletions
at one or both of the first or third amino acid positions of a glycosylation
recognition
site (and/or amino acid deletion at the second position) result in non-
glycosylation at
the modified tripeptide sequence. Thus, the expression of appropriate altered
nucleotide sequences produces variants which are not glycosylated at that
site.
Alternatively, the GDNFR amino acid sequence may be modified to add
glycosylation sites.
One method for identifying GDNFR amino acid residues or regions for
mutagenesis is called "alanine scanning mutagenesis" as described by
Cunningham
and Wells (Science, 244: 1081-1085, 1989). In this method, an amino acid
residue
or group of target residues are identified (e.g., charged residues such as
Arg, Asp,
His, Lys, and Glu) and replaced by a neutral or negatively charged amino acid
(most
3o preferably alanine or polyalanine) to affect the interaction of the amino
acids with
the surrounding aqueous environment in or outside the cell. Those domains
demonstrating functional sensitivity to the substitutions may then be refined
by
introducing additional or alternate residues at the sites of substitution.
Thus, the
target site for introducing an amino acid sequence variation is determined,
alanine
scanning or random mutagenesis is conducted on the corresponding target codon
or
region of the DNA sequence, and the expressed GDNFR variants are screened for
the
optimal combination of desired activity and degree of activity.
CA 02291608 1999-11-26
WO 98154213 PCTi'US98/08486
44
The sites of greatest interest for substitutional mutagenesis include sites
where the amino acids found in GDNFR proteins from various species are
substantially different in terms of side-chain bulk, charge, and/or
hydrophobicity.
Other sites of interest are those in which particular residues of GDNFR-like
proteins,
obtained from various species, are.identical. Such positions are generally
important
for the biological activity of a protein. Initially, these sites are
substituted in a
relatively conservative manner. Such conservative substitutions are shown in
Table 2 under the heading of preferred substitutions. If such substitutions
result in a
change in biological activity, then more substantial changes (exemplary
1 o substitutions) may be introduced, and/or other additions or deletions may
be made,
and the resulting products are screened for activity.
TABLE 2
Amino Acid Substitutions
Original Residue Preferred Substitutions Exemplary Substitutions
Ala (A) Val Val; Leu; Ile
Arg (R) Lys Lys; Gln; Asn
Asn (N) Gln Gln; His; Lys; Arg
Asp (D) Glu Glu
Cys (C) Ser Ser
Gln (Q) Asn Asn
Glu (E) Asp Asp
Gly (G) Pro Pro
His (H) Arg Asn; Gln; Lys; Arg
Ile (I) Leu Leu; Val; Met; Ala; Phe;
norleucine
Leu (L) Ile norleucine; Ile; Val; Met;
Ala;
Phe
Lys (K) Arg Arg; Gln; Asn
Met (M) Leu Leu; Phe; Ile
Phe (F) Leu Leu; Val; Ile; Ala
Pro (P) Gly Gly
Ser (S) Thr Thr
Thr (T) Ser Ser
Trp (W) Tyr Tyr
CA 02291608 1999-11-26
WO 98!54213 PCT/US98108486
Tyr (Y) Phe Trp; Phe; Thr; Ser
Val (V) Leu Ile; Leu; Met; Phe; Ala;
norleucine
Conservative modifications to the amino acid sequence {and the
corresponding modifications to the encoding nucleic acid sequences) are
expected to
produce GDNFR protein products having functional and chemical characteristics
5 similar to those of naturally occurring GDNFR. In contrast, substantial
modifications in the functional and/or chemical characteristics of GDNFR
protein
products may be accomplished by selecting substitutions that differ
significantly in
their effect on maintaining (a) the structure of the polypeptide backbone in
the area
of the substitution, for example, as a sheet or helical conformation, (b) the
charge or
10 hydrophobicity of the molecule at the target site, or (c) the bulk of the
side chain.
Naturally occurnng residues may be divided into groups based on common side
chain properties:
1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;
2) neutral hydrophilic: Cys, Ser, Thr;
15 3) acidic: Asp, Glu;
4) basic: Asn, Gln, His, Lys, Arg;
5) residues that influence chain orientation: Gly, Pro; and
b) aromatic: Trp, Tyr, Phe.
Non-conservative substitutions may involve the exchange of a member of
20 one of these classes for a member from another class. Such substituted
residues may
be introduced into regions of the human GDNFR protein that are homologous with
non-human GDNFR proteins, or into the non-homologous regions of the molecule.
Thus, GDNFR proteins include those biologically active molecules
containing all or part of the amino acid sequences as depicted in the Figures,
as well
25 as consensus and modified sequences in which biologically equivalent amino
acid
residues are substituted for residues within the sequence resulting in a
silent change.
For example, one or more amino acid residues within the sequence can be
substituted
by another amino acid of a similar polarity which acts as a functional
equivalent,
resulting in a silent alteration. Substitutes for an amino acid within the
sequence
3o may be selected from other members of the class to which the amino acid
belongs.
For example, the nonpolar {hydrophobic) amino acids include alanine, leucine,
isoleucine, valine, proiine, phenylalanine, tryptophan and methionine. The
polar
neutral amino acids include glycine, serine, threonine, cysteine, tyrosine,
asparagine,
and glutamine. The positively charged (basic) amino acids include arginine,
lysine
CA 02291608 1999-11-26
WO 98154213 PCTIUS98/08486
46
and histidine. The negatively charged (acidic) amino acids include aspartic
acid and
glutamic acid. It is also contemplated that the GDNFR proteins, analogs, or
fragments or derivatives thereof may be differentially modified during or
after
translation, e.g., by phosphorylation, glycosylation, crosslinking, acylation,
proteolytic cleavage, linkage to an.antibody molecule, membrane molecule or
other
ligand.
B. GDNFR Derivatives
Chemically modified derivatives of GDNFR or GDNFR analogs may be
o prepared by one of skill in the art based upon the present disclosure. The
chemical
moieties most suitable for derivatization include water soluble polymers. A
water
soluble polymer is desirable because the protein to which it is attached does
not
precipitate in an aqueous environment, such as a physiological environment.
Preferably, the polymer will be pharmaceutically acceptable for the
preparation of a
15 therapeutic product or composition. One skilled in the art will be able to
select the
desired polymer based on such considerations as whether the polymer/protein
conjugate will be used therapeutically, and if so, the desired dosage,
circulation time,
resistance to proteolysis, and other considerations. The effectiveness of the
derivatization may be ascertained by administering the derivative, in the
desired
20 form {e.g., by osmotic pump, or, more preferably, by injection or infusion,
or, further
formulated for oral, pulmonary or other delivery routes), and determining its
effectiveness.
Suitable water soluble polymers include, but are not limited to, polyethylene
glycol, copolymers of ethylene glycol/propylene glycol,
carboxymethyIcellulose,
25 dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane,
poly-1,3,6-
trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either
homopolymers or random copolymers), and dextran or poly(n-vinyl
pyrrolidone)polyethylene glycol, propropylene glycol homopolymers,
prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols
(e.g.,
3o glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol
propionaldehyde may have advantages in manufacturing due to its stability in
water.
The polymer may be of any molecular weight, and may be branched or
unbranched. For polyethylene glycol, the preferred molecular weight is between
about 2 kDa and about 100 kDa for ease in handling and manufacturing (the term
35 "about" indicating that in preparations of polyethylene glycol, some
molecules will
weigh more, some less, than the stated molecular weight). Other sizes may be
used,
depending on the desired therapeutic profile (e.g., the duration of sustained
release
t
CA 02291608 2003-O1-29
WO 98/54213
PCT/US98/08486
47
desired; the effects, if any, on biological activity; the ease in handling;
the degree or
lack of antigenicity and other known effects of polyethylene glycol on a
therapeutic
protein or variant). ,
The number of polymer molecules so attached may vary, and one skilled in
the art will be able to ascertain the effect on function. One may mono-
derivatize, or
may provide for a dl-, tri-, tetra- or some combination of derivatizadon, with
the
same or different chemical moieties (e.g., polymers, such as different weights
of
polyethylene glycols). The proportion of polymer molecules to protein (or
peptide)
molecules will vary, as will their concentrations in the reaction mixture. In
general,
1o the optimumratio (in terms of efficiency of reaction in that there is no
excess
unreacted protein or polymer) will be determined by factors such as the
desired ' .
degree of derivatization (e.g., mono, dl-, tri-, etc.), the molecular weight
of the
polymer selected, whether the polymer is branched or unbranched, and the
reaction
conditions.
The polyethylene glycol molecules (or other chemical moieties) should be
attached to the protein with consideration of effects on functional or
antigenic
domains of the protein. There are a number of attachment methods available to
those
skilled in the art. See for example, EP 0 401 3 84~
(coupling PEG to G-CSF), see also Malik et al., Exp.
2o Hematol., 20: 1028-1035, 1992 {reporting pegylation of GM-CSF using tresyl
chloride). For example, polyethylene glycol may be covalently bound through
amino acid residues via a reactive group, such as, a free amino or carboxyl
group.
Reactive groups are those to which an activated polyethylene glycol molecule
may
be bound. The amino acid residues having a free amino group may include lysine
residues and the N-terminal amino acid residue. Those having a free carboxyl
group
may include aspartic acid residues, glutamic acid residues, and the C-terminal
amino
acid residue. Sulfhydrl groups may also be used as a reactive group for
attaching the
polyethylene glycol molecule(s). For therapeutic purposes, attachment at an
amino
group, such as attachment at the N-terminus or lysine group is preferred.
Attachment
34 at residue's important for receptor binding should be avoided if receptor
binding is
desired.
One may specifically desire an N-terminal chemically modified protein.
Using polyethylene glycol as an illustration of the present compositions, one
may
select from a variety of polyethylene glycol molecules (by molecular weight,
branching, etc.), the proportion of polyethylene glycol molecules to protein
(or
peptide) molecules in the reaction mix, the type of pegylation reaction to be
performed, and the method of obtaining the selected N-terminally pegylated
protein.
CA 02291608 2003-O1-29
WO 98/5d213 p~/Ug9g~08486
48
The method of obtaining the N-terminally pegylated preparation (i.e.,
separating this
moiety from other monopegylated moieties if necessary) may be by purification
of
the N-terminally pegylated material from a population of pegylated protein
molecules. Selective N-terminal chemical modification may be accomplished by
reductive alkylation which exploits.differential reactivity of different types
of
primary amino groups (lysine versus the N-terminal) available for
derivatization in a
particular protein. Under the appropriate reaction conditions, substantially
selective
derivatization of the protein at the N-terminus with a carbonyl group
containing
polymer is achieved. For example, one may selectively N-terminally pegylate
the
to protein by performing the reaction at a pH which allows one to take
advantage of the
pKa differences between the e-amino group of the lysine residues and that of
the a-
amino group of the N-terminal residue of the protein. By such selective
derivatization, attachment of a water soluble polymer to a protein is
controlled: the
conjugation with the polymer takes place predominantly at the N-terminus of
the
protein and no significant modification of other reactive groups, such as the
lysine
side chain amino groups, occurs. Using reductive alkyladon, the water soluble
polymer may be of the type described above, and should have a single reactive
aldehyde for coupling to the protein. Polyethylene glycol propionaldehyde,
containing a single reactive aldehyde, may be used.
. The present invention contemplates use of derivatives which are
prokaryote-expressed GDNFR proteins, or variants thereof, linked to at least
one
polyethylene glycol molecule, as well as use of GDNFR proteins, or variants
thereof,
attached to one or more polyethylene glycol molecules,via an acyl or alkyl
linkage.
Pegylation may be carried out by any of the pegylation reactions known in
the art. See, for example: Focus on Growth Factors, 3 {2): 4-10, 1992;
EP 0 154 316, EP 0 401 384;
and the other publications cited herein that relate to pegylation. The
pegylation
may be carried out via an acylation reaction or an alkylation reaction with a
reactive
polyethylene glycol molecule (or an analogous reactive water-soluble polymer).
3o Pegylation by acylation generally involves reacting an active ester
derivative
of polyethylene glycol (PEG) with the GDNFR protein or variant. Any known or
subsequently discovered reactive PEG molecule may be use to carry out the
pegylation of GDNFR protein or variant. A preferred activated PEG ester is PEG
esterified to N-hydroxysuccinimide (NHS). As used herein, "acylation" is
contemplated to include without limitation the following types of linkages
between
the therapeutic protein and a water soluble polymer such as PEG: amide,
carbamate,
urethane, and the like. See Bioconjugate Chem., 5: 133-140, 1994. Reaction
CA 02291608 1999-11-26
WO 98154213 PCTIUS98/08486
49
conditions may be selected from any of those known in the pegylation art or
those
subsequently developed, but should avoid conditions such as temperature,
solvent,
and pH that would inactivate the GDNFR or variant to be modified.
Pegylation by acylation will generally result in a poly-pegylated GDNFR
protein or variant. Preferably, the connecting linkage will be an amide. Also
preferably, the resulting product will be substantially only (e.g., > 95%)
mono, dl- or
tri-pegylated. However, some species with higher degrees of pegylation may be
formed in amounts depending on the specific reaction conditions used. If
desired,
more purified pegylated species may be separated from the mixture,
particularly
1o unreacted species, by standard purification techniques, including, among
others,
dialysis, salting-out, ultrafiltration, ion-exchange chromatography, gel
filtration
chromatography and electrophoresis.
Pegylation by alkylation generally involves reacting a terminal aldehyde
derivative of PEG with the GDNFR protein or variant in the presence of a
reducing
t 5 agent. Pegylation by alkylation can also result in poly-pegylated GDNFR
protein or
variant. In addition, one can manipulate the reaction conditions to favor
pegylation
substantially only at the a-amino group of the N-terminus of the GDNFR protein
or
variant (i.e., a mono-pegylated protein). In either case of monopegylation or
polypegylation, the PEG groups are preferably attached to the protein via a
20 -CH:-NH- group. With particular reference to the -CHz- group, this type of
linkage
is referred to herein as an "alkyl" linkage.
Derivatization via reductive alkylation to produce a monopegylated product
exploits differential reactivity of different types of primary amino groups
(lysine
versus the N-terminal) available for derivatization. The reaction is performed
at a
25 pH which allows one to take advantage of the pKa differences between the e-
amino
groups of the lysine residues and that of the a-amino group of the N-terminal
residue
of the protein. By such selective derivatization, attachment of a water
soluble
polymer that contains a reactive group such as an aldehyde, to a protein is
controlled:
the conjugation with the polymer takes place predominantly at the N-terminus
of the
3o protein and no significant modification of other reactive groups, such as
the lysine
side chain amino groups, occurs. In one important aspect, the present
invention
contemplates use of a substantially homogeneous preparation of
monopolymer/GDNFR protein (or variant) conjugate molecules (meaning GDNFR
protein or variant to which a polymer molecule has been attached substantially
only
35 (i.e., > 95%) in a single location). More specifically, if polyethylene
glycol is used,
the present invention also encompasses use of pegylated GDNFR protein or
variant
lacking possibly antigenic linking groups, and having the polyethylene glycol
CA 02291608 1999-11-26
WO 98/54213 PCT/LTS98108486
molecule directly coupled to the GDNFR protein or variant.
Thus, GDNFR protein products according to the present invention include
pegylated GDNFR protein or variants, wherein the PEG groups) is (are) attached
via
acyl or alkyl groups. As discussed above, such products may be mono-pegylated
or
5 poly-pegylated (e.g., containing 2-6, and preferably 2-5, PEG groups). The
PEG
groups are generally attached to the protein at the a- or e-amino groups of
amino
acids, but it is also contemplated that the PEG groups could be attached to
any amino
group attached to the protein, which is sufficiently reactive to become
attached to a
PEG group under suitable reaction conditions.
1o The polymer molecules used in both the acylation and alkylation approaches
may be selected from among water soluble polymers as described above. The
polymer selected should be modified to have a single reactive group, such as
an
active ester for acylation or an aldehyde for alkylation, preferably, so that
the degree
of polymerization may be controlled as provided for in the present methods. An
15 exemplary reactive PEG aldehyde is polyethylene glycol propionaldehyde,
which is
water stable, or mono C 1-C 10 alkoxy or aryloxy derivatives thereof (see,
U.S. Patent
5,252,714). The polymer may be branched or unbranched. For the acylation
reactions, the polymers) selected should have a single reactive ester group.
For the
present reductive alkylation, the polymers) selected should have a single
reactive
20 aldehyde group. Generally, the water soluble polymer will not be selected
from
naturally-occurring glycosyl residues since these are usually made more
conveniently by mammalian recombinant expression systems. The polymer may be
of any molecular weight, and may be branched or unbranched.
An exemplary water-soluble polymer for use herein is polyethylene glycol.
25 As used herein, polyethylene glycol is meant to encompass any of the forms
of PEG
that have been used to derivatize other proteins, such as mono-(C 1-C 10)
alkoxy- or
aryloxy-polyethylene glycol.
In general, chemical derivatization may be performed under any suitable
condition used to react a biologically active substance with an activated
polymer
30 molecule. Methods for preparing a pegylated GDNFR protein product will
generally
comprise the steps of (a) reacting a GDNFR protein product with polyethylene
glycol (such as a reactive ester or aldehyde derivative of PEG) under
conditions
whereby the protein becomes attached to one or more PEG groups, and (b)
obtaining
the reaction product(s). In general, the optimal reaction conditions for the
acylation
35 reactions will be determined case-by-case based on known parameters and the
desired result. For example, the larger the ratio of PEG:protein, the greater
the
percentage of poly-pegylated product.
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/08486
51
Reductive alkylation to produce a substantially homogeneous population of
mono-polymer/GDNFR protein product will generally comprise the steps of:
(a) reacting a GDNFR protein or variant with a reactive PEG molecule under
reductive alkylation conditions, at a pH suitable to permit selective modif
cation of
the a-amino group at the amino terminus of said GDNFR protein or variant; and
(b) obtaining the reaction product(s).
For a substantially homogeneous population of mono-polymer/GDNFR
protein product, the reductive alkylation reaction conditions are those which
permit
the selective attachment of the water soluble polymer moiety to the N-terminus
of
1o GDNFR protein or variant. Such reaction conditions generally provide for
pKa
differences between the lysine amino groups and the a-amino group at the N-
terminus {the pKa being the pH at which 50% of the amino groups are protonated
and 50% are not). The pH also affects the ratio of polymer to protein to be
used. In
general, if the pH is lower, a larger excess of polymer to protein will be
desired (i.e.,
the less reactive the N-terminal a-amino group, the more polymer needed to
achieve
optimal conditions). If the pH is higher, the polymer:protein ratio need not
be as
large (i.e., more reactive groups are available, so fewer polymer molecules
are
needed). For purposes of the present invention, the pH will generally fall
within the
range of 3-9, preferably 3-6.
2o Another important consideration is the molecular weight of the polymer. In
general, the higher the molecular weight of the polymer, the fewer polymer
molecules may be attached to the protein. Similarly, branching of the polymer
should be taken into account when optimizing these parameters. Generally, the
higher the molecular weight (or the more branches) the higher the
polymer:protein
ratio. In general, for the pegylation reactions contemplated herein, the
preferred
average molecular weight is about 2 kDa to about 100 kDa. The preferred
average
molecular weight is about 5 kDa to about 50 kDa, particularly preferably about
12
kDa to about 25 kDa. The ratio of water-soluble polymer to GDNF protein or
variant will generally range from 1:1 to 100:1, preferably (for
polypegylation) 1:1 to
20:1 and (for monopegylation) 1:1 to S:l .
Using the conditions indicated above, reductive alkylation will provide for
selective attachment of the polymer to any GDNFR protein or variant having an
a-amino group at the amino terminus, and provide for a substantially
homogenous
preparation of monopolymer/GDNFR protein (or variant) conjugate. The term
"monopolymer/GDNFR protein {or variant) conjugate" is used here to mean a
composition comprised of a single polymer molecule attached to a molecule of
GDNFR protein or GDNFR variant protein. The monopolymer/GDNFR protein (or
CA 02291608 1999-11-26
WO 98/542/3 PCT/US98/08486
52
variant) conjugate typically will have a polymer molecule located at the N-
terminus,
but not on lysine amino side groups. The preparation will generally be greater
than
90% monopolymer/GDNFR protein (or variant) conjugate, and more usually greater
than 95% monopolymer/GDNFR protein (or variant) conjugate, with the remainder
of observable molecules being unreacted (i.e., protein lacking the polymer
moiety).
It is also envisioned that the GDNFR protein product may involve the
preparation of
a pegylated molecule involving a fusion protein or linked GDNFR and
neurotrophic
factor, such as GDNFR-a and GDNF molecules or GRR2 and neurturin molecules.
For the present reductive alkylation, the reducing agent should be stable in
1o aqueous solution and preferably be able to reduce only the Schiff base
formed in the
initial process of reductive alkylation. Suitable reducing agents may be
selected
from sodium borohydride, sodium cyanoborohydride, dimethylamine borane,
trimethylamine borane and pyridine borane. A particularly suitable reducing
agent is
sodium cyanoborohydride. Other reaction parameters, such as solvent, reaction
15 times, temperatures, etc., and means of purification of products, can be
determined
case-by-case based on the published information relating to derivatization of
proteins
with water soluble polymers (see the publications cited herein).
C. GDNFR Protein Product Pharmaceutical Compositions
20 GDNFR protein product pharmaceutical compositions typically include a
therapeutically or prophylactically effective amount of GDNFR protein product
in
admixture with one or more pharmaceutically and physiologically acceptable
formulation materials selected for suitability with the mode of
administration.
Suitable formulation materials include, but are not limited to, antioxidants,
25 preservatives, coloring, flavoring and diluting agents, emulsifying agents,
suspending agents, solvents, fillers, bulking agents, buffers, delivery
vehicles,
diluents, excipients and/or pharmaceutical adjuvants. For example, a suitable
vehicle may be water for injection, physiological saline solution, or
artificial
cerebrospinal fluid, possibly supplemented with other materials common in
3o compositions for parenteral administration. Neutral buffered saline or
saline mixed
with serum albumin are further exemplary vehicles. The term "pharmaceutically
acceptable carrier" or "physiologically acceptable carrier" as used herein
refers to a
formulation materials) suitable for accomplishing or enhancing the delivery of
the
GDNFR protein product as a pharmaceutical composition.
35 The primary solvent in a vehicle may be either aqueous or non-aqueous in
nature. in addition, the vehicle may contain other formulation materials for
modifying or maintaining the pH, osmolarity, viscosity, clarity, color,
sterility,
CA 02291608 2003-O1-29
W O 98/54213
PCT/US98/08486
53
stability, rate of dissolution, or odor of the formulation. Similarly, the
vehicle may
contain additional formulation materials for modifying or maintaining the rate
of :._
release of GDNFR protein product, or for promoting the absorption or
penetration of
GDNFR protein product across the blood-brain barrier.
Once the therapeutic pharmaceutical composition has been formulated, it
may be stored in sterile vials as a solution, suspension, gel, emulsion,
solid, or
dehydrated or lyophilized powder. Such formulations may be stored either in a
ready to use form or in a form (e.g., lyophilized) requiring reconstitution
prior to
administration.
1 o The optimal phanmaceutical formulation will be determined by one skilled
in
the art depending upon the intended route of administration and desired
dosage. See
for example, Remington's Pharmaceutical Sciences, 18th Ed. ( 1990, Mack
Publishing Co., Easton, PA 18042) pages 1435-1712.
_ _ Such compositions may influence the physical
15 state, stability, rate of in vivo release, and rate of in vivo clearance of
the present
proteins and derivatives.
Effective administration farms, such as ( 1 ) slow-release formulations, (2)
inhalant mists, or (3} orally active formulations are envisioned. The GDNFR
protein
product pharmaceutical composition also may be formulated for parenteral
20 administration. Such parenterally administered therapeutic compositions are
typically in the form of a pyrogen-free, parenterally acceptable aqueous
solution
comprising the GDNFR protein product in a pharmaceutically acceptable vehicle.
One preferred vehicle is physiological saline. The GDNFR protein product
pharmaceutical compositions also may include particulate preparations of
polymeric
25 compounds such as polylactic acid, polyglycolic acid, etc. or into
liposomes.
Hyaluronic acid may also be used, and this rnay have the effect of promoting
sustained duration in the circulation.
A particularly suitable vehicle for parenteral injection is sterile distilled
water
in which the GDNFR protein product is formulated as a sterile, isotonic
solution,
3o properly preserved. Yet another preparation may involve the formulation of
the
GDNFR protein product with an agent, such as injectable microspheres or
liposomes, that provides for the slow or sustained release of the protein
which may
then be delivered as a depot injection. Other suitable means for the
introduction of
GDNFR protein product include implantable drug delivery devices which contain
the
35 GDNFR protein product.
The preparations of the present invention may include other components, for
example parenterally acceptable preservatives, tonicity agents, cosolvents,
wetting
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/08486
54
agents, complexing agents, buffering agents, antimicrobials, antioxidants and
surfactants, as are well known in the art. For example, suitable tonicity
enhancing
agents include alkali metal halides (preferably sodium or potassium chloride),
mannitol, sorbitol and the like. Suitable preservatives include, but are not
limited to,
benzalkonium chloride, thimerosal, phenethyl alcohol, methylparaben,
propylparaben, chlorhexidine, sorbic acid and the like. Hydrogen peroxide may
also
be used as preservative. Suitable cosolvents are for example glycerin,
propylene
glycol and polyethylene glycol. Suitable complexing agents are for example
caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-
1o cyclodextrin. Suitable surfactants or wetting agents include sorbitan
esters,
polysorbates such as polysorbate 80, tromethamine, lecithin, cholesterol,
tyloxapal
and the like. The buffers can be conventional buffers such as borate, citrate,
phosphate, bicarbonate, or Tris-HCI.
The formulation components are present in concentration that are acceptable
~ 5 to the site of administration. For example, buffers are used to maintain
the
composition at physiological pH or at slightly lower pH, typically within a pH
range
of from about S to about 8.
A pharmaceutical composition may be formulated for inhalation. For
example, the GDNFR protein product may be formulated as a dry powder for
2o inhalation. GDNFR protein product inhalation solutions may also be
formulated in a
liquefied propellant for aerosol delivery. In yet another formulation,
solutions may
be nebulized.
It is also contemplated that certain formulations containing GDNFR protein
product are to be administered orally. GDNFR protein product which is
25 administered in this fashion may be formulated with or without those
carriers
customarily used in the compounding of solid dosage forms such as tablets and
capsules. For example, a capsule may be designed to release the active portion
of the
formulation at the point in the gastrointestinal tract when bioavailability is
maximized and pre-systemic degradation is minimized. Additional formulation
3o materials may be included to facilitate absorption of GDNFR protein
product.
Diluents, flavorings, low melting point waxes, vegetable oils, lubricants,
suspending
agents, tablet disintegrating agents, and binders may also be employed.
Another preparation may involve an effective quantity of GDNFR protein
product in a mixture with non-toxic excipients which are suitable for the
35 manufacture of tablets. By dissolving the tablets in sterile water, or
other
appropriate vehicle, solutions can be prepared in unit dose form. Suitable
excipients
include, but are not limited to, inert diluents, such as calcium carbonate,
sodium
CA 02291608 2003-O1-29
.j
WO 98/54213
PCT/1:J98/08486
carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents,
such as
starch, gelatin, or acacia; or lubricating agents such as magnesium stearate,
stearic-
acid, or talc.
Additional GDNFR protein product formulations will be evident to those ''
skilled in the art, including formulations involving GDNFR protein product in
combination with GDNF protein product. Techniques for formulating a variety of
other sustained- or controlled-delivery means, such as liposome carriers, bio-
erodible
microparticles or porous beads and depot injections, are also known to those
skilled
in the art. See, for example, Supersaxo et al. description of controlled
release porous
1o polymeric microparticles for the delivery of pharmaceutical compositions
(International Publication No. WO 93/15722; International Application No.
PCT/LTS93/00829) .
D. Administration of ~'~_Dj~IFR Protein Prcodu~r
1s The GDNFR protein product may be administered .parenterally via a variety
of routes, including subcutaneous, intramuscular, intravenous, transpulmonary,
transdermal, intrathecal and intracerebral delivery. in addition, protein
factors than
do not readily cross the blood-brain barner may be given directly
intracerebrally or
otherwise in association with other elements that will transport them across
the
2o barrier. For example, the GDNFR protein product may be administered
intracereb~roventricularly or into the brain or spinal cord subarachnoid
space.
GDNFR protein product may also be administered intracerebrally directly into
the
brain parenchyma. GDNFR protein product may be administered extracerebrally in
a form that has been modified chemically or packaged so that it passes the
blood-
25 brain barrier, or with one. or more agents capable of promoting penetration
of
GDNFR protein product across the barrier. For example, a conjugate of NGF and
monoclonal anti-transferrin receptor antibodies has been shown to be
transported to
the brain via binding to transferrin receptors.
To achieve the desired level of GDNFR protein product, rued daily or
30 less frequent injections may be administered, or GDNFR protein product may
be
infused continuously or periodically from a constant- or programmable=flow
implanted pump. Slow-releasing implants containing the neurotrophic factor
embedded in a biodegradable polymer matrix can also deliver GDNFR protein
product. The frequency of dosing will depend on the pharmacokinetic parameters
of
35 the GDNFR protein product as formulated, and the route and site of
administration.
Regardless of the manner of administration, the specific dose may be
calculated according to body weight, body surface area or organ size. Further
CA 02291608 1999-11-26
WO 98/54213 PCT/US98108486
56
refinement of the calculations necessary to determine the appropriate dosage
for
treatment involving each of the above mentioned formulations is routinely made
by
those of ordinary skill in the art and is within the ambit of tasks routinely
performed
by them. Appropriate dosages may be ascertained through use of appropriate
dose-
response data.
The final dosage regimen involved in a method for treating a specific injury
or condition will be determined by the attending physician. Generally, an
effective
amount of the GDNFR protein product will be determined by considering various
factors which modify the action of drugs, e.g., the age, condition, body
weight, sex
to and diet of the patient, the severity of any infection, time of
administration and other
clinical factors. See, Remington's Pharmaceutical Sciences, supra, at pages
697-773,
herein incorporated by reference. For example, it is contemplated that if
GDNFR-a
is used to enhance GDNF action, then the GDNFR-a dose is selected to be
similar to
that required for GDNF therapy; if GDNFR-a is used to antagonize GDNF action,
15 then the GDNFR-a dose would be several times the GDNF dose. Dosing may be
one or more times daily, or less frequently, and may be in conjunction with
other
compositions as described herein. It should be noted that the present
invention is not
limited to the dosages recited herein.
It is envisioned that the continuous administration or sustained delivery of
2o GDNFR protein products may be advantageous for a given treatment. While
continuous administration may be accomplished via a mechanical means, such as
with an infusion pump, it is contemplated that other modes of continuous or
near
continuous administration may be practiced. For example, chemical
derivatization
or encapsulation may result in sustained release forms of the protein which
have the
25 effect of continuous presence in the bloodstream, in predictable amounts,
based on a
determined dosage regimen. Thus, GDNFR protein products include proteins
derivatized or otherwise formulated to effectuate such continuous
administration.
Sustained release forms of the GDNFR protein products will be formulated to
provide the desired daily or weekly effective dosage.
3o It is further contemplated that the GDNFR protein product may be
administered in a combined form with GDNF and/or neurturin. Alternatively, the
GDNFR protein product may be administered separately form a neurotrophic
factor,
either sequentially or simultaneously.
GDNFR protein product of the present invention may also be employed,
35 alone or in combination with other growth factors in the treatment of nerve
disease.
In addition, other factors or other molecules, including chemical
compositions, may
be employed together with a GDNFR protein product. For example, in the
treatment
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/08486
57
of Parkinson's Disease, it is contemplated that GDNFR protein product be used
by
itself or in conjunction with the administration of Levodopa, wherein the
GDNFR
would enhance the activity of endogenous GDNF and thereby enhance the neuronal
uptake of the increased concentration of dopamine.
As stated above, it is also contemplated that additional neurotrophic or
neuron nurturing factors will be useful or necessary to treat some neuronal
cell
populations or some types of injury or disease. Other factors that may be used
in
conjunction with GDNFR or a combination of GDNFR and a neurotrophic factor
such as GDNF or neurturin include, but are not limited to: mitogens such as
insulin,
1~ insulin-like growth factors, epidermal growth factor, vasoactive growth
factor,
pituitary adenylate cyclase activating polypeptide, interferon and
somatostatin;
neurotrophic factors such as nerve growth factor, brain derived neurotrophic
factor,
neurotrophin-3, neurotrophin-4/5, neurotrophin-6, insulin-like growth factor,
ciliary
neurotrophic factor, acidic and basic fibroblast growth factors, fibroblast
growth
factor-5, transforming growth factor-13, cocaine-amphetamine regulated
transcript
(CART); and other growth factors such as epidermal growth factor, leukemia
inhibitory factor, interleukins, interferons, and colony stimulating factors;
as well as
molecules and materials which are the functional equivalents to these factors.
2o GDNFR Protein Product Cell Therapy and Gene Theranv
GDNFR protein product cell therapy, e.g., intracerebral implantation of cells
producing GDNFR protein product, is also contemplated. This embodiment would
involve implanting into patients cells capable of synthesizing and secreting a
biologically active form of GDNFR protein product. Such GDNFR protein product
producing cells may be cells that are natural producers of GDNFR protein
product or
may be recombinant cells whose ability to produce GDNFR protein product has
been
augmented by transformation with a gene encoding the desired GDNFR protein
product. Such a modification may be accomplished by means of a vector suitable
for
delivering the gene as well as promoting its expression and secretion. In
order to
3o minimize a potential immunological reaction in patients being administered
a
GDNFR protein product of a foreign species, it is preferred that the natural
cells
producing GDNFR protein product be of human origin and produce human GDNFR
protein product. Likewise, it is preferred that the recombinant cells
producing
GDNFR protein product be transformed with an expression vector containing a
gene
encoding a human GDNFR protein product.
Implanted cells may be encapsulated to avoid infiltration of surrounding
tissue. Human or non-human animal cells may be implanted in patients in
CA 02291608 2003-O1-29
WO 98/54213 ) PCT/U598/08486
S$
biocompatible, semipermeable polymeric enclosures or membranes that allow
release of GDNFR protein product, but that prevent destruction of the cells by
the
patient's immune system or by other detrimental factors from the surrounding
tissue.
Alternatively, the patient's own cells, transformed to produce GDNFR protein
~=:r
product ex vivo, could be implanted directly into the patient without such
encapsulation.
Techniques for the encapsulation of living cells are familiar to those of
ordinary skill in the art, and the preparation of the encapsulated cells and
their '
implantation in patients may be accomplished without undue experimentation.
For
example, Baetge et al. (International Publication No. W0 95/05452;
International
Application No. PCT/LTS94/09299)
describe biocornpatible capsules containing genetically engineered cells
for the effective delivery of biologically active molecules. In addition, see
U.S.
Patent Numbers 4,892,538, 5,011,472, and 5,106,b27-
A system for encapsulating living cells is
described in PCT Application WO 91/10425 of Aebischer et al
See also, PCT Application WO 91/10470 of .:°:
Aebischer et al., Winn et al., Exper. Neurol., 113:322-329, 1991, Aebischer et
ale,
~Exper. Neurol., 111:269-275, 1991; Tresco et al., ASAIO, 38:17-23, 1992.
In vivo and in vitro gene therapy delivery of GDNFR protein product is also
envisioned. In vitro gene therapy may be accomplished by introducing the gene
coding for GDNFR protein product into targeted cells via local injection of a
nucleic
acid construct or other appropriate delivery vectors. (Hefti, J. Neurobiol,.
25:1418-
1435, 1994). For example, a nucleic acid sequence encoding a GDNFR proteiw
product may be contained in an adeno-associated virus vector for delivery into
the
targeted cells (e.g.; Johnson, International Publication No. WO 95/34670;
International Application No. PCT/LTS95/07178).
Alternative viral vectors include, but are not limited to,
3o retrovirus, adenovirus, herpes simplex virus and papilIoma virus vectors.
Physical
transfer, either in vivo or ex vivo as appropriate, may also be achieved by
liposome-
mediated transfer, direct injection (naked DNA), receptor-mediated transfer
(ligand-
DNA complex), electroporation, calcium phosphate precipitation or nucmparticle
bombardment (gene gun):
It is also contemplated that GDNFR protein product gene therapy or cell
therapy can firrther include the delivery of GDNF protein product. For
example, the
host cell may be modified to express and release both GDNFR-a protein product
and
CA 02291608 2003-O1-29
WO 98/54213
59
PCT/LJS98/08486
GDNF, or GRR2 and neurturin. Alternatively, the GDNFR-a and GDNF protein
products, or GRR2 and neurhuin, may be expressed in and released from separate
cells. Such cells may be separately introduced into the patient or the cells
may be
contained in a single implantable device, such as the encapsulating membrane
described above.
It should be noted that the GDNFR protein product formulations described
herein may be used for veterinary as well as human applications and that the
term
"patient" should not be construed in a limiting manner. In the case of
veterinary
1 o applications, the dosage ranges may be determined ~as described above.
EXAMPLES
15 ~ Example 1
Identification of Cells Expressing High Affinity GDNF Binding Sites
Expression cloning involved the selection of a source of mRNA which is
likely to contain significant levels of the target transcript Retina
photoreceptor cells
20 ' were identified as responsive to GDNF at very low concentrations,
suggesting the
existence of a functional, high affnity receptor. To confum that rat
photoreceptor
cells did express a high affinity receptor for GDNF, [ 1251]GDNF binding and
photographic emulsion analysis were carried out.
25 Rat Retinal Cell Cuttu_~s
The neural-retinas of 5-day-old C57B1f6 mouse pups or 3-day-old Sprague-
'Dawley rat pups (Jackson Laboratories, Bar Harbor, MA) were carefully removed
and dissected free of the pigment epithelium, cut into 1 mm2 fragments and
placed
into ice-cold phosphate-buffered saline (PBS). The retinas were then
transferred into
30 10 mL of Hank's balanced salt solution (HBSS) containing 120 units papain
and
2.000 units DNAase and incubated for 20 minutes at 37°C on a rotary
platform
shaker at about 200 rpm. The cells were then dispersed by trituration through
fire-
polished Pasteur pipettes, sieved through a 20 Wn Niter nylon mesh and
centrifuged
for five minutes at 200 x g . The resulting cell pellet was resuspended into
HBSS
35 containing 1 % ovalbumin and 500 units DNAase, layered on top of a 4 %
ovaIbumin
solution (in HBSS) and centrifuged for 10 minutes at 500 x g. The final pellet
was
resuspended in complete culture medium (see below), adjusted to about 15,000
* trademark
CA 02291608 2003-O1-29
WO 98/54213 '~ ~ j PCT/US98/08486
cells/mL, and seeded in 90 E,i,i aliquots into tissue culture plates coated
with
polyornithine and laminin as previously described (Louis et al., Journal Of
Pharmacology And Experimental Therapeutics, 262, 1274-1283, 1992).
The culture medium consisted of a 1: I mixture of Dulbecco's Modified
Eagle's Medium {DMEM) and F12 medium, and was supplemented with 2.5% heat-
inactivated horse serum (Hyclone, Logan, UT), B27 rnediurn supplement (GIBCO,
Grand Island, NY), D-glucose {final concentration: Smg/mL), L-glutamine (final
concentration: 2mM), 20 mM HEPES, bovine insulin and human transferrin (final
concentrations: 2.5 and 0.1 mg/mL, respectively).
to
Immunocytochemical identification of nhoto~ r,n
Photoreceptors were identified by inununostaining for arrestin, a rod-specific
antigen. Cultures of photoreceptors were fixed for 30 minutes at room
temperature
with 4% paraformaldehyde in PBS; pH 7.4, followed by three washes in PBS. The
v
15 fixed cultures were then incubated in Superblock*blocking buffer (Pierce;
Rockford,
IL) , containing 1 % Nonidet P-40*to increase the penetration of the
antibodies. The
anti-arrestin antibodies (polyclonal rabbit antibody against the synthetic
peptide
sequence of arrestin: Val-Phe-Glu-Glu-Phe-Ala-Arg-Gln-Asn-Leu-Lys-Cys) were
then applied at a dilution of between 1:2000 in the same buffer, and the
cultures were
20 incubated for one hour at 37°C on a rotary shaker. After three
washes with PBS, the
cultures were incubated for one hour at 37°C with goat-anti-rabbit IgG
(Vectastain'~
kit from Vector Laboratories, Burlingame, CA) at a 1:500 dilution. After three
washes with PBS, the secondary antibodies were then labeled with an avidin-
biotin
peroxidase complex diluted at I :500 (45 minutes at 37°C). After three
more washes
25 with PBS, the labeled cell cultures were reacted for 5-20 minutes in a
solution of O.I
M Tris-HCl, pH 7.4, containing 0.04% 3',3'-diaminobenzidine-(HCl)4, 0.06
percent
NiCl2 and 0.02 percent hydrogen peroxide. Based on arrestin-immunoreactivity,
about 90% of the cells in the cultures were rod photoreceptors.
The survival of photoreceptors was determined by examination of arrestin-
3o stained cultures with bright-light optics at 200X magnification. The number
of
arrestin-positive photoreceptors was counted in one diametrical 1 X 6 mm
strip,
representing about 20 percent of the total surface area of a 6 mm-well. Viable
photoreceptors were characterized as having a regularly-shaped cell body, with
a
usually short axon-like process. Photoreceptors showing signs of degeneration,
such
35 as having irregular, vacuolated perikarya or fragmented neurites, were
excluded from
the counts (most of the degenerating photoreceptors, however, detached from
the
" culture substratum). Cell numbers were expressed either as cells/6-mm well.
* trademark
CA 02291608 1999-11-26
WO 98/54213 PCTIUS98/08486
61
Cultured rat retinal cells enriched for photoreceptors { 10,000/6-mm well)
were treated with human recombinant GDNF (ten-fold serial dilutions ranging
from
ng/mL to 1 pglmL). The cultures were fixed after six days and immunostained
5 for arrestin, a rod photoreceptor-specific antigen. In cultures that were
not treated
with GDNF, the number of photoreceptors declined steadily over time to reach
about
25 percent of the initial number after six days in culture. Treatment of the
cultures
with GDNF resulted in an about two-fold higher number of viable arrestin-
positive
photoreceptors after six days in culture. The effect of GDNF was maximal at
about
10 200 pg/mL, with an ED50 of about 30 pg/mL. In addition to promoting
photoreceptor survival, the addition of the GDNF also stimulated the extension
of
their axon-like process, thereby demonstrating an effect on the morphological
development of the photoreceptors (mean neurite length of photoreceptors in
GDNF:
68 pm, compared to 27t I 8 gm in control cultures).
In order to confirm that rat retinal cells express high affinity GDNF
receptors,
[1251~GDNF binding and photographic emulsion analysis were carried out. Post-
natal rat photoreceptor cells were seeded on plastic slide flaskettes (Nunc)
at a
density of 2800 cells/mm2, three to four days before the experiments. The
cells were
washed once with ice-cold washing buffer (Dulbecco's Modified Eagle's Medium
(DMEM) containing 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid
(HEPES), pH 7.5). For competitive binding, the cells were incubated with
various
concentrations of [1251]GDNF in binding buffer (DMEM containing 25 mM
HEPES, pH 7.5, and 2 mg/mL of bovine serum albumin (BSA)) in the presence or
absence of 500 nM unlabeled GDNF at 4oC for four hours. Cells were washed four
times with ice-cold washing buffer, lysed in 1 M NaOH and the radioactivity
associated with the cells was determined in a gamma counter. A significant
amount
of [1251~GDNF bound to the photoreceptor cells even at low ligand
concentrations
(as low as 30 pM), and this binding was inhibited completely by the presence
of
excess unlabeled GDNF.
3o For photographic emulsion detection, cells were incubated with 50 pM of
[ 1251~GDNF in binding buffer in the presence or absence of 500 nM unlabeled
GDNF at 4oC for four hours. Cells were washed six times with ice-cold washing
buffer, fixed with 2.5% glutaraldehyde and dehydrated sequentially with 50%
and
70% ethanol, and dipped in NTB-2 photographic emulsion (Eastman Kodak,
Rochester NY}. After five days of exposure, the slides were developed and
examined. The photographic emulsion analysis demonstrated the association of
[ 125I~GDNF to some of the photoreceptor cells, thereby indicating the
presence of a
CA 02291608 1999-11-26
WO 98/54213 PCT/US98108486
62
receptor for GDNF. This association, however, was efficiently blocked by the
addition of unlabeled GDNF.
Example 2
Expression Cloning of a GDNFR-a from Photoreceptor Cells
Rat photoreceptor cells were selected as a possible source of a high affinity
receptor for GDNF based upon their cell surface binding of radiolabeled GDNF
and
1o their ability to respond to very low concentrations of the ligand, as
described in
Example 1. In order to identify the receptor, a size-selected cDNA library of
approximately 50,000 independent clones was constructed using a mammalian
expression vector (a derivative of pSR, Takebe et al., 1988 supra) and mRNA
isolated from cultured post-natal rat photoreceptor cells, by the methods
described
15 below. The library was divided into pools of approximately 1,500 to 2,000
independent clones and screened using an established expression cloning
approach
(Gearing et al., EMBO Journal, 8, 3667-3676, 1989). Plasmid DNA representing
each pool of the library was prepared and transfected into COS7 cells grown on
plastic microscope slide flaskettes (Nunc, Naperville, IL).
2o The transfected cells were treated with [ 1251]GDNF, fixed with
glutaraldehyde, dehydrated, and dipped in photographic emulsion for
autoradiography. Following exposure for five days, the slides were developed
and
examined for the presence of cells covered by silver grains which indicated
the
binding of [ 1251]GDNF to the cell surface as a result of the cell's
expression of a
25 receptor for GDNF. EGF receptor transfected cells treated with [ 1251]EGF
were
used as a positive control.
One of the 27 pools (F8-11) screened in this manner exhibited 19 positive
cells following transfection. Thus, a single cDNA library pool was identified
which
contained a cDNA clone that expressed GDNFR-a. This pool was divided into 60
3o smaller subpools of 100 clones/pool which were rescreened by the same
procedure
described above. Five of these pools were identified as positive and two of
the five
pools were further subdivided to yield single clones responsible for the GDNF
binding activity. Transfection of plasmid DNA from the single clones into COS7
cells resulted in the binding of [125I]GDNF to approximately 15% of the cells.
This
35 binding was specifically inhibited by competition with excess unlabeled
GDNF.
Construction of Expression cDNA Libraries
~ CA 02291608 2003-O1-29
WO 98/54213 'j PCT/U598/08486
63
Rat retinal cells were harvested from postnatal day 3-7 rats and seeded into
culture dishes coated with laminin and polyomithine at a density of
approximately
s700 cells/mm2. After 3-4 days in culture, the population was estimated to
contain
approximately 80% photoreceptor cells. Total RNA was prepared from this
culture
s by standard methods, and Poly A+.RNA was purified using a polyA-tract kit
(Promega, Madison, WI). A cDNA library was constructed from the rat
photoreceptor poly A+ RNA using the Gibco Superscxipt Choice System
(GibcoBRL, Gaithersburg, MD). Two micrograms of poly A+ RNA were mixed
with 50 ng of random hexamers, heated to 70oC for 10 minutes and then quick-
to chilled on ice. First strand synthesis was carried out with 400U
Superscript II RT at
37oC for one hour. Second strand synthesis was performed in the same tube
after
the addition of dNTPs, 1 OU of E. coli DNA ligase, 40U of E. coli DNA
polymerase
I, and 2U of E. coli RNase H. After two hours at 16oC, the cDNA ends were
blunted
by treatment with 1 OU of T4 polymerase for an additional five minutes at 1
boC.
1s Following isopropanol precipitation, EcoRI cloning sites were added to the
cDNA by
Ligation overnight with 10 ug of unphosphorylated EcoRI adapter
oligonucleotides.
The EcoRl adapted cDNA was then phosphorylated and applied to a
Sephacryl~S-500 HR size fractionation column. Following Loading, the column
was
washed with 100 u1 aliquots of TEN buffer ( 10 mM Tris-HCl pH 7.s, 0.1 mM
20 EDTA, 25 mM NaCI), and 30 p1 fractions were collected. Fractions 6 through
8,
which contained approximately 34 ng of high molecular weight cDNA, were pooled
and precipitated. The recovered EcoRI-adapted cDNA was ligated overnight with
50
ng of EcoRI cut vector pBJS. Aliquots of the ligation mix containing about 15
ng
cDNA each were transformed into competent cells (E. coli strain DH10B;
25 G1BCOBRL, Gaithersburg, MD) by electroporation. The transformation mixture
was titered and then plated' on 27 Amp/LB plates at a density of 1500
colonies/plate.
Colonies were scraped from each plate and collected into 10 mL of Luria broth
(LB)
to make 27 pools of 1 s00 independent clones each. A portion of the cells from
each
pool was frozen in glycerol and the remainder was used to isolate plasmid DNA
3o using a Qiagen tip-500 kit (Qiagen Inc., Chatsworth, CA).
COS Cell Tra~fection and Photog~plic Emulsion A_n l3r~f
COS7 cells were seeded (220,000 cells/slide) on plastic slide flaskettes
(Nunc) coated with ProNectiii (10 ~eg/mL in phosphate buffered saline (PBS))
one
3s day before transfection. For transfection, 700 p1 of Opti MEMI (G1BCOBRL,
Gaithersburg, MD) containing 2_ ug cDNA was mixed gently with 35 ~1 of DEAF
Dextran solution (10 mg/mL, Sigma, St. Louis, MO) in an Eppendorf tube. Cells
* trademark
CA 02291608 1999-11-26
WO 98/54213 PCT/CJS98/0848f>
64
were washed twice with PBS and incubated with the transfection mix for 30
minutes
at 37oC in a 5% C02 atmosphere. Following incubation, 3 mL of DMEM media
containing 10% fetal calf serum (FCS) and 80 nM Chloroquine (Sigma, St. Louis,
MO) were added to each flaskette. Cells were further incubated for 3.5 hours,
shocked with 10% dimethylsulfoxide in DMEM at room temperature for two
minutes, washed once with PBS, and allowed to grow in DMEM containing 10%
FCS. After 48 hours, the transfected COS7 cells were washed once with ice-cold
washing buffer (DMEM containing 25 mM HEPES, pH 7.5) and incubated in ice-
cold binding buffer (DMEM containing 25 mM HEPES, pH 7.5 and 2 mg/mL BSA)
1o supplemented with 50 pM [ 125I~GDNF at 4oC for four hours. Cells were
washed
six times in ice-cold washing buffer, fixed with 2.5% glutaraldehyde at room
temperature for five minutes, dehydrated sequentially with 50% and 70%
ethanol,
and then dipped in NTB-2 photographic emulsion (Eastman Kodak). After 4-5 day
exposure at 4oC in dark, the slides were developed and screened by bright-
field and
15 dark-field microscopy.
Subdivision of Positive Pools
A single pool was identified which contained a putative GDNF receptor
clone. Clones from this pool were plated on 60 plates at a density of 100
2o colonies/platc. Cells were scraped from each plate, collected in LB, and
allowed to
grow for 4-5 hours at 37°C. Frozen stocks and DNA preparations were
made from
each pool, as before, to generate 60 subpools containing 100 independent
clones
each. Two of these 60 subpools were identified as positive by the method
described
above, and clones from those pools were plated at low density to allow
isolation of
25 single colonies. Single colonies (384) were picked from each of the two
subpools
and grown for six hours in 200 p1 LB in 96-well plates. In order to select
single
clones expressing GDNFR-a, the four 96-well plates were arrayed into a single
large
matrix consisting of 16 rows and 24 columns. Cells from the wells in each row
and
in each column were combined to yield a total of 40 mixtures. These mixtures
were
3o grown overnight in 10 mL LB/Amp (100 pg/mL), and DNA was prepared using a
Qiagen tip-20 kit. When analyzed for putative GDNF receptor clones, three row
mixtures and three column mixtures gave positive signals, suggesting nine
potentially positive single clones. DNA from each of the potentially positive
single
clones was prepared and digested with EcoRI and PstI. DNA from three of the
nine
35 single clones exhibited identical restriction patterns while the other six
were
unrelated, suggesting that the three represented the authentic clones
containing
GDNFR-a.
CA 02291608 1999-11-26
WO 98/54213 PCT/IJS98/08486
Example 3
DNA Sequencing and Sequence Analysis
5
DNA from positive, single clones was prepared and sequenced using an
automated ABI373A DNA sequencer (PerkinlElmer Applied Biosystems, Santa
Clara, CA ) and dideoxy-dye-terminators, according to manufacturer's
instructions.
Comparison of GDNFR-a sequence with alI available public databases was
to performed using the FASTA (Pearson and Lipman, Proceedings Of The National
Academy Of Sciences U.S.A., 85, 2444-2448, 1988) program algorithm as
described
in the University of Wisconsin Genetics Computer Group package (Program Manual
for the Wisconsin Package, Version 8, September 1994, Genetics Computer Group,
Madison, WI).
Sequence Characterization of the Rat GDNFR-a
Plasmid DNA from the clones described in Example 2, above, was prepared
and submitted for DNA sequence analysis. Nucleotide sequence analysis of the
cloned 2138 by rat cDNA revealed a single large open reading frame encoding a
2o translation protein of 468 amino acid residues {Figure 3).
The coding sequence is flanked by a S'-untranslated region of 301 by and a
3'-untranslated region of 430 by that does not contain a potential
polyadenylation
site. The presence of an in-frame stop codon upstream of the first ATG at base
pair
302 and its surrounding nucleotide context indicate that this methionine codon
is the
most likely translation initiator site (Kozak, Nucleic Acids Research. 15,
8125-8148,
1987).
No polyadenylation signal is found in the 430 nucleotides of 3' untranslated
sequence in the rat cDNA clone. This is not surprising, since the Northern
blot data
shows the shortest mRNA transcripts to be approximately 3.6 kb.
3o The GDNFR-a polypeptide sequence has an N-terminal hydrophobic region
of approximately 19 residues {methionine-1 to alanine-19, Figure 3) with the
characteristics of a secretory signal peptide (von Heijne, Protein Sequences
And
Data Analysis. 1, 41-42, 1987; von Heijne, Nucleic Acids Research. 14, 4683-
4690,
1986). No internal hydrophobic domain that could serve as a transmembrane
domain was found. Instead, a carboxy-terminal hydrophobic region of 21
residues
(Ieucine-448 to serine-468 in Figure 3) is present and may be involved in a
glycasyl-
phosphatidylinositol (GPI) anchorage of the receptor to the cytoplasmic
membrane.
CA 02291608 1999-11-26
WO 98154213 PCT/US98/08486
66
Except for the presence of three potential N-linked glycosylation sites, no
conserved
sequence or structural motifs were found. The protein is extremely rich in
cysteine
(31 of the 468 amino acid residues) but their spacing is not shared with that
of
cysteine-rich domains found in the extracellular portions of known receptors.
The GDNFR-a sequence was compared to sequences in available public
databases using FASTA. The search did not reveal significant homology to other
published sequences. Once the rat cDNA clone was obtained, it was radiolabeled
and used to probe a cDNA library prepared from human brain substantia nigra as
described below in Example 5.
to
Example 4
GDNF Binding to Cells Expressing GDNFR-a
15 A binding assay was performed in accordance with an assay method
previously described by Jing et al.. (Journal Of Cell Biology, 110, 283-294,
1990).
The assay involved the binding of [ 1251]GDNF to rat photoreceptor cells, COS7
cells or 293T cells which had been transfected to express GDNFR-a. Recombinant
GDNFR-a expressed on the surface of 293T cells was able to bind GDNF
2o specifically and with an affinity comparable to that observed for GDNF
binding sites
on rat retinal cells.
Rat photoreceptor cells were prepared as described in Example 1, above, and
seeded at a density of 5.7 x 105 cells/cm2 two to three days before the assay
in 24-
well Costar tissue culture plates pre-coated with polyornithine and laminin.
COS7
25 cells were seeded at a density of 2.5 x 104 cells/cm2 one day before the
assay and
transfected with 10-20 pg of plasmid DNA using the DEAE-dextran-chloroquine
method (Aruffo and Seed, Proceedings Of The National Academy Of Sciences
U.S.A., 84, 8573-8577, 1987). Cells from each dish were removed and reseeded
into
30 wells of 24-well Costar tissue culture plates 24 hours following the
transfection,
3o and allowed to grow for an additional 48 hours. Cells were then left on ice
for 5 to
minutes, washed once with ice-cold washing buffer and incubated with 0.2 mL of
binding buffer containing various concentrations of [ 1251]GDNF with or
without
unlabeled GDNF at 4oC for four hours. Cells were washed four times with 0.5 mL
ice-cold washing buffer and Iysed with 0.5 mL of 1 M NaOH. The lysates were
35 counted in a 1470 Wizard Automatic Gamma Counter.
For some binding experiments, transiently transfected 293T cells were used
(see below for 293T cell transfection). Two days following transfection, cells
were
CA 02291608 2003-O1-29 _
WO 98/54213 j ') PCT/US98/08486
67
removed from dishes by 2x versine. Cells were pelleted, washed once with ice-
cold
binding buffer and resuspended in ice-cold binding buffer at a density of 3 x
105
cells/mL. The cell suspension was divided into aliquots containing 1.5 x 1 O5
cell/sample. Ceils were then pelleted and incubated with various
concentrations of
s [125IJGDNF in the presence or absence of S00 nM of unlabeled GDNF at 4oC for
four hours with gentle agitation. Cells were washed four times with ice-cold
washing buffer and resuspended in 0.5 mL washing buffer. Two 0.2 mL aliquots
of
the suspension were counted in a gamma counter to determine the amount of
~ 125IJGDNF associated with the cells.
~ In all assays, nonspecific binding was determined by using duplicate
samples,
one of which contained S00 nM of unlabeled GDNF. The level of nonspecific
binding varied from 10% to 20% of the specific binding measured in the absence
of
unlabeled GDNF and was subtracted from the specific binding. The assays
demonstrated that cells did not bind GDNF unless the cell had been transfected
with
is the GDNFR-a cDNA clone.
Example 5
Tissue Distribution of GDNFR-a mRNA
The pattern of expression of GDNFR-a mRNA in embryonic mouse, adult
mouse, rat, and human tissues was examined by Northern blot analysis. The
cloned
rat GDNFR-a cDNA was labeled using the Random Primed DNA Labeling Kit*
(Boehringer Mannheim, Indianapolis, IN) according to the manufacturer's
procedures. Rat, mouse, and~human tissue RNA blots (purchased from Clontech,
Palo Alto, CA) were hybridized with the probe and washed using the reagents of
the
ExpressHyb Kit (Clontech) according to the manufacturer's instructions.
Tissue Northern blots prepared from adult rat, mouse, and human tissues
indicated that GDNFR-a mRNA~is most highly expressed in liver, brain, and
kidney.
3o High mRNA expression was also detected in lung, with lower or non-
detectable
amounts in spleen, intestine, testis, and skeletal muscle. In blots made from
mRNA
isolated from mouse embryo, expression was undetectable at embryonic day 7,
became apparent at day E 1 I, and was very high by day E 17. GDNFR-a mRNA was
expressed in tissue isolated from several subregions of adult human brain at
relatively equal levels. Expression of GDNFR-a mRNA in human adult brain
showed little specificity for aay particular region.
In most tissues, transcripts of two distinct sizes were present. In mouse and
* trademark
CA 02291608 1999-11-26
WO 98/54213 PCT/US9810848(
68
human tissues, transcripts of 8.5 and 4.4 kb were found, while in rat the
transcripts
were 8.5 and 3.6 kb. The relative amounts of the larger and smaller
transcripts
varied with tissue type, the smaller transcript being predominant in liver and
kidney
and the larger being more abundant in brain. The binding of GDNF to 293T cells
transfected with a GDNFR-a cDNA clone in the pBKRSV vector was examined by
Scatchard analysis. Two classes of binding sites were detected, one with a
binding
affinity in the low picomolar range and another with an affinity of about 500
pM.
1 o Example 6
Recombinant Human GDNFR-a
An adult human substantia nigra cDNA library (5'-stretch plus cDNA library,
Clontech, Palo Alto, CA) cloned in bacteriophage gtl0 was screened using the
rat
GDNFR-a cDNA clone of Example I as a probe. The probe was labeled with [32p~_
dNTPs using a Random Primed DNA Labeling Kit (Boehringer Mannheim,
Indianapolis, IN) according to the manufacturer's instructions. Approximately
I .2 x
106 gtl0 phage from the human substantia nigra cDNA library were plated on 15
cm
agarose plates and replicated on duplicate nitrocellulose filters. The filters
were then
screened by hybridization with the radiolabeled probe. The filters were
prehybridized in 200 mL of 6 x SSC, 1 x Denhardts, 0.5% SDS, 50 ug/mI, salmon
sperm DNA at 55°C for 3.5 hours. Following the addition of 2 x 108 cpm
of the
radiolabeled probe, hybridization was continued for 18 hours. Filters were
then
washed twice for 30 minutes each in 0.5x SSC, 0.1 % SDS at 55oC and exposed to
X-ray film overnight with an intensifying screen.
Five positive plaques were isolated whose cDNA inserts represented portions
of the human GDNFR-a cDNA. In comparison to the nucleic acid sequence of rat
GDNFR-a. depicted in Figure 3 (bp 0 through 2140), the five human GDNFR-a
clones were found to contain the following sequences:
TABLE 3
Clone 2 1247 through (SEQ ID N0:21
2330 )
Clone 9 1270 through (SEQ ID N0:23)
2330
Clone 21-A -235 through (SEQ ID N0:9)
1692
Clone 21-B -237 through (SEQ ID NO:11)
1692
Clone 29 805 through 2971(SEQ ID N0:15)
CA 02291608 1999-11-26
r~'O 98154213 PCTIUS98/08486
69
An alignment and comparison of the sequences, as depicted in Figure 5,
provided a
consensus sequence for human GDNFR-a. The translation product predicted by the
human cDNA sequence consists of 465 amino acids and is 93% identical to rat
GDNFR-a.
To generate a human cDNA encoding the full length GDNFR-a, portions of
clones 21B and 2 were spliced together at an internal BgIII site and subcloned
into
the mammalian expression vector pBKRSV (Stratagene, La Jolla, CA}.
Recombinant human GDNFR expression vectors may be prepared for
o expression in mammalian cells. As indicated above, expression may also be in
non-
mammalian cells, such as bacterial cells. The nucleic acid sequences disclosed
herein may be placed into a commercially available mammalian vector (for
example,
CEP4, Invitrogen) for expression in mammalian cells, including the
commercially
available human embryonic kidney cell Iine, "293". For expression in bacterial
cells,
one would typically eliminate that portion encoding the leader sequence (e.g.,
nucleic acids 1-590 of Figure 1 ). One may add an additional methionyl at the
N-
terminus for bacterial expression. Additionally, one may substitute the native
leader
sequence with a different leader sequence, or other sequence for cleavage for
ease of
expression.
Example 7
Soluble GDNFR Constructs
Soluble human GDNFR protein products were made. The following
examples provide four different forms, differing only at the carboxy terminus,
indicated by residue numbering as provided in Figure 2. Two are soluble forms
truncated at different points just upstream from the hydrophobic tail and
downstream
from the last cysteine residue. The other two are the same truncations but
with the
3o addition of the "FLAG" sequence, an octapeptide to which a commercial
antibody is
available (Eastman Kodak). The FLAG sequence is HzN- DYKDDDDK - COOH.
Method
Lambda phage clone #21, containing nearly the entire coding region of
human GDNFR-a, was digested with EcoRI to excise the cDNA insert. This
fragment was purified and ligated into EcoRI cut pBKRSV vector (Stratagene, La
Jolla, CA) to produce the clone 21-B-3/pBKRSV. Primers I and 2 as shown below
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/08486
were used in a PCR reaction with the human GDNFR-a clone 21-B-3/pBKRSV as
template. PCR conditions were 94oC, five minutes followed by 25 cycles of
94oC,
one minute; SSoC, one minute; 72oC, two minutes and a final extension of five
minutes at 72oC. This produced a fragment consisting of nucleotides 1265-1868
of
5 the human GDNFR-a clone plus a termination codon and Hind III restriction
site
provided by primer 2. This fragment was digested with restriction enzymes Hind
III
(contained in primer 2) and BgIII (position 1304 in human GDNFR-a), and the
resulting 572 nucleotide fragment was isolated by gel electrophoresis. This
fragment
contained the hGDNFR-a- coding region from isoleucine-255 to glycine-443. A
1 o similar strategy was used with primers 1 and 3 to produce a fragment with
BgIII and
HindIII ends which coded for isoleucine-255 to proline-446. Primers 4 and 5
were
designed to produce fragments coding for the same regions of hGDNFR-a and
primers 1 and 3, but with the addition of the Flag peptide coding sequence
(IBI/Kodak, New Haven, CN). The Flag peptide sequence consists of eight amino
15 acids (H2N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-COOH) to which antibodies are
commercially available. Primers 1 and 4 or 1 and 5 were used in PCR reactions
with
the same template as before, and digested with HindIII and BgIII as before.
This
procedure produced fragments coding for isoleucine-255 to glycine-443 and
isoleucine-255 to proline-446, but with the addition of the Flag peptide at
their
20 carboxy termini.
Primers
1) 5'-CTGTTTGAATTTGCAGGACTC-3' (SEQ ID N0:30}
2) 5'-CTCCTCTCTAAGCTTCTAACCACAGCTTGGAGGAGC-3' (SEQ ID N0:31)
25 3) 5'-CTCCTCTCTAAGCTTCTATGGGCTCAGACCACAGCTT-3' (SEQ ID N0:32)
4) 5'-CTCCTCTCTAAGCTTCTACTTGTCATCGTCGTCCTTGTAGTCACCACAGCTTGGA
GGAGC-3' (SEQ ID N0:33)
5) 5'-CTCCTCTCTAAGCTTCTACTTGTCATCGTCGTCCTTGTAGTCTGGCTCAGACCAC
AGCTT-3' (SEQ ID N0:34)
All four fragments, produced as described above, were transferred back into
2iB3/pBKRSV. The 21B3/pBKRSV clone was digested with BglII and HindIII,
and treated with calf intestinal alkaline phosphatase (CIAP). The large
fragment
containing the vector and the human GDNFR-a coding region up to the BgIII site
was gel purified and extracted from gel. Each of the four BgIII/HindIII
fragments
produced as described above were ligated into this vector resulting in the
following
constructs in the pBKRSV vector:
CA 02291608 2003-O1-29
wo 9sisaai3 1
PCT/US98/08486
71
TABLE 4
1 ) GDNFR-a/gly-443/pBKRSV hGDNFR-a terminating at glycine 443,
. _ followed by stop codon
2) GDNFR-a/pro-446/pBKRSV hGDNFR-a terminating at proline 44b,
followed by step codon
3) GDNFR-a/gly- hGDNFR-a terminating at glycine 443 with C-
443/Flag/pBKRSV term Flag tag, followed by stop codon
4) GDNFR-a/pro- hGDNFR-a terminating at proline 446 with
446/Flag/pBKRSV C-term Flag tag, followed by stop codon
Correct construction of all clones was confirmed by DNA sequencing.
Inserts from the pBKRSV clones were transferred to other expression vectors
using
enzyme sites present in the pBKRSV polylinker sequence as described below.
Soluble GDNFRs (e.g., sGDNFR-a/gly and sGDNFR-a/pro) have also been
transferred into vectors for transient expression and into pDSR-2 for stable
to expression in CHO cells.
pDSRa2+pL clones:
The appropriate pBICRSV clone is digested with XbaI and SaII. .The insert is
ligated to pDSRa2+PL cut with the same enzymes and treated with CIAP. This
construction may be used for stable expression of GDNFR in CHO cells.
nCEP4 cloneg;
The appropriate pBKRSV clone is digested with SpeI and XhoI. The insert is
ligated to pCEP4 (Invitrogen, San Diego, CA) digested with NheI (SpeI ends)
and
XhoI, and treated with CIAP. This construction may be used for transient of
expression of GDNFR.
The plasmid construct pDSR-2 is prepared substantially in accordance with
~e Process described in U.S. Patent No. 5,714,465 (also see, European Patent
Application No. 90305433; Publicatian No. EP 398 753, filed May 18, 1990 and
WO
90/14363 (1990)). It will be
appreciated,by those skilled in the art that a variety of nucleic acid
sequences
encoding GDNFR analogs may be used.
CA 02291608 1999-11-26
WO 98/54213 PCT/US9tSi08486
72
Another construct is pDSRa2, a derivative of the plasmid pCD (Okayama &
Berg, Mol. Cell Biol. 3: 280-289, 1983) with three main modifications: (i) the
SV40
polyadenylation signal has been replaced with the signal from the a-subunit of
bovine follicular stimulating hormone, a-bFSH (Goodwin et al., Nucleic Acids
Res.
11: 6873-6882, 1983); (ii) a mouse dihydrofolate reductase minigene (Gasser et
al.,
Proc. Natl. Acad. Sci. 79: 6522-6526, 1982) has been inserted downstream from
the
expression cassette to allow selection and amplification of the transformants;
and
(iii) a 267 by fragment containing the "R-element" and part of the "US"
sequences of
the long terminal repeat (LTR) of human T-cell leukemia virus type I (HTLV-I)
has
0 been cloned and inserted between the SV40 promoter and the splice signals as
described previously (Takebe et al., Mol. Cell Biol. 8: 466-472, 1988).
The expression of GDNFR-a in CHO cells has been verified by the binding
of iodinated GDNF to the cell surface. As discussed above, the recombinantly
expressed soluble GDNFR-a protein product may be used to potentiate the
activity
or cell specificity of GDNF. Soluble GDNFR-a attached to a detectable label
also
may be used in diagnostic applications as discussed above.
Example 8
2o Chemical Crosslinking of GDNF with GDNFR-a
In order to study its binding properties and molecular characteristics,
GDNFR-a was transiently expressed on the surface of 293T cells by transfection
of
the rat eDNA clone. Transfection of 293T cells was performed using the Calcium
Phosphate Transfection System (GIBCO/BRL, Gaithersburg, MD) according to the
manufacturers instructions. Two days following transfection, cells were
removed by
2x versine treatment, washed once with washing buffer, and resuspended in
washing
buffer at a density of 2 x 106 cells/mL. A duplicate set of cells were
incubated with
0.5 ulmL PI-PLC at 37oC for 30 minutes before [1251]GDNF binding. These cells
3o were washed three times with ice-cold binding buffer and then incubated
with 1 to 3
nM of [ 125I]GDNF along with other cells at 4oC for four hours. Cells were
washed
four times with ice-cold washing buffer, resuspended in washing buffer
supplemented with 1 mM of Bis suberate for crosslinking (BS3 Pierce, Rockford,
IL)
and incubated at room temperature for 30 minutes. Following three washes with
TBS, a duplicate group of samples was treated by 0.5 u/mL of PI-PLC at 37oC
for
30 minutes. These cells were pelleted and the supernatants were collected. The
cells
were then washed with washing buffer and lysed along with all other cells with
2x
CA 02291608 1999-11-26
WO 98/54213 PCTlUS98/08486
73
SDS-PAGE sample buffer. The cell lysates and the collected supernatants were
resolved on a 7.5% SDS-PAGE.
The cell suspension was divided into aliquots containing 1.5 x l OS
cell/sample. Cells were then pelleted and incubated with various
concentrations of
[ 125I]GDNF in the presence or absence of 500 nM of unlabeled GDNF at 4oC for
four hours with gentle agitation. Cells were washed four times with ice-cold
washing buffer and resuspended in 0.5 mL washing buffer. Two 0.2 mL aliquots
of
the suspension were counted in a gamma counter to determine the amount of
[ 1251]GDNF associated with the cells.
o Although mock transfected 293T cells did not exhibit any GDNF binding
capacity, GDNFR-a transfected cells bound [ 12SI)GDNF strongly even at
picomolar
concentrations. This binding was almost completely inhibited by 500 nM of
unlabeled GDNF, indicating a specific binding of native GDNF to the expressed
receptors.
GDNFR-a expressed by the 293T cells can be released from the cells by
treatment with phosphatidylinositol-specific phospholipase C (PI-PLC,
Boehringer
Mannheim, Indianapolis, IN). The treatment of transfected cells with PI-PLC
prior
to ligand binding almost entirely eliminated the GDNF binding capacity of the
cell.
Additionally, treatment of the transfected cells after cross-linking released
the
2o majority of the cross-linked products into the media. These results
strongly suggest
that GDNFR-a is anchored to the cell membrane through a GPI linkage.
Crosslinking data further indicated that the molecular weight of GDNFR-a is
approximately 50-65 kD, suggesting that there is a low level of glycosylation.
Although the major cross-linked species has a molecular mass consistent with a
monomer of the receptor, a minor species with approximately the mass expected
for
a dimer has been found.
Example 9
3o GDNF Signaling is Mediated by a Complex of GDNFR-a
and the Ret Receptor Protein Tyrosine Kinase
Introduction
Mice carrying targeted null mutations in the GDNF gene exhibit various
defects in tissues derived from neural crest cells, in the autonomic nervous
system,
and in trigeminal and spinal cord motor neurons. The most severe defects are
the
CA 02291608 1999-11-26
WO 98/54213 PCTIUS98/08486
74
absence of kidneys and complete loss of enteric neurons in digestive tract.
The
phenotype of GDNF knockout mice is strikingly similar to that of the c-ret
knockout
animals (Schuchardt et al. 1994), suggesting a possible linkage between the
signal
transduction pathways of GDNF and c-ret.
The proto-oncogene c-ret was identified using probes derived from an
oncogene isolated in gene transfer experiments (Takahashi et al., Cell. 42,
581-588,
1985; Takahashi and Cooper, Mol. Cell. Biol., 7, 1378-1385, 1987). Sequence
analysis of the c-ret cDNA revealed a large open reading frame encoding a
novel
receptor protein tyrosine kinase (PTK). The family of receptor PTKs has been
grouped into sub-families according to extracellular domain structure and
sequence
homology within the intracellular kinase domain (van der Geer et al., 1994).
The
unique extracellular domain structure of Ret places it outside any other known
receptor PTK sub-family; it includes a signal peptide, a cadherin-like motif,
and a
cysteine-rich region (van Heyningen, Nature, 367, 319-320, 1994; Iwamoto et
al.,
1993). In situ hybridization and immunohistochemical analysis showed high
level
expression of ret mRNA and protein in the developing central and peripheral
nervous systems and in the excretory system of the mouse embryo (Pachnis et
al.,
1993; Tsuzuki et al., Oncogene, 10, 191-198, 1995), suggesting a role of the
Ret
receptor either in the development or in the function of these tissues. A
functional
ligand of the Ret receptor has not been identified, thereby limiting a further
understanding of the molecular mechanism of Ret signaling.
Mutations in the c-ret gene are associated with inherited predisposition to
cancer in familial medullary thyroid carcinoma (FMTC), and multiple endocrine
neoplasia type 2A (MEN2A) and 2B (MEN2B). These diseases are probably caused
by "gain of function" mutations that constitutively activate the Ret kinase
(Donis-
Keller et al., Hum. Molec. Genet. 2, 851-856, 1993; Hofstra et al., Nature.
367,
375-376, 1994; Mulligan et al., Nature. 363, 458-460, 1993; Santoro et al.,
Science.
267, 381-383, 1995). They confer a predisposition to malignancy specifically
in
tissues derived from the neural crest, where ret is normally expressed in
early
development. Another ret-associated genetic disorder, Hirschsprung's disease
(HSCR), is characterized by the congenital absence of parasympathetic
innervation
in the lower intestinal tract (Edery et al., Nature. 367, 378-380, 1994; Romeo
et al.,
1994). The most likely causes of HSCR are nonsense mutations that result in
the
production of truncated Ret protein lacking a kinase domain or missense
mutations
that inactivate the Ret kinase. As noted above, targeted disruption of the c-
ret proto-
oncogene in mice results in renal agenesis or severe dysgenesis and lack of
enteric
neurons throughout the digestive tract (Schuchardt et al., 1994). This
phenotype
CA 02291608 1999-11-26
WO 98/54213 PCTIUS98/08486
closely resembles that of GDNF knockout mice. Taken together, these data
suggest
that both Ret and GDNF are involved in signal transduction pathways critical
to the
development of the kidney and the enteric nervous system. How Ret and GDNF are
involved, however, was not known.
5 The isolation and characterization of cDNA for GDNFR-a by expression
cloning, as described above, lead to the expression of GDNFR-a in the
transformed
human embryonic kidney cell line 293T. Transformation resulted in the
appearance
of both high (Kd of approximately 2 pM) and low (Kd of approximately 200 pM)
affinity binding sites. The high affinity binding sites could be composed of
1o homodimers or homo-oligomers of GDNFR-a alone, or of heterodimers or hetero-
oligomers of GDNFR-a with other molecules. As discussed above, because
GDNFR-a lacks a cytoplasmic domain, it must function through one or more
accessory molecules in order to play a role in GDNF signal transduction. In
this
study we confirm that, in the presence of GDNFR-a, GDNF associates with the
Ret
15 protein tyrosine kinase receptor, and quickly induces Ret
autophosphorylation.
Results
Neuro-2a Cells Expressing GDNFR-a Bind GDNF with High Affinity
2o Neuro-2a is a mouse neuroblastoma cell line that endogenously expresses a
high level of Ret protein (Ikeda et al., Oncogene. 5, 1291-1296, 1990; Iwamoto
et
al., Oncogene. 8, 1087-1091, 1993; Takahashi and Cooper, 1987) but does not
express detectable levels of GDNFR-a mRNA as judged by Northern blot. In order
to determine if Ret could associate with GDNF in the presence of GDNFR-a, a
study
2s was performed to examine the binding of [125I)GDNF to Neuro-2a cells
engineered
to express GDNFR-a. Neuro-2a cells were transfected with a mammalian
expression vector containing the rat GDNFR-a cDNA (such as the expression
plasmid described above). Three cional lines, NGR-16, NGR-33, and NGR-38 were
tested for their ability to bind [ 125IjGDNF. The unbound [ 1251]GDNF was
3o removed at the end of the incubation and the amount of radioactivity
associated with
the cells was determined as described in Experimental Procedures. All three
lines
were able to bind [125IJGDNF specifically while parental Neuro-2a cells
exhibited
little or no [ 1251]GDNF binding (Figure 6). Binding could be effectively
competed
by the addition of 500 nM unlabeled GDNF. These results demonstrate that Ret
35 receptor expressed on Neuro-2a cells is unable to bind GDNF in the absence
of
GDNFR-a and are consistent with the previous observation that GDNFR-a is not
expressed at appreciable levels in Neuro-2a cells.
CA 02291608 2003-O1-29
WO 98/54213 - ,~ ~ pCT/US98/08486
76
Equilibrium binding of [ 1251]GDNF to NGR-38 cells was examined over a
wide range of ligand concentrations (0.5 pM to 1 nM of [1251]GDNF in the
presence
or absence o.f 500 nM of unlabeled GDNF) (see Figure 7A). Following
incubation,
unbound [ 1251]GDNF was removed and the radioactivity associated with the
cells
was determined as described in Experimental Procedures. Results are depicted
in
Figure 7: (A) Equilibrium binding of [12SI]GDNF to NGR-38 cells (circles) and
Neuro-2a cells (squares) in the presence (open circles and open squares) or
absence
(filled circles and filled squares) of unlabeled GDNF; (B) Scatchard analysis
of
[1251]GDNF binding to NGR-38 cells. Neuro-2a cells exhibited little binding
even
1o at a concentration of 1 nM [12SI]GDNF, and this binding was not affected by
the
addition of excess unlabeled GDNF. Binding to NGR-38 cells was analyzed by
Scatchard plat as shown in Figure 7B. Two classes of binding sites were
detected,
one with Kd = 1.5 ~ O.S pM and the other with Kd = 332 + 53 pM. These
dissociation constants are very similar to the values obtained for the high
and low
affinity binding sites in 293T cells transiently expressing GDNFR-a, as
described
above.
w.
In order to determine if the Ret receptor PTK could associate with GDNF in
2o cells expressing GDNFR-a, a cross-linking experiment was carried out using
NGR-
38 and parental Neuro-2a cells. NGR-38 cells were incubated with [ 125I]GDNF,
treated with cross-linking reagent, then lysed either.directly in SDS-PAGE
sample
buffer or in Triton X100 lysis buffer and further immunoprecipitated with anti-
Ret
antibody as described in the Experimental Procedures. The immunoprecipitates
were analyzed by SDS-PAGE'in the absence (NR) or presence (R) of -
mercaptoethanol. L;ysates were treated with Ret specific antibody,
immunoprecipitated, and analyzed by SDS-PAGE under reducing conditions (see
l~igure 8, bands are marked as follows: ~75 kD, solid triangle; --1 SO kD,
open
triangle; ~ 185 kD, solid arrow; 250 kD, asterisk; ~400kD, open arrow). The
most
3o prominent cross-linked species were at ~7S kD, and ~ 185 kD, with less
intense
bands of ~ 1 SO kD and 250 kD. A very faint band of 400 kD was also visible
(Figure 8, lane 2). When immunoprecipitates were analyzed by non-reducing
SDS-PAGE, the ~75 kD, 150 and --18S kD bands were present at about the same
intensity as in the reducing gel, but the amount of the --400 kD band
increased
dramatically (Figure 8, lane 4). Also becoming more prominent was the band at
250 kD.
Under both reducing and non-reducing conditions; bands of similar
* trademark
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/08486
77
molecular weight but of greatly reduced intensity were observed when parental
Neuro-2a cells were used instead of NGR-38 (Figure 8, lanes 1 and 3). The ~75
kD
and 150 kD species are likely to represent cross-linked complexes of GDNF and
GDNFR-a, since species with identical molecular weights are produced by cross-
linking in 293T cells that do not express Ret. Furthermore, since the
molecular
weight of Ret is 170 kD, any complex including Ret must be of at least this
size.
The fact that these complexes are immunoprecipitated by anti-Ret antibody
indicates they are products of an association between Ret and the GDNF/GDNFR-a
complex which was disrupted under the conditions of the gel analysis. It is
to envisioned that the broad band at 185 kD probably consists of one molecule
of Ret
( 170 kD) cross-linked with one molecule of monomeric recombinant GDNF ( 15
kD), although some dimeric GDNF may be included. The presence of Ret in this
species was confirmed by a separate experiment in which a band of the same
molecular weight was observed when unlabeled GDNF was cross-linked to NGR-38
15 cells and the products examined by Western blot with anti-Ret antibody
(data not
shown).
The 400 kD band was not reliably identified, partly due to the difficulty in
estimating its molecular weight. The fact that it is prominent only under non-
reducing conditions indicates that it is a disulfide-linked dimer of one or
more of the
2o species observed under reducing conditions. The most likely explanation is
that it
represents a dimer of the 185 kD species, although it may be a mixture of high
molecular weight complexes consisting of two Ret, one or two GDNFR-a, and one
or two GDNF molecules. The exact identity of the 250 kD band has not yet been
determined. One possibility is that it represents cross-linked heterodimers of
the ~75
25 kD (GDNF + GDNFR-a) and 185 kD (GDNF + Ret) complexes.
GDNF Stimulates Autophosphorvlation of Ret in Neuro-2a Cells Expressing
GDNFR-a
The ability of the Ret protein tyrosine kinase receptor to associate with
3o GDNF in the presence of GDNFR-a led to the study of GDNF stimulation of the
autophosphorylation of Ret. NGR-38 cells were treated with GDNF, lysed, and
the
lysates immunoprecipitated with anti-Ret antibody. The immunoprecipitates were
analyzed by Western blot using an anti-phosphotyrosine antibody as described
in the
Experimental Procedures. When NGR-38 cells (Figure 9A, lanes 2-4) were treated
35 with purified recombinant GDNF produced in either mammalian (CHO cells;
Figure 9A, lanes 4) or E. coli cells (Figure 9A, lanes l, 3), a strong band
was
observed at 170 kD, indicating autophosphorylation of tyrosine residues on the
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/08486
78
mature form of Ret. A much weaker corresponding band was observed in GDNF-
treated Neuro-2a cells (Figure 9A, Iane 1 ). No phosphorylation was observed
on the
alternatively glycosylated 150 kD precursor form of Ret (Figure 9A). The
induction
of Ret autophosphorylation by GDNF was dosage dependent. The dose response
and kinetics of GDNF-induced Ret tyrosine phosphorylation in NGR-38 cells are
shown in panels B and C. In all panels, the tyrosine phosphorylated 170 kD Ret
bands are indicated by solid arrows. The amount of Ret protein loaded in each
lane
as determined by reprobing of the immunoblot with anti-Ret antibody (Santa
Cruz,
C-19, Cat. #sc-167) is shown on the right side of panel A. The band at 150 kD
1o represents an alternately glycosylated immature form of Ret that does not
autophosphorylate. As shown in Figure 9B, stimulation of Ret
autophosphorylation
in NGR-38 cells could be detected with 50 pg/mL of GDNF and the response was
saturated at 20-50 ng/mL GDNF. The stimulation of Ret autophosphorylation by
purified recombinant GDNF in NGR-38 cells over times of 0-20 minutes following
treatment is shown in Figure 9C. Increased levels of Ret autophosphorylation
could
be observed within one minute of GDNF treatment and was maximal at 10 minutes
following treatment (Figure 9C).
GDNF and Soluble GDNFR-a Induce Ret Auto~hos~horylation in Neuro-2A Cells
2o As discussed above, GDNFR-a is anchored to the cytoplasmic membrane
through a GPI linkage and can be released by treatment with
phosphatidylinositol-
specific phospholipase C (PI-PLC). When NGR-38 cells were incubated with
PI-PLC, GDNF-induced receptor autophosphorylation of Ret in these cells was
abolished (Figure 10A; PI-PLC treated (lane 1) or untreated (lanes 2 and 3)
NGR-38
cells were incubated with (lanes l and 3) or without (lane 2) GDNF and
analyzed for
Ret autophosphorylation by immunoblotting as described in the Experimental
Procedures).
Figure l OB depicts parental Neuro-2a cells treated with (lanes 2,4,6,8) or
without (lanes 1,3,5,7) GDNF in the presence (lanes 5-8) or absence (lanes 1-
4) of
3o PI-PLC/CM obtained from Neuro-2a or NGR-38 cells, as analyzed for Ret
autophosphorylation by immunoblotting as described in the Experimental
Procedures. NGR-38 cells treated with GDNF were used as a positive control. In
both panels A and B, the autophosphorylated 170 kD Ret bands are marked by
solid
arrows. When conditioned medium containing soluble GDNFR-a released by PI-
PLC treatment (PI-PLC/CM) of NGR-38 cells was added to parental Neuro-2a cells
along with GDNF, autophosphorylation of the Ret receptor comparable to that
obtained with GDNF treatment of NGR-38 cells was observed {Figure l OB, lanes
2
CA 02291608 1999-11-26
WO 98154213 PCT/US98/08486
79
and 8). Only background levels of Ret autophosphorylation were observed when
no
GDNF was added, or when conditioned media derived from PI-PLC treatment of
Neuro-2a cells was tested (Figure l OB, lanes 3-7).
Ret-Fc Fusion Protein Blocks Ret Phosphorylation Induced by GDNF and Soluble
GDNFR-a
To confirm that Ret phosphorylation induced by GDNF in the presence of
GDNFR-a is the result of receptor autophosphorylation, a study was performed
to
determine whether a Ret extracellular domain/Immunoglobulin Fc (Ret-Fc) fusion
1o protein could block Ret activation. Because of the technical difficulty of
blocking
the large number of GDNF alpha receptors expressed on NGR-38 cells, Ret
phosphorylation assays were performed using Neuro-2a as the target cell and
culture
media removed from NGR-38 cells treated with PI-PLC as a source of GDNFR-a.
Cells were treated with mixtures including various combinations of GDNF
15 (50 ng/mL), media containing soluble GDNFR-a (e.g., PI-PLC/CM derived from
NGR-38 cells), and different concentrations of Ret-Fc fusion protein either
alone or
in various combinations as indicated in Figure 11. Neuro-2a cells were treated
with
GDNF, media containing soluble GDNFR-a, Ret-Fc, or the pre-incubated mixtures.
The cells were then lysed, and the lysates were analyzed for c-Ret
2o autophosphorylation by immunoprecipitation using anti-Ret antibody as
described in
the Experimental Procedures. The immunoprecipitates were analyzed by Western
blot using an anti-phosphotyrosine antibody.
The pre-incubated mixture of GDNF and media containing soluble GDNFR-
a induced tyrosine phosphorylation of Ret receptors expressed in Neuro-2a at a
level
25 comparable to GDNF-treated NGR-38 control cells (Figure 11, lanes 7 and 2).
The
position of the autophosphorylated 170 kD Ret bands are marked by a solid
arrow.
When Ret-Fc fusion protein was included in the pre-incubated GDNFIGDNFR-a
mixture, Ret phosphorylation was inhibited in a dose dependent manner (Figure
11,
lanes 8-10). This indicated that Ret phosphorylation is a result of a GDNF/Ret
3o interaction mediated by GDNFR-a. In untreated Neuro-2a cells or in cells
treated
with any combination of GDNF or Ret-Fc fusion protein in the absence of GDNFR-
a, only background levels of Ret phosphorylation were observed (Figure 1 l,
lanes 3-
6).
35 GDNF Induces Autophosphorvlation of c-RET Expressed in Embryonic Motor
Neurons
Spinal cord motor neurons are one of the major targets of GDNF action in
CA 02291608 1999-11-26
WO 98/54213 PCT/US98108486
vivo (Henderson et al., Science. 266, 1062-1064, 1994; Li et al., Proceedings
Of The
National Academy Of Sciences, U.S.A. 92, 9771-9775, 1995; Oppenheim et al.,
Nature. 373, 344-346, 1995; Yan et al., Nature. 373, 341-344, 1995; Zurn et
al.,
Neuroreport. 6, 113-118, 1995). To test the ability of GDNF to induce Ret
5 autophosphorylation in these cells, embryonic rat spinal cord motor neurons
were
treated with (lanes 2 and 4) or without {lanes 1 and 3) 20 ng/mL GDNF followed
by
lysis of the cells, immunoprecipitation with anti-Ret antibody, and analysis
by
Western blotting with anti-phosphotyrosine antibody as described in the
Experimental Procedures. In lysates of cells treated with GDNF, a band of
tyrosine
I o phosphorylated protein with a molecular mass of ~ 170 kD was observed
(Figure 12,
lane 2). No such signal was observed with cells treated with binding buffer
alone
(Figure 12, lane 1 ). When the same Western blot filter was stripped and re-
probed
with anti-Ret antibody (i.e., the amount of c-Ret protein loaded in each lane
was
determined by reprobing the immunoblot with the anti-Ret antibody), bands with
the
15 same molecular mass and similar intensities appeared in both samples
(Figure 12,
lanes 3 and 4). The phosphotyrosine band in GDNF-treated cells co-migrates
with
the Ret protein band, indicating GDNF stimulated autophosphorylation of Ret.
The
autophosphorylated Ret bands (lanes 1 and 2) and the corresponding protein
bands
(lanes 3 and 4) were marked by a solid arrow.
Discussion
Polypeptide growth factors elicit biological effects through binding to their
cognate cell surface receptors. Receptors can be grouped into several classes
based
on their structure and mechanism of action. These classifications include the
protein
tyrosine kinases (PTKs), the serine/threonine kinases, and the cytokine
receptors.
Receptor PTK signaling is initiated by a direct interaction with ligand, which
induces receptor dimerization or oligomerization that in turn leads to
receptor
autophosphorylation. The activated receptor then recruits and phosphorylates
3o intracellular substrates, initiating a cascade of events which culminates
in a
biological response (Schlessinger and UlIrich, Neuron 9, 383-391, 1992). In
contrast, signal transduction by serine/threonine kinase or cytokine receptors
often
involves formation of mufti-component receptor complexes in which the ligand
binding and signal transducing components are distinct. Examples are the TGF-
receptor complex, a serine/threonine kinase receptor consisting of separate
binding
(Type II) and signaling (Type I) components and the CNTF family. CNTF,
interleukin-6 (IL-6) and leukocyte inhibitory factor (LIF) share the common
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/08486
81
signaling components, gp 130 and/or LIFR, in their respective receptor
complexes.
While the ligand specificity of these complexes is determined by a specific
binding
subunit to each individual ligand, signal transduction requires association of
the
initial complex of ligand and ligand binding subunit with other receptor
subunits
which cannot bind ligand directly (Ip et al., Cell. 69, 1121-1132, 1992). In
the
CNTF receptor complex, the ligand binding component is CNTF receptor (CNTFR),
which like GDNFR, is a GPI-anchored membrane protein. The present invention
involves the description of the first example of a receptor PTK whose
autophosphorylation is dependent upon association with a separate ligand-
specific
1 o binding component.
The present study confirms that GDNFR-a, a GPI-linked membrane protein
that binds to GDNF with high affinity, is required for the efficient
association of
GDNF with the Ret receptor PTK. In the absence of GDNFR-a, GDNF is unable to
bind to Ret or stimulate Ret receptor autophosphorylation. In the presence of
15 GDNFR-a, GDNF associates with Ret and rapidly induces Ret
autophosphorylation
in a dose-dependent manner. GDNFR-a is able to function in either membrane
bound or soluble forms (Figure 11), as discussed above. GDNF concentrations of
50
pg/mL ( 1.7 pM) are able activate the Ret tyrosine kinase in cells expressing
GDNFR-a. This is consistent with the dissociation constant ( 1.5 pM) found for
the
2o high affinity GDNF binding sites on NGR-38 cells. The rapid induction of
Ret
phosphorylation by GDNF (detectable one minute after treatment) and the
ability of
Ret-Fc to block autophosphorylation suggest that Ret is being activated
directly
rather than as a downstream consequence of the phosphorylation of some other
receptor.
25 Cross-linking studies support the hypothesis that efficient association of
Ret
with GDNF depends on GDNFR-a. Cross-linking of GDNF to Ret in NGR-38 cells
which express high levels of GDNFR-a is robust, but in parental Neuro-2a cells
cross-linked products are barely detectable. Although conclusive
identification of all
the cross-linked complexes is difficult, the data clearly demonstrates an
association
30 of Ret with GDNF that is dependent on the presence of GDNFR-a, and
demonstrates that GDNFR-a is included in some of the cross-linked products.
The
reason for the presence of minor cross-linked species in Neuro-2a cells is not
clear.
While the expression of GDNFR-a mRNA in Neuro-2a cells could not be detected
by Northern blot, it is possible that GDNFR-a is expressed at very low levels
in
35 these cells.
The fact that Ret can be activated by GDNF in cultured rat embryonic spinal
cord motor neurons further demonstrates the biological relevance of the
RetIGDNF
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/08486
82
interaction. These cells are a primary target of GDNF in vivo, and have been
shown
to respond to low doses of GDNF in vitro (Henderson et al., 1994). Stimulation
of
Ret phosphorylation was abolished when the motor neuron cells were pre-treated
with PI-PLC (data not shown), suggesting that the activation of Ret by GDNF
requires GDNFR-a.
Although binding of ligand to the receptor extracellular domain is the first
step in the activation of other known receptor PTKs, the present data has
shown that
this is not the case for GDNF and Ret. Figure 13 depicts a model for the
binding of
GDNF to GDNFR-a and Ret, and the consequent activation of the Ret PTK in
to response to GDNF. The initial event in this process is the binding of
disulfide-
linked dimeric GDNF to GDNFR-a in either monomeric or dimeric form. Although
there is
currently no direct evidence for the existence of dimeric GDNFR-a, when 293T
cells
were transfected with GDNFR-a cDNA, two classes of binding sites appeared. The
simplest explanation for this observation is the existence of monomeric and
dimeric
GDNFR-a, each with its own ligand binding affinity. This is consistent with
the
Ending that GDNF binding affinities are apparently unaffected by the presence
of
Ret. Since the present experiments do not address the question of whether
dimeric
GDNFR-a is in equilibrium with its monomer in the absence of GDNF or if
2o dimerization is induced by GDNF binding, these possibilities are presented
as
alternate pathways. The complex consisting of dimeric GDNFR-a and dimeric
GDNF can bind two molecules of Ret, forming the active signaling complex. As
for
other PTKs, close contact between the intracellular catalytic domains of two
Ret
molecules is likely to result in receptor autophosphorylation. This notion
that Ret
functions by this mechanism is supported by the fact that the MEN2A mutation
which causes steady state dimerization of Ret results in constituitive
activation of the
Ret kinase (Santoro et al., 1995).
Motor neurons have been reported to respond to GDNF with an EDSp of as
low as 5 fM (Henderson et al., 1994). Although it is difficult to compare
binding
3o affinity with the EDSp for a biological response, it is possible that very
high affinity
GDNF binding sites exist on these cells. Other cells, such as embryonic chick
sympathetic neurons, have been reported to bind GDNF with a Kd of 1-5 nM
(Trupp
et al., Journal Of Cell Biology. 130, 137-148, 1995). It is unlikely that
GDNFR-a is
involved in a receptor complex for such low affinity sites, but a weak direct
interaction between GDNF and Ret may be present.
Expression of c-ret has been observed during embryogenesis in many cell
lineages of the developing central and peripheral nervous systems, including
cells of
CA 02291608 1999-11-26
WO 98/54213 PCTlUS98108486
83
the enteric nervous system (Pachnis, et al., Development, I 19, 1005-1017,
1993;
Tsuzuki et al., 1995). Outside the nervous system, c-ret expression has been
detected in the Wolffian duct, ureteric bud epithelium and collecting ducts of
the
kidney (Pachnis, et al., supra; Tsuzuki et al., 1995). Ret expression has also
been
s detected in all neuroblastoma cell lines derived from the neural crest
(Ikeda et al..
1990) and from surgically resected neuroblastomas (Nagao et al., 1990;
Takahashi &
Cooper, 1987). GDNF expression has been observed in both CNS and PNS, as well
as in non-neuronal tissues during embryonic development. The levels of GDNF
expression found in many non-neuronal tissues were higher than in the nervous
1o system (Choi-Lundberg and Bohn, Brain Res. Dev. Brain Res. 85, 80-88,
1995).
Although expression of GDNFR-a has not been extensively studied, primary
Northern blot analysis detected the presence of high levels of the GDNFR-a
mRNA
in the liver, brain, and kidney of adult rat and mouse. The similarity of the
expression patterns of ret, GDNF, and GDNFR-a in developing nervous system and
t5 kidney is consistent with their combined action during development.
Mammalian kidney development has been postulated to result from
reciprocal interactions between the metanephron and the developing ureter, a
branch
developed from the caudal part of the Wolffian duct (Saxen, Organogenesis of
the
kidney. Development and Cell Biology series, Cambridge University Press,
2o Cambridge, England, 1987). While the expression of Ret has been found at
the
ureteric bud but not in the surrounding mesenchyme in developing embryos, the
expression of GDNF was detected in the undifferentiated but not adult
metanephric
cap of the kidney. These observations suggest that an interaction between GDNF
and Ret is responsible for initiating the development of the ureteric
structure.
25 Further support for this hypothesis is provided by targeted disruptions of
the GDNF
and ret genes, which result in very similar phenotypic defects in kidney
(Schuchardt
et al., Nature. 367, 380-383, 1994; Sanchez, in press). Another major
phenotypic
defect observed in both GDNF (-I-) and ret (-I-) knockout animals is a
complete loss
of the enteric neurons throughout the digestive tract. Hirschsprung's disease,
a
3o genetic disorder characterized by the congenital absence of parasympathetic
innervation in the lower intestinal tract, has also been linked to "loss-of
function"
mutations in ret (Romeo et al., Nature. 367, 377-378, 1994. Edery et al.,
1994). A
later report (Angrist et al., Hum. Mol.Genet. 4, 821-830, 1995) indicated
that,
contrary to earlier observations, some Hirschsprung's patients do not carry
3s mutations in ret. It is now envisioned that such patients may carry
mutations in
GDNF, GDNFR-a or some other critical component of this signaling pathway.
CA 02291608 2003-O1-29
WO 98/54213
pCT/US98108486
84
1125nC,DNF Bindi_n~g to Neuro-2a Cell FxnrPCsing GDNF$~
Neuro-2a cells (ATCC #CCL 131 ) were transfected with an expression
plasmid, as described above, using the Calcium Phosphate Transfection System
(GIBCO/BRL) according to the manufacturer's directions. Transfected cells were
selected for expression of the plasmid by growth in 400 ~g/mL 6418 antibiotic
(Sigma). 6418 resistant clones were expanded and assayed for GDNFR-a
expression by binding to [ 1251]GDNF (Amersham, inc., custom iodination,
catalog
t0 #IMQ1057). Cells from each clone were seeded at a density of 3 x 104
cells/cm2 in
duplicate wells of 24-well tissue culture plates (Becton Dickinson) pre-coated
with
polyomithine. Cells were washed once with ice-cold washing buffer (DMEM
containing 25 mM HEPES, pH 7.5) and were then incubated with 50 pM
[ 1251]GDNF in binding buffer (washing buffer plus 0.2% BSA) at 4oC for four
hours either in the presence or absence of 500 mM unlabeled GDNF. Cells were
then washed four times with ice-cold washing buffer, lysed in 1 M NaOH, and
the
cell-associated radiolabel quantitated in a 1470 Wizard~Automated Gamma
Counter
(Wallac Inc.). The amount of GDNFR-a expressed by individual clones was
estimated by the ratio of [ 125I]GDNF bound to cells in the absence and
presence of
2o unlabeled GDNF. Three clones were chosen as representatives of high,
moderate,
and low level expressors of GDNFR-a for use in binding experiments. The ratios
[ 1251]GDNF bound in the absence and presence of unlabeled GDNF for these
clones
were: NGR-38) 16:1, NGR-lb) 12.8:1, and NGR-33) 8:1. Equilibrium binding of
[ 1251]GDNF to NGR-38 cells was carned out as described above except that
concentrations of labeled GDNF ranged from 0.5 pM to 1 nM. la all assays,
nonspecific binding as estimated by the amount of radiolabel binding to cells
in the
presence of SOO.nM unlabeled GDNF was subtracted from binding in the absence
of
unlabeled GDNF. Binding data was analyzed by Scatchard plot.
Chemical_ C~~ 'nkin
Neuro-2a or NGR-38 cells were washed once with phosphate-buffered saline
(PBS, pH 7.1 ), then treated for four hours at 4oC with 1 or 3 nM [ 1251]GDNF
in
binding buffer in the presence or absence of 500 nM unlabeled GDNF. Following
binding, cells were washed four times with ice-cold washing buffer and
incubated at
room temperature for 45 minutes with 1 mM bis suberate (BS3, Pierce) in
washing
buffer. The cross-linking reaction was quenched by washing the cells three
times
with Tris-buffered saline (T'BS,_pH 7.5). The cells were then either lysed
directly in
* trademark
CA 02291608 2003-O1-29
WO 98/54213 ~ , PCT/US98/08486
SDS-PAGE sample buffer (80 mM Tris HC1 [pH 6.8], 10% glycerol, 1% SDS,
0.025% bromophenol blue) or in Triton X-100 lysis buffei (50 mM Hepes, pH 7.5,
1 % Triton X-100, 50 mM NaCI, 50 mM NaF, 10 mM sodium pyrophosphate, 1
aprotinin (Sigma, Cat.# A-6279), 1 mM PMSF (Sigma, Cat.# P-7626), 0.5 mM
5 Na3V04 (Fisher Cat.# S454-50). The lysates were clarified by centrifugation,
incubated with 5 ltg/mL of anti-Ret antibody (Santa Cruz Antibody, C-19, Cat.
#SC-167), and the resulting immunocomplexes were collected by precipitation
with
protein A-Sepharose CL-4B (Pharmacia). The immunoprecipitates were washed
three times with the lysis buffer, once with 0.5% NP-40 containing 50 mM NaCI
and
10 20 mM Tris-Cl, pH 7.5, and were then resuspended in SDS-PAGE sample buffer.
Both the whole cell lysates and the immunoprecipitates were fractionated by
7.5%
SDS-PAGE with a ratio of Bis:Acrylamide at 1:200.
Wester,~,~lo~ nalv i
15 The autophosphorylation of Ret receptor was examined by Western blot
analysis. Briefly, cells were seeded 24 hours prior to the assay in 6-well
tissue
culture dishes at a density of 1.5 x 106 cells /well. Cells were washed once
with
binding buffer and treated with various concentrations of different reagents ;
(including GDNF, PI-PLC, PI-PLC/CM, and Ret-Fc fusion protein), either alone
or
2o in combination, in binding buffer for various periods of times. Treated
cells and
untreated controls were lysed in Triton X-100 lysis buffer and
immunoprecipitated
with the anti-Ret antibody (Santa Cruz, C-19, Cat. #SC-167) and protein-A
Sepharose as described above. Immunoprecipitates were fractionated by SDS-PAGE
and transferred to nitrocellulose membranes as described by Harlow and Lane
25 (Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory: Cold
Spring
Harbor, New York, 1988). The membranes;were pre-blocked with 5% BSA (Sigma)
and the level of tyrosine phosphorylation of the receptor was determined by
blotting
the membrane with an anti-phosphotyrosine monoclonal antibody 4G 10 (UBI, Cat.
#0S-321 ) at room temperature for two hours. The amount of protein included in
3o each lane was determined by stripping and re-probing the same membrane with
the
anti-Ret antibody. Finally, the membrane was treated with chemilumiaescence
reagents (ECL, Amersham) following the manufacturer's instructions and exposed
to
X-ray films (Hyperfilm*ELC, Amersham).
35 Treatment of Cells with PI-PLC and Generation of PT-PT C' Treated
Cn"r~;t;nnP~
Media
In order to release GPi-linked GDNFR-a from the cell surface, cells were
* trademark
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/08486
86
washed once with washing buffer, then incubated with 1 U/mL
phosphatidylinositol
specific phospholipase C (PI-PLC, Boehringer Mannheim, Cat. # 1143069) in
binding buffer at 37oC for 45 minutes. The cells were then washed three times
with
washing buffer and further processed for Ret autophosphorylation assay or
cross-
linking. For generation of PI-PLC treated conditioned media (PI-PLC/CM), 8 x
106
cells were removed from tissue culture dishes by treating the cells with PBS
containing 2 mM of EDTA at 37oC for S to 10 minutes. Cells were washed once
with washing buffer, resuspended in 1 mL of binding buffer containing 1 U/mL
of
PI-PLC, and incubated at 37oC for 45 minutes. The cells were pelleted, and the
PI-
PLC/CM was collected.
Preparation of the Ret-Fc Fusion Protein
A cDNA encompassing the entire coding region of c-Ret was isolated from a
day 17 rat placenta cDNA library using an oligonucleotide probe corresponding
to
the first 20 amino acids of the mouse c-Ret (Iwamoto et al., 1993; van
Heyningen,
1994). The region coding for the extracellular domain of the Ret receptor
(ending
with the last amino acid, 8636) was fused in-frame with the DNA coding for the
Fc
region of human IgG (IgG I ) and subcloned into the expression vector pDSR2 as
previously described (Bartley et al., Nature. 368, 558-560, 1994). The ret-
2o FcIpDSRa2 plasmid was transfected into Chinese hamster ovary (CHO) cells
and the
recombinant Ret-Fc fusion protein was purified by affinity chromatography
using a
Ni++ column (Qiagen).
Preparation of Embryonic Rat Spinal Cord Motor Neuron Cultures
Enriched embryonic rat spinal cord motor neuron cultures were prepared
from entire spinal cords of E 15 Sprague-Dawley rat fetuses 24 hours before
the
experiments. The spinal cords were dissected, and the meninges and dorsal root
ganglia (DRGs) were removed. The spinal cords were cut into smaller fragments
and digested with papain in L 15 medium (Papain Kit, Worthington). The motor
3o neurons, which are larger than other types of cells included in the
dissociated cell
suspension, were enriched using a 6.8% Metrizamide gradient (Camu and
Henderson, J Neuroscience. 44, 59-70, 1992}. Enriched motor neurons residing
at
the interface between the metrizamide cushion and the cell suspension were
collected, washed, and seeded in tissue culture dishes pre-coated with poly-L-
ornithine and laminin at a density of ~9 x 104 cells/cm2 and were cultured at
37oC.
CA 02291608 1999-11-26
WO 98154213 PCT/US98/08486
87
Example 10
GRR2 Mediation of Neurturin and GDNF-Induced Ret Activation
The present study demonstrates that neurturin binds to both GDNFR-a and
GRR2, a novel receptor related to GDNFR-a. Both GDNFR-a and GRR2 can
mediate neurturin-induced autophosphorylation of the Ret protein tyrosine
kinase.
GDNF also binds both GDNFR-a and GRR2, and activates Ret in the presence of
either binding receptor. However, neuriurin binds GRR2 more effectively than
GDNF, while GDNF binds GDNFR-a more efficiently than neurturin. These data
to indicate that, while there is crosstalk, GDNF is the primary ligand for
GDNFR-a and
neurturin appears to exhibit a preference for GRR2.
Introduction
Recently, Kotzbauer et al. (Nature, 384, 467-470, 1996) reported the cloning
of neurturin, a novel neurotrophic factor that is approximately 42% identical
in
amino acid sequence to GDNF. Both GDNF and neuriurin are synthesized in pre-
pro forms and their precursor molecules are proteolytically processed to yield
mature
proteins of about 100 amino acids that assemble into disulfide-linked
homodimers.
All seven cysteine residues crucial for the structure of GDNF and their
spacing
patterns are conserved in neurturin (Kotzbauer et al., 1996). Although the
biological
activities of neurturin have not yet been thoroughly investigated, they appear
to be
very similar to those of GDNF. Both neurturin and GDNF have been shown to
promote the survival of sympathetic neurons derived from the superior cervical
ganglia (SCG) and of sensory neurons of both the nodose (NG) and dorsal root
ganglia (DRG). Neurturin and GDNF mRNAs are widely distributed in a variety of
both neuronal and non-neuronal tissues of embryos and adults. Both are found
in
brain, kidney, and lung, whereas neurturin mRNA is also expressed at high
levels in
neonatal blood.
3o The structural and biological similarities between GDNF and neurturin
suggest that their action may be mediated by the same or related receptors.
The
receptor for GDNF consists of a complex of GDNF receptor a (GDNFR-a) and the
Ret protein tyrosine kinase (PTK) (Ding et al., Cell, 85, 1113-1124, 1996;
Treanor et
CA 02291608 1999-11-26
W(~ X8154213 PCT/US98/0848G
88
al., Nature, 382, 80-83, 1996). GDNFR-a is a glycosyl-phosphodylinositol (GPI)
anchored cell surface molecule that serves to bind GDNF but cannot signal
independently since it lacks a cytoplasmic domain. GDNF signaling is
accomplished via association of the complex of GDNF and GDNFR-a with Ret,
resulting in activation of the Ret kinase.
GDNFR-a mRNA is widely distributed in neuronal and nonneuronal tissues
and is expressed through embryonic development to adulthood, implying a broad
spectrum of biological functions (Treanor et al., 1996; Fox et al.,
unpublished data).
The other component of the GDNF receptor complex, Ret, is a receptor type PTK
l0 encoded by the ret proto-oncogene. Ret mRNA and protein are highly
expressed in
the CNS and PNS, as well as in the kidney. Various mutations in the ret gene
are
associated with inherited human diseases, including familial medullary thyroid
carcinoma (FMTC), multiple endocrine neoplasia type 2A (MEN2A) and 2B
(MEN2B), and Hirschsprung's disease. Targeted disruption of the ret gene in
15 knockout mice results in severe phenotypic defects, including renal
agenesis or
severe dysgenesis and lack of entire enteric nervous system. These defects are
extremely similar to those caused by GDNF null mutations, implying that GDNF-
mediated signaling through Ret is required for the development of these
tissues.
Much less severe defects, however, were detected in a number of neuronal
structures
2o in which both GDNFR-a and Ret are expressed, such as the trigeminal and
vestibular
ganglia, the facial motor nucleos, the substantial nigra, and the locus
coeruleus
{Schuchardt et al., Nature, 367, 380-383, 1994; Treanor et al., 1996). This
suggests
that either GDNF signaling is not required for the embryonic development of
these
structures, or that some unknown signaling molecules similar to GDNF or Ret
may
25 exist that can substitute for them. Alternatively, the embryonic
development of these
tissues may completely rely on another yet unknown signaling system.
This example describes the cloning of a novel GDNFR-a related receptor,
GRR2, and provides evidence that GRR2 is a receptor for neurturin. Analogous
to
GDNF and GDNFR-a, neurturin effectively binds GRR2 and induces Ret activation.
30 The data also show that both GDNF and neurturin can interact with either
GDNFR-a
or GRR2 and activate the Ret PTK in the presence of either binding receptor.
Results
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/08486
89
Cloning_and Sequence Analysis of GRR2
A human expressed sequence tag (EST) with significant homology to
GDNFR-a was found by a FASTA search of the publicly available nucleic acid
sequence databases (Marra et al., 1996, WashU-HHMI Mouse EST Project,
unpublished}. Oligonucleotides corresponding to the ends of this EST were
synthesized and used in a reverse transcription-polymerase chain reaction (RT-
PCR)
with human fetal brain mRNA as the template. A fragment of the expected length
was isolated and used as a hybridization probe to screen a human fetal brain
cDNA
library. Five positive clones were identified and the longest clone was
sequenced.
This clone contained a large open reading frame coding for a 464 amino acid
protein
related in sequence to GDNFR-a. We have named this protein GDNFR-a Related
Receptor 2 (GRR2}. The oligonucleotides described above were also used to
screen
pools from a rat photoreceptor cDNA library (Jing et al., 199b) by PCR and a
t 5 product of the expected length was obtained from a single pool. An
individual
cDNA clone from this pool was identified by hybridization to the radiolabeled
PCR
product and sequenced. This clone contained a 2.2 kb insert with an open
reading
coding for a 460 amino acid peptide that is nearly identical to human GRR2.
A comparison of the amino acid sequences of human and rat GDNFR-a and
2o GRR2 is shown in Figure 20. Shaded areas indicate amino acid sequence
conservation between all four receptors while boxes indicate conservation only
between the same receptor from different species. The amino acid sequences of
both GDNFR-a and GRR2 are extremely well-conserved between species, each
human receptor being 92% identical to its rat counterpart. The overall amino
acid
25 sequence identity between human GDNFR-a (hGDNFR-a) and human GRR2
(hGRR2) is 48%. The sequence is most divergent in the C-terminal region--amino
acids 350-465 of hGDNFR-a are only 22% identical to amino acids 361-464 of
hGRR2. In the N-terminal region, hGDNFR-a and hGRR2 are more closely related,
sharing 56% amino acid identity. The corresponding identities between the rat
3o GDNFR-a and GRR2 (rGDNFR-a and rGRR2) are very similar: 48% overall, 26%
in the C-terminal region, and 55% in the N-terminal region. The sequence
comparison indicates that GDNFR-a and GRR2 are likely to be structurally very
similar. The positions of 30 of the 31 cysteine residues (shown in boldface,
Figure
CA 02291608 1999-11-26
WO 98/54213 PC'1'/US98108486
20) found in GDNFR-a are conserved in both human and rat GRR2 (one additional
cysteine residue is present near the N-terminus of hGRR2). In addition, the
hydrophobic C-terminus involved in GPI-linkage of GDNFR-a to the cell membrane
(Jing et al., 1996; Treanor et al., 1996) is also present in GRR2.
Figure 20. Comparison of GDNFR-a And GRR2 Peptide Sequences
The amino acid sequences of human and rat GDNFR-a and GRR2 are
aligned. Shaded areas indicate amino acids that are identical in all four
sequences.
Boxes indicate conservation between rat and human orthologs of the same
receptor,
to but not between GDNFR-a and GRR2.
Both Neurturin And GDNF Bind to LA-N-5 And NGR-38 Cells
LA-N-5 is a human neuroblastoma cell line (Sonnenfeld and Ishii, J.
Neuroscience Research, 8:375-391, 1982) that expresses high levels of ret mRNA
15 {Bunone et al., Exp. Cell. Res., 217:92-99, 1995). RT-PCR experiments using
primers specific to GDNFR-a and GRR2 showed that these cells express GRR2
mRNA, but GDNFR-a mRNA was not detected (data not shown). NGR-38 is a cell
line derived from mouse Neuro-2a cells (Ding et al., 1996). It expresses high
levels
of both GDNFR-a and Ret (Jing et al., 1996), but no detectable GRR2 (data not
2o shown), and binds GDNF specifically. LA-N-5 and NGR-38 cells were incubated
with ['z5I]-labeled recombinant human neurturin {NTN) or GDNF in the absence
or
presence of excess unlabeled ligand. As shown in Figure 21A, ['z'I]NTN bound
to
LA-N-5 cells more strongly than ['ZSI]GDNF, although bpth bound at detectable
levels. The binding of ['25I]NTN to LA-N-5 cells was significantly inhibited
by
25 unlabeled neurturin, but not by GDNF. ['25I]GDNF also bound to LA-N-5
cells,
however, the binding was inhibited by either cold GDNF or neurturin.
Figure 2/B depicts the binding of ['BSI]NTN and ['2SI]GDNF to the GDNFR-a
expressing cell line NGR-38. Although both ['z5I]NTN and ['z5I]GDNF bound to
NGR-38 cells, ['25I]GDNF bound more strongly. As was observed for LA-N-5
cells,
3o the binding of ['ZSI]GDNF to NGR-38 cells was inhibited by both unlabeled
neurturin
and GDNF, while binding of ['BSI]NTN was only replaceable by neurturin
(Figure 21 B).
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/08486
91
Figure 21 Binding of Neurturin and GDNF to LA-N-S and NGR-38 Cells
LA-N-5 (A) and NGR-38 (B) cells were incubated with 50 pM of either
[,25I]NTN or ['25I]GDNF in the absence (light gray bars) or presence of
unlabeled
GDNF (dark gray bars) or neurturin {black bars) at 4°C for 2 hours. The
unbound
ligands were removed at the end of the incubation and the radioactivity
associated
with the cells was determined as described.
Cross-Linking of Neurturin and GDNF to GDNFR-a and GRR2
The binding experiments suggest that both neurturin and GDNF interact with
t o GDNFR-a and GRR2. However, lack of a GRR2 specific antibody made further
study of these interactions difficult. To overcome this diff culty, plasmids
were
generated that transiently express GDNFR-a/Fc and GRR2/Fc fusion proteins when
transfected into 293T cells. Conditioned medium (CM) containing either GDNFR-
a/Fc or GRR2/Fc fusion proteins was incubated with [ 1251]NTN or [ 1251]GDNF,
15 chemically cross-linked, and then precipitated directly using Protein-A
Sepharose
beads. The immunoprecipitates were analyzed by SDS-PAGE (Figure 22). Major
species of 100-120 kD and 90-l 10 kD were observed when [1251]GDNF or
[1251]NTN were used, respectively (Figure 22). Strong bands with higher
molecular
mass, 300 kD for GDNFR-a/Fc and 280 kD for GRR2/Fc, were also observed
20 (Figure 22). In addition, minor bands of ~15 kD, 35 kD, and 60 kD in the
[ 125I]GDNF lanes and ~ 12 kD, 26 kD, and 50 kD in the [ 1251]NTN lanes, were
visible (Figure 22). When CM from mock transfected cells were used, no cross-
linked band was precipitated by Protein-A Sepharose {data not shown). None or
much weaker radio-labeled bands were detected when excess unlabeled ligands
were
25 added in the control samples (Figure 22).
Figure 22. Chemical Cross-Linking-of Neurturin And GDNF to GDNFR-a and
GRR2 Receptors.
CM containing GDNFR-a/Fc (GDNFR-a.) or GRR2/Fc (GRR2) fusion
3o proteins were incubated with either 10 nM of ['z5I]NTN (N) or 5 nM of
['ZSI]GDNF
(G) in the presence (+ unlabeled) or absence (- unlabeled) neurturin (N) or
GDNF
(G). The bound receptor-ligand complexes were chemically cross-linked by 1 mM
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/08486
92
of BS3, precipitated with Protein-A Sepharose and analyzed by SDS-PAGE as
described. The solid arrow indicates the 90-110 kD and the 100-120 kD cross-
linked
species. The open arrow depicts the 280 kD and 300 kD complexes.
Neurturin Induces Ret Autophosphorylation in Cells That Express GDNFR-a
The ability of neurturin to associate with GDNFR-a indicates that neurturin,
like GDNF, may activate Ret through GDNFR-a. In order to examine this
possibility, the ability of neurturin to induce Ret autophosphorylation in NGR-
38
cells was tested. NGR-38 cells were treated with concentrations of neurturin
ranging
l0 from 0 to 50 nM, lysed, and the lysates immunoprecipitated with anti-Ret
antibody.
The immunoprecipitates were analyzed by SDS-PAGE followed by immunoblotting
using an anti-phosphotyrosine antibody. A 170 kD band, indicating
autophosphorylation of tyrosine residues on the mature form of Ret, was
observed in
all lanes (Figure 23, lanes 8-14 from left). A much weaker corresponding band
was
i5 observed in neurturin-treated Neuro-2a cells (data not shown). The
induction of Ret
autophosphorylation by neurturin was dose-dependent. Stimulation of Ret
autophosphorylation in NGR-38 cells could be detected with 500 pM neurturin
(Figure 23). In a parallel experiment using GDNF in place of neurturin, an
increase
in the level of phosphorylation of the 170 kD Ret band over background could
be
20 seen at a GDNF concentration of 5 pM (Figure 23, lanes 1-7 from left). When
the
filters were stripped and re-probed with the anti-Ret antibody, the 170 kD Ret
protein band appeared in all lanes with approximately equal intensity (data
not
shown).
25 Figure 23. Neurturin and GDNF Induce Ret Autophosphorylation in NGR-38
Cells
NGR-38 cells were treated with various concentrations of GDNF or neurturin
as described. The cells were lysed, immunoprecipitated with anti-Ret antibody,
fractionated by SDS-PAGE, and blotted with anti-phosphotyrosine antibody for
Ret
phosphorylation. The bands of phosphorylated Ret are indicated by an arrow.
Neurturin And GDNF Induce Ret Autophosphorylation in LA-N-5 Cells
Both neurturin and GDNF bind to GRR2, and the Ret PTK can be activated
by either neurturin or GDNF through GDNFR-a. These observations suggest that
CA 02291608 1999-11-26
WO 98/54213 PCTNS98/08486
93
GRR2 may also be able to mediate neurturin and/or GDNF activation of Ret. To
assess this possibility, human LA-N-5 neuroblastoma cells expressing GRR2 and
Ret
were treated with various concentrations of neurturin or GDNF and processed
for
immunoblotting as described in the previous section (Figure 24). As shown,
both
neurturin and GDNF induced Ret autophosphorylation (Figure 24).
Fisure 24. Neurturin And GDNF Induced Ret Autophosphor~rlation in LA-N-5 Cells
LA-N-5 cells were treated with various concentrations of GDNF or neurturin
as described. The cells were lysed, immunoprecipitated with anti-Ret antibody,
o fractionated by SDS-PAGE, and blotted with anti-phosphotyrosine antibody for
Ret
phosphorylation. The bands of phosphorylated Ret are indicated by an arrow.
Neurturin And GDNF Induce MAP Kinase activation in LA-N-5 And NGR-38 Cells
We have demonstrated that both neurturin and GDNF can induce Ret
15 autophosphorylation in cells expressing either GDNFR-a or GRR2. We then
tested
if the activation of Ret kinase by neurturin and/or GDNF could lead to
activation of
the downstream signaling molecule MAP kinase. Both LA-N-5 and NGR-38 cells
were treated with either neurturin, GDNF, or NGF. Treated cells were lysed
directly
in SDS-PAGE sample buffer, fractionated by SDS-PAGE, and immunoblotted using
2o an anti-phosphorylated MAP kinase antibody {New England Biolabs, Beverly,
MA}.
As shown in Figure 25, both p44 and p42 isoforms of MAP kinase are apparently
activated by both neurturin and GDNF in either LA-N-5 or NGR-38 cells. MAP
kinase activation by NGF (used as a positive control} was also observed.
25 Fi rye 25 (Panels A and B7. Neurturin And GDNF Induced MAP Kinase
Activation
in LA-N-5 And NGR-38 Cells
25A. LA-N-5 cells were treated with various concentrations of GDNF or
neurturin as described. The cells were lysed directly in 2 X SDS-PAGE sample
buffer containing 0.5 mM NaV04, fractionated by SDS-PAGE, and blotted with an
30 antibody against phosphorylated MAP kinase (MAPK-P). 25B. The membrane was
stripped and reprobed with an anti-MAP kinase antibody for the amount of MAP
kinase proteins loaded in each lane (MAPK).
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/08486
94
Discussion
Signal transduction by most receptor PTKs starts by direct interaction with
their ligands and consequent activation of the receptors. Cloning and
characterization of GDNFR-a, an accessory molecule for ligand binding,
revealed a
novel mechanism by which Ret receptor PTK transduces the GDNF signal. GDNF
does not bind Ret alone, instead, it first binds to GDNFR-a and then interacts
with
Ret as a part of the GDNF-GDNFR-a complex. The newly cloned GRR2 is related
to GDNFR-a at both the amino acid level and the three dimensional structure.
It
shares 48% identical amino acid residues with GDNFR-a, among which are 30 of
the
31 cysteines.
We have demonstrated that both neurturin and GDNF bind to GDNFR-a and
GRR2. Binding of GDNF or neuriurin to either GDNFR-a or GRR2 results in
further association of the ligand with Ret and consequent activation of the
Ret PTK
and the MAP kinase, a downstream signaling molecule. However, each of the
ligands appears to bind to one receptor preferentially. Neurturin binds GRR2
expressing LA-N-5 cells more efficiently than GDNF, and GDNF binds GDNFR-a
expressing NGR-38 cells more efficiently than neurturin. It is not clear at
this time
why the binding of [ 125I]GDNF to both GDNFR-a and GRR2 can be replaced by
2o both unlabeled GDNF and neurturin, but that of [ 125I]NTN can only be
inhibited by
cold neurturin.
Consistent with the binding study, GDNF is more effectively cross-linked to
GDNFR-a/Fc fusion receptors than to GRR2/Fc, while neurturin cross-linking
shows
the opposite result.
Experimental Procedures
cDNA Cloning of GRR2
A search of the GenBank database for sequences related to GDNFR-a
3o resulted in the identification of EST, H12981.Gb_Estl. Primers
corresponding to
nucleotides 47 to 65 (5'-CTGCAAGAAGCTGCGCTCC-3') and 244 to 265 (5'-
CTTGTCCTCATAGGAGCAGC-3') of H12981.Gb_Estl were synthesized and
used for RT-PCR with human fetal brain mRNA (Clontech, Cat. #64019-1) as the
S
CA 02291608 2003-O1-29
WO 98/54213 j ) PCT/US98/08486
template. A 218 nt fragment was amplified, subcloned into pBlue-Script
(Stratagene, La Jolla, CA), and sequenced to verify its correspondence with
the
. original EST. The fragment was then radiolabeled with ['~P]-dCTP using a
Raqdom
' Primed DNA Labeling Kit (Stratagene, La Jolla, CA) according to the ,r
5 manufacturer's instructions. The radio-labeled probe was used to screen a
human
fetal brain cDNA library (Stratagene, La Jolla, CA). Two million clones were
plated
on I S cm agarose plates and replicated on duplicate nitrocellulose filters.
The filters
were prehybridized at 55°C for 3.5 hours in 200 ml of 6 x SSC, 1 x
Denhardts, 0.5%
SDS; and 50 ~cg/ml salmon sperm DNA. Following the addition of 2 x 10a cpm of
1 o the radiolabeled probe, hybridization was continued for I 8 hours. Filters
were then
washed twice for 30 minutes each at 55°C in 0.2 x SSC, O.I% SDS and
exposed to
X-ray film overnight with an intensifying screen. Five positive clones were ;:
identified and their DNA sequences were determined.
15 The oligonucleotide primers described above were also used for PCR . .
screening of DNAs isolated from 27 pools ( 1500 clones- each) of a rat
photoreceptor
cDNA library (Jing et al., 1996). A single positive pool was identified and
screened
by hybridization to the same radio-labeled probe as described above. An
individual
cDNA clone from this pool was identified and sequenced.
DNA Seauencing and Sg~~rnce Ana~,3rsi s
DNA sequencing was performed using an automated Applied Biosystems
373A DNA sequences and Taq DyeDeoxy ~Terminator~cycle sequencing kits
(Applied Biosystems, Foster City CA). Comparison of the GDNFR-a and GRR2
sequences with public databases was carried out using the FASTA computer
algorithm (Pearson and Lipman, Proceedings Of The National Academy Of Sciences
Of The United States Of America. 85, 2444-2448, 1988). The peptide sequences
of
GDNFR-a and GRR2 were aligned using the Lineup program. All sequence analysis
programs used were included in the Wisconsin sequence analysis package
(Wisconsin Package Version 9.0, Genetics Computer Group, Madison, WI).
* trademark
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/08486
96
Recombinant human neurturin was expressed in E. coli as insoluble protein.
The inclusion bodies were solubilized, and the neurturin protein was re-folded
and
purified by ion exchange and hydrophobic interaction chromatography.
[ 1251]NTN (2000 Ci/mmole) was prepared using purified E. coli expressed
protein (Amersham, Inc. , Arlington Heights, IL; custom iodination, catalog
#IMQ1057). Recombinant human GDNF was also radio-iodinated (Ding et al.,
1996). Binding of [1251]NTN and [1251]GDNF to LA-N-5 and NGR-38 cells were
carried out as previously described (Jing et al., 1990). Briefly, cells were
seeded one
day before the assay in 24-well Costar tissue culture plates pre-coated with
polyornithine at a density of 3 x 104 cells/cm2. Cells were placed on ice for
5 to 10
minutes, washed once with ice-cold buffer (DMEM containing 25 mM HEPES [pH
7.0]) and incubated at 4°C in 0.2 ml binding buffer (washing buffer
containing 2
mg/ml bovine serum albumin) containing various concentrations of [ 1251]NTN or
[ 1251]GDNF in the absence or presence of 500 nM unlabeled Iigands for 4
hours.
Cells were washed 4 times with 0.5 ml ice-cold washing buffer and Iysed with
0.5 ml
of 1 M NaOH. The lysates were counted in a 1470 Wizard Automatic Gamma
Counter (Wallac Inc., Gaithersburg, MD).
Chemical Cross-Linking
The coding regions of the first 455 amino acids of human GDNFR-a and the
first 451 residues of human GRR2 cDNAs were fused in frame with a DNA
fragment encoding the Fc region of human IgG 1 tagged with 6 histidine
residues at
the carboxy terminus (Culouscou et al., J. Biochem., 270:12857-12863, 1995).
This
construct was then inserted into the expression vector pBK RSV (Stratagene, La
Jolla, CA) as previously described (Jing et al., 1996). The GDNFR-a/Fc and
GRR2lFc fusion constructs were transfected into 293T cells, and conditioned
media
(CM, DMEM supplied with 0.5% fetal calf serum) containing the fusion proteins
were collected 4 days after transfection. Aliquots of 1 ml CM plus 50 ~1 of 1
M
HEPES, pH 7.5 were incubated at 4°C with 10 nM of ['ZSI]NTN or 5
nM
[1251]GDNF in the presence or absence of 1 ~M of unlabeled ligand for 4 hours.
Bis
suberate (BS3 Pierce, Rockford, IL) stock solution in washing buffer (40 mM)
was
added to each binding mixture to a final concentration of 1 mM, mixed and
CA 02291608 2003-O1-29
WO 98/54213
PCT/US98/08486
97
incubated at room temperature for 30 minutes. The reaction was quenched by
adding 50 u1 of 1 M glycine and incubating at room temperature for 15 minutes.
Triton X-100 was added to a final concentration of I %, and the cross-linked
product
was precipitated directly with 200 p,1 of Protein-A Sepharose CL-4B
(Pharmacia).
The cross-linked products were analyzed by 7.5% SDS-PAGE under reducing
conditions.
Ret autophosphorylation was examined by immunoblot analysis as
t o previously described (Jing et al., 1996). Briefly, cells were seeded 24
hours prior to
the assay in 6-well tissue culture dishes at a density of 1.5 x 106 cells
/well. Cells
were washed once with binding buffer and treated with various concentrations
of
neurturin or GDNF (0.5 pM - SO nM) in binding buffer at 37°C for 10
minutes.
Treated cells a,nd untreated controls were lysed in Triton X-100 lysis buffer
(50 mM
15 HEPES, pH 7.5, 1 % Triton X-100, 50 mM NaCI, 50 mM NaF, 10 mM sodium
pyrophosphate, 1% aprotinin (Sigma, Cat.# A-6279), 1 mM PMSF (Sigma, Cat.# P-
7626), 0.5 mM Na3V04 (Fisher Cat.# S454-SO) and immunoprecipitated with an
anti-Ret antibody (Santa Cruz Biotechnology),and protein-A Sepharose as
described
(Ding et al., 1996). Immunoprecipitates were fractionated by 7.5% SDS-PAGE and
20 transferred to nitrocellulose membranes as described by Harlow and Lane
(Antibodies LAboratory Manual, Spring Harbor Laboratory, Spring Harbor Press,
1988). The membranes were blocked with 5% BSA (Sigma) and tyrosine
phosphorylation of the Ret receptor was detected by probing with an anti-
phosphotyrosine monoclonal antibody 4610 (ITBI, Cat #0S-321 ) at room
25 temperature for 2 hours. The amount of Ret protein in each lane was
determined by
stripping and re-probing the same .membrane with the anti-Ret antibody.
Detection
was accomplished using a sheep anti-mouse secondary antibody or protein-A
conjugated to horseradish peroxidase (Amersham, cat.#NA931 ) in conjunction
with
chemiluminescence reagents (ECL, Amershsm) following the manufacturer's
30 instructions.
Activation of the MAP kinases was analyzed using a PhosphoPl~s MAPK
Antibody Kit (New England Biolabs, Beverly, MA, Cat. #9100) following
manufacturer's instructions. LA-N-5 and NGR-38 cells were seeded in 6-well
dishes
* trademark
CA 02291608 2003-O1-29
WO 98/54213 '~ ~~ PCT/US98/08486
98
as described above. Cells were quiesced in DMEM containing 0.5% fetal calf
serum
(FCS) at 37°C for 24 hours. The cells were then incubated with fresh
media for 2
hours, treated with 50 ng/ml of NGF, GDNF, or neuriurin at 37°C for 5
minutes, and
lysed directly in 150 p,1 of 2 X SDS-PAGE sample buffer containing 0.5 mM
NaV04. The cell lysates were fractionated by 10% SDS-PAGE and transferred to a
nitrocellulose filter. The filter was blocked with 5% non-fat dry milk at
4°C
overnight and then incubated overnight at 4°C with a 1:1000 dilution of
anti-
phosphorylated MAP kinase antibody in the same buffer (New England Biolabs).
Bands were detected using a horseradish peroxidase conjugated anti-rabbit
antibody
to and the LumiGLO cherriilu~inescent reagents according to the manufacturer's
recommendations.' After exposure to X-ray film, the filter was stripped and
reprobed
by the anti-MAPK antibody . ,
Figure 25 (Panels A and B7 Neiurturin An_d GDNF Induced IVLAP I~n~ce
a~r;vation
in LA-N-S And NGR-38 Cells
25A. LA-N-5 cells were treated with various concentrations of GDNF or
neurturin as described. The cells were lysed directly in 2 X SDS-PAGE sample
buffer containing 0.5 mM NaVO,, fractionated by SDS-PAGE, and blotted with an
antibody against phosphorylated MAP kinase, (MAPK-P). 25B. The membrane was
2o stripped and reprobed with an anti-MAP kinase antibody for the amount of
MAP
kinase proteins loaded in each lane (MAPK).
Example 11
Cloning and Expression of GRR2 and GRR3
Signaling'by glial cell line-derived neurotrophic factor (GDNF) is mediated
by two receptor components. GDNF receptor-a (GDNFR-a) binds GDNF
specifically, leading to the association of GDNF with Ret and the activation
of the
3o Ret kinase. Similarly, neuriurin induces Ret activation through association
with .
GRR2, a GDNFR-a-related receptor. Both GDNFR-a and GRR2 are capable of
binding either GDNF or neurturin, but each exhibits a marked preference for
its
cognate ligand. A third molecule was cloned~and is related in structure and
primary
* trademark
CA 02291608 1999-11-26
WO 98154213 PCT/US98/08486
99
amino acid sequence to GDNFR-a and GRR2. This molecule has been named
GDNFR-a-related receptor 3 (GRR3). Analysis of the tissue distribution of
GDNFR-a, GRR2, GRR3, and Ret by mRNA blot and in situ hybridization reveals
overlapping but distinct patterns of expression. Consistent with their role in
GDNF
function, GDNFR-a and ret are co-expressed at known sites of GDNF action. GRR2
and GRR3 transcripts are also co-localized with those of ret in some cases,
suggesting that GRR3 may also mediate Ret activation by GDNF or a related
ligand.
Introduction
to
Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor
for midbrain dopaminergic neurons, motor neurons, and several other types of
neuronal cells. Targeted disruption of the GDNF gene in mice causes complete
renal
agenesis and the absence of enteric neurons (Moore et al., Nature, 382, 76-79,
1996;
is Pichel et al., Nature, 382, 73-76, 1996; Sanchez et al., Nature, 382, 70-
73, 1996; and
Hudson et al., Brain Research Bulletin, 36, 425-32, 1995), indicating an
essential
role for GDNF in the development of the renal and the enteric nervous systems.
The
GDNF receptor was discovered to consist of a novel ligand binding component,
GDNFR-a, and a signaling component, the Ret receptor protein tyrosine kinase.
2o GDNFR-a is attached to the cell membrane through a glycosyl-
phosphatidylinositol (GPI) linkage but has no cytoplasmic domain. It binds
GDNF
specifically and with high affinity regardless of whether or not Ret is
present. Ret is
a receptor protein tyrosine kinase {PTK) originally discovered as a large open
reading frame in the ret proto-oncogene. Its unique extraceilular domain
structure,
25 which includes a signal peptide, a cadherin-like motif, and a cysteine-rich
region,
places it outside any other known receptor PTK sub-family. Ret alone does not
bind
GDNF, but was found to form a complex with GDNF and GDNFR-a that results in
Ret activation. Activation of the Ret kinase appears to be associated with the
biological effects of GDNF. Targeted disruption of the Ret PTK gene results in
a
3o phenotype nearly identical to that resulting from the disruption of GDNF
(Schuchardt et aL, Nature, 367, 380-383, 1994). In situ hybridization and
immunohistochemical analysis detects high level expression of ret mRNA and
protein in the developing central and peripheral nervous systems and in the
excretory
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/08486
100
system of the mouse embryo. This expression pattern is similar to that of GDNF
and
is consistent with Ret's role in GDNF signaling.
The expression pattern of GDNFR-a is also consistent with its involvement
in GDNF signaling. GDNFR-a mRNA has been found in a number of GDNF-
responsive cell types and structures of the nervous system, often colocalized
with ret.
In the central nervous system, GDNFR-a mRNA has been observed in both
developing and adult rat ventral midbrain, facial nucleus and ventral spinal
cord. In
addition, some specific cells in the superior colliculus, the lateral septum,
the
molecular layer of cerebellum adjacent to Purkinje cells, and some nuclei in
cerebral
cortex and the dorsomedial tegmental area have been shown to express GDNFR-a.
In the peripheral nervous system, GDNFR-a mRNA expression has been found in
subpopulations of neurons in dorsal root ganglia, in enteric neurons, and in
neurons
from sympathetic ganglia. High levels of GDNFR-a mRNA expression were also
observed in other regions of the nervous system, including the retina,
thalamus, pons,
t5 and medulla oblongata. Expression has also been seen in non-neuronal
tissues such
as the developing nephrons, pituitary, urogenital tract and pancreatic
primordium.
Neurturin is a molecule which has similarities to GDNF in both amino acid
sequence and biological activity. The GRR2 protein (GDNFR-a-Related Receptor
2), is a novel protein related in amino acid sequence to GDNFR-a. GRR2 is
capable
2o of binding both GDNF and neurturin, and like GDNFR-a, mediates the
activation of
the Ret PTK in response to these ligands. Although both GDNF and neurturin can
bind both GDNFR-a and GRR2, GDNF exhibits a marked preference for GDNFR-a
while neurturin interacts more strongly with GRR2. GDNFR-a-Related Receptor 3
(GRR3) a third member of this receptor family has also been found. The present
25 study examines the tissue and cell-specific mRNA expression of GDNFR-a,
GRR2,
GRR3, and ret.
Results
30 Molecular Cloning and Sequence Comparison of GRR3 with GRR2 and GDNFR-a
Examination of publicly available sequence databases revealed the presence
of a short expressed sequence tag (EST) with sequence homology to the GDNFR-a
and GRR2 cDNA clones (WashU-HHMI Mouse EST Project). Oligonucleotides
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/08486
141
corresponding to the ends of this EST were used as primers in a reverse
transcription-polymerase chain reaction (RT-PCR) with total rat embryo RNA as
the
template. A 225 nucleotide (nt) fragment was amplified, cloned into a plasmid
vector, and sequenced to verify that it corresponded to the original GDNFR-
a/GRR2-related EST. Plasmid DNAs isolated from pools of an E 15 rat embryo
cDNA library were screened by PCR and a single positive pool was found. Clones
from this pool were screened by hybridization to the radiolabeled 225 nt PCR
fragment and a single positive clone was isolated. Sequence analysis of the
1.8 kb
insert from this clone revealed an open reading frame coding for a 397 amino
acid
o peptide related to both GDNFR-a and GRR2. This protein was designated GDNFR-
a-related receptor 3 (GRR3).
An alignment of the amino acid sequences of rat GDNFR-a, GRR2, and
GRR3 is shown in Figure 26. The overall amino acid sequence identity among the
three receptors is in the range of 30%-50%. GDNFR-a and GRR2 are somewhat
more closely related to each other (48% identity) than they are to GRR3 (35%
and
33% identity, respectively). Hydrophobic regions are found at both the amino
and
carboxy termini of all three molecules, except for the amino terminus of GRR2
{underlined, Figure 26). The amino terminal regions of both GDNFR-a and GRR3
have the characteristics expected for signal peptide sequences. Although the
GRR2
2o N-terminal sequence does not fit the criteria for a classical signal
peptide, there is
evidence that GRR2 is secreted. The carboxy terminal hydrophobic region of
GDNFR-a is known to be involved in GPI-linkage to the cell membrane, and it is
likely that the corresponding regions in GRR2 and GRR3 serve the same purpose.
The most striking feature of the sequence alignment is the conservation of 28
cysteine residues among all three receptors (highlighted, Figure 26),
indicating that
these proteins probably have similar three-dimensional structures. Several
potential
N-glycosylation sites are present in the receptors (shown in boldface, Figure
26), but
none are found at the same position in all three receptors. GDNFR-a and GRR2
share sites at positions 365 and 427 that are not found in GRR3, and GRR2
shares a
3o possible site with GRR3 at positions 322-323 (Figure 26).
CA 02291608 1999-11-26
WO 98154213 PCT/US98/08486
102
Expression of GDNFR-a, GRR2. and GRR3 in Adult Rat
The expression of GDNFR-a, GRR2 and GRR3 mRNAs in adult rat tissues
was examined by blot hybridization analysis. GDNFR-a mRNA is widely
expressed, with high levels found in lung, brain, liver, kidney and spleen.
Expression is also detectable in heart and among the tissues examined is
absent only
in muscle and testis. Two distinct size transcripts are observed and their
relative
amounts vary among the tissues. The 3.6 kb transcript is predominant in liver,
lung,
heart, and spleen while comparable amounts of the 3.6 kb and 8.5 kb
transcripts are
present in brain and kidney. The tissue distribution of GRR2 mRNA is similar
to
o that of GDNFR-a. GRR2 expression is highest in lung, spleen and brain, with
lesser
amounts in kidney and heart. One difference is the lack of GRR2 expression in
liver.
The size of the GRR2 transcripts is approximately 3.6 kb, similar to the
smaller of
the two GDNFR-a transcripts. The expression of GRR3 mRNA is highest in kidney
and is absent in brain. Detectable expression of GRR3 is also present in
spleen,
~5 lung, liver, and heart. The transcript size for GRR3 is somewhat smaller
(~2.1 kb)
than that observed for GDNFR-a and GRR2.
Expression of GDNFR-a, GRR2 and GRR3 in Mouse Embr~ro
Developmental expression of GDNFR-a, GRR2, and GRR3 mRNA was
2o examined in the mouse on embryonic days 7, 11, 15, and 17. Expression of
the 3.6
kb transcript of GDNFR-a is first apparent at El 1, seems to decrease somewhat
at
E 15, but then increases dramatically by E 17. A minor amount of the 8.5 kb
GDNFR-a mRNA can be detected on El 1, but no expression of this transcript is
detected thereafter. The expression of the 3.6 kb GRR2 transcript is barely
25 detectable at E11, but increases gradually through E17. Expression of the
2.1 kb
GRR3 mRNA is not detected at E7, but is quite strong by Ei 1. After E11,
expression decreases and remains constant from E 15-E 17.
he situ Hybridization Analysis of the Exuression of GDNFR-a. GRR2, and GRR3
3o In order to provide clues to the potential roles and functional sites of
GDNFR-a, GRR2 and GRR3, their expression was examined in regions where
biological effects of GDNF have been demonstrated. In the E 18 rat embryo,
GDNF
is highly expressed in the growing ureteric buds and maturing nephrons of the
CA 02291608 1999-11-26
WO 98/54213 PCT/US98i08486
103
kidney as well as in the enteric neurons of the intestine. GDNFR-a is found in
the
same regions of the kidney and intestine as GDNF, but is also expressed at
moderate
levels in both the dorsal and ventral spinal cord. ret is expressed in the
kidney and
intestine as well, although its expression in the kidney seems to be confined
to the
ureteric buds. Expression of ret is high in the ventral motor neurons, but low
in the
dorsal region of the spinal cord. Like ret, expression of GRR2 in the kidney
is
restricted to the ureteric buds. GRRZ is expressed in both the dorsal and
ventral
regions of the spinal cord. A weak, diffuse hybridization signal was detected
in the
liver for GDNF, ret, and GDNFR-a.
1o In the postnatal day 7 rat, ret expression can be detected at substantial
levels
in the substantia nigra, trigeminal ganglia, and at a lower level in the
reticular
thalamic nucleus. GDNFR-a expression is high in both the reticular and
ventromedial thalamic nuclei as well as in the medial habenular nucleus.
Moderate
expression of GDNFR-a is observed in the substantia nigra and lower but
detectable
levels are found in the hippocampus. GRR2 is expressed at moderate levels in
the
reticular thalamic nucleus, ventromedial thalamic nucleus, cerebral cortex
(especially
the cingulate cortex), and the substantia nigra. We could detect no expression
of
GRR3 in the P7 rat brain, but significant expression could be detected in the
trigeminal ganglia.
Discussion
This study describes the isolation of GRR3, a novel molecule related to
GDNFR-a and GRR2 and compares the tissue expression of ret with that of ail
three
members of the GDNFR receptor family. GRR2 is 48% identical in amino acid
sequence to GDNFR-a, while GRR3 is somewhat more distantly related at 35%
identity. The position of 28 cysteine residues are conserved in all three
molecules.
Like GDNFR-«, both GRR2 and GRR3 have hydrophobic C-termini that are likely
to be involved in GPI linkage to the cell membrane, and neither has a
cytoplasmic
domain. This strong conservation of sequence and structural features suggests
that
GDNFR-a, GRR2, and GRR3 define a new family of receptors for GDNF and
related ligands. GDNF signaling is initiated by binding to GDNFR-a and
accomplished by association and consequent activation of the Ret PTK. Based
upon
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/0848fi
104
its sequence and structural similarities to GDNFR-a and GRR2, GRR3 is likely
to
function as a binding partner for GDNF, neurturin, and/or some other as yet
undiscovered member of this ligand family.
The expression patterns of GDNFR-a, GRR2, and GRR3 in adult rat tissues
are similar but distinct. All three mRNAs are found in lung, spleen, heart,
and
kidney while none of the three show significant expression in muscle or
testis. Adult
brain exhibits high expression of GDNFR-a and GRR2 mRNAs, but little or no
GRR3 is detected. Expression of GDNFR-a mRNA is high in liver while GRR2
mRNA is almost nonexistent. If GDNF, neurturin and other as yet undiscovered
1o GDNF-like ligands signal exclusively through Ret, differences in expression
patterns
of the ligand-specific binding receptors could provide a mechanism for ligand
tissue
specificity. Since the expression of c-ret can be detected throughout the
period from
E8.5 to E1 b.5, differences in the temporal expression of the receptor
proteins could
also define ligand specificity during development.
Expression of all the receptors and of c-ret is high in the adult kidney, the
site
of the most severe defects found in Ret knockout animals. In situ
hybridization
analyses indicate that ret, GDNFR-a, GRR2 and GRR3 are colocalized in several
tissues, suggesting that GRR2 and GRR3 may also exert their in vivo effects
through
interaction with Ret (Table 5).
Table 5
Expression of ret, GDNFR-a, GRR2, and GRR3
in embryonic day 18 rat
ret GDNFR-a GRR2 GRR3
Kidney/Intestine +++ +++ ++ -
Brain:
Thalamic Nuclei:
Reticular ++ +++ ++ -
Ventral medial + +++ ++ -
CA 02291608 1999-11-26
W() 98/54213 PCT/US98/o8486
105
Substantia Nigra +++ +++ +++ -
Habenular nucleus - +++ - -
Hippocampus +/- ++ - -
Spinal cord:
Dorsal + ++ ++ -
Ventral ++ +++ ++ -
Trigeminal Ganglia +++ +++ - +++
* High levels of expression were detected in the adult kidney.
Both GDNFR-a and GRR2 are transcribed along with ret in the kidney and
intestine, in the substantia nigra, in the thalamus, and in ventral spinal
motor
neurons. This finding is consistent with GDNF's ability to promote the
survival of
dopaminergic and motor neurons and with the phenotypes of the Ret and GDNF
knockout animals. Although little expression of GRR3 was found in the brain,
it is
co-expressed with ret and GDNFR-a in the trigeminal ganglia in E 18 and P7
rats.
o These observations indicate that GDNF action may be regulated by association
with
different binding components depending on the tissue and developmental stage,
while always signaling through Ret.
Although expression of ret is often co-localized with that of GDNFR-a,
GRR2 and GRR3, there are several sites that express one or more of the binding
receptors at high levels while ret expression is undetectable. Little or no
ret is
expressed in the spleen or lung where all three receptors are expressed at
high levels.
High levels of GDNFR-a mRNA are found in the liver, medial habenular nucleus,
and the hippocampus, and GRR2 expression is prominent in the cortex. Little
ret
expression was observed in either of these regions. The lack of ret expression
at
2o some sites of substantial GDNFR expression suggests that either a signaling
partner
other than Ret may be employed by the GDNFRs in these tissues or that the
receptors have an alternate mechanism of action. Two possibilities are that
the
receptors may act to sequester ligands of the GDNF family or that some
fraction of
CA 02291608 1999-11-26
WO 98/54213 PCTIUS98/08486
106
the membrane bound receptors are released and mediate ligand function as
soluble
receptors.
Experimental Procedures
Cloning of GRR3
The GenBank database was searched for sequences related to GDNFR-a and
GRR2 using the Wisconsin sequence analysis package (Wisconsin Package Version
9.0, Genetics Computer Group, Madison, WI). Oligonucleotide primers
t0 corresponding to regions near the ends of the EST AA238748.Gb_New2 were
synthesized. Primers corresponding to AA238748.Gb New2 were used for PCR
screening of 83 pools of 1000 clones each from a rat E15 embryonic cDNA
library.
A single positive pool was identified by this method. The DNA fragment
amplified
from this pool was subcloned into a plasmid vector, and the insert was
sequenced
using an Applied Biosystems 373A automated DNA sequencer with Taq DyeDeoxy
Terminator cycle sequencing kits (Applied Biosystems, Foster City, CA). The
insert
was then labeled with ['ZP]-dCTP using a Random Primed DNA Labeling Kit
(Stratagene, La 3olla, CA) according to the manufacturer's instructions.
Clones from
the cDNA library pool that had been identified as positive by PCR were plated
on 15
2o cm agarose plates and replicated on duplicate nitrocellulose filters for
screening by
hybridization to the radiolabeled insert. Filters were prehybridized at
55°C for 3.5
hours in 200 ml of 6 x SSC, 1 x Denhardts, 0.5% SDS, and 50 pg/ml salmon sperm
DNA. Following the addition of 2 x 10g cpm of the radiolabeled probe,
hybridization was continued for 18 hours. Filters were then washed twice for
30
minutes each at 55°C in 0.2 x SSC, 0.1 % SDS and exposed to X-ray film
overnight
with an intensifying screen.
DNA Sequencing and Sequence Anal
DNA from clones that screened positively by hybridization was prepared and
sequenced using an automated Applied Biosystems 373A DNA sequencer and Taq
DyeDeoxy Terminator cycle sequencing kits (Applied Biosystems, Foster City,
CA).
The peptide sequences of GDNFR-a, GRR2, and GRR3 were aligned using the
CA 02291608 2003-O1-29
~1
_. , WO 98/54213 PCT/US98/08486
107
Lineup program (Wisconsin Package Version 9.0, Genetics Computer Group,
Madison, WI).
Blot Hybridization Analy~
For blot hybridization analysis, the cloned rat GRR3 cDNA was labeled
using the Random Primed DNA Labeling Kit (Boehringer Mannheim, Indianapolis,
IN) according to the manufacturer's instructions. Rat and mouse RNA blots
{Clontech) were hybridized with the probe and washed at high stringency using
the
reagents of the ExpressHyb Kit (Clontech, Palo Alto, CA) according to the
1o instructions of the.manufacturer. Following exposure on X-ray film, the
filters were
stripped of probe by boiling in 0.5% SDS for 10 minutes and rehybridized with
a p- ,
actin probe (CIontech, Palo Alto, CA) as a control for total RNA loading. ==
In situ Hybridization
~s In situ hybridization using anti-sense riboprobes of GDNF; ret, GDNFR-a,
GRR2, and GRR3, was done according to Zhou et al. (Journal Of Neuroscience
Research, 37, 129-143, 1994). The ret probe is a 316 nt fragment derived from
the
extracellular domain of the rat ret cDNA. GDNF mRNA was detected using a 303
nt fragment of a rat GDNF cDNA clone (nucleotide #50 to 352, Lin et al.,
1993).
2o GDNFR-a transcripts were detected with a 396 nt riboprobe (nucleotides 1072
to
1468). GRR2 transcripts were detected with a 205 nt antisense riboprobe
corresponding to amino acids 339-413 (Figure 26). GRR3 transcripts were
detected
with a 225 nt antissnse riboprobe corresponding to amino acids 239-315 (Figure
26).
While the present invention bas been described in terms of preferred
embodiments and exemplary nucleic acid and amino acid sequences, it is
understood
that variations and modifications will occur to those skilled in the art.
Therefore, it
is intended that the appended claims cover all such equivalent variations
which come
3o within the scope of the invention as claimed.
* trademark
CA 02291608 1999-11-26
WO 98/54213 PCTIUS98/08486
108
References
Angrist, M., Bolk, S., Thiel, B., Puffenberger, E.G., Hofstra, R.M., Buys,
C.H.,
Cass, D.T., and Chakravarti, A. (1995). Mutation analysis of the RET receptor
tyrosine kinase in Hirschsprung disease. Hum. Mol.Genet.4, 821-830.
Arenas, E., Trupp, M., Akerud, P., and Ibanez, C.F. ( 1995). GDNF Prevents
degeneration and promotes the phenotype of brain noradrenergic neurons in
vivo.
Neuron 15, 1465-1473.
Aruffo, A. and Seed, B. ( 1987). Molecular cloning of a CD28 cDNA by a high-
efficiency COS cell expression system. Proceedings Of The National Academy Of
Sciences Of The United States Of America. 84, 8573-8577.
Bartley, T.D., Hunt, R.W., WeIcher, A.A., Boyle, W.J., Parker, V.P., Lindberg,
R.A., Lu, H.S., Colombero, A.M., Elliott, R.L., Guthrie, B.A., Holst, P.L.,
Skrine,
J.D., Toso, R.J., Zhang, M., Fernandez, E., Trail, G., Varnum, B., Yarden, Y.,
Hunter, T., and Fox, G.M. ( 1994). B61 is a Ligand for the ECK Receptor
protein-
tyrosine kinase. Nature. 368, 558-560.
Beck, K.D., Valverde, J., Alexi, T., Poulsen, K., Moffat, B., Vandlen, R.A.,
Rosenthal, A., and Hefti, F. (1995). Mesencephalic dopaminergic neurons
protected
by GDNF from axotomy-induced degeneration in the adult brain. Nature. 373, 339-
341.
Camu, W. and Henderson, C. (1992). Purification of embryonic rat motoneurons
by
panning on a monoclonal antibody to the low-affinity NGF receptor. J
Neuroscience. 44, 59-70.
Choi-Lundberg, D.L. and Bohn, M.C. (1995). Ontogeny and distribution of glial
cell line-derived neurotrophic factor (GDNF) mRNA in rat. Brain Res. Dev.
Brain
Res. 85, 80-88.
Davis, S., Aldrich, T.H., Valenzuela, D.M., Wong, V.V., Furth, M.E., Squinto,
S.P.,
and Yancopoulos, G.D. { 1991). The receptor for ciliary neurotrophic factor.
Science.
253, 59-63.
CA 02291608 1999-11-26
WO 98154213 PCT/US98/08486
109
Donis-Keller, H., Dou, S., Chi, D., Carlson, K., Toshima, K., Lairmore, T.,
Howe,
J., Moley, J., Goodfellow, P. and Wells, S. (1993). Mutations in the ret proto-
oncogene are associated with MEN 2A and FMTC. Hum. Molec. Genet. 2, 851-
856.
Ebendal, T., Tomac, A., Hoffer, B.J., and Olson, L. ( 1995). Glial cell line-
derived
neurotrophic factor stimulates fiber formation and survival in cultured
neurons from
peripheral autonomic ganglia. Journal Of Neuroscience Research. 40, 276-284.
1o Economides, A.N., Ravetch, J.V., Yancopoulos, G.D., and Stahl, N. (1995).
Desilmer cytokines: targeting actions to cells of choice. Science 270, 1351-
1353.
Edery, P., Lyonnet, S., Mulligan, L., Pelet, A., Dow, E., Abel, L., Holder,
S.,
NihouI-Fekete, C., Ponder, B. and Munnich, A. ( 1994). Mutations of the ret
proto-
~s oncogene in Hirschsprug's disease. Nature. 367, 378-380.
Gearing, D.P., King, J.A., Gough, N.M., and Nicola, N.A. (1989). Expression
cloning of a receptor for human granulocyte-macrophage colony-stimulating
factor.
EMBO Journal 8, 3667-3676.
Henderson, C.E., Phillips, H.S., Pollock, R.A., Davies, A.M., Lemeulle, C.,
Armanini, M., Simpson, L.C., Moffet, B., Vandlen, R.A., Koliatsos, V.E., and
et al
(1994). GDNF: a potent survival factor for motoneurons present in peripheral
nerve
and muscle. Science. 266, 1062-1064.
Hoffer, B.J., Hoffinan, A., Bowenkamp, K., Huettl, P., Hudson, J., Martin, D.,
Lin,
L.F., and Gerhardt, G.A. ( 1994). Glial cell line-derived neurotrophic factor
reverses
toxin-induced injury to midbrain dopaminergic neurons in vivo. Neuroscience
Letters. 182, 107-111.
Hofstra, R., Landsvater, R., Ceccherini, L, Stulp, R., Stelwagen, T., Luo, Y.,
Pasini,
B., Hoppener, J., van Amstel, H., Romeo, G., Lips, C. and Buys, C. ( 1994). A
mutation in the ret proto-oncogene associated with multipleendocrine neoplasia
type 2B and sporadic medullary thyroid carcinoma. Nature. 367, 375-376.
Ikeda, L, Ishizaka, Y., Tahira, T., Suzuki, T., Onda, M., Sugimura, T., and
Nagao,
M. (1990). Specific expression of the ret proto-oncogene in human
neuroblastoma
CA 02291608 1999-11-26
WO 98/54213 PCT/US98I08486
110
cell lines. Oncogene. 5, 1291-1296.
Ip, N.Y., Nye, S.H., Boulton, T.G., Davis, S., Yasukawa, K., Kishimoto, T.,
Anderson, D.J., and et al (1992). CNTF and LIF act on neuronal cells via
shared
signaling pathways that involve the IL-6 signal transducing receptor component
gp130. Cell. 69, 1121-1132.
Iwamoto, T., Taniguchi, M., Asia, N., Ohkusu, K., Nakashima, I. and Takahashi,
M. ( 1993). cDNA cloning of mouse ret proto-oncongene and its sequence
similarity
1o to the cadherin superfamily. Oncogene. 8, 1087-1091.
Jing, S.Q., Spencer, T., Miller, K., Hopkins, C., and Trowbridge, LS. (1990).
Role of
the human transferrin receptor cytoplasmic domain in endocytosis: localization
of a
specific signal sequence for internalization. Journal Of Cell Biology. 110,
283-294.
Kearns, C.M. and Gash, D.M. (1995}. GDNF protects nigral dopamine neurons
against 6-hydroxydopamine in vivo. Brain Research. 672, 104-111.
Kozak, M. (1987). An analysis of 5'-noncoding sequences from 699 vertebrate
2o messenger RNAs. Nucleic Acids Research. 15, 8125-8148.
Li, L., Wu, W., Lin, L.F., Lei, M., Oppenheim, R.W., and Houenou, L.J. (1995).
Rescue of adult mouse motoneurons from injury-induced cell death by glial cell
line-
derived neurotrophic factor. Proceedings Of The National Academy Of Sciences
Of
The United States Of America. 92, 9771-9775.
Lin, L-F.H., Doherty, D.H., Lile, J.D., Bektesh, S., and Collins, F. (1993).
GDNF: a
glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons.
Science. 260, 1130-1132.
Louis, J.C., Magal, E., and Varon, S. ( 1992). Receptor-mediated toxicity of
norepinephrine on cultured catecholaminergic neurons of the rat brain stem.
Journal
Of Pharmacology And Experimental Therapeutics. 262, 1274-1283.
Mount, H.T., Dean, D.O., Alberch, J., Dreyfus, C.F., and Black, LB. ( 1995).
Glial
cell line-derived neurotrophic factor promotes the survival and morphologic
differentiation of Purkinje cells. Proceedings Of The National Academy Of
Sciences
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/08486
111
Of The United States Of America. 92, 9092-9096.
Mulligan, L., Kwok, J., Healey, C., Elsdon, M., Eng, C., Gardner, E., Love,
D.,
Mole, S., Moore, J., Papi, L., Ponder, M., Telenius, H., Tunnacliffe, A. and
Ponder,
A. ( 1993). Germ-line mutations of the ret proto-oncongene in mutiple
endocrine
neoplasia type 2A. Nature. 363, 458-460.
Oppenheim, R.W., Houenou, L.J., Johnson, J.E., Lin, L.F., Li, L., Lo, A.C.,
Newsome, A.L., Prevette, D.M., and Wang, S. (1995). Developing motor neurons
rescued from programmed and axotomy-induced cell death by GDNF. Nature. 373,
344-346.
Pachnis, V., Mankoo, B., and Costantini, F. (1993). Expression of the c-ret
proto-
oncogene during mouse embryogenesis. Development, 119, 1005-1017.
Pearson, W.R. and Lipman, D.J. (1988). Improved tools for biological sequence
comparison. Proceedings Of The National Academy Of Sciences Of The United
States Of America. 85, 2444-2448.
2o Poulsen, K.T., Armanini, M.P., Klein, R.D., Hynes, M.A., Phillips, H.S.,
and
Rosenthal, A. ( 1994). TGF beta 2 and TGF beta 3 are potent survival factors
for
midbrain dopaminergic neurons. Neuron. 13, 1245-1252.
Romeo, G., Patrizia, R, Luo, Y., Barone, V., Seri, M., Ceccherini, L, Pasini,
B.,
Bocciardi, R., Lerone, M., Kaariainen, H. and Maartucciello, G. ( 1994). Point
mutations affecting the tyrosine kinase domain of the ret proto-oncogene in
Hirschsprung's disease. Nature. 367, 377-378.
Santoro, M., Carlomagno, F., Romeo, A., Bottaro, D., Dathan, N., Grieco, M.,
3o Fusco, A., Vecchio, G., Matoskova, B., Kraus, M. and Di Fiore, P. (1995).
Activation of ret as a dominant transforming gene by germline mutations of
MEN2A and MEN2B. Science. 267, 381-383.
Sauer, H., Rosenblad, C., and Bjoerklund, A. (1995). Glial cell line-derived
neurotrophic factor but not transforming growth factor beta 3 prevents delayed
degeneration of nigral dopaminergic neurons following striatal 6-
hydroxydopamine
lesion. Proceedings Of The National Academy Of Sciences Of The United States
Of
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/08486
112
America. 92, 8935-8939.
Saxen, L. ( 1987). Organogenesis of the kidney. Development and Cell Biology
series, Cambridge University Press, Cambridge, England.
Schaar, D.G., Sieber, B.A., Dreyfus, C.F., and Black, LB. (1993). Regional and
cell-
specific expression of GDNF in rat brain. Experimental Neurology. 124, 368-
371.
Schaar, D.G., Sieber, B.A., Sherwood, A.C., Dean, D., Mendoza, G.,
Ramakrishnan,
1o L., Dreyfus, C.F., and Black, LB. (1994). Multiple astrocyte transcripts
encode nigral
trophic factors in rat and human. Experimental Neurology. 130, 387-393.
Schlessinger, J. and Ullrich, A. ( 1992). Growth factor signaling by receptor
tyrosine
kinases. Neuron 9, 383-391.
Schuchardt, A., D'Agati, V., Larsson-Blomberg, L., Costantini, F. and Pachnis,
V.
(1994). Defects in the kidney and enteric nervous system of mice lacking the
tyrosine kinase receptor ret. Nature. 367, 380-383.
2o Segarini, P.R., Ziman, J.M., Kane, C.J., and Dasch, J.R. ( 1992). Two novel
patterns
of transforming growth factor beta (TGF-beta) binding to cell surface proteins
are
dependent upon the binding of TGF-beta I and indicate a mechanism of positive
cooperativity. Journal Of Biological Chemistry. 267, 1048-1053.
Springer, J.E., Mu, X., Bergmann, L.W., and Trojanowski, J.Q. (1994).
Expression
of GDNF mRNA in rat and human nervous tissue. Experimental Neurology. 127,
167-170.
Stroemberg, L, Bjoerklund, L., Johansson, M., Tomac, A., Collins, F., Olson,
L.,
3o Hoffer, B., and Humpel, C. (1993). Glial cell line-derived neurotrophic
factor is
expressed in the developing but not adult striatum and stimulates developing
dopamine neurons in vivo. Experimental Neurology. 124, 401-412.
Takahashi, M., Ritz, J. and Cooper, G. {1985}. Activation of a novel human
tranforming gene, ret, by DNA rearrangement. Cell. 42, 581-588.
Takahashi, M. and Cooper, G. (1987). Ret trasnforming gene encodes a fusion
CA 02291608 1999-11-26
WO 98/54213 PCT/US98/0848G
113
protein homologous to tyrosine kinases. Mol. Cell. Biol., 7, 1378-1385.
Takebe, Y., Seiki, M., Fujisawa, J., Hoy, P., Yokota, K., Arai, K., Yoshida,
M., and
Arai, N. (1988). SRa promoter: an efficient and versatile mammalian cDNA
expression system composed of the simian virus 40 early promoter and the R-U5
segment of human T-cell leukemia virus type 1 long terminal repeat. Mol. Cell.
Biol.
8, 466-472.
Tomac, A., Lindqvist, E., Lin, L.F., Ogren, S.O., Young, D., Hoffer, B.J., and
Olson,
to L. (1995a). Protection and repair of the nigrostriatal dopaminergic system
by GDNF
in vivo. Nature. 373, 335-339.
Tomac, A., Widenfalk, J., Lin, L.F., Kohno, T., Ebendal, T., Hoffer, B.J., and
Olson,
L. (1995b). Retrograde axonal transport of glial cell line-derived
neurotrophic factor
in the adult nigrostriatal system suggests a trophic role in the adult.
Proceedings Of
The National Academy Of Sciences Of The United States Of America. 92, 8274-
8278.
Trupp, M., Ryden, M., Joernvall, H., Funakoshi, H., Timmusk, T., Arenas, E.,
and
2o Ibanez, C.F. ( 1995). Peripheral expression and biological activities of
GDNF, a new
neurotrophic factor for avian and mammalian peripheral neurons. Journal Of
Cell
Biology. 130, 137-148.
Tsuzuki, T., Takahashi, M., Asai, N., Iwashita, T., Matsuyama, M. and Asai, J.
(1995). Spatial and temporal expression of the ret proto-oncongene product in
embryonic, infant and adult rat tissues. Oncogene, 10, 191=198.
Ullrich, A and Schlessinger, J. ( I 990). Signal transduction by receptors
with
tyrosine kinase activity. Cell, 61, 203-211.
van der Geer, P., Hunter, T., and Lindberg, R.A. (1994). Receptor protein-
tyrosine
kinases and their signal transduction pathways. 10, 251-337.
van Heyningen, V. (/994). One gene-four syndromes. Nature, 367, 319-320.
von Heijne, G. ( 1986). A new method for predicting signal sequence cleavage
sites.
Nucleic Acids Research. 14, 4683-4690.
CA 02291608 1999-11-26
WO 98/54213 PCTIUS98/08486
114
Yan, Q., Matheson, C., and Lopez, O.T. (1995). In vivo neurotrophic effects of
GDNF on neonatal and adult facial motor neurons. Nature. 373, 34I-344.
Zurn, A.D., Baetge, E.E., Hammang, J.P., Tan, S.A., and Aebischer, P. (1994).
Glial
cell line-derived neurotrophic factor {GDNF), a new neurotrophic factor for
motoneurones. Neuroreport. 6, 113-118.
CA 02291608 1999-11-26
115
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: AMGEN INC.
(ii) TITLE OF INVENTION: NEUROTROPHIC FACTOR RECEPTORS
(iii) NUMBER OF SEQUENCES: 44
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Gowling, Strathy & Henderson
(B) STREET: 160 Elgin Street, Suite 2600
(C) CITY: Ottawa
(D) STATE: Ontario
(E} COUNTRY: CA
(F) ZIP: K1P 1C3
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: 27-APR-1998
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/866,354
(B} FILING DATE: 30-MAY-1997
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Cowling, Strathy & Henderson
(B) REGISTRATION NUMBER:
(C) REFERENCE/DOCKET NUMBER: 08-885021CA
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2568 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 540..1934
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
AATCTGGCCTCGGAACACGCCATTCTCCGC GCCGCTTCCAATAACCACTAACATCCCTAA 60
CGAGCATCCGAGCCGAGGGCTCTGCTCGGA AATCGTCCTGGCCCAACTCGGCCCTTCGAG 120
CTCTCGAAGATTACCGCATCTATTTTTTTT TTCTTTTTTTTCTTTTCCTAGCGCAGATAA 180
CA 02291608 1999-11-26
116
AGTGAGCCCG GAAAGGGAAG GAGGGGGCGG GGACACCATT GCCCTGAAAG AATAAATAAG 240
TAAATAAACA AACTGGCTCC TCGCCGCAGC TGGACGCGGT CGGTTGAGTC CAGGTTGGGT 300
CGGACCTGAA CCCCTAAAAG CGGAACCGCC TCCCGCCCTC GCCATCCCGG AGCTGAGTCG 360
CCGGCGGCGG TGGCTGCTGC CAGACCCGGA GTTTCCTCTT TCACTGGATG GAGCTGAACT 420
TTGGGCGGCC AGAGCAGCAC AGCTGTCCGG GGATCGCTGC ACGCTGAGCT CCCTCGGCAA 480
GACCCAGCGG CGGCTCGGGA TTTTTTTGGG GGGGCGGGGA CCAGCCCCGC GCCGGCACC 539
ATGTTC CTGGCG ACCCTGTAC TTCGCGCTG CCGCTCTTG GACTTG CTC
587
MetPhe LeuAla ThrLeuTyr PheAlaLeu ProLeuLeu AspLeu Leu
1 5 10 15
CTGTCG GCCGAA GTGAGCGGC GGAGACCGC CTGGATTGC GTGAAA GCC 635
LeuSer AlaGlu ValSerGly GlyAspArg LeuAspCys ValLys Ala
20 25 30
AGTGAT CAGTGC CTGAAGGAG CAGAGCTGC AGCACCAAG TACCGC ACG 683
SerAsp GlnCys LeuLysGlu GlnSerCys SerThrLys TyrArg Thr
35 40 45
CTAAGG CAGTGC GTGGCGGGC AAGGAGACC AACTTCAGC CTGGCA TCC 731
LeuArg GlnCys ValAlaGly LysGluThr AsnPheSer LeuAla Ser
50 55 60
GGC CTGGAGGCC AAGGAT GAGTGCCGC AGCGCCATG GAGGCC CTGAAG 779
Gly LeuGluAla LysAsp GluCysArg SerAlaMet GluAla LeuLys
65 70 75 80
CAG AAGTCGCTC TACAAC TGCCGCTGC AAGCGGGGT ATGAAG AAGGAG 827
G1n LysSerLeu TyrAsn CysArgCys LysArgGly MetLys LysGlu
85 90 95
AAG AACTGCCTG CGCATT TACTGGAGC ATGTACCAG AGCCTG CAGGGA 875
Lys AsnCysLeu ArgIle TyrTrpSer MetTyrGln SerLeu GlnGly
100 105 110
AAT GATCTGCTG GAGGAT TCCCCATAT GAACCAGTT AACAGC AGATTG 923
Asn AspLeuLeu GluAsp SerProTyr GluProVal AsnSer ArgLeu
115 120 125
TCA GATATATTC CGGGTG GTCCCATTC ATATCAGAT GTTTTT CAGCAA 971
Ser AspIlePhe ArgVal ValProPhe IleSerAsp ValPhe GlnGln
130 135 140
GTG GAGCACATT CCCAAA GGGAACAAC TGCCTGGAT GCAGCG AAGGCC 1019
Val GluHisIle ProLys GlyAsnAsn CysLeuAsp AlaAla LysAla
145 150 155 160
TGC AACCTCGAC GACATT TGCAAGAAG TACAGGTCG GCGTAC ATCACC 1067
Cys AsnLeuAsp AspIle CysLysLys TyrArgSer AlaTyr IleThr
165 170 175
CCG TGCACCACC AGCGTG TCCAACGAT GTCTGCAAC CGCCGC AAGTGC 1115
Pro CysThrThr SerVal SerAsnAsp ValCysAsn ArgArg LysCys
180 185 190
CAC AAGGCCCTC CGGCAG TTCTTTGAC AAGGTCCCG GCCAAG CACAGC 1163
His LysAlaLeu ArgGln PhePheAsp LysValPro AlaLys HisSer
195 200 205
CA 02291608 1999-11-26
117
TAC GGA CTCTTCTGC TGC GAC TGC GAG
ATG TCC CGG ATC ACA CGG
GCC
1211
Tyr Gly LeuPheCys SerCysArg AspIle CysThr Glu
Met Ala Arg
210 215 220
AGG CGA ACCATCGTG GTGTGC TCCTAT GAGAGG GAGAAG 1259
CAG CCT GAA
Arg ArgGln ThrIleVal ProValCys SerTyrGlu GluArg GluLys
225 230 235
240
CCC AACTGT TTGAATTTG CAGGACTCC TGCAAGACG AATTAC ATCTGC 1307
Pro AsnCys LeuAsnLeu GlnAspSer CysLysThr AsnTyr IleCys
245 250 255
AGA TCTCGC CTTGCGGAT TTTTTTACC AACTGCCAG CCAGAG TCAAGG 1355
Arg SerArg LeuAlaAsp PhePheThr AsnCysGln ProGlu SerArg
260 265 270
TCT GTCAGC AGCTGTCTA AAGGAAAAC TACGCTGAC TGCCTC CTCGCC
1403
Ser ValSer SerCysLeu LysGluAsn TyrAlaAsp CysLeu LeuAla
275 280 285
TAC TCGGGG CTTATTGGC ACAGTCATG ACCCCCAAC TACATA GACTCC 1451
Tyr SerGly LeuIleGly ThrValMet ThrProAsn TyrIle AspSer
290 295 300
AGT AGCCTC AGTGTGGCC CCATGGTGT GACTGCAGC AACAGT GGGAAC 1499
Ser SerLeu SerValAla ProTrpCys AspCysSer AsnSer GlyAsn
305 310 315 320
GAC CTAGAA GAGTGCTTG AAATTTTTG AATTTCTTC AAGGAC AATACA 1547
Asp LeuGlu GluCysLeu LysPheLeu AsnPhePhe LysAsp AsnThr
325 330 335
TGT CTTAAA AATGCAATT CAAGCCTTT GGCAATGGC TCCGAT GTGACC
1595
Cys LeuLys AsnAlaIle GlnAlaPhe GlyAsnGly SerAsp ValThr
340 345 350
GTG TGGCAG CCAGCCTTC CCAGTACAG ACCACCACT GCCACT ACCACC 1643
Val TrpGln ProAlaPhe ProValGln ThrThrThr AlaThr ThrThr
355 360 365
ACT GCCCTC CGGGTTAAG AACAAGCCC CTGGGGCCA GCAGGG TCTGAG 1691
Thr AlaLeu ArgValLys AsnLysPro LeuGlyPro AlaGly SerGlu
370 375 380
AAT GAAATT CCCACTCAT GTTTTGCCA CCGTGTGCA AATTTA CAGGCA 1739
Asn GluIle ProThrHis ValLeuPro ProCysAla AsnLeu GlnAla
385 390 395 400
CAG AAGCTG AAATCCAAT GTGTCGGGC AATACACAC CTCTGT ATTTCC 1787
Gln LysLeu LysSerAsn ValSerGly AsnThrHis LeuCys IleSer
405 410 415
AAT GGTAAT TATGAAAAA GAAGGTCTC GGTGCTTCC AGCCAC ATAACC
1835
Asn GlyAsn TyrGluLys GluGlyLeu GlyAlaSer SerHis IleThr
420 425 430
ACA TCA ATGGCTGCT CCTCCAAGC TGTGGTCTG AGCCCA CTGCTG 1883
AAA
Thr LysSer Met Ala ProProSer CysGlyLeu SerPro LeuLeu
Ala
435 440 445
GTC CTGGTG GTA GCT CTGTCCACC CTATTATCT TTAACA GAAACA 1931
ACC
Val LeuVal Val Ala LeuSerThr LeuLeuSer LeuThr Thr
Thr Glu
450 455 460
CA 02291608 1999-11-26
11g
TCA TAGCTGCATT 1984
AAA.AAAATAC
AATATGGACA
TGTAAAAAGA
CAAAAACCAA
Ser
465
GTTATCTGTTTCCTGTTCTCTTGTATAGCTGAAATTCCAGTTTAGGAGCTCAGTTGAGAA2044
ACAGTTCCATTCAACTGGAACATTTTTTTTTTTNCCTTTTAAGAAAGCTTCTTGTGATCC2104
TTNGGGGCTTCTGTGAAAAACCTGATGCAGTGCTCCATCCAAACTCAGAAGGCTTTGGGA2164
TATGCTGTATTTTAAAGGGACAGTTTGTAACTTGGGCTGTAAAGCAAACTGGGGCTGTGT2224
TTTCGATGATGATGATNATCATGATNATGATNNNNNNNNNn~SNr~INN
NNN nfNNNNNNNNN2284
n~JNNNNNNNNGATTTTAACAGTTTTACTTCTGGCCTTTCCTAGCTAGAGAAGGAGTTAAT2344
ATTTCTAAGGTAACTCCCATATCTCCTTTAATGACATTGATTTCTAATGATATAAATTTC2404
AGCCTACATTGATGCCAAGCTTTTTTGCCACAAAGAAGATTCTTACCAAGAGTGGGCTTT2464
GTGGAAACAGCTGGTACTGATGTTCACCTTTATATATGTACTAGCATTTTCCACGCTGAT2524
GTTTATGTACTGTAAACAGTTCTGCACTCTTGTACAAAAGAAAA 2568
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 465 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Phe Leu Ala Thr Leu Tyr Phe Ala Leu Pro Leu Leu Asp Leu Leu
1 5 10 15
Leu Ser Ala Glu Val Ser Gly Gly Asp Arg Leu Asp Cys Val Lys Ala
20 25 30
Ser Asp Gln Cys Leu Lys Glu Gln Ser Cys Ser Thr Lys Tyr Arg Thr
35 40 45
Leu Arg Gln Cys Val Ala Gly Lys Glu Thr Asn Phe Ser Leu Ala Ser
50 55 60
Gly Leu Glu Ala Lys Asp G1u Cys Arg Ser Ala Met Glu Ala Leu Lys
65 70 75 80
Gln Lys Ser Leu Tyr Asn Cys Arg Cys Lys Arg Gly Met Lys Lys Glu
85 90 95
Lys Asn Cys Leu Arg Ile Tyr Trp Ser Met Tyr Gln Ser Leu Gln Gly
100 105 110
Asn Asp Leu Leu Glu Asp Ser Pro Tyr Glu Pro Val Asn Ser Arg Leu
115 120 125
Ser Asp Ile Phe Arg Val Val Pro Phe Ile Ser Asp Val Phe Gln Gln
130 135 140
CA 02291608 1999-11-26
119
Val Glu His Ile Pro Lys Gly Asn Asn Cys Leu Asp Ala Ala Lys Ala
145 150 155 160
Cys Asn Leu Asp Asp Ile Cys Lys Lys Tyr Arg Ser Ala Tyr Ile Thr
165 170 175
Pro Cys Thr Thr Ser Val Ser Asn Asp Val Cys Asn Arg Arg Lys Cys
180 185 190
His Lys Ala Leu Arg Gln Phe Phe Asp Lys Val Pro Ala Lys His Ser
195 200 205
Tyr Gly Met Leu Phe Cys Ser Cys Arg Asp Ile Ala Cys Thr Glu Arg
210 215 220
Arg Arg Gln Thr Ile Val Pro Val Cys Ser Tyr Glu G1u Arg Glu Lys
225 230 235 240
Pro Asn Cys Leu Asn Leu Gln Asp Ser Cys Lys Thr Asn Tyr Ile Cys
245 250 255
Arg Ser Arg Leu Ala Asp Phe Phe Thr Asn Cys Gln Pro Glu Ser Arg
260 265 270
Ser Val Ser Ser Cys Leu Lys Glu Asn Tyr Ala Asp Cys Leu Leu Ala
275 280 285
Tyr Ser Gly Leu Ile Gly Thr Val Met Thr Pro Asn Tyr Ile Asp Ser
290 295 300
Ser Ser Leu Ser Val Ala Pro Trp Cys Asp Cys Ser Asn Ser Gly Asn
305 310 315 320
Asp Leu Glu Glu Cys Leu Lys Phe Leu Asn Phe Phe Lys Asp Asn Thr
325 330 335
Cys Leu Lys Asn Ala Ile Gln Ala Phe Gly Asn Gly Ser Asp Val Thr
340 345 350
Val Trp Gln Pro Ala Phe Pro Val Gln Thr Thr Thr Ala Thr Thr Thr
355 360 365
Thr Ala Leu Arg Val Lys Asn Lys Pro Leu Gly Pro Ala Gly Ser Glu
370 375 380
Asn Glu Ile Pro Thr His Val Leu Pro Pro Cys Ala Asn Leu Gln Ala
385 390 395 400
Gln Lys Leu Lys Ser Asn Val Ser Gly Asn Thr His Leu Cys Ile Ser
405 410 415
Asn Gly Asn Tyr Glu Lys Glu Gly Leu Gly Ala Ser Ser His Ile Thr
420 425 430
Thr Lys Ser Met Ala Ala Pro Pro Ser Cys G1y Leu Ser Pro Leu Leu
435 440 445
Val Leu Val Val Thr Ala Leu Ser Thr Leu Leu Ser Leu Thr Glu Thr
450 455 460
Ser
465
CA 02291608 1999-11-26
120
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2138 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 302..1705
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:3:
AGCTCGCTCTCCCGGGGCAG TGGTGTGGAT GCACCGGAGTTCGGGCGCTGGGCAAGTTGG 60
GTCGGAACTGAACCCCTGAA AGCGGGTCCG CCTCCCGCCCTCGCGCCCGCCCGGATCTGA 120
GTCGCTGGCGGCGGTGGGCG GCAGAGCGAC GGGGAGTCTGCTCTCACCCTGGATGGAGCT 180
GAACTTTGAGTGGCCAGAGG AGCGCAGTCG CCCGGGGATCGCTGCACGCTGAGCTCTCTC 240
CCCGAGACCGGGCGGCGGCT TTGGATTTTG GGGGGGCGGGGACCAGCTGCGCGGCGGCAC 300
C ATG TA GCC ACT CTG TAC TTC GCG AT TTG 346
TTC C CTG CCA CTC CTG G
Met Phe eu Ala Thr Leu Tyr Phe Ala sp Leu
L Leu Pro Leu Leu A
1 5 10 15
CTG ATG GCC GAG GTG AGT GGT GGA CTG GAC GTG AAA 394
TCC GAC CGT TGT
Leu Met Ala Glu Val Ser Gly Gly Leu Asp Val Lys
Ser Asp Arg Cys
20 25 30
GCC AGC CAG TGC CTG AAG GAA CAG AGC ACC TAC CGC 442
GAT AGC TGC AAG
Ala Ser Gln Cys Leu Lys Glu Gln Ser Thr Tyr Arg
Asp Ser Cys Lys
35 40 45
ACA CTA CAG TGC GTG GCG GGC AAG AAC TTC CTG ACA 490
AGG GAA ACC AGC
Thr Leu Gln Cys Val Ala Gly Lys Asn Phe Leu Thr
Arg Glu Thr Ser
50 55 60
TCC GGC GAG GCC AAG GAT GAG TGC GCC ATG GCC TTG 538
CTT CGT AGC GAG
Ser Gly Glu Ala Lys Asp Glu Cys Ala Met Ala Leu
Leu Arg Ser Glu
65 70 75
AAGCAGAAGTCT CTGTACAAC TGCCGCTGC AAGCGG GGCATGAAG AAA 586
LysGlnLysSer LeuTyrAsn CysArgCys LysArg GlyMetLys Lys
80 85 90 95
GAGAAGAATTGT CTGCGTATC TACTGGAGC ATGTAC CAGAGCCTG CAG 634
GluLysAsnCys LeuArgIle TyrTrpSer MetTyr GlnSerLeu Gln
100 105 110
GGAAATGACCTC CTGGAAGAT TCCCCGTAT GAGCCG GTTAACAGC AGG 682
GlyAsnAspLeu LeuGluAsp SerProTyr GluPro ValAsnSer Arg
115 120 125
TTGTCAGATATA TTCCGGGCA GTCCCGTTC ATATCA GATGTTTTC CAG
730
LeuSerAspIle PheArgAla ValProPhe IleSer AspValPhe Gln
130 135 140
CA 02291608 1999-11-26
121
CAAGTGGAA CACATTTCC AAAGGGAAC AACTGCCTG GACGCA GCCAAG 778
GlnValGlu HisIleSer LysGlyAsn AsnCysLeu AspAla AlaLys
145 150 155
GCCTGCAAC CTGGACGAC ACCTGTAAG AAGTACAGG TCGGCC TACATC 826
AlaCysAsn LeuAspAsp ThrCysLys LysTyrArg SerAla TyrIle
160 165 170 175
ACCCCCTGC ACCACCAGC ATGTCCAAC GAGGTCTGC AACCGC CGTAAG 874
ThrProCys ThrThrSer MetSerAsn GluValCys AsnArg ArgLys
180 185 190
TGCCACAAG GCCCTCAGG CAGTTCTTC GACAAGGTT CCGGCC AAGCAC 922
CysHisLys AlaLeuArg GlnPhePhe AspLysVal ProAla LysHis
195 200 205
AGCTACGGG ATGCTCTTC TGCTCCTGC CGGGACATC GCCTGC ACCGAG 970
SerTyrGly MetLeuPhe CysSerCys ArgAspIle AlaCys ThrGlu
210 215 220
CGGCGGCGA CAGACTATC GTCCCCGTG TGCTCCTAT GAAGAA CGAGAG 1018
ArgArgArg GlnThrIle ValProVal CysSerTyr GluGlu ArgGlu
225 230 235
AGGCCCAAC TGCCTGAGT CTGCAAGAC TCCTGCAAG ACCAAT TACATC 1066
ArgProAsn CysLeuSer LeuGlnAsp SerCysLys ThrAsn TyrIle
240 245 250 255
TGCAGATCT CGCCTTGCA GATTTTTTT ACCAACTGC CAGCCA GAGTCA 1114
CysArgSer ArgLeuAla AspPhePhe ThrAsnCys GlnPro GluSer
260 265 270
AGGTCTGTC AGCAACTGT CTTAAGGAG AACTACGCA GACTGC CTCCTG 1162
ArgSerVal SerAsnCys LeuLysGlu AsnTyrAla AspCys LeuLeu
275 280 285
GCCTACTCG GGACTGATT GGCACAGTC ATGACTCCC AACTAC GTAGAC 1210
AlaTyrSer G1yLeuIle GlyThrVal MetThrPro AsnTyr ValAsp
290 295 300
TCCAGCAGC CTCAGCGTG GCACCATGG TGTGACTGC AGCAAC AGCGGC 1258
SerSerSer LeuSerVal AlaProTrp CysAspCys SerAsn SerGly
305 310 315
AATGACCTG GAAGACTGC TTGAAATTT CTGAATTTT TTTAAG GACAAT 1306
AsnAspLeu GluAspCys LeuLysPhe LeuAsnPhe PheLys AspAsn
320 325 330 335
ACTTGTCTC AAAAATGCA ATTCAAGCC TTTGGCAAT GGCTCA GATGTG 1354
ThrCysLeu LysAsnAla IleGlnAla PheGlyAsn GlySer AspVal
340 345 350
ACCATGTGG CAGCCAGCC CCTCCAGTC CAGACCACC ACTGCC ACCACT 1402
ThrMetTrp GlnProAla ProProVal GlnThrThr ThrAla ThrThr
355 360 365
ACCACTGCC TTCCGGGTC AAGAACAAG CCTCTGGGG CCAGCA GGGTCT 1450
ThrThrAla PheArgVal LysAsnLys ProLeuGly ProAla GlySer
370 375 380
GAGAATGAG ATCCCCACA CACGTTTTA CCACCCTGT GCGAAT TTGCAG 1498
GluAsnGlu IleProThr HisValLeu ProProCys AlaAsn LeuGln
385 390 395
CA 02291608 1999-11-26
122
GCTCAG AAGCTG AAATCCAAT GTGTCGGGT AGCACA CACCTCTGT CTT 1546
AlaGln LysLeu LysSerAsn ValSerGly SerThr HisLeuCys Leu
400 405 410 415
TCTGAT AGTGAT TTCGGAAAG GATGGTCTC GCTGGT GCCTCCAGC CAC
1594
SerAsp SerAsp PheGlyLys AspGlyLeu AlaGly AlaSerSer His
420 425 430
ATAACC ACAAAA TCAATGGCT GCTCCTCCC AGCTGC AGTCTGAGC TCA 1642
IleThr ThrLys SerMetAla AlaProPro SerCys SerLeuSer Ser
435 440 445
CTGCCG GTGCTG ATGCTCACC GCCCTTGCT GCCCTG TTATCTGTA TCG 1690
LeuPro ValLeu MetLeuThr AlaLeuAla AlaLeu LeuSerVal Ser
450 455 460
TTGGCA GAAACG TCGTAGCTGCATC ACAAAAGAGA 1745
CGGGAAAACA
GTATGAAAAG
LeuAla GluThr Ser
465
ACCAAGTATTCTGTCCCTGTCCTCTTGTATATCTGAAAATCCAGTTTTAA 1805
AAGCTCCGTT
GAGAAGCAGTTTCACCCAACTGGAACTCTTTCCTTGTTTTTAAGAAAGCTTGTGGCCCTC 1865
AGGGGCTTCTGTTGAAGAACTGCTACAGGGCTAATTCCAAACCCATAAGGCTCTGGGGCG 1925
TGGTGCGGCTTAAGGGGACCATTTGCACCATGTAAAGCAAGCTGGGCTTATCATGTGTTT 1985
GATGGTGAGGATGGTAGTGGTGATGATGATGGTAATTTTAACAGCTTGAACCCTGTTCTC 2045
TCTACTGGTTAGGAACAGGAGATACTATTGATAAAGATTCTTCCATGTCTTACTCAGCAG 2105
CATTGCCTTCTGAAGACAGGCCCGCAGCCGTCG 2138
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 468 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Met Phe Leu Ala Thr Leu Tyr Phe Ala Leu Pro Leu Leu Asp Leu Leu
1 5 10 15
Met Ser Ala Glu Val Ser Gly Gly Asp Arg Leu Asp Cys Val Lys Ala
20 25 30
Ser Asp Gln Cys Leu Lys Glu Gln Ser Cys Ser Thr Lys Tyr Arg Thr
35 40 45
Leu Arg Gln Cys Val Ala Gly Lys Glu Thr Asn Phe Ser Leu Thr Ser
50 55 60
Gly Leu Glu Ala Lys Asp Glu Cys Arg Ser Ala Met Glu Ala Leu Lys
65 70 75 80
Gln Lys Ser Leu Tyr Asn Cys Arg Cys Lys Arg Gly Met Lys Lys Glu
85 90 95
CA 02291608 1999-11-26
123
Lys Asn Cys Leu Arg Ile Tyr Trp Ser Met Tyr Gln Ser Leu Gln Gly
100 105 110
Asn Asp Leu Leu Glu Asp Ser Pro Tyr Glu Pro Val Asn Ser Arg Leu
115 120 125
Ser Asp Ile Phe Arg Ala Val Pro Phe Ile Ser Asp Val Phe Gln Gln
130 135 140
Val Glu His Ile Ser Lys Gly Asn Asn Cys Leu Asp Ala Ala Lys Ala
145 150 155 160
Cys Asn Leu Asp Asp Thr Cys Lys Lys Tyr Arg Ser Ala Tyr Ile Thr
165 170 175
Pro Cys Thr Thr Ser Met Ser Asn Glu Val Cys Asn Arg Arg Lys Cys
180 185 190
His Lys Ala Leu Arg Gln Phe Phe Asp Lys Val Pro Ala Lys His Ser
195 200 205
Tyr Gly Met Leu Phe Cys Ser Cys Arg Asp Ile Ala Cys Thr Glu Arg
210 215 220
Arg Arg Gln Thr Ile Val Pro Val Cys Ser Tyr Glu Glu Arg Glu Arg
225 230 235 240
Pro Asn Cys Leu Ser Leu Gln Asp Ser Cys Lys Thr Asn Tyr Ile Cys
245 250 255
Arg Ser Arg Leu Ala Asp Phe Phe Thr Asn Cys Gln Pro Glu Ser Arg
260 265 270
Ser Val Ser Asn Cys Leu Lys Glu Asn Tyr Ala Asp Cys Leu Leu Ala
275 280 285
Tyr Ser Gly Leu Ile Gly Thr Val Met Thr Pro Asn Tyr Val Asp Ser
290 295 300
Ser Ser Leu Ser Val Ala Pro Trp Cys Asp Cys Ser Asn Ser Gly Asn
305 310 315 320
Asp Leu Glu Asp Cys Leu Lys Phe Leu Asn Phe Phe Lys Asp Asn Thr
325 330 335
Cys Leu Lys Asn Ala Ile Gln Ala Phe Gly Asn Gly Ser Asp Val Thr
340 345 350
Met Trp Gln Pro Ala Pro Pro Val Gln Thr Thr Thr Ala Thr Thr Thr
355 360 365
Thr Ala Phe Arg Val Lys Asn Lys Pro Leu Gly Pro Ala Gly Ser Glu
370 375 380
Asn Glu Ile Pro Thr His Val Leu Pro Pro Cys Ala Asn Leu Gln Ala
385 390 395 400
Gln Lys Leu Lys Ser Asn Val Ser Gly Ser Thr His Leu Cys Leu Ser
405 410 415
Asp Ser Asp Phe Gly Lys Asp Gly Leu Ala Gly Ala Ser Ser His Ile
420 425 430
CA 02291608 1999-11-26
124
Thr Thr Lys Ser Met Ala Ala Pro Pro Ser Cys Ser Leu Ser Ser Leu
435 440 445
Pro Val Leu Met Leu Thr Ala Leu Ala Ala Leu Leu Ser Val Ser Leu
450 455 460
Ala Glu Thr Ser
465
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3209 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: CDNA
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..539
(D) OTHER INFORMATION: /note= "1 to 539 is -237 to 301 of
Figure 5 Gdnfr"
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 540..1937
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:5:
AATCTGGCCTCGGAACACGC CATTCTCCGC GCCGCTTCCAATAACCACTAACATCCCTAA 60
CGAGCATCCGAGCCGAGGGC TCTGCTCGGA AATCGTCCTGGCCCAACTCGGCCCTTCGAG 120
CTCTCGAAGATTACCGCATC TATTTTTTTT TTCTTTTTTTTCTTTTCCTAGCGCAGATAA 180
AGTGAGCCCGGAAAGGGAAG GAGGGGGCGG GGACACCATTGCCCTGAAAGAATAAATAAG 240
TAAATAAACAAACTGGCTCC TCGCCGCAGC TGGACGCGGTCGGTTGAGTCCAGGTTGGGT
300
CGGACCTGAACCCCTAAAAG CGGAACCGCC TCCCGCCCTCGCCATCCCGGAGCTGAGTCG 360
CCGGCGGCGGTGGCTGCTGC CAGACCCGGA GTTTCCTCTTTCACTGGATGGAGCTGAACT 420
TTGGGCGGCCAGAGCAGCAC AGCTGTCCGG GGATCGCTGCACGCTGAGCTCCCTCGGCAA 480
GACCCAGCGGCGGCTCGGGA TTTTTTTGGG GGGGCGGGGACCAGCCCCGCGCCGGCACC 539
ATG TTC GCG ACC CTG TAC TTC GCG CTC TTG TTG CTC
CTG CTG CCG GAC
587
Met Phe Ala Thr Leu Tyr Phe Ala Leu Leu Leu Leu
Leu Leu Pro Asp
1 5 10 15
CTG TCG GAA GTG AGC GGC GGA GAC GAT TGC AAA GCC
GCC CGC CTG GTG
635
Leu Ser Glu Val Ser Gly Gly Asp Asp Cys Lys Ala
Ala Arg Leu Val
20 25 30
AGT GAT TGC CTG AAG GAG CAG AGC ACC AAG CGC ACG 683
CAG TGC AGC TAC
Ser Asp Cys Leu Lys Glu Gln Ser Thr Lys Arg Thr
Gln Cys Ser Tyr
35 40 45
CA 02291608 1999-11-26
125
CTA CAG TGCGTGGCG AAGGAG ACCAAC AGCCTG GCA
AGG GGC TTC TCC
731
Leu ArgGln CysValAla GlyLysGlu ThrAsnPhe SerLeu AlaSer
50 55 60
GGC CTGGAG GCCAAGGAT GAGTGCCGC AGCGCCATG GAGGCC CTGAAG
779
Gly LeuGlu AlaLysAsp GluCysArg SerAlaMet GluAla LeuLys
65 70 75 80
CAG AAGTCG CTCTACAAC TGCCGCTGC AAGCGGGGT ATGAAG AAGGAG 827
Gln LysSer LeuTyrAsn CysArgCys LysArgGly MetLys LysGlu
85 90 95
AAG AACTGC CTGCGCATT TACTGGAGC ATGTACCAG AGCCTG CAGGGA 875
Lys AsnCys LeuArgIle TyrTrpSer MetTyrGln SerLeu GlnGly
100 105 110
AAT GATCTG CTGGAGGAT TCCCCATAT GAACCAGTT AACAGC AGATTG 923
Asn AspLeu LeuGluAsp SerProTyr GluProVal AsnSer ArgLeu
115 120 125
TCA GATATA TTCCGGGTG GTCCCATTC ATATCAGAT GTTTTT CAGCAA 971
Ser AspIle PheArgVal ValProPhe IleSerAsp ValPhe GlnGln
130 135 140
GTG GAGCAC ATTCCCAAA GGGAACAAC TGCCTGGAT GCAGCG AAGGCC 1019
Val GluHis IleProLys GlyAsnAsn CysLeuAsp AlaAla LysAla
145 150 155 160
TGC AACCTC GACGACATT TGCAAGAAG TACAGGTCG GCGTAC ATCACC 1067
Cys AsnLeu AspAspIle CysLysLys TyrArgSer AlaTyr IleThr
165 170 175
CCG TGCACC ACCAGCGTG TCCAANGAT GTCTGCAAC CGCCGC AAGTGC 1115
Pro CysThr ThrSerVal SerXaaAsp ValCysAsn ArgArg LysCys
180 185 190
CAC AAGGCC CTCCGGCAG TTCTTTGAC AAGGTCCCG GCCAAG CACAGC 1163
His LysAla LeuArgGln PhePheAsp LysValPro AlaLys HisSer
1g5 200 205
TAC GGAATG CTCTTCTGC TCCTGCCGG GACATCGCC TGCACA GAGCGG 1211
Tyr GlyMet LeuPheCys SerCysArg AspIleAla CysThr GluArg
210 215 220
AGG CGACAG ACCATCGTG CCTGTGTGC TCCTATGAA GAGAGG GAGAAG 1259
Arg ArgGln ThrIleVal ProValCys SerTyrGlu GluArg GluLys
225 230 235 240
CCC AACTGT TTGAATTTG CAGGACTCC TGCAAGACG AATTAC ATCTGC 1307
Pro AsnCys LeuAsnLeu GlnAspSer CysLysThr AsnTyr IleCys
245 250 255
AGA TCTCGC CTTGCGGAT TTTTTTACC AACTGCCAG CCAGAG TCAAGG 1355
Arg SerArg LeuAlaAsp PhePheThr AsnCysGln ProGlu SerArg
260 265 270
TCT GTCAGC AGCTGTCTA AAGGAAAAC TACGCTGAC TGCCTC CTCGCC 1403
Ser ValSer SerCysLeu LysGluAsn TyrAlaAsp CysLeu LeuAla
275 280 285
TAC TCGGGG CTTATTGGC ACAGTCATG CCCAAC TACATA GACTCC 1451
ACC
Tyr SerGly LeuIleGly Thr Met ProAsn TyrIle Ser
Val Thr Asp
290 295 300
CA 02291608 1999-11-26
126
AGTAGCCTC AGTGTGGCC CCATGGTGT GACTGCAGC AACAGTGGG AAC 1499
SerSerLeu SerValAla ProTrpCys AspCysSer AsnSerGly Asn
305 310 315 320
GACCTAGAA GAGTGCTTG AAATTTTTG AATTTCTTC AAGGACAAT ACA 1547
AspLeuGlu GluCysLeu LysPheLeu AsnPhePhe LysAspAsn Thr
325 330 335
TGTCTTAAA AATGCAATT CAAGCCTTT GGCAATGGC TCCGATGTG ACC
1595
CysLeuLys AsnAlaIle GlnAlaPhe GlyAsnGly SerAspVal Thr
340 345 350
GTGTGGCAG CCAGCCTTC CCAGTACAG ACCACCACT GCCACTACC ACC 1643
ValTrpGln ProAlaPhe ProValGln ThrThrThr AlaThrThr Thr
355 360 365
ACTGCCCTC CGGGTTAAG AACAAGCCC CTGGGGCCA GCAGGGTCT GAG 1691
ThrAlaLeu ArgValLys AsnLysPro LeuGlyPro AlaGlySer Glu
370 375 380
AATGAAATT CCCACTCAT GTTTTGCCA CCGTGTGCA AATTTACAG GCA 1739
AsnGluIle ProThrHis ValLeuPro ProCysAla AsnLeuGln Ala
385 390 395 400
CAGAAGCTG AAATCCAAT GTGTCGGGC AATACACAC CTCTGTATT TCC 1787
GlnLysLeu LysSerAsn ValSerGly AsnThrHis LeuCysIle Ser
405 410 415
AATGGTAAT TATGAAAAA GAAGGTCTC GGTGCTTCC AGCCACATA ACC
1835
AsnGlyAsn TyrGluLys GluGlyLeu GlyAlaSer SerHisIle Thr
420 425 430
ACAAAATCA ATGGCTGCT CCTCCAAGC TGTGGTCTG AGCCCACTG CTG
1883
ThrLysSer MetAlaAla ProProSer CysGlyLeu SerProLeu Leu
435 440 445
GTCCTGGTG GTAACCGCT CTGTCCACC CTATTATCT TTAACAGAA ACA 1931
ValLeuVal ValThrAla LeuSerThr LeuLeuSer LeuThrGlu Thr
450 455 460
TCATAGCTGCATTAAA GGACATGT AAAAAGA CAAAAACCAAG TT 1987
AAAATACAAT
AT
Ser
465
ATCTGTTTCCTGTTCTCTTGTATAGCTGAA AGGAGCTCAGTTGAGAAACA 2047
ATTCCAGTTT
GTTCCATTCAACTGGAACATTTTTTTTTTTNCCTTTTAAGAAAGCTTCTTGTGATCCTTC 2107
GGGGCTTCTGTGAAAAACCTGATGCAGTGCTCCATCCAAACTCAGAAGGCTTTGGGATAT 2167
GCTGTATTTTAAAGGGACAGTTTGTAACTTGGGCTGTAAAGCAAACTGGGGCTGTGTTTT 2227
CGATGATGATGATCATCATGATCATGATNNNr~~NNNNNNNNNNNNNNNNNn~~VnfNNNNNN2287
NNNNNNNGATTTTAACAGTTTTACTTCTGGCCTTTCCTAGCTAGAGAAGGAGTTAATATT 2347
TCTAAGGTAACTCCCATATCTCCTTTAATGACATTGATTTCTAATGATATAAATTTCAGC 2407
CTACATTGATGCCAAGCTTTTTTGCCACAAAGAAGATTCTTACCAAGAGTGGGCTTTGTG 2467
GAAACAGCTGGTACTGATGTTCACCTTTATATATGTACTAGCATTTTCCACGCTGATGTT 2527
TATGTACTGTAAACAGTTCTGCACTCTTGTACAAAAGAAAAAACACCTGTCACATCCAAA 2587
CA 02291608 1999-11-26
127
TATAGTATCTGTCTTTTCGT CAAAATAGAG AGTGGGGAATGAGTGTGCCGATTCAATACC2647
TCAATCCCTGAACGACACTC TCCTAATCCT AAGCCTTACCTGAGTGAGAAGCCCTTTACC2707
TAACAAAAGTCCAATATAGC TGAAATGTCG CTCTAATACTCTTTACACATATGAGGTTAT2767
ATGTAGAAAAAAATTTTACT ACTAAATGAT TTCAACTATTGGCTTTCTATATTTTGAAAG2827
TAATGATATTGTCTCATTTT TTTACTGATG GTTTAATACAAAATACACAGAGCTTGTTTC2887
CCCTCATAAGTAGTGTTCGC TCTGATATGA ACTTCACAAATACAGCTCATCAAAAGCAGA2947
CTCTGAGAAGCCTCGTGCTG TAGCAGAAAG TTCTGCATCATGTGACTGTGGACAGGCAGG3007
AGGAAACAGAACAGACAAGC ATTGTCTTTT GTCATTGCTCGAAGTGCAAGCGTGCATACC3067
TGTGGAGGGAACTGGTGGCT GCTTGTAAAT GTTCTGCAGCATCTCTTGACACACTTGTCA3127
TGACACAATCCAGTACCTTG GTTTTCAGGT TATCTGACAAAGGCAGCTTTGATTGGGACA3187
TGGAGGCATGGGCAGGCCGG AA 3209
(2) INFORMATION
FOR SEQ
ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 465 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ
ID N0:6:
Met Phe Ala Thr Leu Tyr Phe Ala Leu Leu Leu Leu
Leu Leu Pro Asp
1 5 10 15
Leu Ser Glu Val Ser Gly Gly Asp Asp Cys Lys Ala
Ala Arg Leu Val
20 25 30
Ser Asp Gln Cys Leu Lys Glu Gln Ser Cys Ser Thr Lys Tyr Arg Thr
35 40 45
Leu Arg Gln Cys Val Ala Gly Lys Glu Thr Asn Phe Ser Leu Ala Ser
50 55 60
Gly Leu Glu Ala Lys Asp Glu Cys Arg Ser Ala Met Glu Ala Leu Lys
65 70 75 80
Gln Lys Ser Leu Tyr Asn Cys Arg Cys Lys Arg Gly Met Lys Lys Glu
85 90 95
Lys Asn Cys Leu Arg Ile Tyr Trp Ser Met Tyr Gln Ser Leu Gln Gly
100 105 110
Asn Asp Leu Leu Glu Asp Ser Pro Tyr Glu Pro Val Asn Ser Arg Leu
115 120 125
Ser Asp Ile Phe Arg Val Val Pro Phe Ile Ser Asp Val Phe Gln Gln
130 135 140
Val Glu His Ile Pro Lys Gly Asn Asn Cys Leu Asp Ala Ala Lys Ala
145 150 155 160
CA 02291608 1999-11-26
128
Cys Asn Leu Asp Asp Ile Cys Lys Lys Tyr Arg Ser Ala Tyr Ile Thr
165 170 175
Pro Cys Thr Thr Ser Val Ser Xaa Asp Val Cys Asn Arg Arg Lys Cys
180 185 190
His Lys Ala Leu Arg Gln Phe Phe Asp Lys Val Pro Ala Lys His Ser
195 200 205
Tyr Gly Met Leu Phe Cys Ser Cys Arg Asp Ile Ala Cys Thr Glu Arg
210 215 220
Arg Arg Gln Thr Ile Val Pro Val Cys Ser Tyr Glu Glu Arg Glu Lys
225 230 235 240
Pro Asn Cys Leu Asn Leu Gln Asp Ser Cys Lys Thr Asn Tyr Ile Cys
245 250 255
Arg Ser Arg Leu Ala Asp Phe Phe Thr Asn Cys Gln Pro Glu Ser Arg
260 265 270
Ser Val Ser Ser Cys Leu Lys Glu Asn Tyr Ala Asp Cys Leu Leu Ala
275 280 285
Tyr Ser Gly Leu Ile Gly Thr Val Met Thr Pro Asn Tyr Ile Asp Ser
290 295 300
Ser Ser Leu Ser Val Ala Pro Trp Cys Asp Cys Ser Asn Ser Gly Asn
305 310 315 320
Asp Leu Glu Glu Cys Leu Lys Phe Leu Asn Phe Phe Lys Asp Asn Thr
325 330 335
Cys Leu Lys Asn Ala Ile Gln Ala Phe Gly Asn Gly Ser Asp Val Thr
340 345 350
Val Trp Gln Pro Ala Phe Pro Val Gln Thr Thr Thr Ala Thr Thr Thr
355 360 365
Thr Ala Leu Arg Val Lys Asn Lys Pro Leu Gly Pro Ala Gly Ser Glu
370 375 380
Asn Glu Ile Pro Thr His Val Leu Pro Pro Cys Ala Asn Leu Gln Ala
385 390 395 400
Gln Lys Leu Lys Ser Asn Val Ser Gly Asn Thr His Leu Cys Ile Ser
405 410 415
Asn Gly Asn Tyr Glu Lys Glu Gly Leu Gly Ala Ser Ser His Ile Thr
420 425 430
Thr Lys Ser Met Ala Ala Pro Pro Ser Cys Gly Leu Ser Pro Leu Leu
435 440 445
Val Leu Val Val Thr Ala Leu Ser Thr Leu Leu Ser Leu Thr G1u Thr
450 455 460
Ser
465
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
CA 02291608 1999-11-26
129
(A) LENGTH: 508 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: CDNA
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..508
(D) OTHER INFORMATION: /note= "1 to 508 is -237 to 272 of
Figure 5 Hsgr-2laf"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
TCTGGCCTCG GAACACGCCA TTCTCCGCGC CGCTTCCAAT AACCACTAAC ATCCCTAACG 60
AGCATCCGAG CCGAGGGCTC TGCTCGGAAA TCGTCCTGGC CCAACTCGGC CCTTCGAGCT 120
CTCGAAGATT ACCGCATCTA TTTTTTTTTT CTTTTTTTTC TTTTCCTAGC GCAGATAAAG 180
TGAGCCCGGA AAGGGAAGGA GGGGGCGGGG ACACCATTGC CCTGAAAGAA TAAATAAGTA 240
AATAAACAAA CTGGCTCCTC GCCGCAGCTG GACGCGGTCG GTTGAGTCCA GGTTGGGTCG 300
GACCTGAACC CCTAAAAGCG GAACCGCCTC CCGCCCTCGC CATCCCGGAG CTGAGTCGCC 360
GGCGGCGGTG GCTGCTGCCA GACCCGGAGT TTCCTCTTTC ACTGGATGGA GCTGAACTTT 420
GGGCGGCCAG AGCAGCACAG CTGTCCGGGG ATCGCTGCAC GCTGAGCTCC CTCGGCAAGA 480
CCCAGCGGCG GCTCGGGATT TTTTTGGG 508
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 510 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..510
(D) OTHER INFORMATION: /note= "1 to 510 is -237 to 272 of
Figure 5 Hsgr-2lbf"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: B:
AATCTGGCCT CGGAACACGC CATTCTCCGC GCCGCTTCCA ATAACCACTA ACATCCCTAA 60
CGAGCATCCG AGCCGAGGGC TCTGCTCGGA AATCGTCCTG GCCCAACTCG GCCCTTCGAG 120
CTCTCGAAGA TTACCGCATC TATTTTTTTT TTCTTTTTTT TCTTTTCCTA GCGCAGATAA 180
AGTGAGCCCG GAAAGGGAAG GAGGGGGCGG GGACACCATT GCCCTGAAAG AATAAATAAG 240
CA 02291608 1999-11-26
130
TAAATAAACA AACTGGCTCC TCGCCGCAGC TGGACGCGGT CGGTTGAGTC CAGGTTGGGT 300
CGGACCTGAA CCCCTAAAAG CGGAACCGCC TCCCGCCCTC GCCATCCCGG AGCTGAGTCG 360
CCGGCGGCGG TGGCTGCTGC CAGACCCGGA GTTTCCTCTT TCACTGGATG GAGCTGAACT 420
TTGGGCGGCC AGAGCAGCAC AGCTGTCCGG GGATCGCTGC ACGCTGAGCT CCCTCGGCAA 480
GACCCAGCGG CGGCTCGGGA TTTTTTTGGG 510
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1927 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 538..1926
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..537
(D) OTHER INFORMATION: /note= "1 to 537 is -235 to 301 of
Figure 5 2lacon"
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:9:
TCTGGCCTCGGAACACGCCA TTCTCCGCGC CGCTTCCAATAACCACTAACATCCCTAACG 60
AGCATCCGAGCCGAGGGCTC TGCTCGGAAA TCGTCCTGGCCCAACTCGGCCCTTCGAGCT 120
CTCGAAGATTACCGCATCTA TTTTTTTTTT CTTTTTTTTCTTTTCCTAGCGCAGATAAAG 180
TGAGCCCGGAAAGGGAAGGA GGGGGCGGGG ACACCATTGCCCTGAAAGAATAAATAAGTA 240
AATAAACAAACTGGCTCCTC GCCGCAGCTG GACGCGGTCGGTTGAGTCCAGGTTGGGTCG 300
GACCTGAACCCCTAAAAGCG GAACCGCCTC CCGCCCTCGCCATCCCGGAGCTGAGTCGCC 360
GGCGGCGGTGGCTGCTGCCA GACCCGGAGT TTCCTCTTTCACTGGATGGAGCTGAACTTT 420
GGGCGGCCAGAGCAGCACAG CTGTCCGGGG ATCGCTGCACGCTGAGCTCCCTCGGCAAGA 480
CCCAGCGGCGGCTCGGGATT TTTTTGGGGG GGCGGGGACCAGCCCCGCGCCGGCACC 537
ATG TTC GCG NCC CTG TAC TTC GCG CTC TTG TTG CTC 585
CTG CTG CCG GAC
Met Phe Ala Xaa Leu Tyr Phe Ala Leu Leu Leu Leu
Leu Leu Pro Asp
1 5 10 15
CTG TCG GAA GTG AGC GGC GGA GAC GAT TGC AAA GCC 633
GCC CGC CTG GTG
Leu Ser Glu Val Ser Gly Gly Asp Asp Cys Lys Ala
Ala Arg Leu Val
20 25 30
AGT GAT TGC CTG AAG GAG CAG AGC ACC AAG CGC ACG 681
CAG TGC AGC TAC
Ser Asp Cys Leu Lys Glu Gln Ser Thr Lys Arg Thr
Gln Cys Ser Tyr
35 40 45
CA 02291608 1999-11-26
131
CTAAGGCAG TGCGTGGCG GGCAAGGAG ACCAACTTC AGCCTG GCA 729
TCC
LeuArgGln CysValAla GlyLysGlu ThrAsn SerLeu AlaSer
Phe
50 55 60
GGCCTGGAG GCCAAGGAT GAGTGCCGC AGCGCCATG GAGGCC CTGAAG 777
GlyLeuGlu AlaLysAsp GluCysArg SerAlaMet GluAla LeuLys
65 70 75 80
CAGAAGTCG CTCTACAAC TGCCGCTGC AAGCGGGGT ATGAAG AAGGAG 825
GlnLysSer LeuTyrAsn CysArgCys LysArgGly MetLys LysGlu
85 90 95
AAGAACTGC CTGCGCATT TACTGGAGC ATGTACCAG AGCCTG CAGGGA 873
LysAsnCys LeuArgIle TyrTrpSer MetTyrGln SerLeu GlnGly
100 105 110
AATGATCTG CTGGAGGAT TCCCCATAT GAACCAGTT AACAGC AGATTG 921
AsnAspLeu LeuGluAsp SerProTyr GluProVal AsnSer ArgLeu
115 120 125
TCAGATATA TTCCGGGTG GTCCCATTC ATATCAGAT GTTTTT CAGCAA 969
SerAspIle PheArgVal ValProPhe IleSerAsp ValPhe GlnGln
130 135 140
GTGGAGCAC ATTCCCAAA GGGAACAAC TGCCTGGAT GCAGCG AAGGCC 1017
ValGluHis IleProLys GlyAsnAsn CysLeuAsp AlaAla LysAla
145 150 155 160
TGCAACCTC GACGACATT TGCAAGAAG TACAGGTCG GCGTAC ATCACC 1065
CysAsnLeu AspAspIle CysLysLys TyrArgSer AlaTyr IleThr
165 170 175
CCGTGCACC ACCAGCGTG TCCAACGAT GTCTGCAAC CGCCGC AAGTGC 1113
ProCysThr ThrSerVal SerAsnAsp ValCysAsn ArgArg LysCys
180 185 190
CACAAGGCC CTCCGGCAG TTCTTTGAC AAGGTCCCG GCCAAG CACAGC 1161
HisLysAla LeuArgGln PhePheAsp LysValPro AlaLys HisSer
195 200 205
TACGGAATG CTCTTCTGC TCCTGCCGG GACATCGCC TGCACA GAGCGG 1209
TyrGlyMet LeuPheCys SerCysArg AspIleAla CysThr GluArg
210 215 220
AGGCGACAG ACCATCGTG CCTGTGTGC TCCTATGAA GAGAGG GAGAAG 1257
ArgArgGln ThrIleVal ProValCys SerTyrGlu GluArg GluLys
225 230 235 240
CCCAACTGT TTGAATTTG CAGGACTCC TGCAAGACG AATTAC ATCTGC 1305
ProAsnCys LeuAsnLeu GlnAspSer CysLysThr AsnTyr IleCys
245 250 255
AGATCTCGC CTTGCGGAT TTTTTTACC AACTGCCAG CCAGAG TCAAGG 1353
ArgSerArg LeuAlaAsp PhePheThr AsnCysGln ProGlu SerArg
260 265 270
TCTGTCAGC AGCTGTCTA GAAAAC TACGCTGAC TGCCTC CTCGCC 1401
AAG
SerValSer SerCysLeu LysGluAsn TyrAlaAsp CysLeu LeuAla
275 280 285
TACTCGGGG CTTATTGGC GTCATG CCCAAC TACATA GACTCC 1449
ACA ACC
TyrSerGly LeuIleGly ValMet ThrProAsn TyrIle Ser
Thr Asp
290 295 300
CA 02291608 1999-11-26
132
AGTAGCCTC AGTGTG GCCCCATGG TGTGACTGC AGCAACAGT GGGAAC
1497
SerSerLeu SerVal AlaProTrp CysAspCys SerAsnSer GlyAsn
305 310 315
320
GACCTAGAA GAGTGC TTGAAATTT TTGAATTTC TTCAAGGAC AATACA 1545
AspLeuGlu GluCys LeuLysPhe LeuAsnPhe PheLysAsp AsnThr
325 330 335
TGTCTTAAA AATGCA ATTCAAGCC TTTGGCAAT GGCTCCGAT GTGACC 1593
CysLeuLys AsnAla IleGlnAla PheGlyAsn GlySerAsp ValThr
340 345 350
GTGTGGCAG CCAGCC TTCCCAGTA CAGACCACC ACTGCCACT ACCACC 1641
ValTrpGln ProAla PheProVal GlnThrThr ThrAlaThr ThrThr
355 360 365
ACTGCCCTC CGGGTT AAGAACAAG CCCCTGGGG CCAGCAGGG TCTGAG 1689
ThrAlaLeu ArgVal LysAsnLys ProLeuGly ProAlaGly SerGlu
370 375 380
AATGAAATT CCCACT CATGTTTTG CCACCGTGT GCAAATTTA CAGGCA 1737
AsnGluIle ProThr HisValLeu ProProCys AlaAsnLeu GlnAla
385 390 395 400
CAGAAGCTG AAATCC AATGTGTCG GGCAATACA CACCTCTGT ATTTCC 1785
GlnLysLeu LysSer AsnValSer GlyAsnThr HisLeuCys IleSer
405 410 415
AATGGTAAT TATGAA AAAGAAGGT CTCGGTGCT TCCAGCCAC ATAACC 1833
AsnGlyAsn TyrGlu LysGluGly LeuGlyAla SerSerHis IleThr
420 425 430
ACAAAATCA ATGGCT GCTCCTCCA AGCTGTGGT CTGAGCCCA CTGCTG
1881
ThrLysSer MetAla AlaProPro SerCysGly LeuSerPro LeuLeu
435 440 445
GTCCTGGTG GTAACC GCTCTGTCC ACCCTATTA TCTTTAACA GAA 1926
ValLeuVal ValThr AlaLeuSer ThrLeuLeu SerLeuThr Glu
450 455 460
A
1927
(2)INFORMATIONFOR SEQID :
NO:10
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 463 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Met Phe Leu Ala Xaa Leu Tyr Phe Ala Leu Pro Leu Leu Asp Leu Leu
1 5 10 15
Leu Ser Ala Glu Val Ser Gly Gly Asp Arg Leu Asp Cys Val Lys Ala
20 25 30
Ser Asp Gln Cys Leu Lys Glu Gln Ser Cys Ser Thr Lys Tyr Arg Thr
35 40 45
CA 02291608 1999-11-26
133
Leu Arg Gln Cys Val Ala Gly Lys Glu Thr Asn Phe Ser Leu Ala Ser
50 55 60
Gly Leu Glu Ala Lys Asp G1u Cys Arg Ser Ala Met Glu Ala Leu Lys
65 70 75 80
Gln Lys Ser Leu Tyr Asn Cys Arg Cys Lys Arg Gly Met Lys Lys Glu
85 90 95
Lys Asn Cys Leu Arg Ile Tyr Trp Ser Met Tyr Gln Ser Leu Gln Gly
100 105 110
Asn Asp Leu Leu Glu Asp Ser Pro Tyr Glu Pro Val Asn Ser Arg Leu
115 120 125
Ser Asp Ile Phe Arg Val Val Pro Phe I1e Ser Asp Val Phe Gln Gln
130 135 140
Val Glu His Ile Pro Lys Gly Asn Asn Cys Leu Asp Ala Ala Lys Ala
145 150 155 160
Cys Asn Leu Asp Asp Ile Cys Lys Lys Tyr Arg Ser Ala Tyr Ile Thr
165 170 175
Pro Cys Thr Thr Ser Val Ser Asn Asp Val Cys Asn Arg Arg Lys Cys
180 185 190
His Lys Ala Leu Arg Gln Phe Phe Asp Lys Val Pro Ala Lys His Ser
195 200 205
Tyr Gly Met Leu Phe Cys Ser Cys Arg Asp Ile Ala Cys Thr Glu Arg
210 215 220
Arg Arg Gln Thr Ile Val Pro Val Cys Ser Tyr Glu Glu Arg Glu Lys
225 230 235 240
Pro Asn Cys Leu Asn Leu Gln Asp Ser Cys Lys Thr Asn Tyr Ile Cys
245 250 255
Arg Ser Arg Leu Ala Asp Phe Phe Thr Asn Cys Gln Pro Glu Ser Arg
260 265 270
Ser Val Ser Ser Cys Leu Lys Glu Asn Tyr Ala Asp Cys Leu Leu Ala
275 280 285
Tyr Ser Gly Leu Ile Gly Thr Val Met Thr Pro Asn Tyr Ile Asp Ser
290 295 300
Ser Ser Leu Ser Val Ala Pro Trp Cys Asp Cys Ser Asn Ser Gly Asn
305 310 315 320
Asp Leu Glu Glu Cys Leu Lys Phe Leu Asn Phe Phe Lys Asp Asn Thr
325 330 335
Cys Leu Lys Asn Ala Ile Gln Ala Phe Gly Asn Gly Ser Asp Val Thr
340 345 350
Val Trp Gln Pro Ala Phe Pro Val Gln Thr Thr Thr Ala Thr Thr Thr
355 360 365
Thr Ala Leu Arg Val Lys Asn Lys Pro Leu Gly Pro Ala Gly Ser Glu
370 375 380
CA 02291608 1999-11-26
134
Asn Glu Ile Pro Thr His Val Leu Pro Pro Cys Ala Asn Leu Gln Ala
385 390 395 400
Gln Lys Leu Lys Ser Asn Val Ser Gly Asn Thr His Leu Cys Ile Ser
405 410 415
Asn Gly Asn Tyr Glu Lys Glu Gly Leu Gly Ala Ser Ser His Ile Thr
420 425 430
Thr Lys Ser Met Ala Ala Pro Pro Ser Cys Gly Leu Ser Pro Leu Leu
435 440 445
Val Leu Val Val Thr Ala Leu Ser Thr Leu Leu Ser Leu Thr Glu
450 455 460
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1929 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 540..1928
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..539
(D) OTHER INFORMATION: /note= "1 to 539 is -237 to 301 of
Figure 5 2lbcon"
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:11:
AATCTGGCCTCGGAACACGC CATTCTCCGC GCCGCTTCCAATAACCACTAACATCCCTAA 60
CGAGCATCCGAGCCGAGGGC TCTGCTCGGA AATCGTCCTGGCCCAACTCGGCCCTTCGAG 120
CTCTCGAAGATTACCGCATC TATTTTTTTT TTCTTTTTTTTCTTTTCCTAGCGCAGATAA 180
AGTGAGCCCGGAAAGGGAAG GAGGGGGCGG GGACACCATTGCCCTGAAAGAATAAATAAG 240
TAAATAAACAAACTGGCTCC TCGCCGCAGC TGGACGCGGTCGGTTGAGTCCAGGTTGGGT 300
CGGACCTGAACCCCTAAAAG CGGAACCGCC TCCCGCCCTCGCCATCCCGGAGCTGAGTCG 360
CCGGCGGCGGTGGCTGCTGC CAGACCCGGA GTTTCCTCTTTCACTGGATGGAGCTGAACT 420
TTGGGCGGCCAGAGCAGCAC AGCTGTCCGG GGATCGCTGCACGCTGAGCTCCCTCGGCAA 480
GACCCAGCGGCGGCTCGGGA TTTTTTTGGG GGGGCGGGGACCAGCCCCGCGCCGGCACC 539
ATG TTC GCG ACC CTG TAC TTC GCG CTC TTG TTG CTC 587
CTG CTG CCG GAC
Met Phe Ala Thr Leu Tyr Phe Ala Leu Leu Leu Leu
Leu Leu Pro Asp
1 5 10 15
CTG TCG GAA GTG AGC GGC GGA GAC GAT TGC AAA GCC 635
GCC CGC CTG GTG
Leu Ser Glu Val Ser Gly Gly Asp Asp Cys Lys Ala
Ala Arg Leu Val
CA 02291608 1999-11-26
135
20 25 30
AGT CAG TGCCTGAAG CAGAGC AGCACC AAGTAC ACG 683
GAT GAG TGC CGC
Ser Gln CysLeuLys GluGlnSer SerThr LysTyr Thr
Asp Cys Arg
35 40 45
CTA CAG TGCGTGGCG GGCAAGGAG ACCAACTTC AGCCTG TCC 731
AGG GCA
Leu ArgGln CysValAla GlyLysGlu ThrAsnPhe SerLeu AlaSer
50 55 60
GGC CTGGAG GCCAAGGAT GAGTGCCGC AGCGCCATG GAGGCC CTGAAG 779
Gly LeuGlu AlaLysAsp GluCysArg SerAlaMet GluAla LeuLys
65 70 75 80
CAG AAGTCG CTCTACAAC TGCCGCTGC AAGCGGGGT ATGAAG AAGGAG 827
Gln LysSer LeuTyrAsn CysArgCys LysArgGly MetLys LysGlu
85 90 95
AAG AACTGC CTGCGCATT TACTGGAGC ATGTACCAG AGCCTG CAGGGA 875
Lys AsnCys LeuArgIle TyrTrpSer MetTyrGln SerLeu GlnGly
100 105 110
AAT GATCTG CTGGAGGAT TCCCCATAT GAACCAGTT AACAGC AGATTG 923
Asn AspLeu LeuGluAsp SerProTyr GluProVal AsnSer ArgLeu
115 120 125
TCA GATATA TTCCGGGTG GTCCCATTC ATATCAGAT GTTTTT CAGCAA 971
Ser AspIle PheArgVal ValProPhe IleSerAsp ValPhe GlnGln
130 135 140
GTG GAGCAC ATTCCCAAA GGGAACAAC TGCCTGGAT GCAGCG AAGGCC 1019
Val GluHis IleProLys GlyAsnAsn CysLeuAsp AlaAla LysAla
145 150 155 160
TGC AACCTC GACGACATT TGCAAGAAG TACAGGTCG GCGTAC ATCACC 1067
Cys AsnLeu AspAspIle CysLysLys TyrArgSer AlaTyr IleThr
165 170 175
CCG TGCACC ACCAGCGTG TCCAACGAT GTCTGCAAC CGCCGC AAGTGC 1115
Pro CysThr ThrSerVal SerAsnAsp ValCysAsn ArgArg LysCys
180 185 190
CAC AAGGCC CTCCGGCAG TTCTTTGAC AAGGTCCCG GCCAAG CACAGC 1163
His LysAla LeuArgGln PhePheAsp LysValPro AlaLys HisSer
195 200 205
TAC GGAATG CTCTTCTGC TCCTGCCGG GACATCGCC TGCACA GAGCGG 1211
Tyr GlyMet LeuPheCys SerCysArg AspIleAla CysThr GluArg
210 215 220
AGG CGACAG ACCATCGTG CCTGTGTGC TCCTATGAA GAGAGG GAGAAG 1259
Arg ArgGln ThrIleVal ProValCys SerTyrGlu GluArg GluLys
225 230 235 240
CCC AACTGT TTGAATTTG CAGGACTCC TGCAAGACG AATTAC ATCTGC 1307
Pro AsnCys LeuAsnLeu GlnAspSer CysLysThr Tyr IleCys
Asn
245 250 255
AGA TCTCGC CTTGCGGAT TTTTTTACC AACTGCCAG CCAGAG TCAAGG 1355
Arg SerArg LeuAla PhePheThr AsnCysGln ProGlu SerArg
Asp
260 265 270
TCT GTC TGT AAC TAC GAC C
AGC CTA GCT TGC
AGC AAG
GAA
TC CTCGCC 1403
CA 02291608 1999-11-26
136
Ser ValSerSer CysLeu LysGluAsn TyrAlaAsp CysLeuLeu Ala
275 280 285
TAC TCGGGGCTT ATTGGC ACAGTCATG ACCCCCAAC TACATAGAC TCC
1451
Tyr SerGlyLeu IleGly ThrValMet ThrProAsn TyrIleAsp Ser
290 295 300
AGT AGCCTCAGT GTGGCC CCATGGTGT GACTGCAGC AACAGTGGG AAC 1499
Ser SerLeuSer ValAla ProTrpCys AspCysSer AsnSerGly Asn
305 310 315 320
GAC CTAGAAGAG TGCTTG AAATTTTTG AATTTCTTC AAGGACAAT ACA 1547
Asp LeuGluGlu CysLeu LysPheLeu AsnPhePhe LysAspAsn Thr
325 330 335
TGT CTTAAAAAT GCAATT CAAGCCTTT GGCAATGGC TCCGATGTG ACC 1595
Cys LeuLysAsn AlaIle GlnAlaPhe G1yAsnGly SerAspVal Thr
340 345 350
GTG TGGCAGCCA GCCTTC CCAGTACAG ACCACCACT GCCACTACC ACC 1643
Val TrpGlnPro AlaPhe ProValGln ThrThrThr AlaThrThr Thr
355 360 365
ACT GCCCTCCGG GTTAAG AACAAGCCC CTGGGGCCA GCAGGGTCT GAG
1691
Thr AlaLeuArg ValLys AsnLysPro LeuGlyPro AlaGlySer Glu
370 375 380
AAT GAAATTCCC ACTCAT GTTTTGCCA CCGTGTGCA AATTTACAG GCA 1739
Asn GluIlePro ThrHis ValLeuPro ProCysAla AsnLeuGln Ala
385 390 395 400
CAG AAGCTGAAA TCCAAT GTGTCGGGC AATACACAC CTCTGTATT TCC
1787
Gln LysLeuLys SerAsn ValSerGly AsnThrHis LeuCysIle Ser
405 410 415
AAT GGTAATTAT GAAAAA GAAGGTCTC GGTGCTTCC AGCCACATA ACC 1835
Asn GlyAsnTyr GluLys GluGlyLeu GlyAlaSer SerHisIle Thr
420 425 430
ACA AAATCAATG GCTGCT CCTCCAAGC TGTGGTCTG AGCCCACTG CTG 1883
Thr LysSerMet AlaAla ProProSer CysGlyLeu SerProLeu Leu
435 440 445
GTC CTGGTGGTA ACCGCT CTGTCCACC CTATTATCT TTAACAGAA 1928
Val LeuValVal ThrAla LeuSerThr LeuLeuSer LeuThrGlu
450 455 460
A
1929
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 463 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
Met Phe Leu Ala Thr Leu Tyr Phe Ala Leu Pro Leu Leu Asp Leu Leu
1 5 10 15
CA 02291608 1999-11-26
137
Leu Ser Ala Glu Val Ser Gly Gly Asp Arg Leu Asp Cys Val Lys Ala
20 25 30
Ser Asp Gln Cys Leu Lys Glu Gln Ser Cys Ser Thr Lys Tyr Arg Thr
35 40 45
Leu Arg Gln Cys Val Ala Gly Lys Glu Thr Asn Phe Ser Leu Ala Ser
50 55 60
Gly Leu Glu Ala Lys Asp Glu Cys Arg Ser Ala Met Glu Ala Leu Lys
65 70 75 80
Gln Lys Ser Leu Tyr Asn Cys Arg Cys Lys Arg Gly Met Lys Lys Glu
85 90 95
Lys Asn Cys Leu Arg Ile Tyr Trp Ser Met Tyr Gln Ser Leu Gln Gly
100 105 110
Asn Asp Leu Leu Glu Asp Ser Pro Tyr Glu Pro Val Asn Ser Arg Leu
115 120 125
Ser Asp Ile Phe Arg Val Val Pro Phe Ile Ser Asp Val Phe Gln Gln
130 135 140
Val Glu His Ile Pro Lys Gly Asn Asn Cys Leu Asp Ala Ala Lys Ala
145 150 155 160
Cys Asn Leu Asp Asp Ile Cys Lys Lys Tyr Arg Ser Ala Tyr Ile Thr
165 170 175
Pro Cys Thr Thr Ser Val Ser Asn Asp Val Cys Asn Arg Arg Lys Cys
180 185 190
His Lys Ala Leu Arg Gln Phe Phe Asp Lys Val Pro Ala Lys His Ser
195 200 205
Tyr Gly Met Leu Phe Cys Ser Cys Arg Asp Ile Ala Cys Thr Glu Arg
210 215 220
Arg Arg Gln Thr Ile Val Pro Val Cys Ser Tyr Glu Glu Arg Glu Lys
225 230 235 240
Pro Asn Cys Leu Asn Leu Gln Asp Ser Cys Lys Thr Asn Tyr Ile Cys
245 250 255
Arg Ser Arg Leu Ala Asp Phe Phe Thr Asn Cys Gln Pro Glu Ser Arg
260 265 270
Ser Val Ser Ser Cys Leu Lys Glu Asn Tyr Ala Asp Cys Leu Leu Ala
275 280 285
Tyr Ser Gly Leu Ile Gly Thr Val Met Thr Pro Asn Tyr Ile Asp Ser
290 295 300
Ser Ser Leu Ser Val Ala Pro Trp Cys Asp Cys Ser Asn Ser Gly Asn
305 310 315 320
Asp Leu Glu Glu Cys Leu Lys Phe Leu Asn Phe Phe Lys Asp Asn Thr
325 330 335
Cys Leu Lys Asn Ala Ile Gln Ala Phe Gly Asn Gly Ser Asp Val Thr
340 345 350
CA 02291608 1999-11-26
138
Val Trp Gln Pro Ala Phe Pro Val Gln Thr Thr Thr Ala Thr Thr Thr
355 360 365
Thr Ala Leu Arg Val Lys Asn Lys Pro Leu Gly Pro Ala Gly Ser Glu
370 375 380
Asn Glu Ile Pro Thr His Val Leu Pro Pro Cys Ala Asn Leu Gln Ala
385 390 395
400
Gln Lys Leu Lys Ser Asn Val Ser Gly Asn Thr His Leu Cys Ile Ser
405 410 415
Asn Gly Asn Tyr Glu Lys Glu Gly Leu Gly Ala Ser Ser His Ile Thr
420 425 430
Thr Lys Ser Met Ala Ala Pro Pro Ser Cys Gly Leu Ser Pro Leu Leu
435 440 445
Val Leu Val Val Thr Ala Leu Ser Thr Leu Leu Ser Leu Thr Glu
450 455 460
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 699 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..699
(D) OTHER INFORMATION: /note= "1 to 699 is 814 to 1512 of
Figure 5 Hsgr-29a"
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..697
(xi)SEQUENCE SEQID
DESCRIPTION: N0:13:
G CC CC AT TC
TCG A AGC GAT
GCG GTG G
TAC TCC
ATC A
ACC
CCG
TGC
A
46
Ser hr hr er al
Ala T S Val
Tyr Ser
Ile Asn
Thr Asp
Pro V
Cys
T
1 5 10 15
TGCAAC CGCCGCAAG TGCCAC AAGGCCCTC CGGCAGTTC TTTGACAAG 94
CysAsn ArgArgLys CysHis LysAlaLeu ArgGlnPhe PheAspLys
20 25 30
GTCCCG GCCAAGCAC AGCTAC GGAATGCTC TTCTGCTCC TGCCGGGAC 142
ValPro AlaLysHis SerTyr GlyMetLeu PheCysSer CysArgAsp
35 40 45
ATCGCC TGCACAGAG CGGAGG CGACAGACC ATCGTGCCT GTGTGCTCC 190
IleAla CysThrGlu ArgArg ArgGlnThr IleValPro ValCysSer
50 55 60
TATGAA GAGAGGGAG AAGCCC AACTGTTTG AATTTGCAG GACTCCTGC
238
TyrGlu GluArgGlu LysPro AsnCysLeu AsnLeuGln AspSerCys
CA 02291608 1999-11-26
139
65 70 75
AAGACG AATTAC ATCTGC AGATCTCGC CTTGCGGAT TTTTTTACC AAC
286
LysThr AsnTyr IleCys ArgSerArg LeuAlaAsp PhePheThr Asn
80 85 90 95
TGCCAG CCAGAG TCAAGG TCTGTCAGC AGCTGTCTA AAGGAAAAC TAC
334
CysGln ProGlu SerArg SerValSer SerCysLeu LysGluAsn Tyr
100 105 110
GCTGAC TGCCTC CTCGCC TACTCGGGG CTTATTGGC ACAGTCATG ACC 382
AlaAsp CysLeu LeuAla TyrSerGly LeuIleGly ThrValMet Thr
115 120 125
CCCAAC TACATA GACTCC AGTAGCCTC AGTGTGGCC CCATGGTGT GAC
430
ProAsn TyrIle AspSer SerSerLeu SerValAla ProTrpCys Asp
130 135 140
TGCAGC AACAGT GGGAAC GACCTAGAA GAGTGCTTG AAATTTTTG AAT
478
CysSer AsnSer GlyAsn AspLeuGlu GluCysLeu LysPheLeu Asn
145 150 155
TTCTTC AAGGAC AATACA TGTCTTAAA AATGCAATT CAAGCCTTT GGC
526
PhePhe LysAsp AsnThr CysLeuLys AsnAlaIle GlnAlaPhe Gly
160 165 170
175
AATGGC TCCGAT GTGACC GTGTGGCAG CCAGCCTTC CCAGTACAG ACC
574
AsnGly SerAsp ValThr ValTrpGln ProAlaPhe ProValGln Thr
180 185 190
ACCACT GCCGCT ACCACC ACTGCCCTC CGGGTTAAG AACAAGCCC CTG 622
ThrThr AlaAla ThrThr ThrAlaLeu ArgValLys AsnLysPro Leu
195 200 205
GGGCCA GCAGGG TCTGAG AATGAAATT CCCACTCAT GTTTTGCCA CCG 670
GlyPro AlaGly SerGlu AsnGluIle ProThrHis ValLeuPro Pro
210 215 220
TGTGCA AATTTA CAGGCA CAGAAGCTG AA
699
CysAla AsnLeu GlnAla GlnLysLeu
225 230
(2)INFORMATION FORSEQ ID :
N0:14
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 232 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
Ser Ala Tyr Ile Thr Pro Cys Thr Thr Ser Val Ser Asn Asp Val Cys
1 5 10 15
Asn Arg Arg Lys Cys His Lys Ala Leu Arg Gln Phe Phe Asp Lys Val
20 25 30
Pro Ala Lys His Ser Tyr Gly Met Leu Phe Cys Ser Cys Arg Asp Ile
35 40 45
CA 02291608 1999-11-26
140
Ala Cys Thr Glu Arg Arg Arg Gln Thr Ile Val Pro Val Cys Ser Tyr
SO 55 60
Glu Glu Arg Glu Lys Pro Asn Cys Leu Asn Leu Gln Asp Ser Cys Lys
65 70 75 80
Thr Asn Tyr Ile Cys Arg Ser Arg Leu Ala Asp Phe Phe Thr Asn Cys
85 90 95
Gln Pro Glu Ser Arg Ser Val Ser Ser Cys Leu Lys Glu Asn Tyr Ala
100 105 110
Asp Cys Leu Leu A1a Tyr Ser Gly Leu Ile Gly Thr Val Met Thr Pro
115 120 125
Asn Tyr Ile Asp Ser Ser Ser Leu Ser Val Ala Pro Trp Cys Asp Cys
130 135 140
Ser Asn Ser Gly Asn Asp Leu Glu Glu Cys Leu Lys Phe Leu Asn Phe
145 150 155
160
Phe Lys Asp Asn Thr Cys Leu Lys Asn Ala Ile Gln Ala Phe Gly Asn
165 170 175
Gly Ser Asp Val Thr Val Trp Gln Pro Ala Phe Pro Val Gln Thr Thr
180 185 190
Thr Ala Ala Thr Thr Thr Ala Leu Arg Val Lys Asn Lys Pro Leu Gly
195 200 205
Pro Ala Gly Ser Glu Asn Glu Ile Pro Thr His Val Leu Pro Pro Cys
210 215 220
Ala Asn Leu Gln Ala Gln Lys Leu
225 230
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2157 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..886
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..2157
(D) OTHER INFORMATION: /note= "1 to 2157 is 814 to 2971 of
Figure 5 29brc"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
G TCG GCG TAC ATC ACC CCG TGC ACC ACC AGC GTG TCC AAT GAT GTC 46
Ser Ala Tyr Ile Thr Pro Cys Thr Thr Ser Val Ser Asn Asp Val
1 5 10 15
CA 02291608 1999-11-26
141
TGC CGC AAG CAC GCC CTCCGGCAG TTT AAG
AAC CGC TGC AAG TTC GAC
94
Cys AsnArg LysCys HisLysAla LeuArgGln Phe AspLys
Arg Phe
20 25 30
GTC CCGGCCAAG CACAGC TACGGAATG CTCTTCTGC TCCTGC CGGGAC
142
Val ProAlaLys HisSer TyrGlyMet LeuPheCys SerCys ArgAsp
35 40 45
ATC GCCTGCACA GAGCGG AGGCGACAG ACCATCGTG CCTGTG TGCTCC
190
Ile AlaCysThr GluArg ArgArgGln ThrIleVal ProVal CysSer
50 55 60
TAT GAAGAGAGG GAGAAG CCCAACTGT TTGAATTTG CAGGAC TCCTGC
238
Tyr GluGluArg GluLys ProAsnCys LeuAsnLeu GlnAsp SerCys
65 70 75
AAG ACGAATTAC ATCTGC AGATCTCGC CTTGCGGAT TTTTTT ACCAAC 286
Lys ThrAsnTyr IleCys ArgSerArg LeuAlaAsp PhePhe ThrAsn
80 85 90 95
TGC CAGCCAGAG TCAAGG TCTGTCAGC AGCTGTCTA AAGGAA AACTAC 334
Cys GlnProGlu SerArg SerValSer SerCysLeu LysGlu AsnTyr
100 105 110
GCT GACTGCCTC CTCGCC TACTCGGGG CTTATTGGC ACAGTC ATGACC 382
Ala AspCysLeu LeuAla TyrSerGly LeuIleGly ThrVal MetThr
115 120 125
CCC AACTACATA GACTCC AGTAGCCTC AGTGTGGCC CCATGG TGTGAC
430
Pro AsnTyrIle AspSer SerSerLeu SerValAla ProTrp CysAsp
130 135 140
TGC AGCAACAGT GGGAAC GACCTAGAA GAGTGCTTG AAATTT TTGAAT
47g
Cys SerAsnSer GlyAsn AspLeuGlu GluCysLeu LysPhe LeuAsn
145 150 155
TTC TTCAAGGAC AATACA TGTCTTAAA AATGCAATT CAAGCC TTTGGC 526
Phe PheLysAsp AsnThr CysLeuLys AsnAlaIle GlnAla PheGly
160 165 170 175
AAT GGCTCCGAT GTGACC GTGTGGCAG CCAGCCTTC CCAGTA CAGACC 574
Asn GlySerAsp ValThr ValTrpGln ProAlaPhe ProVal GlnThr
180 185 190
ACC ACTGCCGCT ACCACC ACTGCCCTC CGGGTTAAG AACAAG CCCCTG
622
Thr ThrAlaAla ThrThr ThrAlaLeu ArgValLys AsnLys ProLeu
195 200 205
GGG CCAGCAGGG TCTGAG AATGAAATT CCCACTCAT GTTTTG CCACCG 670
Gly ProAlaGly SerGlu AsnGluIle ProThrHis ValLeu ProPro
210 215 220
TGT GCAAATTTA CAGGCA CAGAAGCTG AAATCCAAT GTGTCG GGCAAT 718
Cys AlaAsnLeu GlnAla GlnLysLeu LysSerAsn ValSer GlyAsn
225 230 235
ACA CACCTCTGT ATTTCC AATGGTAAT TATGAAAAA GAAGGT CTCGGT
766
Thr HisLeuCys IleSer AsnGly Tyr Lys GluGly LeuGly
Asn Glu
240 245 250 255
GCT TCC CAC ACC AAA ATG GCT CCTCCA AGC 814
AGC ATA ACA TCA GCT TGT
Ala SerSerHis IleThr ThrLys Met ProPro Ser
Ser Ala Cys
Ala
260 265 270
CA 02291608 1999-11-26
142
GGT CTG AGC CCA CTG CTG GTC CTG GTG GTA ACC GCT CTG TCC ACC CTA 862
Gly Leu Ser Pro Leu Leu Val Leu Val Val Thr Ala Leu Ser Thr Leu
275 280 285
TTA TCT CTGCATTAAA ATGGACATGT916
TTA ACA AAAATACAAT
GAA ACA
TCA TAG
Leu Ser
Leu Thr
Glu Thr
Ser
290
AAAAAGACAA ATCTGTTTCCTGTTCTCTTGTATAGCTGAA 976
AAACCAAGTT ATTCCAGTTT
AGGAGCTCAGTTGAGAAACAGTTCCATTCAACTGGAACATTTTTTTTTTTCCTTTTAAGA1036
AAGCTTCTTGTGATCCTTCGGGGCTTCTGTGAAAAACCTGATGCAGTGCTCCATCCAAAC1096
TCAGAAGGCTTTGGGATATGCTGTATTTTAAAGGGACAGTTTGTAACTTGGGCTGTAAAG1156
CAAACTGGGGCTGTGTTTTCGATGATGATGATCATCATGATCATGATNNNNNNNNNNNNN1216
NI'INNNNNNNNnfL~7VrfNNNNNNNNNNNNGATTTTAACAGTTTTACTTCTGGCCTTTCCTAGC1276
TAGAGAAGGAGTTAATATTTCTAAGGTAACTCCCATATCTCCTTTAATGACATTGATTTC1336
TAATGATATAAATTTCAGCCTACATTGATGCCAAGCTTTTTTGCCACAAAGAAGATTCTT1396
ACCAAGAGTGGGCTTTGTGGAAACAGCTGGTACTGATGTTCACCTTTATATATGTACTAG1456
CATTTTCCACGCTGATGTTTATGTACTGTAAACAGTTCTGCACTCTTGTACAAAAGAAAA1516
AACACCTGTCACATCCAAATATAGTATCTGTCTTTTCGTCAAAATAGAGAGTGGGGAATG1576
AGTGTGCCGATTCAATACCTCAATCCCTGAACGACACTCTCCTAATCCTAAGCCTTACCT1636
GAGTGAGAAGCCCTTTACCTAACAAAAGTCCAATATAGCTGAAATGTCGCTCTAATACTC1696
TTTACACATATGAGGTTATATGTAGAAAAAAATTTTACTACTAAATGATTTCAACTATTG1756
GCTTTCTATATTTTGAAAGTAATGATATTGTCTCATTTTTTTACTGATGGTTTAATACAA1816
AATACACAGAGCTTGTTTCCCCTCATAAGTAGTGTTCGCTCTGATATGAACTTCACAAAT1876
ACAGCTCATCAAAAGCAGACTCTGAGAAGCCTCGTGCTGTAGCAGAAAGTTCTGCATCAT1936
GTGACTGTGGACAGGCAGGAGGAAACAGAACAGACAAGCATTGTCTTTTGTCATTGCTCG1996
AAGTGCAAGCGTGCATACCTGTGGAGGGAACTGGTGGCTGCTTGTAAATGTTCTGCAGCA2056
TCTCTTGACACACTTGTCATGACACAATCCAGTACCTTGGTTTTCAGGTTATCTGACAAA2116
GGCAGCTTTGATTGGGACATGGAGGCATGGGCAGGCCGGAA 2157
(2) INFORMATION
FOR SEQ
ID N0:16:
(i) SEQUENCE CS:
CHARACTERISTI
(A) LENGTH: o acids
294 amin
(B) TYPE: amino
acid
(D) TOPOLOGY:
linear
(ii) MOLECULE
TYPE:
protein
(xi) SEQUENCE SEQ ID
DESCRIPTION: N0:16:
Ser Ala Ile Thr Val Cys
Tyr Pro Cys
Thr Thr
Ser Val
Ser Asn
Asp
CA 02291608 1999-11-26
143
1 5 10
Asn Arg Arg Lys Cys His Lys Ala Leu Arg Gln Phe Phe Asp Lys Val
25 30
Pro Ala Lys His Ser Tyr Gly Met Leu Phe Cys Ser Cys Arg Asp Ile
35 40 45
Ala Cys Thr Glu Arg Arg Arg Gln Thr Ile Val Pro Val Cys Ser Tyr
50 55 60
Glu Glu Arg Glu Lys Pro Asn Cys Leu Asn Leu Gln Asp Ser Cys Lys
65 70 75 80
Thr Asn Tyr Ile Cys Arg Ser Arg Leu Ala Asp Phe Phe Thr Asn Cys
85 90 95
Gln Pro Glu Ser Arg Ser Val Ser Ser Cys Leu Lys Glu Asn Tyr Ala
100 105 110
Asp Cys Leu Leu Ala Tyr Ser Gly Leu Ile Gly Thr Val Met Thr Pro
115 120 125
Asn Tyr Ile Asp Ser Ser Ser Leu Ser Val Ala Pro Trp Cys Asp Cys
130 135 140
Ser Asn Ser Gly Asn Asp Leu Glu Glu Cys Leu Lys Phe Leu Asn Phe
145 150 155
160
Phe Lys Asp Asn Thr Cys Leu Lys Asn Ala Ile Gln Ala Phe Gly Asn
165 170 175
Gly Ser Asp Val Thr Val Trp Gln Pro Ala Phe Pro Val Gln Thr Thr
180 185 190
Thr Ala Ala Thr Thr Thr Ala Leu Arg Val Lys Asn Lys Pro Leu Gly
195 200 205
Pro Ala Gly Ser Glu Asn Glu Ile Pro Thr His Val Leu Pro Pro Cys
210 215 220
Ala Asn Leu Gln Ala Gln Lys Leu Lys Ser Asn Val Ser Gly Asn Thr
225 230 235
240
His Leu Cys Ile Ser Asn Gly Asn Tyr Glu Lys Glu Gly Leu Gly Ala
245 250 255
Ser Ser His I1e Thr Thr Lys Ser Met Ala Ala Pro Pro Ser Cys Gly
260 265 270
Leu Ser Pro Leu Leu Val Leu Val Val Thr Ala Leu Ser Thr Leu Leu
275 280 285
Ser Leu Thr Glu Thr Ser
290
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 659 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 02291608 1999-11-26
144
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..658
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..659
(D) OTHER INFORMATION: /note= "1 to 659 is 1033 to 1691 of
Figure 5 Hsgr-2lar"
(xi) SEQID
SEQUENCE N0:17:
DESCRIPTION:
G AG CC TC CT
AAT GAC TGC TGC CGC
TTG T AAG AGA
C ACG T
AAT
TAC
A
46
A sn 1n er ys sn yr 1e ys er
Leu Asp Cys Thr T I C Arg Arg
G S L A S
1 5 10 15
CTTGCG GATTTT ACCAAC TGCCAGCCA GAGTCA AGGTCTGTC AGC
TTT
94
LeuAla AspPhePhe ThrAsn CysGlnPro GluSer ArgSerVal Ser
20 25 30
AGCTGT CTAAAGGAA AACTAC GCTGACTGC CTCCTC GCCTACTCG GGG
142
SerCys LeuLysGlu AsnTyr AlaAspCys LeuLeu AlaTyrSer Gly
35 40 45
CTTATT GGCACAGTC ATGACC CCCAACTAC ATAGAC TCCAGTAGC CTC
190
LeuIle GlyThrVal MetThr ProAsnTyr IleAsp SerSerSer Leu
50 55 60
AGTGTG GCCCCATGG TGTGAC TGCAGCAAC AGTGGG AACGACCTA GAA 238
SerVal AlaProTrp CysAsp CysSerAsn SerGly AsnAspLeu Glu
65 70 75
GAGTGC TTGAAATTT TTGAAT TTCTTCAAG GACAAT ACATGTCTT AAA 286
GluCys LeuLysPhe LeuAsn PhePheLys AspAsn ThrCysLeu Lys
80 85 90 95
AATGCA ATTCAAGCC TTTGGC AATGGCTCC GATGTG ACCGTGTGG CAG
334
AsnAla IleGlnAla PheGly AsnGlySer AspVal ThrValTrp Gln
100 105 110
CCAGCC TTCCCAGTA CAGACC ACCACTGCC ACTACC ACCACTGCC CTC
382
ProAla PheProVal GlnThr ThrThrAla ThrThr ThrThrAla Leu
115 120 125
CGGGTT AAGAACAAG CCCCTG GGGCCAGCA GGGTCT GAGAATGAA ATT
430
ArgVal LysAsnLys ProLeu GlyProAla GlySer GluAsnGlu Ile
130 135 140
CCCACT CATGTTTTG CCACCG TGTGCAAAT TTACAG GCACAGAAG CTG
47g
ProThr HisValLeu ProPro CysAlaAsn LeuGln AlaGlnL Leu
s
145 150 y
155
AAATCC AATGTGTCG GGCAAT ACACACCTC TGTATT TCCAATGGT AAT
526
LysSer AsnValSer GlyAsn ThrHisLeu CysIle SerAsnGl Asn
160 165 y
170
175
TATGAA AAAGAAGGT CTCGGT GCTTCCAGC CAC ACCACAAAA TCA
ATA
574
TyrGlu LysGluGly LeuGly AlaSerSer HisIle ThrThrLys Ser
180 185 190
CA 02291608 1999-11-26
145
ATG GCT GCT CCT CCA AGC TGT GGT CTG AGC CCA CTG CTG GTC CTG GTG 622
Met Ala Ala Pro Pro Ser Cys Gly Leu Ser Pro Leu Leu Val Leu Val
195 200 205
GTA ACC GCT CTG TCC ACC CTA TTA TCT TTA ACA GAA A 659
Val Thr Ala Leu Ser Thr Leu Leu Ser Leu Thr Glu
210 215
(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 219 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
Asn Leu Gln Asp Ser Cys Lys Thr Asn Tyr Ile Cys Arg Ser Arg Leu
1 5 10 15
Ala Asp Phe Phe Thr Asn Cys Gln Pro Glu Ser Arg Ser Val Ser Ser
20 25 30
Cys Leu Lys Glu Asn Tyr Ala Asp Cys Leu Leu Ala Tyr Ser Gly Leu
35 40 45
Ile Gly Thr Val Met Thr Pro Asn Tyr Ile Asp Ser Ser Ser Leu Ser
50 55 60
Val Ala Pro Trp Cys Asp Cys Ser Asn Ser Gly Asn Asp Leu Glu Glu
65 70 75 80
Cys Leu Lys Phe Leu Asn Phe Phe Lys Asp Asn Thr Cys Leu Lys Asn
85 90 95
Ala Ile Gln Ala Phe Gly Asn Gly Ser Asp Val Thr Val Trp Gln Pro
100 105 110
Ala Phe Pro Val Gln Thr Thr Thr Ala Thr Thr Thr Thr Ala Leu Arg
115 120 125
Val Lys Asn Lys Pro Leu Gly Pro Ala Gly Ser Glu Asn Glu Ile Pro
130 135 140
Thr His Val Leu Pro Pro Cys Ala Asn Leu Gln Ala Gln Lys Leu Lys
145 150 155 160
Ser Asn Val Ser Gly Asn Thr His Leu Cys Ile Ser Asn Gly Asn Tyr
165 170 175
Glu Lys Glu Gly Leu Gly Ala Ser Ser His Ile Thr Thr Lys Ser Met
180 185 190
Ala Ala Pro Pro Ser Cys Gly Leu Ser Pro Leu Leu Val Leu Val Val
195 200 205
Thr Ala Leu Ser Thr Leu Leu Ser Leu Thr Glu
210 215
(2) INFORMATION FOR SEQ ID N0:19:
CA 02291608 1999-11-26
146
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 630 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..629
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..630
(D) OTHER INFORMATION: /note= "1 to 630 is 1062 to 1691 of
Figure 5 Hsgr-2lbr"
{xi)SEQUENCE SEQID
DESCRIPTION: N0:19:
AC TCT CTT TTT 47
ATC CGC GCG TTT
TGC GAT ACC
AGA AAC
TGC
CAG
CCA
Ile Ser Leu Phe Phe Cys Pro
Cys Arg Ala Thr Gln
Arg Asp Asn
1 5 10 15
GAGTCAAGG TCTGTCAGCAGC TGTCTAAAG GAA AACTACGCT GACTGC 95
GluSerArg SerValSerSer CysLeuLys Glu AsnTyrAla AspCys
20 25 30
CTCCTCGCC TACTCGGGGCTT ATTGGCACA GTC ATGACCCCC AACTAC 143
LeuLeuAla TyrSerGlyLeu IleGlyThr Val MetThrPro AsnTyr
35 40 45
ATAGACTCC AGTAGCCTCAGT GTGGCCCCA TGG TGTGACTGC AGCAAC 191
IleAspSer SerSerLeuSer ValAlaPro Trp CysAspCys SerAsn
50 55 60
AGTGGGAAC GACCTAGAAGAG TGCTTGAAA TTT TTGAATTTC TTCAAG 239
SerGlyAsn AspLeuGluGlu CysLeuLys Phe LeuAsnPhe PheLys
65 70 75
GACAATACA TGTCTTAAAAAT GCAATTCAA GCC TTTGGCAAT GGCTCC 287
AspAsnThr CysLeuLysAsn AlaIleGln Ala PheGlyAsn GlySer
80 85 90 95
GATGTGACC GTGTGGCAGCCA GCCTTCCCA GTA CAGACCACC ACTGCC 335
AspValThr ValTrpGlnPro AlaPhePro Val GlnThrThr ThrAla
100 105 110
ACTACCACC ACTGCCCTCCGG GTTAAGAAC AAG CCCCTGGGG CCAGCA 383
ThrThrThr ThrAlaLeuArg ValLysAsn Lys ProLeuGly ProAla
115 120 125
GGGTCTGAG AATGAAATTCCC ACTCATGTT TTG CCACCGTGT GCAAAT 431
GlySerGlu AsnGluIlePro ThrHisVal Leu ProProCys AlaAsn
130 135 140
TTACAGGCA CAGAAGCTGAAA TCCAATGTG TCG GGCAATACA CACCTC 479
LeuGlnAla GlnLysLeuLys SerAsnVal Ser GlyAsnThr HisLeu
145 150 155
TGTATTTCC AATGGTAATTAT GAAAAAGAA GGT CTCGGTGCT TCCAGC 527
CA 02291608 1999-11-26
147
Cys Ile Ser Asn Gly Asn Tyr Glu Lys Glu Gly Leu Gly Ala Ser Ser
160 165 170 175
CAC ATA ACC ACA AAA TCA ATG GCT GCT CCT CCA AGC TGT GGT CTG AGC 575
His Ile Thr Thr Lys Ser Met Ala Ala Pro Pro Ser Cys Gly Leu Ser
180 185 190
CCA CTG CTG GTC CTG GTG GTA ACC GCT CTG TCC ACC CTA TTA TCT TTA 623
Pro Leu Leu Val Leu Val Val Thr Ala Leu Ser Thr Leu Leu Ser Leu
195 200 205
ACA GAA A 630
Thr Glu
(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 209 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
Ile Cys Arg Ser Arg Leu Ala Asp Phe Phe Thr Asn Cys Gln Pro Glu
1 5 10 15
Ser Arg Ser Val Ser Ser Cys Leu Lys Glu Asn Tyr Ala Asp Cys Leu
20 25 30
Leu Ala Tyr Ser Gly Leu Ile Gly Thr Val Met Thr Pro Asn Tyr Ile
35 40 45
Asp Ser Ser Ser Leu Ser Val Ala Pro Trp Cys Asp Cys Ser Asn Ser
50 55 60
Gly Asn Asp Leu Glu Glu Cys Leu Lys Phe Leu Asn Phe Phe Lys Asp
65 70 75 80
Asn Thr Cys Leu Lys Asn Ala Ile Gln Ala Phe Gly Asn Gly Ser Asp
85 90 95
Val Thr Val Trp Gln Pro Ala Phe Pro Val Gln Thr Thr Thr Ala Thr
100 105 110
Thr Thr Thr Ala Leu Arg Val Lys Asn Lys Pro Leu Gly Pro Ala Gly
115 120 125
Ser Glu Asn Glu Ile Pro Thr His Val Leu Pro Pro Cys Ala Asn Leu
130 135 140
Gln Ala Gln Lys Leu Lys Ser Asn Val Ser Gly Asn Thr His Leu Cys
145 150 155 160
Ile Ser Asn Gly Asn Tyr Glu Lys Glu Gly Leu Gly Ala Ser Ser His
165 170 175
Ile Thr Thr Lys Ser Met Ala Ala Pro Pro Ser Cys Gly Leu Ser Pro
180 185 190
CA 02291608 1999-11-26
148
Leu Leu Val Leu Val Val Thr Ala Leu Ser Thr Leu Leu Ser Leu Thr
195 200 205
Glu
(2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1075 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..445
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..1075
(D) OTHER INFORMATION: /note= "1 to 1075 is 1255 to 2330
of Figure 5 Hsgr-2"
(xi)SEQUENCE SEQ
DESCRIPTION: ID
N0:21:
T TG AA 46
GGG A TTT
AAC TTG
GAC AAT
CTA TTC
GAA TTC
GAG AAG
TGC
T
Gly
Asn
Asp
Leu
Glu
Glu
Cys
Leu
Lys
Phe
Leu
Asn
Phe
Phe
Lys
1 5 10 15
GACAATACA TGTCTTAAA AATGCAATT CAAGCC TTTGGCAAT GGCTCC 94
AspAsnThr CysLeuLys AsnAlaIle GlnAla PheGlyAsn GlySer
20 25 30
GATGTGACC GTGTGGCAG CCAGCCTTC CCAGTA CAGACCACC ACTGCC 142
AspValThr ValTrpGln ProAlaPhe ProVal GlnThrThr ThrAla
35 40 45
ACTACCACC ACTGCCCTC CGGGTTAAG AACAAG CCCCTGGGG CCAGCA 190
ThrThrThr ThrAlaLeu ArgValLys AsnLys ProLeuGly ProAla
50 55 60
GGGTCTGAG AATGAAATT CCCACTCAT GTTTTG CCACCGTGT GCAAAT 238
GlySerGlu AsnGluIle ProThrHis ValLeu ProProCys AlaAsn
65 70 75
TTACAGGCA CAGAAGCTG AAATCCAAT GTGTCG GGCAATACA CACCTC 286
LeuGlnAla GlnLysLeu LysSerAsn ValSer GlyAsnThr HisLeu
80 85 90 95
TGTATTTCC AATGGTAAT TATGAAAAA GAAGGT CTCGGTGCT TCCAGC 334
CysIleSer AsnGlyAsn TyrGluLys GluGly LeuGlyAla SerSer
100 105 110
CACATAACC ACAAAATCA ATGGCTGCT CCTCCA AGCTGTGGT CTGAGC 382
HisIleThr ThrLysSer MetAlaAla ProPro SerCysGly LeuSer
115 120 125
CCACTGCTG GTCCTGGTG GTAACCGCT CTGTCC ACCCTATTA TCTTTA 430
CA 02291608 1999-11-26
149
Pro Leu Leu Val Leu Val Val Thr Ala Leu Ser Thr Leu Leu Ser Leu
130 135 140
ACA GAA TCA TAG CTGCATTAAA AAAATACAAT 485
ACA ATGGACATGT AAAAAGACAA
Thr Glu Ser
Thr
145
AAACCAAGTTATCTGTTTCC TGTTCTCTTG TATAGCTGAAATTCCAGTTTAGGAGCTCAG545
TTGAGAAACAGTTCCATTCA ACTGGAACAT TTTTTTTTTTCCTTTTAAGAAAGCTTCTTG605
TGATCCTTCGGGGCTTCTGT GAAAAACCTG ATGCAGTGCTCCATCCAAACTCAGAAGGCT665
TTGGGATATGCTGTATTTTA AAGGGACAGT TTGTAACTTGGGCTGTAAAGCAAACTGGGG725
CTGTGTTTTCGATGATGATG ATCATCATGA TCATGATNNNnff~7VnfNNNNNNTfl~hlNN~INNNN785
Tf~SN2~ NNNNNNGATT TTAACAGTTT TACTTCTGGCCTTTCCTAGCTAGAGAAGGA845
GTTAATATTTCTAAGGTAAC TCCCATATCT CCTTTAATGACATTGATTTCTAATGATATA905
AATTTCAGCCTACATTGATG CCAAGCTTTT TTGCCACAAAGAAGATTCTTACCAAGAGTG965
GGCTTTGTGGAAACAGCTGG TACTGATGTT CACCTTTATATATGTACTAGCATTTTCCAC1025
GCTGATGTTTATGTACTGTA AACAGTTCTG CACTCTTGTACAAAAGAAAA 1075
(2) INFORMATION
FOR SEQ
ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 147 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
Gly Asn Asp Leu Glu Glu Cys Leu Lys Phe Leu Asn Phe Phe Lys Asp
1 5 10 15
Asn Thr Cys Leu Lys Asn Ala Ile Gln Ala Phe Gly Asn Gly Ser Asp
20 25 30
Val Thr Val Trp Gln Pro Ala Phe Pro Val Gln Thr Thr Thr Ala Thr
35 40 45
Thr Thr Thr Ala Leu Arg Val Lys Asn Lys Pro Leu Gly Pro Ala Gly
50 55 60
Ser Glu Asn Glu Ile Pro Thr His Val Leu Pro Pro Cys Ala Asn Leu
65 70 75 80
Gln Ala Gln Lys Leu Lys Ser Asn Val Ser Gly Asn Thr His Leu Cys
85 90 95
Ile Ser Asn Gly Asn Tyr Glu Lys Glu Gly Leu Gly Ala Ser Ser His
100 105 110
Ile Thr Thr Lys Ser Met Ala Ala Pro Pro Ser Cys Gly Leu Ser Pro
115 120 125
CA 02291608 1999-11-26
I5~
Leu Leu Val Leu Val Val Thr Ala Leu Ser Thr Leu Leu Ser Leu Thr
130 135 140
Glu Thr Ser
145
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1059 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..428
(ix) FEATURE:
(A} NAME/KEY: misc_feature
(B) LOCATION: 1..1059
(D) OTHER INFORMATION: /note= "1 to 1059 is 1272 to 2330
of Figure 5 Hsgr-9"
(xi)SEQUENCE SEQ
DESCRIPTION: ID
N0:23:
AG AA TTT AAT TTC TGT AAA 47
TGC TTG TTC AAG CTT
TTG GAC
A AAT
ACA
Cys Phe Asn Phe Cys Lys
Leu Leu Phe Lys Leu
Lys Asp
Asn
Thr
1 5 10 15
AATGCAATT CAAGCCTTTGGC AATGGC TCCGAT GTGACCGTG TGGCAG 95
AsnAlaIle GlnAlaPheGly AsnGly SerAsp ValThrVal TrpGln
20 25 30
CCAGCCTTC CCAGTACAGACC ACCACT GCCACT ACCACCACT GCCCTC 143
ProAlaPhe ProValGlnThr ThrThr AlaThr ThrThrThr AlaLeu
35 40 45
CGGGTTAAG AACAAGCCCCTG GGGCCA GCAGGG TCTGAGAAT GAAATT 191
ArgValLys AsnLysProLeu GlyPro AlaGly SerGluAsn GluIle
50 55 60
CCCACTCAT GTTTTGCCACCG TGTGCA AATTTA CAGGCACAG AAGCTG 239
ProThrHis ValLeuProPro CysAla AsnLeu GlnAlaGln LysLeu
65 70 75
AAATCCAAT GTGTCGGGCAAT ACACAC CTCTGT ATTTCCAAT GGTAAT 287
LysSerAsn ValSerGlyAsn ThrHis LeuCys IleSerAsn GlyAsn
80 85 90 95
TATGAAAAA GAAGGTCTCGGT GCTTCC AGCCAC ATAACCACA AAATCA 335
TyrGluLys GluGlyLeuGly AlaSer SerHis IleThrThr LysSer
100 105 110
ATGGCTGCT CCTCCAAGCTGT GGTCTG AGCCCA CTGCTGGTC CTGGTG 383
MetAlaAla ProProSerCys GlyLeu SerPro LeuLeuVal LeuVal
115 120 125
GTAACCGCT CTGTCCACCCTA TTATCT TTAACA GAAACATCA TAG 428
CA 02291608 1999-11-26
ISI
Val Thr Ala Leu Ser Thr Leu Leu Ser Leu Thr Glu Thr Ser
130 135 140
CTGCATTAAAAAAATACAATATGGACATGTAAAAAGACAAAAACCAAGTTATCTGTTTCC488
TGTTCTCTTGTATAGCTGAAATTCCAGTTTAGGAGCTCAGTTGAGAAACAGTTCCATTCA548
ACTGGAACATTTTTTTTTTTTCCTTTTAAGAAAGCTTCTTGTGATCCTTTGGGGCTTCTG608
TGAAAAACCTGATGCAGTGCTCCATCCAAACTCAGAAGGCTTTGGGATATGCTGTATTTT668
AAAGGGACAGTTTGTAACTTGGGCTGTAAAGCAAACTGGGGCTGTGTTTTCGATGATGAT728
GATGATCATGATGATGATCATCATGATCATGATGATGATCATCATGATCATGATGATGAT788
TTTAACAGTTTTACTTCTGGCCTTTCCTAGCTAGAGAAGGAGTTAATATTTCTAAGGTAA848
CTCCCATATCTCCTTTAATGACATTGATTTCTAATGATATAAATTTCAGCCTACATTGAT908
GCCAAGCTTTTTTGCCACAAAGAAGATTCTTACCAAGAGTGGGCTTTGTGGAAACAGCTG968
GTACTGATGTTCACCTTTATATATGTACTAGCATTTTCCACGCTGATGTTTATGTACTGT1028
AAACAGTTCTGCACTCTTGTACAAAAGAAAA 1059
(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 141 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
Cys Leu Lys Phe Leu Asn Phe Phe Lys Asp Asn Thr Cys Leu Lys Asn
1 5 10 15
Ala Ile Gln Ala Phe Gly Asn Gly Ser Asp Val Thr Val Trp Gln Pro
20 25 30
Ala Phe Pro Val Gln Thr Thr Thr Ala Thr Thr Thr Thr Ala Leu Arg
35 40 45
Val Lys Asn Lys Pro Leu Gly Pro Ala Gly Ser Glu Asn Glu Ile Pro
50 55 60
Thr His Val Leu Pro Pro Cys Ala Asn Leu Gln Ala Gln Lys Leu Lys
65 70 75 80
Ser Asn Val Ser Gly Asn Thr His Leu Cys Ile Ser Asn Gly Asn Tyr
85 90 95
Glu Lys Glu Gly Leu Gly Ala Ser Ser His Ile Thr Thr Lys Ser Met
100 105 110
Ala Ala Pro Pro Ser Cys Gly Leu Ser Pro Leu Leu Val Leu Val Val
115 120 125
Thr Ala Leu Ser Thr Leu Leu Ser Leu Thr Glu Thr Ser
130 135 140
CA 02291608 1999-11-26
152
(2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
Gln Ser Cys Ser Thr Lys Tyr Arg Thr Leu
1 5 10
(2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCE CHARACTERISTICS:
{A) LENGTH: 10 amino acids
(B) TYPE: amino acid
{C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:
Cys Lys Arg Gly Met Lys Lys Glu Lys Asn
1 5 10
(2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
Leu Leu Glu Asp Ser Pro Tyr Glu Pro Val
1 5 10
(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
{A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
CA 02291608 1999-11-26
153
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
Cys Ser Tyr Glu Glu Arg Glu Arg Pro Asn
1 5 10
(2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
Pro Ala Pro Pro Val Gln Thr Thr Thr Ala Thr Thr Thr Thr
1 5 10
(2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
CTGTTTGAAT TTGCAGGACT C 21
(2} INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
CTCCTCTCTA AGCTTCTAAC CACAGCTTGG AGGAGC 36
(2) INFORMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
CA 02291608 1999-11-26
154
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
CTCCTCTCTA AGCTTCTATG GGCTCAGACC ACAGCTT 37
(2) INFORMATION FOR SEQ ID N0:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:
CTCCTCTCTA AGCTTCTACT TGTCATCGTC GTCCTTGTAG TCACCACAGC TTGGAGGAGC 60
(2) INFORMATION FOR SEQ ID N0:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:34:
CTCCTCTCTA AGCTTCTACT TGTCATCGTC GTCCTTGTAG TCTGGCTCAG ACCACAGCTT 60
(2) INFORMATION FOR SEQ ID N0:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4232 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
CA 02291608 1999-11-26
155
(B) LOCATION: 1587..2978
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:35:
CATGAAGAAACCTCAGTAAGTCTCAGACTTGGCCCAAAGGAGCCCAACTAGTTACTCCCT60
GGTCTGTTACAGAGGATCTGGCTATTACACTCAACAGCAAAAATTCAATTCAATCCCGCT120
AAAGATATAAGAATCACTAGGAAKAATAAGCCAGAACTCAAGACAGAAATAGCATTAAGT180
AGTTCCTTCAGTACAGTGAGCAGAAGCTGGCCACTCTACGACTCTAWAAGACTCAGAAAA240
GCTTACTAGGGACCWCTGGGCATWCCGGTGTCCTATGTGGGGATTTCGTAACGTCTTTGA300
GTCAGAAGCTGCCCTCAAAATAGTTTCTTCTCAAAACGGTTTCAGGCTTTGTTAGAAAGG360
GAAGACTTCACTGCCACTTTACCCAGATCATCTACCCCATCCTTGGAATGAATGGGGAAG420
CTTCAGCCACCCTACCAGGCTCCTAAAATCACCAACTTGAGAGAAAAACTATAACGTTGC480
TCTACCAGTACTTCAGGAGGTTAAAGAAAGTCACAGAAGAAAAGAACTCTGGGGAAAACA540
GTCAAATTCGGCTATTAAGACATTAGTTACAGGCCCCTGTACCTCTCCTCTAGAAACCCT600
GGGAGTACACCCGCAGAGGAGAGAGAGCCCAAGCCACCAAGCAAAGTCAACCAATCTGGC660
AAAGGGGCGTCCCACTGCGGCTTTCAGTCCAAGAAGTGGATCCTGCTGGTTCGCAGTCTC720
TCTTCTATCTCCTCACTTCCTATTTACCCTTTGAAGTGGGTACTGAATAGCCCGTTCCCA780
AGCAGAGGCCCTTTGTATACGGGGTGCTACAGTCGCCTGGTGGAAACACCTTGGCAGAGT840
TGTTTGGTGCCAGGATGGGCCACTGAAGGCATCTGCTGTGGACACACACACACACACACA900
CACACACACACACACACACAGAGAGAGGAGAGAGAAAGACACACGCACGCAGAGACACAC960
GGTCACTGGAATTCCATTAGAAAAAAGTGAGCCGAGCAAGGGTTAGCGGGAGAAGATTTT1020
TTTGAATCTTGTCTTCGTCTTGGTGCGAAAGAAGCGACTCCAGTCTCTCGTCCTCGAAGC1080
TCCGACTGGATTGTTCTTGGGCGCTGACACCCGTCTGTGGATTTCTTTTCTATTTGCATT1140
TTATTCCGACCCCCTCCCTCGCCGCTTCCTTCCAGCCCTTCACTCGCAAATCGCCTCTCT1200
CCCCACCTCCCCAGGCCCCTCCTGGGAAGCGCAGGGGAATTGGACCCGCGGGGACTCACG1260
CCTTCCCGGACGATTGGAGGGGAGGGCTGACCCCAGGACTGGGCTGTTGGCTTAGAAAGC1320
CGATACACAGATACGCGTATATTTGATTGTAGCGGGCAAGGGGGGCGTCGAGAGGCAGCA1380
GCCCATCGCCCGCCTCTCACCCCACCCCCTCCAGCCAGAGGCGAGAATCGCAGGACTCGG1440
GATCTTCATCGGGTGGACTAGCTGGGATCTCCGCATTGGATTTGGGGCTGATTACCACTG1500
CTTGGCTATTATTATTGTTGTTGTTACTACTATTATTTTTTTTTACCCAAGGGAGAAAGA1560
CAAAAAAACGGTGGGATTTATTTAAC ATC TTG 1613
ATG GCA AAC
GTC TTC
TGC CTC
Met Ile Leu
Ala Asn
Val Phe
Cys Leu
1 5
TTC TTC GCC AGC 1661
TTT CTA CCT TCC
GAC GAC TCC
ACC CTC
CGC TCT
TTG
Phe Phe Ala Ser
Phe Leu Pro Ser
Asp Asp Ser
Thr Leu
Arg Ser
Leu
CA 02291608 1999-11-26
156
15 20 25
CTGCAGGGC CCCGAGCTC CACGGC TGGCGCCCC CCAGTGGAC TGTGTC 1709
LeuGlnG1y ProGluLeu HisGly TrpArgPro ProValAsp CysVal
30 35 40
CGGGCCAAT GAGCTGTGT GCCGCC GAATCCAAC TGCAGCTCT CGCTAC 1757
ArgAlaAsn GluLeuCys AlaAla GluSerAsn CysSerSer ArgTyr
45 50 55
CGCACTCTG CGGCAGTGC CTGGCA GGCCGCGAC CGCAACACC ATGCTG 1805
ArgThrLeu ArgGlnCys LeuAla GlyArgAsp ArgAsnThr MetLeu
60 65 70
GCCAACAAG GAGTGCCAG GCGGCC TTGGAGGTC TTGCAGGAG AGCCCG 1853
AlaAsnLys GluCysGln AlaAla LeuGluVal LeuGlnGlu SerPro
75 80 85
CTGTACGAC TGCCGCTGC AAGCGG GGCATGAAG AAGGAGCTG CAGTGT 1901
LeuTyrAsp CysArgCys LysArg GlyMetLys LysGluLeu GlnCys
90 95 100 105
CTGCAGATC TACTGGAGC ATCCAC CTGGGGCTG ACCGAGGGT GAGGAG 1949
LeuGlnIle TyrTrpSer IleHis LeuGlyLeu ThrGluGly GluGlu
110 115 120
TTCTACGAA GCCTCCCCC TATGAG CCGGTGACC TCCCGCCTC TCGGAC 1997
PheTyrGlu AlaSerPro TyrGlu ProValThr SerArgLeu SerAsp
125 130 135
ATCTTCAGG CTTGCTTCA ATCTTC TCAGGGACA GGGGCAGAC CCGGTG 2045
IlePheArg LeuAlaSer IlePhe SerGlyThr GlyAlaAsp ProVal
140 145 150
GTCAGCGCC AAGAGCAAC CATTGC CTGGATGCT GCCAAGGCC TGCAAC 2093
ValSerAla LysSerAsn HisCys LeuAspAla AlaLysAla CysAsn
155 160 165
CTGAATGAC AACTGCAAG AAGCTG CGCTCCTCC TACATCTCC ATCTGC 2141
LeuAsnAsp AsnCysLys LysLeu ArgSerSer TyrIleSer IleCys
170 175 180 185
AACCGCGAG ATCTCGCCC ACCGAG CGCTGCAAC CGCCGCAAG TGCCAC 2189
AsnArgGlu IleSerPro ThrGlu ArgCysAsn ArgArgLys CysHis
190 195 200
AAGGCCCTG CGCCAGTTC TTCGAC CGGGTGCCC AGCGAGTAC ACCTAC 2237
LysAlaLeu ArgGlnPhe PheAsp ArgValPro SerGluTyr ThrTyr
205 210 215
CGCATGCTC TTCTGCTCC TGCCAA GACCAGGCG TGCGCTGAG CGCCGC 2285
ArgMetLeu PheCysSer CysGln AspGlnAla CysAlaGlu ArgArg
220 225 230
CGGCAAACC ATCCTGCCC AGCTGC TCCTATGAG GACAAGGAG AAGCCC 2333
ArgGlnThr IleLeuPro SerCys SerTyrGlu AspLysGlu LysPro
235 240 245
AACTGCCTG GACCTGCGT GGCGTG TGCCGGACT GACCACCTG TGTCGG 2381
AsnCysLeu AspLeuArg GlyVal CysArgThr AspHisLeu CysArg
250 255 260 265
TCCCGGCTG GCCGACTTC CATGCC AATTGTCGA GCCTCCTAC CAGACG 2429
CA 02291608 1999-11-26
157
SerArgLeu AlaAspPhe HisAla AsnCysArg AlaSerTyr GlnThr
270 275 280
GTCACCAGC TGCCCTGCG GACAAT TACCAGGCG TGTCTGGGC TCTTAT 2477
ValThrSer CysProAla AspAsn TyrGlnAla CysLeuGly SerTyr
285 290 295
GCTGGCATG ATTGGGTTT GACATG ACACCTAAC TATGTGGAC TCCAGC 2525
AlaGlyMet IleG1yPhe AspMet ThrProAsn TyrValAsp SerSer
300 305 310
CCCACTGGC ATCGTGGTG TCCCCC TGGTGCAGC TGTCGTGGC AGCGGG 2573
ProThrGly IleValVal SerPro TrpCysSer CysArgGly SerGly
315 320 325
AACATGGAG GAGGAGTGT GAGAAG TTCCTCAGG GACTTCACC GAGAAC 2621
AsnMetGlu GluG1uCys GluLys PheLeuArg AspPheThr GluAsn
330 335 340 345
CCATGCCTC CGGAACGCC ATCCAG GCCTTTGGC AACGGCACG AACGTG 2669
ProCysLeu ArgAsnAla IleGln AlaPheGly AsnGlyThr AsnVal
350 355 360
AACGTGTCC CCAAAAGGC CCCTCG TTCCAGGCC ACCCAGGCC CCTCGG 2717
AsnValSer ProLysGly ProSer PheGlnAla ThrGlnAla ProArg
365 370 375
GTGGAGAAG ACGCCTTCT TTGCCA GATGACCTC AGTGACAGT ACCAGC 2765
ValGluLys ThrProSer LeuPro AspAspLeu SerAspSer ThrSer
380 385 390
TTGGGGACC AGTGTCATC ACCACC TGCACGTCT GTCCAGGAG CAGGGG 2813
LeuGlyThr SerValIle ThrThr CysThrSer ValGlnGlu GlnGly
395 400 405
CTGAAGGCC AACAACTCC AAAGAG TTAAGCATG TGCTTCACA GAGCTC 2861
LeuLysAla AsnAsnSer LysGlu LeuSerMet CysPheThr GluLeu
410 415 420 425
ACGACAAAT ATCATCCCA GGGAGT AACAAGGTG ATCAAACCT AACTCA 2909
ThrThrAsn IleIlePro GlySer AsnLysVal IleLysPro AsnSer
430 435 440
GGCCCCAGC AGAGCCAGA CCGTCG GCTGCCTTG ACCGTGCTG TCTGTC 2957
GlyProSer ArgAlaArg ProSer AlaAlaLeu ThrValLeu SerVal
445 450 455
CTGATGCTG AAACTGGCC TTGTAGGCTGTGG 3008
GAACCGAGTC
AGAAGATTTT
LeuMetLeu LysLeuAla Leu
460
TGAAAGCTAC AAACACACAC 3068
GCAGACAAGA AGACACACAC
ACAGCCGCCT
GACGAAATGG
ACACCTTGCA ,AAAAAAAAT TGAACCTGTC 3128
P TGTTTTTCCC TCCTCCCAGG
ACCTTGTCGC
TTTCTTCTCT CAGGCAGGCA 3188
GGAGAAGTTT GCCTGAGAGC
TTGTAAACCA
AACAGACAAG
TGGCCCAGGGGTCCCCTGGCAGGGGAAACTCTGGTGCCGGGGAGGGCACGAGGCTCTAGA3248
AATGCCCTTCACTTTCTCCTGGTGTTTTTCTCTCTGGACCCTTCTGAAGCAGAGACCGGA3308
CAAGAGCCTGCAGCGGAAGGGACTCTGGGCTGTGCCTGAGGCTGGCTGGGGGCAGGACAA3368
CA 02291608 1999-11-26
158
CACAGCTGCTTCCCCAGGCTGCCCACTCTGGGGACCCGCTGGGGGCTGGCAGAGGGCATC3428
GGTCAGCGGGGCAGCGGGGCTGGCCATGAGGGTCCACCTTCAGCCCTTTGGCTTCAAGGA3488
TGGAGATGGTTTTGCCCTCCCTCTCTGCCCTCGGGTGGGGCTGGTGGGTCTGCAGCTGGT3548
GTGGGAACTTCCCCACGGATGGCGGTGGAGGGGGTTCGCACCGTGCTGGGCTCCCCCTGA3608
CTGTAGCACGGAGTGTTGGGGCTGGGGGCCAGCTCCAGGAGGGCTTGAGAGCTCAGCCTG3668
CCTGGGAGAGCCCTTGTGGCGAGGCATTAAAACTTGGGCACCAGCTTCTTTCTCGGTGGC3728
AGAAATTTTGAAGTCAGAGAGAAACGGTCCTTTGTTGGCTTCTTTGCTTTCTCGTGGGTC3788
CTTTGGCAGGCCTCCCTTTGGGGAGAGGGAGGGGAGAGACCACAGCCGGGTGTGTGTCTG3848
CAGCACCGTGGGCCCTCAAGCTTTCCTGCTGTCTTCTCCCTCCTCCTCCTTTCCCCTTTC3908
TCTTTCCTCATTTCCTAGACGTACGTCAACTGTATGTACATACCGGGGCTCCTCTCCTAA3968
CATATATGTATATACACATCCATATACATATATTGTGTGGTTTCCCCTTTCTTTCCTTTT4028
TTTAAGCAACAAAACTATGGAAATAATACCCCAACAGATGAGCGAAAATGTATTATTGTA4088
AAGTTTATTTTTTTTAATACTGTTGTCTATAATGGGGAAAAAGGACATTGGCCCCGCAGT4148
GCCCTGCCCCAGTCAGCCTGGCTGGGCTCTGGTGGGGGCTCCTGATCCGCATCCAAGCTT4208
AACCAAGGCTCCAATAAACGTGCG 4232
(2) INFORMATION FOR SEQ ID N0:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 464 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:36:
Met Ile Leu Ala Asn Val Phe Cys Leu Phe Phe Phe Leu Asp Asp Thr
1 5 10 15
Leu Arg Ser Leu Ala Ser Pro Ser Ser Leu Gln Gly Pro Glu Leu His
20 25 30
Gly Trp Arg Pro Pro Val Asp Cys Val Arg Ala Asn Glu Leu Cys Ala
35 40 45
Ala Glu Ser Asn Cys Ser Ser Arg Tyr Arg Thr Leu Arg Gln Cys Leu
50 55 60
Ala Gly Arg Asp Arg Asn Thr Met Leu Ala Asn Lys Glu Cys Gln Ala
65 70 75 80
Ala Leu Glu Val Leu Gln Glu Ser Pro Leu Tyr Asp Cys Arg Cys Lys
85 90 95
Arg Gly Met Lys Lys Glu Leu Gln Cys Leu Gln Ile Tyr Trp Ser Ile
100 105 110
CA 02291608 1999-11-26
159
His Leu Gly Leu Thr Glu Gly Glu Glu Phe Tyr Glu Ala Ser Pro Tyr
115 120 125
Glu Pro Val Thr Ser Arg Leu Ser Asp Ile Phe Arg Leu Ala Ser Ile
130 135 140
Phe Ser Gly Thr Gly Ala Asp Pro Val Val Ser Ala Lys Ser Asn His
145 150 155 160
Cys Leu Asp Ala Ala Lys Ala Cys Asn Leu Asn Asp Asn Cys Lys Lys
165 170 175
Leu Arg Ser Ser Tyr Ile Ser Ile Cys Asn Arg Glu Ile Ser Pro Thr
180 185 190
Glu Arg Cys Asn Arg Arg Lys Cys His Lys Ala Leu Arg Gln Phe Phe
195 200 205
Asp Arg Val Pro Ser Glu Tyr Thr Tyr Arg Met Leu Phe Cys Ser Cys
210 215 220
Gln Asp Gln Ala Cys Ala Glu Arg Arg Arg Gln Thr Ile Leu Pro Ser
225 230 235 240
Cys Ser Tyr Glu Asp Lys Glu Lys Pro Asn Cys Leu Asp Leu Arg Gly
245 250 255
Val Cys Arg Thr Asp His Leu Cys Arg Ser Arg Leu Ala Asp Phe His
260 265 270
Ala Asn Cys Arg Ala Ser Tyr Gln Thr Val Thr Ser Cys Pro Ala Asp
275 280 285
Asn Tyr Gln Ala Cys Leu Gly Ser Tyr Ala Gly Met Ile Gly Phe Asp
290 295 300
Met Thr Pro Asn Tyr Val Asp Ser Ser Pro Thr Gly Ile Val Val Ser
305 310 315 320
Pro Trp Cys Ser Cys Arg Gly Ser Gly Asn Met Glu Glu Glu Cys Glu
325 330 335
Lys Phe Leu Arg Asp Phe Thr Glu Asn Pro Cys Leu Arg Asn Ala Ile
340 345 350
Gln Ala Phe Gly Asn Gly Thr Asn Val Asn Val Ser Pro Lys Gly Pro
355 360 365
Ser Phe Gln Ala Thr Gln Ala Pro Arg Val Glu Lys Thr Pro Ser Leu
370 375 380
Pro Asp Asp Leu Ser Asp Ser Thr Ser Leu Gly Thr Ser Val Ile Thr
385 390 395 400
Thr Cys Thr Ser Val Gln Glu Gln Gly Leu Lys Ala Asn Asn Ser Lys
405 410 415
Glu Leu Ser Met Cys Phe Thr Glu Leu Thr Thr Asn Ile Ile Pro Gly
420 425 430
Ser Asn Lys Val Ile Lys Pro Asn Ser Gly Pro Ser Arg Ala Arg Pro
435 440 445
CA 02291608 1999-11-26
160
Ser Ala Ala Leu Thr Val Leu Ser Val Leu Met Leu Lys Leu Ala Leu
450 455 460
(2) INFORMATION FOR SEQ ID N0:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1991 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 203..1402
(xi)SEQUENCE SEQ ID
DESCRIPTION: N0:37:
CAAGTCAAAG CTTTAGGACA 60
GTTTAATCAT ATAATAGGAA
GATCCAAGAG
CCCAGAGAGA
TAAAGCAAGG GTTCGGCAGG 120
CCCACAGGCT ATCCGGGGAC
CCAGCTCCTG
ATGCCCAGAT
AGGGCAGTGC GAGGAGCGAG 180
AGGCAGTAGT GGGAGCGCGG
TTTCCATCCT
CCATCCAGGG
AGCCCGGCGC 232
CTACAGCTCG
CC
ATG
GTG
CGC
CCC
CTG
AAC
CCG
CGA
CCG
CTG
MetVa l Pro
Arg Arg
Pro Pro
Leu Leu
Asn
1 5 10
CCGCCCGTA GTCCTGATG TTGCTGCTG CTGCTG CCGCCGTCG CCGCTG 280
ProProVal ValLeuMet LeuLeuLeu LeuLeu ProProSer ProLeu
15 20 25
CCTCTCGCA GCCGGAGAC CCCCTTCCC ACAGAA AGCCGACTC ATGAAC 328
ProLeuAla AlaGlyAsp ProLeuPro ThrGlu SerArgLeu MetAsn
30 35 40
AGCTGTCTC CAGGCCAGG AGGAAGTGC CAGGCT GATCCCACC TGCAGT 376
SerCysLeu GlnAlaArg ArgLysCys GlnAla AspProThr CysSer
45 50 55
GCTGCCTAC CACCACCTG GATTCCTGC ACCTCT AGCATAAGC ACCCCA 424
AlaAlaTyr HisHisLeu AspSerCys ThrSer SerIleSer ThrPro
60 65 70
CTGCCCTCA GAGGAGCCT TCGGTCCCT GCTGAC TGCCTGGAG GCAGCA 472
LeuProSer GluGluPro SerValPro AlaAsp CysLeuGlu AlaAla
75 80 85 90
CAGCAACTC AGGAACAGC TCTCTGATA GGCTGC ATGTGCCAC CGGCGC 520
GlnGlnLeu ArgAsnSer SerLeuIle GlyCys MetCysHis ArgArg
95 100 105
ATGAAGAAC CAGGTTGCC TGCTTGGAC ATCTAT TGGACCGTT CACCGT 568
MetLysAsn GlnValAla CysLeuAsp IleTyr TrpThrVal HisArg
110 115 120
GCCCGCAGC CTTGGTAAC TATGAGCTG GATGTC TCCCCCTAT GAAGAC 616
AlaArgSer LeuGlyAsn TyrGluLeu AspVal SerProTyr GluAsp
125 130 135
CA 02291608 1999-11-26
161
ACAGTGACC AGCAAACCC TGGAAA AATCTC AGCAAACTG AACATG 664
ATG
ThrValThr SerLysPro TrpLysMet AsnLeu SerLysLeu AsnMet
140 145 150
CTCAAACCA GACTCAGAC CTCTGCCTC AAGTTT GCCATGCTG TGTACT 712
LeuLysPro AspSerAsp LeuCysLeu LysPhe AlaMetLeu CysThr
155 160 165 170
CTCAATGAC AAGTGTGAC CGGCTGCGC AAGGCC TACGGGGAG GCGTGC 760
LeuAsnAsp LysCysAsp ArgLeuArg LysAla TyrGlyGlu AlaCys
175 180 185
TCCGGGCCC CACTGCCAG CGCCACGTC TGCCTC AGGCAGCTG CTCACT 808
SerGlyPro HisCysGln ArgHisVal CysLeu ArgGlnLeu LeuThr
190 195 200
TTCTTCGAG AAGGCCGCC GAGCCCCAC GCGCAG GGCCTGCTA CTGTGC 856
PhePheGlu LysAlaAla GluProHis AlaGln GlyLeuLeu LeuCys
205 210 215
CCATGTGCC CCCAACGAC CGGGGCTGC GGGGAG CGCCGGCGC AACACC 904
ProCysAla ProAsnAsp ArgGlyCys GlyGlu ArgArgArg AsnThr
220 225 230
ATCGCCCCC AACTGCGCG CTGCCGCCT GTGGCC CCCAACTGC CTGGAG 952
IleAlaPro AsnCysAla LeuProPro ValAla ProAsnCys LeuGlu
235 240 245 250
CTGCGGCGC CTCTGCTTC TCCGACCCG CTTTGC AGATCACGC CTGGTG 1000
LeuArgArg LeuCysPhe SerAspPro LeuCys ArgSerArg LeuVal
255 260 265
GATTTCCAG ACCCACTGC CATCCCATG GACATC CTAGGAACT TGTGCA 1048
AspPheGln ThrHisCys HisProMet AspIle LeuGlyThr CysAla
270 275 280
ACAGAGCAG TCCAGATGT CTACGAGCA TACCTG GGGCTGATT GGGACT 1096
ThrGluGln SerArgCys LeuArgAla TyrLeu GlyLeuIle G1yThr
285 290 295
GCCATGACC CCCAACTTT GCCAGCAAT GTCAAC ACCAGTGTT GCCTTA 1144
AlaMetThr ProAsnPhe AlaSerAsn ValAsn ThrSerVal AlaLeu
300 305 310
AGCTGCACC TGCCGAGGC AGTGGCAAC CTGCAG GAGGAGTGT GAAATG 1192
SerCysThr CysArgGly SerGlyAsn LeuGln GluGluCys GluMet
315 320 325 330
CTGGAAGGG TTCTTCTCC CACAACCCC TGCCTC ACGGAGGCC ATTGCA 1240
LeuGluGly PhePheSer HisAsnPro CysLeu ThrGluAla IleAla
335 340 345
GCTAAGATG CGTTTTCAC AGCCAACTC TTCTCC CAGGACTGG CCACAC 1288
AlaLysMet ArgPheHis SerGlnLeu PheSer GlnAspTrp ProHis
350 355 360
CCTACCTTT GCTGTGATG GCACACCAG AATGAA AACCCTGCT GTGAGG 1336
ProThrPhe AlaValMet AlaHisGln AsnGlu AsnProAla ValArg
365 370 375
CCACAGCCC TGGGTGCCC TCTCTTTTC TCCTGC ACGCTTCCC TTGATT 1384
ProGlnPro TrpValPro SerLeuPhe SerCys ThrLeuPro LeuIle
380 385 390
CA 02291608 1999-11-26
162
CTG CTC 1432
CTG AGC
CTA TGG
TAGCTGGACT
TCCCCAGGGC
CCTCTTCCCC
Leu Leu
Leu Ser
Leu Trp
395 400
TCCACCACACCCAGGTGGACTTGCAGCCCACAAGGGGTGAGGAAAGGACAGCAGCAGGAA1492
GGAGGTGCAGTGCGCAGATGAGGGCACAGGAGAAGCTAAGGGTTATGACCTCCAGATCCT1552
TACTGGTCCAGTCCTCATTCCCTCCACCCCATCTCCACTTCTGATTCATGCTGCCCCTCC1612
TTGGTGGCCACAATTTAGCCATGTCATCTGGTGGTGACCAGCTCCACCAAGCCCCTTTGT1672
GAGCCCTTCCTCTTGACTACCAGGATCACCAGAATCTAATAAGTTAGCCTTTCTCTATTG1732
CATTCCAGATTAGGGTTAGGGTAGGGAGGACTGGGTGTTCTGAGGCAGCCTAGAAAGTCA1792
TTCTCCTTTGTGAAGAAGGCTCCTGCCCCCTCGTCTCCTCCTCTGAGTGGAGGATGGAAA1852
ACTACTGCCTGCACTGCCCTGTCCCCGGATCCTGCCGAACATCTGGGCATCAGGAGCTGG1912
AGCCTGTGGGCCTTGCTTTATTCCTATTATTGTCCTAAAGTCTCTCTGGGCTCTTGGATC1972
ATGATTAAACCTTTGACTG 1991
(2) INFORMATION FOR SEQ ID N0:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 400 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:38:
Met Val Arg Pro Leu Asn Pro Arg Pro Leu Pro Pro Val Val Leu Met
1 5 10 15
Leu Leu Leu Leu Leu Pro Pro Ser Pro Leu Pro Leu Ala Ala Gly Asp
20 25 30
Pro Leu Pro Thr Glu Ser Arg Leu Met Asn Ser Cys Leu Gln Ala Arg
35 40 45
Arg Lys Cys Gln Ala Asp Pro Thr Cys Ser Ala Ala Tyr His His Leu
50 55 60
Asp Ser Cys Thr Ser Ser Ile Ser Thr Pro Leu Pro Ser Glu Glu Pro
s5 70 75 so
Ser Val Pro Ala Asp Cys Leu Glu Ala Ala Gln Gln Leu Arg Asn Ser
85 90 95
Ser Leu Ile Gly Cys Met Cys His Arg Arg Met Lys Asn Gln Val Ala
100 105 110
Cys Leu Asp Ile Tyr Trp Thr Val His Arg Ala Arg Ser Leu Gly Asn
115 120 125
Tyr Glu Leu Asp Val Ser Pro Tyr Glu Asp Thr Val Thr Ser Lys Pro
130 135 140
CA 02291608 1999-11-26
163
Trp Lys Met Asn Leu Ser Lys Leu Asn Met Leu Lys Pro Asp Ser Asp
145 150 155 160
Leu Cys Leu Lys Phe Ala Met Leu Cys Thr Leu Asn Asp Lys Cys Asp
165 170 175
Arg Leu Arg Lys Ala Tyr Gly Glu Ala Cys Ser Gly Pro His Cys Gln
180 185 190
Arg His Val Cys Leu Arg Gln Leu Leu Thr Phe Phe Glu Lys Ala Ala
195 200 205
Glu Pro His Ala Gln Gly Leu Leu Leu Cys Pro Cys Ala Pro Asn Asp
210 215 220
Arg Gly Cys Gly Glu Arg Arg Arg Asn Thr Ile Ala Pro Asn Cys Ala
225 230 235 240
Leu Pro Pro Val Ala Pro Asn Cys Leu Glu Leu Arg Arg Leu Cys Phe
245 250 255
Ser Asp Pro Leu Cys Arg Ser Arg Leu Val Asp Phe Gln Thr His Cys
260 265 270
His Pro Met Asp Ile Leu Gly Thr Cys Ala Thr Glu Gln Ser Arg Cys
275 280 285
Leu Arg Ala Tyr Leu Gly Leu Ile Gly Thr Ala Met Thr Pro Asn Phe
290 295 300
Ala Ser Asn Val Asn Thr Ser Val Ala Leu Ser Cys Thr Cys Arg Gly
305 310 315 320
Ser Gly Asn Leu Gln Glu Glu Cys Glu Met Leu Glu Gly Phe Phe Ser
325 330 335
His Asn Pro Cys Leu Thr Glu Ala Ile Ala Ala Lys Met Arg Phe His
340 345 350
Ser Gln Leu Phe Ser Gln Asp Trp Pro His Pro Thr Phe Ala Val Met
355 360 365
Ala His Gln Asn Glu Asn Pro Ala Val Arg Pro Gln Pro Trp Val Pro
370 375 380
Ser Leu Phe Ser Cys Thr Leu Pro Leu Ile Leu Leu Leu Ser Leu Trp
385 390 395 400
(2) INFORMATION FOR SEQ ID N0:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2215 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 684..2063
CA 02291608 1999-11-26
164
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:39:
GCGGCCGCGT CGACCTTGAC CATGCAGACA CTTTTTCAGG CCTCTGTCTG GTGTGAAGTT 60
GGCAGATACA AGCAAGGCCC GAAAGGGGTC TCAGCTTCTC TCTCCTGGGC CTCCTGGACT 120
GAGTTAGGCT TGCTTCTGGT TGTCTTCTAA AGGCACGGTG ATACAGAATG ATGAGACTAG 180
GCTGGAGGGG GCTTTCTGCT TCTCTGTGTG TGACCTTGAG TTATCTCCCT TCGTTGGATC 240
CGAGCTTTCC TGGAATATGA TCTGCCTAAG GTCCAGACAG
300
TGTTGAATAT
GAATATGAGT
GCTCTGAGGG TTAACTGACT ACCTTGGATG GAGTGGGGGT
360
TTTGGAGCCT
TCAAATCAAT
TTGTCCAATG GGAGTTGAGG CTTGCCACAT CATGTTGAAG
420
CAAGATCCCT
TTGCATAAGC
CCATGCCATT CTGTCTGGAC AGCAGTTTCA GTGAAGGCCT
480
TATTGGCATC
TTACCTTTCC
TCCTGGATTT ATCATTCTGT GCTCAAGAGG AAATGAATGT
540
GTTCCACTGC
CTAGGATTGT
GAACCATGGT TGTAGGGGAG CTGTGTTGAC CTTGGTCTTG
600
TATGGCCAAC
CAGGTTGGGT
GTGTTCTTTT GTGTAAAGTG CTTAGGCCTA CATTGGGGTC
660
GGTGAGAAGT
TCCTTCAAAC
AGAGACTGTG GTGGCCCTCA AC 710
TTC CCA
ATG
CTT
GTC
TTC
CCT
TCC
CAC
T
Met yr
Leu Pro
Val
Phe
Pro
Ser
His
T
1 5
GACGAAACCCTC CGCTCT TTGGCC AGCCCT TCCCTGCAGGGC TCT 758
TCC
AspGluThrLeu ArgSer LeuAla SerPro SerLeuGlnGly Ser
Ser
15 20 25
GAGCTCCACGGC TGGCGC CCCCAA GTGGAC GTCCGGGCCAAT GAG 806
TGT
GluLeuHisGly TrpArg ProGln ValAsp ValArgAlaAsn Glu
Cys
30 35 40
CTGTGTGCGGCT GAATCC AACTGC AGCTCC TACCGCACCCTT CGG 854
AGG
LeuCysAlaAla GluSer AsnCys SerSer TyrArgThrLeu Arg
Arg
45 50 55
CAGTGCCTGGCA GGCCGG GATCGC AATACC CTGGCCAATAAG GAG 902
ATG
GlnCysLeuAla G1yArg AspArg AsnThr LeuAlaAsnLys Glu
Met
60 65 70
TGCCAGGCAGCC CTGGAG GTCTTG CAGGAA CCACTGTATGAC TGC 950
AGC
CysGlnAlaAla LeuGlu ValLeu GlnGlu ProLeuTyrAsp Cys
Ser
75 80 85
CGCTGCAAGCGG GGCATG AAGAAG GAGCTG TGTCTGCAGATC TAC 998
CAG
ArgCysLysArg GlyMet LysLys GluLeu CysLeuGlnIle Tyr
Gln
90 95 100 105
TGGAGCATCCAT CTGGGG CTGACA GAGGGT GAGTTCTATGAA GCT 1046
GAG
TrpSerIleHis LeuGly LeuThr GluGly GluPheTyrGlu Ala
Glu
110 115 120
TCCCCCTATGAG CCTGTG ACCTCG CGCCTC GACATCTTCAGG CTC 1094
TCG
SerProTyrGlu ProVal ThrSer ArgLeu AspIlePheArg Leu
Ser
125 130 135
GCTTCAATCTTC TCAGGG ACAGGG ACAGAC GCGGTCAGTACC AAA 1142
CCG
AlaSerIlePhe SerGly ThrGly ThrAsp AlaValSerThr Lys
Pro
CA 02291608 1999-11-26
165
140 145 150
AGCAACCAC TGCCTGGAT GCCGCCAAG GCCTGC AACCTGAAT GACAAC 1190
SerAsnHis CysLeuAsp AlaAlaLys AlaCys AsnLeuAsn AspAsn
155 160 165
TGCAAGAAG CTTCGCTCC TCTTATATC TCCATC TGCAACCGT GAGATC 1238
CysLysLys LeuArgSer SerTyrIle SerIle CysAsnArg GluIle
170 175 180 185
TCTCCCACC GAACGCTGC AACCGCCGC AAGTGC CACAAGGCT CTGCGC 1286
SerProThr GluArgCys AsnArgArg LysCys HisLysAla LeuArg
190 195 200
CAGTTCTTT GACCGTGTG CCCAGCGAG TATACC TACCGCATG CTCTTC 1334
GlnPhePhe AspArgVal ProSerGlu TyrThr TyrArgMet LeuPhe
205 210 215
TGCTCCTGT CAGGACCAG GCATGTGCT GAGCGT CGCCGGCAA ACCATC 1382
CysSerCys GlnAspGln AlaCysAla GluArg ArgArgGln ThrIle
220 225 230
CTGCCCAGT TGCTCCTAT GAGGACAAG GAGAAG CCCAACTGC CTGGAC 1430
LeuProSer CysSerTyr GluAspLys GluLys ProAsnCys LeuAsp
235 240 245
CTGCGCAGC CTGTGTCGT ACAGACCAC CTGTGC CGGTCCCGA CTGGCA 1478
LeuArgSer LeuCysArg ThrAspHis LeuCys ArgSerArg LeuAla
250 255 260 265
GATTTCCAC GCCAACTGT CGAGCCTCC TACCGG ACAATCACC AGCTGT 1526
AspPheHis AlaAsnCys ArgAlaSer TyrArg ThrIleThr SerCys
270 275 280
CCTGCGGAC AACTACCAG GCATGTCTG GGCTCC TATGCTGGC ATGATT 1574
ProAlaAsp AsnTyrGln AlaCysLeu GlySer TyrAlaGly MetIle
285 290 295
GGGTTTGAT ATGACACCC AACTATGTG GACTCC AACCCCACG GGCATC 1622
GlyPheAsp MetThrPro AsnTyrVal AspSer AsnProThr GlyIle
300 305 310
GTGGTGTCT CCCTGGTGC AATTGTCGT GGCAGT GGGAACATG GAAGAA 1670
ValValSer ProTrpCys AsnCysArg GlySer GlyAsnMet GluGlu
315 320 325
GAGTGTGAG AAGTTCCTC AGGGACTTC ACGGAA AACCCATGC CTCCGG 1718
GluCysGlu LysPheLeu ArgAspPhe ThrGlu AsnProCys LeuArg
330 335 340 345
AATGCCATT CAGGCCTTT GGTAATGGC ACAGAT GTGAACATG TCTCCC 1766
AsnAlaIle GlnAlaPhe GlyAsnGly ThrAsp ValAsnMet SerPro
350 355 360
AAAGGCCCC TCACTCCCA GCTACCCAG GCCCCT CGGGTGGAG AAGACT 1814
LysGlyPro SerLeuPro AlaThrGln AlaPro ArgValGlu LysThr
365 370 375
CCTTCACTG CCAGATGAC CTCAGTGAC AGCACC AGCCTGGGG ACCAGT 1862
ProSerLeu ProAspAsp LeuSerAsp SerThr SerLeuGly ThrSer
380 385 390
GTC ATC ACC ACC TGC ACA TCT ATC CAG GAG CAA GGG CTG AAG GCC AAC 1910
CA 02291608 1999-11-26
166
Val Ile Thr Thr Cys Thr Ser Ile Gln Glu Gln Gly Leu Lys Ala Asn
395 400 405
AACTCCAAA GAGTTA ATG TGCTTC ACAGAG CTCACGACA AACATC 1958
AGC
AsnSerLys GluLeu Met CysPhe ThrGlu LeuThrThr AsnIle
Ser
410 415 420 425
AGTCCAGGG AGTAAA GTG ATCAAA CTTAAC TCAGGCTCC AGCAGA 2006
AAG
SerProGly SerLys Val IleLys LeuAsn SerGlySer SerArg
Lys
430 435 440
GCCAGACTG TCGGCT TTG ACTGCC CTCCCA CTCCTGATG CTGACC 2054
GCC
AlaArgLeu SerAla Leu ThrAla LeuPro LeuLeuMet LeuThr
Ala
445 450 455
TTGGCCTTG TAGGCCTTTG GAACCCAGCA TCAAGCAACC
2103
CAAAAGTTCT
LeuAlaLeu
460
CAGATATGAA CTCCCGCCTG ACAAAATGGA AACACACGCA TACACACATG CCACACACAG 2163
ACACACACAC AGACACACAC ACACACACAC ATACAGACGT CGACGCGGCC GC 2215
(2) INFORMATION FOR SEQ ID N0:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 460 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:40:
Met Leu Val Phe Pro Ser His Tyr Pro Asp Glu Thr Leu Arg Ser Leu
1 5 10 15
Ala Ser Pro Ser Ser Leu Gln Gly Ser Glu Leu His Gly Trp Arg Pro
20 25 30
Gln Val Asp Cys Val Arg Ala Asn Glu Leu Cys Ala Ala Glu Ser Asn
35 40 45
Cys Ser Ser Arg Tyr Arg Thr Leu Arg Gln Cys Leu Ala Gly Arg Asp
50 55 60
Arg Asn Thr Met Leu Ala Asn Lys Glu Cys Gln Ala Ala Leu Glu Val
65 70 75 80
Leu Gln Glu Ser Pro Leu Tyr Asp Cys Arg Cys Lys Arg Gly Met Lys
85 90 95
Lys Glu Leu Gln Cys Leu Gln Ile Tyr Trp Ser Ile His Leu Gly Leu
100 105 110
Thr Glu Gly Glu Glu Phe Tyr Glu Ala Ser Pro Tyr Glu Pro Val Thr
115 120 125
Ser Arg Leu Ser Asp Ile Phe Arg Leu Ala Ser Ile Phe Ser Gly Thr
130 135 140
Gly Thr Asp Pro Ala Val Ser Thr Lys Ser Asn His Cys Leu Asp Ala
CA 02291608 1999-11-26
167
145 150 155 160
Ala Lys Ala Cys Asn Leu Asn Asp Asn Cys Lys Lys Leu Arg Ser Ser
165 170 175
Tyr Ile Ser Ile Cys Asn Arg Glu Ile Ser Pro Thr Glu Arg Cys Asn
180 185 190
Arg Arg Lys Cys His Lys Ala Leu Arg Gln Phe Phe Asp Arg Val Pro
195 200 205
Ser Glu Tyr Thr Tyr Arg Met Leu Phe Cys Ser Cys Gln Asp Gln Ala
210 215 220
Cys Ala Glu Arg Arg Arg Gln Thr Ile Leu Pro Ser Cys Ser Tyr Glu
225 230 235 240
Asp Lys Glu Lys Pro Asn Cys Leu Asp Leu Arg Ser Leu Cys Arg Thr
245 250 255
Asp His Leu Cys Arg Ser Arg Leu Ala Asp Phe His Ala Asn Cys Arg
260 265 270
Ala Ser Tyr Arg Thr Ile Thr Ser Cys Pro Ala Asp Asn Tyr Gln Ala
275 280 285
Cys Leu Gly Ser Tyr Ala Gly Met Ile Gly Phe Asp Met Thr Pro Asn
290 295 300
Tyr Val Asp Ser Asn Pro Thr Gly Ile Val Val Ser Pro Trp Cys Asn
305 310 315 320
Cys Arg Gly Ser Gly Asn Met Glu Glu Glu Cys Glu Lys Phe Leu Arg
325 330 335
Asp Phe Thr Glu Asn Pro Cys Leu Arg Asn Ala Ile Gln Ala Phe Gly
340 345 350
Asn Gly Thr Asp Val Asn Met Ser Pro Lys Gly Pro Ser Leu Pro Ala
355 360 365
Thr Gln Ala Pro Arg Val Glu Lys Thr Pro Ser Leu Pro Asp Asp Leu
370 375 380
Ser Asp Ser Thr Ser Leu Gly Thr Ser Val Ile Thr Thr Cys Thr Ser
385 390 395 400
Ile Gln Glu Gln Gly Leu Lys Ala Asn Asn Ser Lys Glu Leu Ser Met
405 410 415
Cys Phe Thr Glu Leu Thr Thr Asn Ile Ser Pro Gly Ser Lys Lys Val
420 425 430
Ile Lys Leu Asn Ser Gly Ser Ser Arg Ala Arg Leu Ser Ala Ala Leu
435 440 445
Thr Ala Leu Pro Leu Leu Met Leu Thr Leu Ala Leu
450 455 460
(2) INFORMATION FOR SEQ ID N0:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1699 base pairs
CA 02291608 1999-11-26
16g
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 67..1257
(xi)SEQUENCE SEQ
DESCRIPTION: ID
N0:41:
GCGGCCGCGT CGACGC CAGCACAGG CCGGGTCCGC
60
CGAC C CAGAGCGCTG GGCGTCCAGA
CCCGCC CTC CGG CCG CCGCCG CTA 108
ATG TCC AGC CGA CCG GTG
GGG ATC
Met Leu Pro ProPro Leu Ile
Gly Ser Arg Pro Val
Arg
Ser
1 5 10
CTGCTACTG GTGCTGTCGCTG TGGCTA CCCCTTGGA ACAGGA AACTCC 156
LeuLeuLeu ValLeuSerLeu TrpLeu ProLeuGly ThrGly AsnSer
15 20 25 30
CTTCCCACA GAGAACAGGCTT GTGAAC AGCTGTACC CAGGCC AGAAAA 204
LeuProThr GluAsnArgLeu ValAsn SerCysThr GlnAla ArgLys
35 40 45
AAATGCGAG GCTAATCCCGCT TGCAAG GCTGCCTAC CAGCAC CTGGAC 252
LysCysGlu AlaAsnProAla CysLys AlaAlaTyr GlnHis LeuAsp
50 55 60
TCCTGCACC CCCAGTCTCAGC AGTCCA CTGCCCTCA GGGGAG TCTGCC 300
SerCysThr ProSerLeuSer SerPro LeuProSer GlyGlu SerAla
65 70 75
ACATCTGCA GCGTGCCTTGAA GCAGCA CAGCAACTC AGGAAC AGCTCT 348
ThrSerAla AlaCysLeuGlu AlaAla GlnGlnLeu ArgAsn SerSer
80 85 90
CTCATAGAC TGCAGGTGCCAC CGGCGC ATGAAGCAC CAAGCT ACCTGT 396
LeuIleAsp CysArgCysHis ArgArg MetLysHis GlnAla ThrCys
95 100 105 110
CTGGACATT TATTGGACCGTT CACCCT GTCCGAAGC CTTGGT GACTAC 444
LeuAspIle TyrTrpThrVal HisPro ValArgSer LeuGly AspTyr
115 120 125
GAGTTGGAC GTCTCACCCTAT GAAGAC ACAGTGACC AGCAAA CCCTGG 492
GluLeuAsp ValSerProTyr GluAsp ThrValThr SerLys ProTrp
130 135 140
AAAATGAAT CTCAGCAAGCTG AGCATG CTCAAACCA GACTCC GACCTC 540
LysMetAsn LeuSerLysLeu SerMet LeuLysPro AspSer AspLeu
145 150 155
TGCCTCAAA TTTGCTATGCTG TGTACT CTTAACGAC AAGTGC GACCGC 588
CysLeuLys PheAlaMetLeu CysThr LeuAsnAsp LysCys AspArg
160 165 170
CTCCGAAAG GCCTACGGGGAG GCGTGC TCAGGGATC CGCTGC CAGCGC 636
LeuArgLys AlaTyrGlyGlu AlaCys SerGlyIle ArgCys GlnArg
175 180 185 190
CA 02291608 1999-11-26
169
CACCTCTGC CTAGCTCAG CTGCGC TCCTTCTTC GAGAAGGCG GCAGAG 684
HisLeuCys LeuAlaGln LeuArg SerPhePhe GluLysA1a AlaGlu
195 200 205
TCCCACGCT CAGGGCCTG CTGCTG TGTCCCTGT GCACCCGAA GATGCG 732
SerHisAla GlnGlyLeu LeuLeu CysProCys AlaProGlu AspAla
210 215 220
GGCTGTGGG GAGCGCCGG CGCAAC ACCATCGCC CCCAGTTGC GCCCTC 780
GlyCysGly GluArgArg ArgAsn ThrIleAla ProSerCys AlaLeu
225 230 235
CCGTCTGTG GCCCCCAAC TGCCTA GATCTTCGG AGCTTCTGC CGTGCG 828
ProSerVal AlaProAsn CysLeu AspLeuArg SerPheCys ArgAla
240 245 250
GACCCTCTG TGCAGATCA CGCCTG ATGGACTTC CAGACCCAC TGCCAC 876
AspProLeu CysArgSer ArgLeu MetAspPhe GlnThrHis CysHis
255 260 265 270
CCTATGGAC ATCCTCGGG ACTTGT GCAACTGAG CAGTCCAGA TGTCTG 924
ProMetAsp IleLeuGly ThrCys AlaThrGlu GlnSerArg CysLeu
275 280 285
CGGGCATAC CTGGGGCTA ATTGGG ACTGCCATG ACCCCAAAC TTCATC 972
ArgAlaTyr LeuGlyLeu IleGly ThrAlaMet ThrProAsn PheIle
290 295 300
AGCAAGGTC AACACTACT GTTGCC TTAGGCTGT ACCTGCCGA GGCAGT 1020
SerLysVal AsnThrThr ValAla LeuGlyCys ThrCysArg GlySer
305 310 315
GGCAACCTG CAGGACGAG TGTGAA CAGCTGGAA AAGTCCTTC TCCCAG 1068
GlyAsnLeu GlnAspGlu CysGlu GlnLeuGlu LysSerPhe SerGln
320 325 330
AACCCCTGC CTCATGGAG GCCATT GCGGCTAAA ATGCGTTTC CACAGA 1116
AsnProCys LeuMetGlu AlaIle AlaAlaLys MetArgPhe HisArg
335 340 345 350
CAACTCTTC TCCCAGGAC TGGGCG GACTCTACT TTTTCTGTG ATGCAG 1164
GlnLeuPhe SerGlnAsp TrpAla AspSerThr PheSerVal MetGln
355 360 365
CAGCAGAAC AGCAGCCCT GCTCTG AGGCCCCAG CTCAGGCTA CCCGTT 1212
GlnGlnAsn SerSerPro AlaLeu ArgProGln LeuArgLeu ProVal
370 375 380
CTGTCTTTC TTCATCCTT ACCTTG ATTCTGCTG CAGACCCTC TGG 1257
LeuSerPhe~PheIleLeu ThrLeu IleLeuLeu GlnThrLeu Trp
385 390 395
TAACTGGGCTCCCTCAGGGTCCTTTGTCCTCTCCACCACACCCAGACCGA CTTGCAGCCT1317
GTGATGGGAGAGAAAATGCTGGCCTCTGGAAGAAGATGCAACCAGGCTCA CTGCACATCC1377
TGTCTGCTCCAGATGAGGTCTTGGAAGAAGCGAGGGCTGTGACCGTTCAG AATCCTGAGC1437
GGCCAGCTTTCAAACCTCTCCTACTTACTCCTGCTTGGGCTGCTCCTCCCTAGGACCTTG1497
TACTCCAGTTTGGCTGTATATTGTGGTGGTGATTAGCTTCCCACCTCCAGCCCTTCTTCC1557
TGTTTCCCAGGACCACCCAGGGCTAATGACTCACTCATTCCTGGTTGCCTTCTCCAGGAA1617
CA 02291608 1999-11-26
1~~
GGCAGGCTGA GGGTTCTGAG GCAGCTGAGA AAGATGGTCC CTTTGTGAGG AAGGCTGGTG 1677
GTCCAACCGT CGACGCGGCC GC 1699
(2) INFORMATION FOR SEQ ID N0:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 397 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:42:
Met Gly Leu Ser Arg Ser Pro Arg Pro Pro Pro Leu Val Ile Leu Leu
1 5 10 15
Leu Val Leu Ser Leu Trp Leu Pro Leu Gly Thr Gly Asn Ser Leu Pro
20 25 30
Thr Glu Asn Arg Leu Val Asn Ser Cys Thr Gln Ala Arg Lys Lys Cys
35 40 45
Glu Ala Asn Pro Ala Cys Lys Ala Ala Tyr Gln His Leu Asp Ser Cys
50 55 60
Thr Pro Ser Leu Ser Ser Pro Leu Pro Ser Gly Glu Ser Ala Thr Ser
65 70 75 80
Ala Ala Cys Leu Glu Ala Ala Gln Gln Leu Arg Asn Ser Ser Leu Ile
85 90 95
Asp Cys Arg Cys His Arg Arg Met Lys His Gln Ala Thr Cys Leu Asp
100 105 110
Ile Tyr Trp Thr Val His Pro Val Arg Ser Leu Gly Asp Tyr Glu Leu
115 120 125
Asp Val Ser Pro Tyr Glu Asp Thr Val Thr Ser Lys Pro Trp Lys Met
130 135 140
Asn Leu Ser Lys Leu Ser Met Leu Lys Pro Asp Ser Asp Leu Cys Leu
145 150 155 160
Lys Phe Ala Met Leu Cys Thr Leu Asn Asp Lys Cys Asp Arg Leu Arg
165 170 175
Lys Ala Tyr Gly Glu Ala Cys Ser Gly Ile Arg Cys Gln Arg His Leu
180 185 190
Cys Leu Ala Gln Leu Arg Ser Phe Phe Glu Lys Ala Ala Glu Ser His
195 200 205
Ala Gln Gly Leu Leu Leu Cys Pro Cys Ala Pro Glu Asp Ala Gly Cys
210 215 220
Gly Glu Arg Arg Arg Asn Thr Ile Ala Pro Ser Cys Ala Leu Pro Ser
225 230 235 240
Val Ala Pro Asn Cys Leu Asp Leu Arg Ser Phe Cys Arg Ala Asp Pro
245 250 255
CA 02291608 1999-11-26
l~l
Leu Cys Arg Ser Arg Leu Met Asp Phe Gln Thr His Cys His Pro Met
260 265 270
Asp Ile Leu Gly Thr Cys Ala Thr Glu Gln Ser Arg Cys Leu Arg Ala
275 280 285
Tyr Leu Gly Leu Ile Gly Thr Ala Met Thr Pro Asn Phe Ile Ser Lys
290 295 300
Val Asn Thr Thr Val Ala Leu Gly Cys Thr Cys Arg Gly Ser Gly Asn
305 310 315 320
Leu Gln Asp Glu Cys Glu Gln Leu Glu Lys Ser Phe Ser Gln Asn Pro
325 330 335
Cys Leu Met Glu Ala Ile Ala Ala Lys Met Arg Phe His Arg Gln Leu
340 345 350
Phe Ser Gln Asp Trp Ala Asp Ser Thr Phe Ser Val Met Gln Gln Gln
355 360 365
Asn Ser Ser Pro Ala Leu Arg Pro Gln Leu Arg Leu Pro Val Leu Ser
370 375 380
Phe Phe Ile Leu Thr Leu Ile Leu Leu Gln Thr Leu Trp
385 390 395
(2) INFORMATION FOR SEQ ID N0:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 498 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:43:
Met Val Xaa Xaa Leu Xaa Xaa Xaa Pro Xaa Pro Pro Xaa Xaa Xaa Met
1 5 10 15
Xaa Leu Xaa Leu Leu Ser Leu Ala Leu Pro Leu Xaa Xaa Xaa Leu Gln
20 25 30
Gly Ala Glu Leu Xaa Gly Xaa Xaa Arg Leu Xaa Xaa Asp Cys Val Xaa
35 40 45
Ala Xaa Xaa Xaa Cys Xaa Ala Glu Xaa Xaa Cys Ser Xaa Xaa Tyr Arg
50 55 60
Thr Leu Arg Gln Cys Xaa Ala Gly Xaa Xaa Xaa Asn Thr Xaa Leu Ala
65 70 75 80
Ser Gly Xaa Glu Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Ala Xaa Glu
85 90 95
Xaa Leu Xaa Xaa Ser Ser Leu Tyr Asp Cys Arg Cys Lys Arg Gly Met
100 105 110
CA 02291608 1999-11-26
172
Lys Lys Glu Xaa Xaa Cys Leu Xaa Ile Tyr Trp Ser Xaa His Xaa Xaa
115 120 125
Leu Xaa Xaa Gly Asn Xaa Xaa Leu Glu Xaa Ser Pro Tyr Glu Pro Xaa
130 135 140
Val Thr Ser Arg Leu Ser Asp Ile Phe Arg Xaa Xaa Ser Xaa Xaa Ser
145 150 155 160
Xaa Xaa Xaa Xaa Asp Xaa Xaa Xaa Xaa Xaa Lys Ser Asn Xaa Cys Leu
165 170 175
Asp Ala Ala Lys Ala Cys Asn Leu Asn Asp Xaa Cys Lys Lys Leu Arg
180 185 190
Ser Ala Tyr Ile Xaa Xaa Cys Xaa Xaa Xaa Xaa Ser Xaa Xaa Glu Arg
195 200 205
Cys Asn Arg Arg Lys Cys His Lys Ala Leu Arg Gln Phe Phe Asp Lys
210 215 220
Val Pro Xaa Xaa His Xaa Tyr Gly Met Leu Phe Cys Ser Cys Xaa Xaa
225 230 235 240
Xaa Asp Xaa Ala Cys Xaa Glu Arg Arg Arg Gln Thr Ile Xaa Pro Ser
245 250 255
Cys Ser Tyr Glu Xaa Xaa Glu Lys Pro Asn Cys Leu Asp Leu Arg Xaa
260 265 270
Xaa Cys Arg Thr Asp Xaa Leu Cys Arg Ser Arg Leu Ala Asp Phe Xaa
275 280 285
Thr Asn Cys Xaa Xaa Xaa Xaa Arg Xaa Val Xaa Ser Cys Xaa Ala Xaa
290 295 300
Asn Tyr Xaa Xaa Cys Leu Xaa Ala Tyr Xaa Gly Leu Ile Gly Thr Xaa
305 310 315 320
Met Thr Pro Asn Tyr Val Asp Ser Ser Xaa Thr Xaa Xaa Xaa Val Ala
325 330 335
Pro Trp Cys Xaa Cys Arg Gly Ser Gly Asn Xaa Xaa Glu Glu Cys G1u
340 345 350
Lys Phe Leu Xaa Phe Phe Xaa Xaa Asn Pro Cys Leu Xaa Asn Ala Ile
355 360 365
Gln Ala Phe Gly Asn Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
370 375 380
Xaa Pro Xaa Phe Ser Val Xaa Xaa Xaa Xaa Xaa Thr Xaa Thr Xaa Ala
385 390 395 400
Xaa Arg Val Xaa Xaa Xaa Pro Ser Leu Xaa Xaa Xaa Xaa Ser Xaa Xaa
405 410 415
Xaa Xaa Leu Xaa Thr Xaa Val Xaa Xaa Xaa Cys Xaa Xaa Leu Gln Xaa
420 425 ' 430
Gln Xaa Leu Lys Xaa Asn Xaa Ser Xaa Glu Xaa Xaa Xaa Cys Phe Xaa
435 440 445
CA 02291608 1999-11-26
173
Glu Leu Thr Thr Asn Xaa Xaa Xaa Xaa Ser Gly Xaa Xaa Xaa Xaa Ile
450 455 460
Xaa Xaa Xaa Ser Xaa Xaa Ala Xaa Pro Ser Xaa Ala Leu Xaa Xaa Leu
465 470 475 480
Pro Val Leu Met Leu Thr Ala Leu Ala Xaa Leu Leu Ser Xaa Xaa Xaa
485 490 495
Xaa Ser
(2) INFORMATION FOR SEQ ID N0:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 489 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi)SEQUENCE
DESCRIPTION.
SEQ
ID
N0:44:
XaaXaa XaaXaa XaaXaaXaa XaaXaaPro XaaXaa XaaXaaLeu Xaa
1 5 10 15
ThrLeu XaaSer LeuXaaXaa ProLeuXaa LeuXaa XaaSerXaa Xaa
20 25 30
XaaXaa XaaArg XaaXaaXaa AspCysVal XaaAla XaaXaaXaa Cys
35 40 45
XaaAla GluXaa XaaCysSer XaaXaaTyr ArgThr LeuArgGln Cys
50 55 60
XaaAla GlyXaa XaaXaaAsn XaaXaaXaa XaaXaa XaaXaaXaa Ala
65 70 75 80
XaaXaa GluCys XaaXaaAla XaaGluXaa LeuXaa XaaSerSer Leu
85 90 95
TyrAsp CysArg CysLysArg GlyMetLys LysGlu XaaXaaCys Leu
100 105 110
XaaIle TyrTrp SerXaaHis XaaXaaLeu XaaXaa GlyXaaXaa Xaa
115 120 125
LeuGlu XaaSer ProTyrGlu XaaProVal ThrSer ArgLeuSer Asp
130 135 140
IlePhe ArgXaa XaaSerXaa XaaSerXaa XaaXaa XaaAspXaa Xaa
145 150 155 160
XaaXaa XaaLys SerAsnXaa CysLeuAsp AlaAla LysAlaCys Asn
165 170 175
LeuAsn AspXaa CysLysLys LeuArgSer AlaTyr IleXaaXaa Cys
180 185 190
CA 02291608 1999-11-26
174
Xaa Xaa Xaa Xaa Ser Xaa Xaa Glu Arg Cys Asn Arg Arg Lys Cys His
195 200 205
Lys Ala Leu Arg Gln Phe Phe Asp Lys Val Pro Xaa Xaa His Xaa Tyr
210 215 220
Gly Met Leu Phe Cys Ser Cys Xaa Xaa Xaa Asp Xaa Ala Cys Xaa Glu
225 230 235 240
Arg Arg Arg Gln Thr Ile Xaa Pro Ser Cys Ser Tyr Glu Xaa Xaa Glu
245 250 255
Xaa Pro Asn Cys Leu Asp Leu Arg Ser Xaa Cys Arg Thr Asp Xaa Leu
260 265 270
Cys Arg Ser Arg Leu A1a Asp Phe Xaa Thr Asn Cys Xaa Pro Xaa Xaa
275 280 285
Arg Xaa Xaa Thr Xaa Cys Xaa Ala Xaa Asn Tyr Xaa Xaa Cys Leu Xaa
290 295 300
Ala Tyr Xaa Gly Leu Ile Gly Thr Xaa Met Thr Pro Asn Tyr Val Asp
305 310 315 320
Ser Xaa Xaa Thr Xaa Xaa Xaa Val Ala Pro Trp Cys Xaa Cys Arg Gly
325 330 335
Ser Gly Asn Xaa Xaa Glu Glu Cys Glu Lys Phe Leu Xaa Xaa Phe Xaa
340 345 350
Xaa Asn Pro Cys Leu Xaa Asn Ala Ile Gln Ala Phe Gly Asn Gly Xaa
355 360 365
Asp Val Xaa Met Ser Gln Xaa Xaa Pro Xaa Xaa Xaa Xaa Thr Xaa Ala
370 375 380
Xaa Xaa Xaa Xaa Xaa Xaa Arg Val Xaa Xaa Xaa Pro Xaa Leu Xaa Xaa
385 390 395 400
Xaa Xaa Ser Xaa Xaa Xaa Xaa Xaa Xaa Thr Xaa Val Xaa Xaa Xaa Cys
405 410 415
Xaa Xaa Xaa Gln Xaa Gln Xaa Leu Lys Xaa Asn Xaa Ser Xaa Xaa Xaa
420 425 430
Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
435 440 445
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Xaa Xaa Ala Xaa Xaa Ser Xaa
450 455 460
Xaa Leu Xaa Xaa Leu Pro Val Leu Met Leu Thr Xaa Leu Xaa Xaa Xaa
465 470 475 480
Leu Xaa Xaa Xaa Leu Xaa Glu Thr Ser
485
