Note: Descriptions are shown in the official language in which they were submitted.
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~ RECEPTORS FOR TGF-(3-RELATED NEUROTROPHIC FACTORS
Reference to Government Grants
This invention was made with government support
under National Institutes of Health Grant Numbers RO1
AG13729 and RO1 AG13730. The government has certain
rights in this invention.
Related Applications
This application claims the benefit of U.S.
Provisional Application entitled TrnR2, A Novel Receptor
Which Mediates Neurturin And GDNF Signaling Through Ret
filed April 17, 1997. -
Background of the Invention
(1) Field of the Invention
This invention relates generally to receptors for
trophic or growth factors and, more particularly, to a
novel receptor for TGF-~i-related neurotrophic factors.
(2) Description of the Related Art
The development and maintenance of tissues in
complex organisms requires precise control over the
processes of cell proliferation, differentiation,
survival and function. A major mechanism whereby these
processes are controlled is through the actions of
polypeptides known as "growth factors". These
structurally diverse molecules act through specific cell
surface receptors to produce these actions.
Of particular importance are those growth factors,
termed "neurotrophic factors", that promote the
differentiation, growth and survival of neurons and
reside in the nervous system or in innervated tissues.
Nerve growth factor (NGF) was the first neurotrophic
factor to be identified and characterized (Levi-
Montalcini et al., J. Exp. Zool. 116:321, 1951 which is
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2
incorporated by reference). NGF exists as a non-
covalently bound homodimer that promotes the survival and
growth of sympathetic, neural crest-derived sensory, and
basal forebrain cholinergic neurons.
In recent years it has become apparent that growth
factors fall into classes, i.e. families or superfamilies
based upon the similarities in their amino acid
sequences. Examples of such families that have been
identified include the fibroblast growth factor family,
the neurotrophin family and the transforming growth
factor-beta (TGF-B) family. As an example of family
member sequence similarities, TGF-a family members have 7
canonical framework cysteine residues which identify
members of this superfamily.
NGF is the prototype member of the neurotrophin
family. Brain-derived neurotrophic factor (BDNF), the
second member of this family to be discovered, was shown
to be related to NGF by virtue of the conservation of all
six cysteines that form the three internal disulfides of
the NGF monomer (Barde, Prog Growth Factor Res 2:237-248,
1990 and Liebrock et al. Nature 341:149-152, 1989 which
are incorporated by reference). By utilizing the
information provided by BDNF of the highly conserved
portions of two factors, additional members (NT-3, NT-
4/5) of this neurotrophin family were rapidly found by
several groups (Klein, FASEH J 8:738-44, 1994 which is
incorporated by reference).
Signal transduction for neurotrophins is mediated
by a family of closely related tyrosine kinase receptors
(Trk). The known members of the Trk family of receptors
are: TrkA, identified as the NGF receptor; TrkB, which
mediates signaling by BDNF and NT-4/5; and TrkC, which
transduces the signals of NT-3 (for review, see
(Tuszynski et al., Ann Neurol 35:59-S12, 1994 which is
incorporated by reference). In addition to these
preferential specificities, there is evidence of cross-
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3
talk between different members of the neurotrophin and
Trk families, particularly in fibroblast-based model
systems. For example, NT-3 stimulates phosphorylation of
TrkH expressed in fibroblasts with a dose-response
relationship equivalent to that of HDNF and NT-4/5 (Ip et
al, Neuron 10:137-149, 1993, incorporated herein by
reference), while in PC12 cells, the TrkB receptor is
100-fold more sensitive to stimulation by BDNF and NT-4/5
than by NT-3. NT-3 may also signal through TrkA,
although with different specificities than NGF (Ip et al,
supra and Cordon-Cardo et al, Cell 66:173-183, 1991,
incorporated herein by reference).
Recently, a new family of neurotrophic factors has
been identified whose members are not structurally
related to NGF and other neurotrophins but are
structurally similar to TGF-Vii. As described in copending
applications 08/519,777 and 08/615,944, which are
incorporated herein by reference, the known members of
this new family, which has been named TRN (TGF-(3 Related
Neurotrophic factors), are glial cell line-derived
neurotrophic factor (GDNF), neurturin (NTN), and
persephin (PSP).
The placement of human GDNF and NTN into the same
growth factor family is based on the similarities of
their physical structures and biological activities.
These two proteins have 42% identity in their amino acid
sequences including seven cysteine residues whose
positions are exactly conserved in neurturin and GDNF.
The biological activities of GDNF and NTN include
supporting the survival of rat superior cervical, nodose,
and dorsal root ganglion neurons in vitro, although NTN
is more potent than GDNF in promoting SCG survival
(Kotzbauer et al., supra). In addition, as disclosed in
the copending international patent application, WO
97/08196, which is incorporated herein by reference, the
accumulation of radiolabeled NTN in the sensory neurons
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following injection into partially crushed sciatic nerves
of rats can be blocked by a 100 fold excess of unlabeled
NTN or unlabeled GDNF, suggesting that NTN and GDNF
compete for the same receptor.
Recently, it was reported that GDNF acts through a
multicomponent receptor complex in which a transmembrane
signal transducing component, the Ret protein-tyrosine
kinase (Ret or Ret PTK), is activated upon the binding of
GDNF with another protein, called GDNF Receptor a (GDNFR-
a,) which has no transmembrane domain and is attached to
the cell surface via a glycosyl-phosphatidylinositol
(GPI) linkage (Durbec et al., Nature 381:789-793, 1996;
Jing et al., Cell 85:1113-1124, 1996; Treanor et al.,
Nature 382:80-83, 1996; Trupp et al., Nature 381:785-789,
1996, which are incorporated herein by reference). GDNF
also induces activation of the Ret PTK when a soluble
form of GDNFR-a is added to the culture medium along with
GDNF, demonstrating that GDNFR-a does not need to be
anchored to the cell membrane to interact with Ret (Jing
et al., supra). The formation of a functional GDNF
receptor complex by GDNFR-a and Ret is supported, in
part, by the observations that mice deficient in either
GDNF or Ret are phenotypically similar and that GDNFR-a
and Ret are expressed together in the developing nephron,
midbrain, and motor neurons, all known targets of GDNF
action.
Neuronal degeneration and death occur during
development, during senescence, and as a consequence of
pathological events throughout life. It is now generally
believed that neurotrophic factors regulate many aspects
of neuronal function, including survival and development
in fetal life, and structural integrity and plasticity in
adulthood. Since both acute nervous system injuries as
well as chronic neurodegenerative diseases are
characterized by structural damage and, possibly, by
disease-induced apoptosis, it is likely that neurotrophic
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factors play some role in these afflictions. Indeed, a
considerable body of evidence suggests that neurotrophic
factors may be valuable therapeutic agents for treatment
of these neurodegenerative conditions, which are perhaps
5 the most socially and economically destructive diseases
now afflicting our society. Nevertheless, because
different neurotrophic factors can act preferentially
through different receptors and on different neuronal
cell types, there remains a continuing need for the
identification and characterization of these receptors of
growth factors in the diagnosis and treatment of a
variety of acute and chronic diseases of the nervous
system.
Summary of the Invention:
Briefly, therefore, the present invention is
directed to the identification and isolation of
substantially purified polypeptides that mediate the
survival and growth promoting effects of neurotrophic
factors on neurons. Accordingly, the inventors herein
have succeeded in discovering that members of the TRN
growth factor family share receptors and signal
transduction pathways. In particular, the inventors have
discovered that signaling of NTN and GDNF through the Ret
tyrosine kinase receptor is mediated by a novel family of
co-receptors, referenced herein as TrnR (TGF-(3-related
neurotrophic factor Receptors). The TrnR co-receptor
family includes the known co-receptor protein GDNFR-a,
referred to herein as TrnRl, and a novel protein, TrnR2,
either of which can form a functional receptor complex
with Ret for both NTN and GDNF.
The existence of this co-receptor family was
established by the isolation and characterization of a
cDNA encoding TrnR2 which shows significant homology with
TrnRl. In particular, a comparison of their respective
predicted amino acid sequences revealed that human TrnRl
and human TrnR2 are 48~ identical at the amino acid level
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and share 30 of 31 cysteine residues with nearly
identical spacing, indicating a conserved cysteine
backbone structure. Both co-receptors also contain a
predicted N-terminal signal sequence, a putative C-
terminal GPI linkage signal peptide (GPIsp), and three
potential N-linked glycosylation sites.
Recently, new nomenclature for this family of GPI-
linked co-receptors for GDNF and neurturin has been
adopted by scientists in the field such that the official
name for the TrnR family is now GFRa, with individual
members of the family being named GFRal (previously known
as GDNFRa, TrnRl and RetLl), GFRa2 (previously TrnR2,
NTNRa. and RetL2) and GFRa3 (previously TrnR3).
Nomenclature Committee, Neuron 19(3):485, 1997. However,
the older TrnR nomenclature will be used herein.
Accordingly, the invention provides a
substantially purified TrnR2 polypeptide. It is believed
that TrnR2 homologs of different mammalian species have
at least 85~ amino acid sequence identity while amino
acid sequence identity may be as low as 65~ in TrnR2
homologs of non-mammalian species such as avian species.
TrnR2 polypeptides identified herein include predicted
precursor and mature forms of TrnR2 protein in which the
predicted mature protein lacks the N-terminal signal
sequence and the C-terminal GPIsp but is otherwise
identical to the precursor protein. Human precursor and
mature proteins have the predicted amino acid sequences
set forth in SEQ ID NOS:2 and 3, respectively. The
corresponding predicted precursor and mature forms of
mouse TrnR2 protein have the amino acid sequences shown
in SEQ ID NOS:5 and 6 (Figure 2). In addition, TrnR2
polypeptides of the invention include variants of human
and mouse precursor proteins translated from
alternatively spliced TrnR2 mRNA having the amino acid
sequences shown in SEQ ID NOS:7 and 8. Soluble TrnR2
polypeptides which lack a GPI anchor are also
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contemplated by the invention. Such soluble TrnR2
polypeptides include soluble forms of alternatively
spliced variants of TrnR2. TrnR2 polypeptides also
include biologically active fragments of the full-length
precursor or mature proteins which are capable of binding
a TRN growth factor, or which are capable of activating
the Ret PTK in the presence of the TRN growth factor, or
which are capable of eliciting in a host animal
antibodies specific for TrnR2.
The present invention also provides nucleotide
sequences that encode a TrnR2 polypeptide. Human
precursor and mature TrnR2 proteins are encoded by
residues 36 to 1427 and residues 99 to 1331,
respectively, of the nucleotide sequence set forth in SEQ
ID NO:1. Mouse precursor and mature TrnR2 proteins are
encoded by residues i to 1389 and residues 64 to 1296,
respectively, of the nucleotide sequence set forth in SEQ
ID N0:4.
Expression vectors and stably transformed cells
are also provided. The transformed cells can be used in
a method for producing a TrnR2 polypeptide.
In another embodiment, the present invention
provides a method for preventing or treating neuronal
degeneration comprising administering to a patient in
need thereof a therapeutically effective amount of a
TrnR2 polypeptide, optionally along with a
therapeutically effective amount of NTN or GDNF. A
patient may also be treated by implanting transformed
cells which express a TrnR2 polypeptide or a DNA sequence
which encodes TrnR2 into a patient's tissues which would
benefit from increased sensitivity to a TRN such as NTN
or GDNF. In another embodiment, a patient with neuronal
degeneration is treated by implanting neuronal cells
cultured and expanded by growth in the presence of TrnR2
and NTN or GDNF.
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Another embodiment provides a method for treating
tumor cells by administering a composition comprising an
effective amount of TrnR2 and an effective amount of NTN
or GDNF or a composition comprising DNA sequences
encoding TrnR2 and NTN or GDNF to produce a maturation
and differentiation of the cells.
Yet another embodiment involves the use of a
soluble TrnR2 polypeptide as an agonist of TRN growth
factors.
In another embodiment the present invention
provides isolated and purified TrnR2 antisense
polynucleotides.
The present invention also provides compositions
and methods for detecting TrnR2 expression. One method
detects TrnR2 protein using anti-TrnR2 antibodies and
other methods are based upon detecting TrnR2 mRNA using
recombinant DNA techniques.
Among the several advantages found to be achieved
by the present invention, therefore, may be noted the
provision of a new co-receptor for neurturin and GDNF
which mediates the ability of these growth factors to
maintain and prevent the atrophy, degeneration or death
of certain cells, in particular neurons; the provision of
other members of the TrnR family of growth factor
receptors by making available new methods capable of
obtaining said other family members; the provision of
methods for obtaining TrnR2 by recombinant techniques;
the provision of methods for preventing or treating
diseases producing cellular degeneration and,
particularly, neuronal degeneration; the provision of
methods for limiting the effects of TRN growth factors in
a patient; and the provision of methods that can detect
and monitor TrnR2 levels in a patient.
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Brief Description of the Drawinys
Figure 1 illustrates the homology of the amino
acid sequences for the predicted precursor forms of TrnRl
(human, SEQ ID N0:12; rat, SEQ ID N0:13) and TrnR2
(human, SEQ ID N0:2; mouse, SEQ ID N0:5) with identical
amino acid residues enclosed in boxes and shared cysteine
residues shaded;
Figure 2 illustrates the nucleotide sequence (SEQ
ID N0:4) and amino acid translation (SEQ ID N0:5) of the
long splice variant of precursor mouse TrnR2 with the
predicted N-terminal signal sequence and C-terminal
hydrophobic domain underlined, a potential GPI attachment
site indicated by a asterisk, the potential N-linked
glycosylation sites enclosed in boxes, and the amino acid
region missing in the short splice variant shaded;
Figures 3A-C illustrate the effect of NTN and GDNF
on Ret phosphorylation as detected by an immunoassay
using antibodies specific for phosphotyrosine and Ret in
(Fig. 3A) fibroblasts stably transfected with Ret alone
(Ret) or both Ret and the long splice variant of TrnR2
(Ret/TrnR2) and treated with GDNF or NTN or not treated
(-), (Fig. 3B) fibroblasts expressing both Ret and TrnR2-
LV which were pre-treated (+) or not treated (-) with
phosphatidylinositol-specific phospholipase C (PIPLC)
before growth factor treatment, and (Fig. 3C) fibroblasts
stably expressing both Ret and TrnR2-LV (TrnR2/Ret) or
Ret and TrnRl (TrnRl/Ret) and treated with increasing
amounts of NTN or GDNF;
Figure 3D illustrates the effect of GDNF, NTN, and
persephin (PSP) on Ret tyrosine phosphorylation as
detected by an immunoassay using antibodies specific for
phosphotyrosine and Ret in fibroblasts coexpressing Ret
and either the long splice variant of TrnR2 (TrnR2-LV) or
the short splice variant (TrnR2-SV);
Figure 3E illustrates the binding affinities of
soluble TrnR2-LV fused with the Fc region of human IgGl
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(R2-Ig) for GDNF, NTN and PSP as measured in an ELISA
binding assay;
Figure 4 illustrates the tissue distribution of
TrnR2 mRNA in adult mouse showing a Northern blot of
5 total RNA probed with a 3ZP-labeled TrnR2 cDNA fragment;
Figures 5A-D illustrates the expression of TrnRl,
TrnR2, and Ret in known sites of GDNF and/or NTN action
showing in situ hybridization analysis using 33P-labeled
RNA probes of tissue samples from (Fig. 5A) E14 mouse
10 (developing) ventral mesencephalon (vm), (Fig. 5B) adult
mouse spinal cord, (Fig. 5C) E14 mouse (developing)
kidney (k), gut (g) and dorsal root ganglia (drg), and
(Fig. 5D) adult rat superior cervical ganglion (SCG);
Figure 6 illustrates TrnRl, TrnR2, and Ret
expression in primary SCG cultures containing a
contaminating population of non-neuronal cells showing
the amount of different mRNAs at varying times after
removal of nerve growth factor as measured by reverse
transcription-polymerase chain reaction (RT-PCR) using
primers specific for Ret, TrnR2, neuron-specific enolase
NSE, TrnRl, and a Schwann cell marker (S100);
Figure 7 illustrates that expression of TrnRl, but
not TrnR2, is up-regulated in the distal sciatic nerve
after nerve injury as shown by Northern blot analysis of
total RNA isolated from normal sciatic nerve (N) and the
distal segment of sciatic nerve seven days post-
transection (7D) using 32P-labeled TrnRl and TrnR2 probes
and brain RNA as a positive control fox the detection of
TrnR2 mRNA.
Figure 8 illustrates the expression of GF (TRN)
receptors and neurturin in the adult mouse forebrain
showing darkfield photographs of coronal sections
analyzed by in situ hybridization using 33P-labeled
riboprobes to detect expression of GFRa-1 (TrnRl) (Fig.
8A), GFRa-2 (TrnR2) (Fig. 8B), Ret (Fig. 8C) and NTN
(Fig. 8D), in which the various regions are abbreviated
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as Cg-eingulate cortex, C1-claustrum, DHB-nucleus of the
diagonal band of Broca, DEn-dorsal endopiriform nucleus,
LS~.ateral septal nucleus, MS-medial septal nucleus,
Pir-piriform cortex, Tu-olfactory tubercle, and
VPwentral pallidum;
Figure 9 illustrates the expression of GF (TRN)
receptors and neurturin in in the neocortex, hippocampus,
thalamus, and hypothalamus showing darkfield photographs
of coronal sections of the adult mouse brain analyzed by
in situ hybridization using 33P-labeled riboprobes to
detect expression of (Fig. 8) GFRa-1 (TrnRl), (Fig. 8B)
GFRa-2 (TrnR2), (Fig. 8C) Ret, and (Fig. 8D) NTN, in
which the various regions are abbreviated as Th-thalamic
nuclei, A~-amygdala, H-hypothalamus, LD-3aterodorsal
nucleus of the thalamus, MD-mediodorsal nucleus of the
thalamus, MHb-medial habenula, Rt-reticular thalamic
nucleus, STh~ubthalamic nucleus, and ZI-zona incerta;
Figure 10 illustrates the expression of GF (TRN)
receptor components in the adult mouse midbrain showing
darkfield photographs of coronal sections of adult mouse
midbrain analyzed by in situ hybridization using 33P-
labeled riboprobes to detect expression of (Fig. l0A)
GFRa-1 (TrnRl) in the compacta region of the substantia
nigra, the VTA, the oculomotor nucleus and the
superficial layers of the superior colliculus, (Fig. lOB)
GFRa-2 in the compacta region of the substantia nigra, in
the VTA, and the oculomotor nucleus, (Fig. lOC) Ret mRNA
in the SNc and the VTA, in which the various regions are
abbreviated as 3-oculomotor nucleus, SN-substantia nigra,
SuN~-supramammillary nucleus, MGN-medial geniculate
nucleus, and VTA-central tegmental area;
Figure 11 illustrates the expression of NTN mRNA
in the supraoptic and paraventricular nuclei of the
hypothalamus showing (Fig. 11A) a darkfield photograph
and (Fig. 11B-11C) brightfield photographs at higher
magnification which show detection of NTN expression in
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magnocellular neurons in the supraoptic (Fig. 11B) and
paraventricular (Fig. 11C) nuclei with the various
regions abbreviated as PV--paraventricular nucleus,
SO-supraoptic nucleus, and 3V-third ventricle:
Figure 12 illustrates expression of GDNF and GF
(TRN) receptor mRNA in adult mouse midbrain and brainstem
showing darkfield photographs of coronal sections
analyzed by in situ hybridization using 33P-labeled
riboprobes to detect expression of (Fig. 12A) Ret mRNA in
cranial nerve nuclei 10 and 12, and in the
gigantocellular reticular nucleus (Gi), (Fig. 12B) GFRa-2
(TrnR2) in cranial nerve nuclei Sp5, 6, 7 and ventral
cochlear nucleus (VC). (Fig. 12C) GDNF in the VC and the
facial motor nucleus, (Fig. 12D) GFRa-1 in the facial
motor nucleus and in the dorsal cochlear nucleus (DC),
(Fig. 12E) GFRa-2 in the inferior colliculus the
tegmental nuclei, and the locus coeruleus, (Fig. 12F) Ret
mRNA in the trigeminal motor nucleus and the inferior
colliculus, with the various regions being identified as
6-~abducens nucleus, ~-Facial nucleus, 10-wagal motor
nucleus, 12-hypoglossal nucleus, Gi-gigantocellular
reticular nucleus, IC-inferior colliculus, LC~.ocus
coeruleus, Sp5-spinal trigeminal nucleus, Mo5-motor
trigeminal nucleus, Tg~egmental nuclei, VG-ventral
cochlear nucleus;
Figure 13 illustrates GF (TRN) receptor expression
in adult mouse cervical spinal cord showing darkfield
photographs of transverse sections analyzed by in situ
hybridization using 33P-labeled riboprobes to detect
expression of (Fig. 13A) GFRa-1 (TrnRl), (Fig. 13B) GFRa-
2 (TrnR2), and (Fig. 13C) Ret, with various regions
identified as DID-dorsal horn, and VH-ventral horn;
Figure 14 illustrates GF (TRN) receptor and NTN
mRNA expression in adult cerebellum showing darkfield
photographs of sagittal sections analyzed by in situ
hybridization using 33P-labeled riboprobes to detect
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expression of (Fig. 14A) GFRa-1 (TrnRl) in cells adjacent
to Purkinje neurons in the Purkinje layer, (Fig. 14B)
GFRa-2 (TrnR2) in the granule cell layer and in neurons
that appear to be Purkinje cells in the Purkinje layer,
(Fig. 14C) Ret in the Purkinje layer in cells surrounding
Purkinje neurons and in the molecular layer, and (Fig.
14D) NTN in the Purkinje and granule cell layers, with
the various regions abbreviated as Gr-granule cell layer,
P~urkinje layer, Mol--molecular layer.
Description of the Preferred Embodiments
The present invention is based upon the surprising
discovery that NTN, like GDNF, can stimulate Ret PTK
through the known co-receptor GDNFR-a and thereby cause
the activation of the mitogen-activated protein kinase
(MAPK) and phosphatidylinositol 3-kinase (PI-3-K)
intracellular signaling pathways. As the first co-
receptor known to mediate signaling by at least two
members of the TRN family of growth factors, GDNFR-a is
referred to herein as TrnRl. The unexpected discovery
that members of the TRN family of growth factors share a
receptor complex and signal transduction pathways led to
the identification, isolation and sequencing of a cDNA
encoding a novel second co-receptor for NTN and GDNF,
TrnR2. Prior to this invention, TrnR2 was unknown and
had not been identified as a discrete biologically active
substance, nor had it been isolated in pure form.
TrnR2 was identified by searching a database of
Expressed Sequence Tags (dbEST database) using the Basic
local alignment search tool (BLAST, Altschul et al.,
J.Mol.Biol. 215:403-410, 1990 incorporated herein by
reference) and the full length rat TrnRl protein sequence
(Gen8ank Accession No. U59486) as a query. Three human
ESTs (H12981, 802135, W73681) which showed only partial,
but significant, homology to rat TrnRl were identified by
the BLAST search. The determination and alignment of the
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14
complete sequences of these three ESTs, obtained from the
WashU-Merck EST project, indicated that they all encoded
partial cDNAs of an identical transcript.
The 5' end of the cDNA was obtained by the rapid
amplification of cDNA ends (RACE) technique using as
templates human brain and placenta cDNA libraries
(Marathon RACE libraries, Clontech, Palo Alto, CA) and
the Klentaq LA polymerase chain reaction (PCR) technique
described by Barnes, Proc.Natl.Acad.Sci.U.S.A. 91:2216-
2220, 1994, incorporated herein by reference. Two
alternatively spliced forms of TrnR2 mRNA were identified
in both brain and placenta, the short splice variant
(TrnR2-SV) is missing 399 nucleotides of the coding
sequence from the 5' end of the long splice variant
(TrnR2-LV) (residues 75 to 473 of SEQ ID NO:1). The
predicted amino acid sequence of the long splice variant
of human precursor TrnR2 contains 464 amino acids and is
shown in SEQ ID N0:2, the predicted amino acid sequence
for the mature protein is shown in SEQ ID N0:3. The
short splice variant has a predicted amino acid sequence
of 331 amino acids as set forth in SEQ ID N0:7,~, which
would be encoded by nucleotides 36-74 and 474-1427 of SEQ
ID NO:1. The corresponding mouse cDNAs for both the long
and short splice variants were also obtained by PCR,
using a brain cDNA template. The full length precursor
murine cDNA is set forth in SEQ ID N0:4 and contains a
single long open reading frame (ORF) encoding a predicted
protein of 463 amino acids (SEQ ID N0:5); the short
splice variant identified has an ORF encoding a predicted
330 amino acid poiypeptide (SEQ ID N0:8) which would be
encoded by nucleotides 1-39 and 438-1389 of SEQ ID N0:4.
All physical features of TrnR2 indicate that it is
closely related to TrnRl. As shown in Figure 1, the
predicted amino acid sequence for TrnR2-LV shows
significant homology with TrnRl. (The human and rat TrnRl
sequences (SEQ ID Nos. 12 and 13, respectively) are those
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reported by Jing et al., supra.) The predicted protein
for precursor TrnR2 contains a putative 21 amino acid
signal sequence (residues 1-21 of SEQ ID N0:2 and SEQ ID
N0:5 for human and mouse proteins, respectively) at the
5 amino terminus, three potential N-linked glycosylation
sites, and has a stretch of 16 carboxyl-terminal
hydrophobic amino acids (residues 449-464 of SEQ ID N0:2
and 448-463 of SEQ ID N0:5 for human and mouse proteins,
respectively). The presence of the N- and C-terminal
10 hydrophobic regions indicates that mature TrnR2 is
potentially a GPI-linked protein (Udenfriend and
Kodukula, Ann. Rev. Biochem. 64:563-591, 1995,
incorporated herein by reference), as has been
demonstrated for TrnRl (Treanor et al. supra; Jing et
15 al., supra). A potential GPI attachment site for the
human and mouse long splice variants is the glycine
residue at position 411 of SEQ ID N0:2 and 3,
respectively. Accordingly, the predicted GPI attachment
site in the short splice variant is the glycine residue
at position 299 of SEQ ID N0:7 for the human protein and
at position 299 of SEQ ID N0:8 for the mouse protein.
The inventors herein have found significant
functional similarities and dissimilarities between TrnR2
and TrnRl. Experimental data which is discussed below
indicate that either NTN or GDNF can activate Ret in the
presence of either TrnR2 or TrnRl. In addition, Ret
activation in the presence of either co-receptor responds
to stimulation with either NTN or GDNF in a dose-
dependent manner. However, while the TrnRl/Ret receptor
complex responds equivalently to the same amounts of each
ligand, the TrnR2/Ret complex is more sensitive to NTN
than GDNF, indicating that, at least in the model system
examined, the latter complex may function preferentially
as a NTN receptor. Also, TrnRl and TrnR2 are expressed
in a partially overlapping manner. While both co-
receptors are expressed in the dorsal root ganglia (DRG)
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and the brain, TrnR2 is not expressed or expressed at
very low levels in several known targets of GDNF action
in which both TrnRl and Ret are expressed, including
embryonic and adult nigra, motor neurons, gut and kidney.
In contrast, TrnR2 and Ret appear to comprise the
expressed receptor complex in SCG neurons.
Although the data indicate that a physiological
pairing of ligand and receptor may exist for the TrnR
family as it does for the Trk family, there is also in
vivo evidence of cross-talk between the TRN ligands and
their receptors as observed in several ligand-receptor
systems having multiple family members, including between
members of the neurotrophin growth factor and Trk
receptor families. For example, the inventors herein
have found that neuroblastoma cell lines may express
either TrnRl, TrnR2 or both, but despite this
heterogeneity, those cell lines which respond to GDNF or
NTN always respond to both factors. Thus, it is believed
that TrnR2, TrnRl or other as yet unidentified members of
the TrnR family can combine in vivo with Ret to form a
functional receptor complex for NTN and GDNF, and
possibly for persephin and other as yet unidentified
members of the TRN growth factor family as well.
Accordingly, the invention provides a
substantially purified TrnR2 polypeptide. A TrnR2
polypeptide of the invention includes growth factor
receptors of any origin which are substantially
homologous to and which are biologically equivalent to
the human or mouse TrnR2 polypeptides characterized and
described herein. Such substantially homologous growth
factor receptors may be native to any tissue or species
and, similarly, biological activity can be characterized
in any of a number of biological assay systems.
The term "biologically equivalent" is intended to
mean that the compositions of the present invention are
capable of demonstrating some or all of the same signal
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17
mediating properties in a similar fashion, not
necessarily to the same degree, as the recombinantly
produced human or mouse TrnR2.
Hy "substantially homologous" it is meant that the
degree of amino acid homology of human or mouse TrnR2 to
a TrnR2 from any species is greater than that between
TrnR2 and TrnRl (GDNFR-a).
Sequence identity or percent identity is intended
to mean the percentage of identical residues between two
sequences, referenced to human TrnR2 when determining
percent identity with non-human TrnR2, referenced to
TrnR2 when determining percent identity with non-TrnR2
growth factor receptors and referenced to human TrnRl
when determining percent identity of non-TrnR2 growth
factor receptors with TrnRl, when-the two sequences are
aligned using the Clustlal method (Higgins et al, Cabios
8:189-191, 1992) of multiple sequence alignment in the
Lasergene biocomputing software (DNASTAR, INC, Madison,
WI). In this method, multiple alignments are carried out
in a progressive manner, in which larger and larger
alignment groups are assembled using similarity scores
calculated from a series of pairwise alignments. Optimal
sequence alignments are obtained by finding the maximum
alignment score, which is the average of all scores
between the separate residues in the alignment,
determined from a residue weight table representing the
probability of a given amino acid change occurring in two
related proteins over a given evolutionary interval.
Penalties for opening and lengthening gaps in the
alignment contribute to the score. The default
parameters used with this program are as follows: gap
penalty for multiple alignment = 10; gap length penalty
for multiple alignment = 10; k-tuple value in pairwise
alignment = 1; gap penalty in pairwise alignment = 3;
window value in pairwise alignment = 5; diagonals saved
in pairwise alignment = 5. The residue weight table used
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18
for the alignment program is PAM250 (Dayhoff et al., in
Atlas of Protein Sequence and Structure, Dayhoff, Ed.,
NHRF, Washington, Vol. 5, suppl. 3, p. 345, 1978).
Percent conservation is calculated from the above
alignment by adding the percentage of identical residues
to the percentage of positions at which the two residues
represent a conservative substitution (defined as having
a log odds value of greater than or equal to 0.3 in the
PAM250 residue weight table). Conservation is referenced
to human TrnR2 when determining percent conservation with
non-human TrnR2, and referenced to TrnR2 when determining
percent conservation with non-TrnR2 growth factor
receptors. Conservative amino acid changes satisfying
this requirement are: R-K; E-D, Y-F, L-M; V-I, Q-H. The
calculations of identity (I) and conservation (C) between
human and mouse TrnR2 (hTrnR2 and mTrnR2, respectively)
and between each of these and human and rat TrnRl (hTrnRl
and rTrnRl, respectively) are shown in Table 1.
Table 1
COMPARISON $ IDENTITY $ CONSERVATION
hTrnR2 v. mTrnR2 94 95
hTrnR2 v. hTrnRl 48 53
hTrnR2 v. rTrnRl 47 52
mTrnR2 v. hTrnRl 43 47
mTrnR2 v. rTrnRl 47 52
The degree of homology between the predicted
precursor mouse and human TrnR2 proteins is about 94$
sequence identity and all TrnR2 homologs of non-human
mammalian species are believed to have at least about 85$
sequence identity with human TrnR2. For non-mammalian
species such as avian species, it is believed that the
degree of homology with TrnR2 is at least about 65$
identity. By way of comparison, the variations between
members of the TrnR family of receptors can be seen by
comparing TrnRl and TrnR2 (Fig. 1). Human and mouse
precursor TrnR2 share about 94$ identical amino acids and
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19
have about 53% and 52% sequence conservation with human
_ and rat precursor TrnRl, respectively. It is believed
that the different TrnR family members similarly have a
sequence identity of about 40% to that of TrnR2 and about
40% to that of TrnRl and within a range of about 30% to
about 85% identity with TrnR2 and within a range of about
30% to about 85$ sequence identity with TrnRl. Thus, a
given non-TrnR2 and non-TrnRl family member from one
species would be expected to show lesser sequence
identity with TrnR2 and with TrnRl from the same species
than the sequence identity between human TrnR2 and TrnR2
from a non-human mammalian species, but greater sequence
identity than that between human TrnR2 and any other
known growth factor receptor except TrnRl.
A TrnR2 polypeptide of the invention can also
include hybrid and modified forms of TrnR2 and fragments
thereof in which certain amino acids have been deleted or
replaced and modifications such as where one or more
amino acids have been changed to a modified amino acid or
unusual amino acid and modifications such as
glycosylations so long as the hybrid or modified form
retains TrnR2 biological activity. By TrnR2 biological
activity, it is meant that Ret PTK is activated in the
presence of the hybrid or modified TrnR2 and NTN or GDNF
or other TRN growth factor, although not necessarily at
the same level of potency as that of TrnR2 isolated from
tissues or cells which naturally produce TrnR2 such as
SCG neurons or that of the recombinantly produced human
or mouse TrnR2.
Also included within the meaning of substantially
homologous is any TrnR2 polypeptide which may be isolated
by virtue of cross-reactivity with antibodies to the
TrnR2 described herein or whose encoding nucleotide
sequences including genomic DNA, mRNA or cDNA may be
isolated through degenerate PCR or by hybridization with
the complementary sequence of genomic or subgenomic
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nucleotide sequences or cDNA of the human or mouse TrnR2
described herein or fragments thereof. It will also be
appreciated by one skilled in the art that allelic
variants of TrnR2 are included within the present
5 invention.
Isolation of cDNAs corresponding to two
alternatively spliced TrnR2 mRNAs of different lengths
indicate a TrnR2 protein product for each spliced variant
may exist. Thus, any and all TrnR2 proteins encoded by
10 alternatively spliced TrnR2 mRNAs are intended to be
included within the term "TrnR2 polypeptide" as used
herein.
The predicted amino acid sequence and biological
function of TrnR2 indicate that it is an externally
15 disposed plasma membrane protein anchored to the
extracellular surface of the cell membrane by a glycosyl-
phosphatidyl (GPI) linkage. It is well-known in the art
that GPI anchored membrane proteins are synthesized as a
precursor protein with an N-terminal signal sequence and
20 a C-terminal GPIsp which are cleaved during the cellular
processing events leading to the mature protein. Thus,
all precursor TrnR2 proteins containing either or both of
the signal sequence and GPIsp as well as mature TrnR2
proteins which contain neither signal peptide are
embraced by the term "TrnR2 polypeptide". It is also
well-recognized in the art that GPI anchored proteins may
exist in soluble form following cleavage of the GPI
linkage with phospholipases. Therefore, the term "TrnR2
polypeptide also includes soluble TrnR2 polypeptides
generated by phospholipase cleavage of anchored TrnR2
polypeptides.
A preferred TrnR2 polypeptide has an amino acid
sequence selected from the group consisting of SEQ ID
N0:2, SEQ ID N0:3, SEQ ID N0:5, and SEQ ID N0:6. A more
preferred TrnR2 polypeptide is human mature TrnR2 protein
which has the amino acid sequence set forth in SEQ ID
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- 21
N0:3.
The term "TrnR2 polypeptide" is intended to include
fragments which have one or more of the biological
activities of precursor or mature TrnR2 protein. Such
activities include binding a member of the TRN growth
factor family, particularly NTN or GDNF, and binding to
Ret in the presence of NTN, GDNF, or other TRN family
member, with such binding leading to Ret phosphorylation.
It is believed that by using the nucleotide sequences
encoding precursor TrnR2, which are provided herein,
those skilled in the art can readily construct multiple
TrnR2 fragments and screen them for the desired
biological activity.
A preferred TrnR2 fragment is one which lacks the
hydrophobic domain for the GPI-attachment site which are
referred to herein as soluble TrnR2 fragments. For
example, soluble TrnR2 fragments include, but are not
limited to, polypeptides having an amino acid sequence
encoded by nucleotide residues 99 to 1331 of SEQ ID NO:1
(human long splice variant), nucleotides 36-74 and 474-
1331 of SEQ ID NO:1 (human short splice variant),
nucleotide residues 64-1296 of SEQ ID N0:4 (mouse long
splice varaint), nucleotides 1-39 and 438-1296 (mouse
short splice variant), and fra~nts thereof.
TrnR2 fragments also included in the scope of the
invention are antigenic fragments which are capable of
eliciting TrnR2 specific antibodies when administered to
a host animal as conjugated to a carrier molecule or in
nonconjugated form.
A TrnR2 protein of the present invention may be
- isolated in purified form from tissues or cells which
naturally produce TrnR2. Such tissues or cells may
originate from any eukaryotic organism that naturally
produce TrnR2. Alternatively, a substantially pure TrnR2
polypeptide may be prepared by recombinant DNA
technology. Hy "pure form" or "purified form" or
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. 22
"substantially purified form" it is meant that a TrnR2
composition is substantially free of other proteins which
are not TrnR2.
One skilled in the art can readily follow known
methods for isolating proteins in order to obtain the
TrnR2 polypeptide substantially free of other proteins,
including immunochromatography, size-exclusion
chromatography, HPLC, ion-exchange chromatography, and
ligand affinity chromatography. As readily appreciated
by those skilled in the art, an example of one way to
obtain TrnR2 protein naturally produced by cells in
culture would be to treat the cells with PI-PLC to cleave
the GPI-linked TrnR2 protein from the cell surface, and
then purifying the soluble TrnR2 protein from the media
by ligand affinity chromatography using NTN or GDNF as
the ligand or by immunochromatography using an antibody
raised against TrnR2 protein or an antigenic TrnR2
peptide.
A recombinant TrnR2 polypeptide may be made by
expressing the DNA sequences encoding TrnR2 in a suitable
transformed host cell. Using methods well known in the
art, the DNA encoding the TrnR2 polypeptide may be linked
to an expression vector, transformed into a host cell and
conditions established that are suitable for expression
of the TrnR2 polypeptide by the transformed cell.
Any suitable expression vector may be employed to
produce recombinant TrnR2 such as, for example, the
mammalian expression vector pCMV-neo (Brewer, Meth.Cel1
Hiol. 43:233-245, 1994, incorporated herein by reference)
which was used herein or the E. coli pET expression
vectors, in particular, pET-30a (Studier et al., Methods
Enzymol. 185:60-89, 1990 which is incorporated by
reference). Other suitable expression vectors for
expression in mammalian and bacterial cells are known in
the art as are expression vectors for use in yeast or
insect cells. Baculovirus expression systems can also be
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. 23
employed.
In another embodiment, the present invention
provides an isolated and purified polynucleotide
comprising a nucleotide sequence that encodes a TrnR2
polypeptide. Nucleotide sequences included in the
invention are those encoding human or mouse precursor and
mature TrnR2 proteins. Preferred nucleotide sequences
encoding human proteins are as set forth in SEQ ID NO:1:
nucleotides 36-1427 encode a precursor TrnR2 and
nucleotides 99-1331 encode a human mature TrnR2.
Similarly, preferred nucleotide sequences which encode a
mouse precursor and a mouse mature protein are
nucleotides 1-1389 and nucleotides 64-1296 of SEQ ID
N0:4, respectively. Nucleotide sequences encoding TrnR2
fragments are also contemplated, particularly soluble
TrnR2 fragments. Preferred nucleotide sequences encoding
a soluble TrnR2 fragment are residues 99 to 1331 of SEQ
ID NO:l (human long splice variant), nucleotides 36-74
and 474-1427 of SEQ ID NO:1 (human short splice variant),
residues 64-1296 of SEQ ID N0:4 (mouse long splice
variant) and nucleotides 1-39 and 438-1389 of SEQ ID N0:4
(mouse short splice variant). It is understood by the
skilled artisan that degenerate DNA sequences can encode
the TrnR2 polypeptides described herein and these are
also intended to be included within the present
invention.
Based upon the high sequence conservation between
the human and mouse TrnR2 coding sequences, it is
believed that DNA probes and primers can be made and used
to readily obtain TrnR2-encoding cDNA clones from
a different species. Thus, a cDNA encoding a TrnR2 from a
species other than human or mouse is embraced by the
invention.
Also included within the scope of this invention are
nucleotide sequences that are substantially the same as a
nucleic acid sequence encoding TrnR2. Substantially the
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24
same sequences may, for example, be substituted with
codons more readily expressed in a given host cell such
as E. coli according to well known and standard
procedures. Such modified nucleic acid sequences would
be included within the scope of this invention.
Specific nucleic acid sequences can be modified by
those skilled in the art and, thus, all nucleic acid
sequences which encode for the amino acid sequences of
TrnR2 or biologically active fragments thereof can
likewise be so modified. The present invention thus also
includes polynucleotides containing a nucleic acid
sequence which will hybridize with all such nucleic acid
sequences -- or complements of the nucleic acid sequences
where appropriate -- and encode for a polypeptide having
biological activity as a coreceptor for NTN or GDNF. The
present invention also includes nucleic acid sequences
which encode for polypeptides that have one or more of
the biological activities of TrnR2 and those that are
recognized by antibodies that bind to TrnR2.
The cDNA sequences provided herein allow genomic
clones for the TrnR2 gene to be readily isolated. One
use for genomic clones is far chromosome localization
studies. For example, human and mouse genomic clones for
the TrnR2 gene were obtained by screening P1 (mouse) and
PAC (human) genomic libraries (Genome Systems, St. Louis,
MO) with a PCR assay using primers derived from the TrnR2
coding region. A human PAC genomic clone containing the
TrnR2 gene in a 120 kb genomic fragment was used to
localize the TrnR2 gene to the short arm of human
chromosome 8 in region p12-21 by fluorescence in situ
hybridization analysis (FISH). The mouse chromosomal
location can readily be determined in a similar fashion
using a mouse genomic clone.
A search of the database for neurological diseases
genetically mapped to the human locus revealed only one
such disease, SPGSA, an autosomal recessive form of
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spastic paraplegia, localized to the paracentric region
of chromosome 8 (Hentati et al., Hum.Molec.Genet. 3:1263-
.. 1267, 1994, incorporated herein by reference). Also, an
amplification event on 8p12 has been observed in some
5 cases of breast and ovarian cancer (Imbert et al.,
Genomics 32:29-38, 1996, incorporated herein by
reference). Genomic clones for the TrnR2 gene are also
useful for surveying for possible gene or chromosome
rearrangements in patients suffering from a neurological
10 disease with no identified cause.
The present invention also encompasses vectors
comprising expression regulatory elements operably linked
to any of the nucleic acid sequences included within the
scope of the invention. This invention also includes
15 host cells, of any variety, that have been transformed
with vectors comprising expression regulatory elements
operably linked to any of the nucleic acid sequences
included within the scope of the present invention.
In one embodiment, recombinant cells expressing both
20 Ret and TrnR2 are provided which are useful for screening
compounds for TRN growth factor agonistic or antagonistic
activity. The recombinant cells may be produced by
transforming a suitable host cell such as fibroblasts
with nucleotide sequences encoding for expression Ret and
25 TrnR2 proteins. The protein-encoding nucleotide
sequences may be on the same or on different vectors.
Ret-encoding nucleotide sequences may be readily isolated
by screening a suitable cDNA library using an
oligonucleotide probe corresponding to a region of the
known human and/or mouse amino acid sequences (Iwamoto,
_ et al., Oncogene 8, 1087-1091, 1993 incorporated herein
by reference). Suitable cDNA libraries would be those
prepared from tissues known to express Ret, including but
not limited to placental tissue.
Agonistic or antagonistic activity of a test
compound would be determined by incubating the target
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26
Ret/TrnR2-expressing cells with the test compound in the
absence or presence of a TRN growth factor such as NTN,
GDNF, or persephin and assaying for Ret protein tyrosine
kinase activity. Compounds which increase Ret PTK
activity in the absence of a TRN have agonistic activity,
while compounds which reduce or block Ret PTK activity in
the presence of a TRN are TRN antagonists.
The Ret PTK activity may be assayed by looking for
tyrosine phosphorylation of Ret as described herein.
Alternatively, or additionally, the target cell may be
engineered to include a reporter gene whose expression a.s
under the control of a TRN-responsive enhancer/promoter
region. NTN and GDNF are known to cause an increase in
mitogen-activated protein kinase (MAPK) activation in SCG
neurons (Kotzbauer, et al., Nature 384:467-470, 1996
incorporated herein by reference) and the inventors
herein have also discovered that phosphatidylinositol 3-
kinase (PI-3-K) is also activated by NTN and GDNF. Thus,
expression of a reporter gene operably linked to the
enhancer/promoter regions of genes downstream in the MAPK
or PI-3-K intracellular signalling pathways would be
expected to be increased in the presence of a TRN agonist
and decreased in the presence of a TRN antagonist. It is
believed that such enhancer/promoter regions are known to
those skilled in the art and can be readily isolated.
Known reporter genes which encode for readily detectable
products include, but are not limited to, a-
galactosidase, chloramphenicol acetyl transferase,
luciferase and a-glucuronidase. Detection of the
expression of known reporter genes, which is well known
to those skilled in the art, may serve as a sensitive
indicator for any NTN or GDNF agonistic activity of test
compounds.
Methods are also provided herein for producing TrnR2
polypeptides. Preparation can be by isolation from a
variety of cell types so long as the cell type expresses
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. 27
TrnR2 protein. Examples of biological material suitable
_ for TrnR2 isolation include, but are not limited to,
brain tissue, human neuroblastoma cell lines, and
superior cervical ganglion cells. A second and preferred
method involves utilization of recombinant methods by
isolating a nucleic acid sequence encoding a TrnR2
polypeptide, cloning the sequence along with appropriate
regulatory sequences into suitable vectors and cell
types, and expressing the sequence to produce TrnR2. In
one embodiment, the nucleotide sequence does not encode
the C-terminal hydrophobic domain containing the GPI-
attachment site, thus producing a soluble TrnR2 fragment
that is secreted into the growth medium.
The present invention also provides probes which may
be used to identify cells and tissues which may be
responsive to NTN or GDNF in normal or disease conditions
by detecting TrnR2 expression in such cells. Detection
of TrnR2 expression may also be useful to determine if a
patient suffering from a NTN- or GDNF-related disorder
has aberrant TrnR2 expression or expresses a biologically
inactive TrnR2 mutant. TrnR2 expression may be detected
with probes which react with TrnR2 mRNA or TrnR2 protein.
For example, to detect the presence of mRNA encoding
a TrnR2 polypeptide or biologically inactive mutant
thereof, a sample is obtained from a patient. The sample
may be from blood or a tissue biopsy. The sample may be
treated to extract the nucleic acids contained therein
which may then be subjected to gel electrophoresis or
other size separation techniques.
The mRNA of the sample is contacted with a
- polynucleotide probe comprising a nucleic acid sequence
complementary to TrnR2 mRNA. The polynucleotide probe
may be an oligonucieotide containing a minimum of about 8
to 12, preferably at least about 20, contiguous
nucleotides which are complementary to the TrnR2 target
sequence. Oligonucleotide probes may be prepared by any
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28
method known in the art such as, for example, excision,
transcription or chemical synthesis. Alternatively, the
polynucleotide probe may comprise a cDNA encoding TrnR2
or a fragment thereof as a probe.
To enable detection of hybridization between the
polynucleotide probe and the target sequence, the probe
may be labelled with any detectable label known in the
art such as, for example, radioactive or fluorescent
labels or enzymatic markers. Labeling of the probe can
be accomplished by any method known in the art such as by
PCR, random priming, end labelling, nick translation or
the like. One skilled in the art will also recognize
that other methods not employing a labelled probe can be
used to determine the hybridization. Examples of methods
that can be used for detecting hybridization include
Southern blotting, fluorescence in situ hybridization,
and single-strand conformation polymorphism with PCR
amplification.
Hybridization conditions for the type of probe used
may be readily determined by those skilled in the art.
High stringency conditions are preferred in order to
prevent false positives. The stringency of hybridization
is determined by a number of factors in the hybridization
and washing steps. Such factors are well known to those
skilled in the art and outlined in, for example, Sambrook
et al. (Sambrook, et al., 1989, supra).
The sensitivity of detection in a sample of TrnR2
mRNA may be increased using the technique of reverse
transcription/polymerization chain reaction (RT/PCR) to
amplify cDNA transcribed from TrnR2 mRNA using primers
specific for a TrnR2-encoding nucleotide sequence (see
example 4 and Fig. 6 below). The method of RT/PCR is
well known and routinely performed by those skilled in
the art.
The present invention further provides for methods
to detect the presence of the TrnR2 protein or
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29
biologically inactive mutants thereof in a sample
_ obtained from a patient. Any method known in the art for
detecting proteins can be used. Such methods include,
but are not limited to immunodiffusion,
immunoelectrophoresis, immunochemical methods, binder-
ligand assays, immunohistochemical techniques,
agglutination and complement assays. (For example, see
Basic and Clinical. Immunology, Sites and Terr, eds.,
Appleton & Lange, Norwalk, Conn. pp 217-262, 1991 which
is incorporated by reference). Preferred are binder-
ligand immunoassay methods which involve reacting
antibodies with an epitope or epitopes of a TrnR2 protein
or derivative thereof to competitively displace a labeled
TrnR2 polypeptide.
As used herein, a derivative_of the TrnR2 protein is
intended to include a polypeptide in which certain amino
acids have been deleted or replaced or changed to
modified or unusual amino acids wherein the derivative is
biologically equivalent to TrnR2 and wherein the
polypeptide derivative cross-reacts with antibodies
raised against the TrnR2 protein. By cross-reaction it
is meant that an antibody reacts with an antigen other
than the one that induced its formation.
Numerous competitive and non-competitive protein
binding immunoassays are well known in the art.
Antibodies as used herein are intended to include full-
length anti-TrnR2 antibody molecules and TrnR2 binding
fragments of such antibody molecules. The anti-TrnR2
antibody may be unlabeled, for example as used in
agglutination tests, or labeled for use in a wide variety
r of assay methods. Labels that can be used include
radionuclides, enzymes, fluorescers, chemiluminescers,
enzyme substrates or co-factors, enzyme inhibitors,
particles, dyes and the like for use in radioimmunoassay
(RIA), enzyme immunoassays, e.g., enzyme-linked
immunosorbent assay (ELISA), fluorescent immunoassays and
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the like.
Polyclonal or monoclonal antibodies to the TrnR2
protein or an epitope thereof can be made for use in
immunoassays by any of a number of methods known in the
5 art. By epitope reference is made to an antigenic
determinant of a polypeptide. An epitope could comprise
3 amino acids in a spacial conformation which is unique
to the epitope. Generally an epitope consists of at
least 5 such amino acids. Methods of determining the
10 spatial conformation of amino acids are known in the art,
and include, for example, x-ray crystallography and 2
dimensional nuclear magnetic resonance.
One approach for preparing antibodies to a protein
is the selection and preparation of an amino acid
15 sequence of all or part of the protein, chemically
synthesizing the sequence and injecting it into an
appropriate animal, usually a rabbit or a mouse (See
Example 11).
Oligopeptides can be selected as candidates for the
20 production of an antibody to the TrnR2 protein based upon
the oligopeptides lying in hydrophilic regions, which are
thus likely to be exposed in the mature protein.
Antibodies to TrnR2 can also be raised against
oligopeptides that include one or more of the conserved
25 regions identified herein such that the antibody can
cross-react with other family members. Such antibodies
can be used to identify and isolate the other family
members.
Methods for preparation of the TrnR2 protein or an
30 epitope thereof include, but are not limited to chemical
synthesis, recombinant DNA techniques or isolation from
biological samples. Chemical synthesis of a peptide can
be performed, for example, by the classical Merrifeld
method of solid phase peptide synthesis (Merrifeld, J Am
Chem Soc 85:2149, 1963 which is incorporated by
reference) or the FMOC strategy on a Rapid Automated
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31
Multiple Peptide Synthesis system (DuPont Company,
Wilmington, DE) (Caprino and Han, J Org Chem 37:39:04,
1972 which is incorporated by reference).
Polyclonal antibodies can be prepared by immunizing
rabbits or other animals by injecting antigen followed by
subsequent boosts at appropriate intervals. The animals
are bled and sera assayed against purified TrnR2 protein,
usually by ELISA or by bioassay based upon the ability to
block one or more of the biological activities of TrnR2.
When using avian species, e.g. chicken, turkey and the
like, the antibody can be isolated from the yolk of the
egg. Monoclonal antibodies can be prepared after the
method of Milstein and Kohler by fusing splenocytes from
immunized mice with continuously replicating tumor cells
such as myeloma or lymphoma cells. (Milstein and Kohler
Nature 256:495-497, 1975; Gulfre and Milstein, Methods in
Enzymology: Immunochemical Techniques 73:1-46, Langone
and Banatis eds., Academic Press, 19$1 which are
incorporated by reference). The hybridoma cells so
formed are then cloned by limiting dilution methods and
supernates assayed for antibody production by ELISA, RIA
or bioassay.
If a patient suffering from a GDNF or NTN-related
disorder expresses apparently normal levels of TrnR2, a.t
may be possible that the expressed TrnR2 may be a
biologically inactive mutant. To verify this, cDNA
obtained from mRNA isolated from a sample of a relevant
target tissue may be sequenced using methods known in the
art.
The present invention also includes therapeutic or
pharmaceutical compositions comprising an effective
amount of a TrnR2 polypeptide for treating patients with
cellular degeneration and a method for promoting cell
survival which comprises administering to a patient in
need thereof a therapeutically effective amount of a
TrnR2 polypeptide.
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Certain degeneration disorders may be related to a
lack of or reduced expression of biologically active
TrnR2, while NTN or GDNF expression is normal. In
addition, it may be desirable under certain circumstances
to increase TrnR2 levels even where TrnR2 expression is
not decreased. It has been shown that soluble TrnRl
added to Ret-expressing cells, i.e., the TrnRl is not
bound to the membrane, can activate Ret in the presence
of GDNF (Ding et al, supra). Thus, it is believed that
administering a TrnR2 polypeptide will increase the
number of cells which have a functional TrnR2/Ret
receptor complex and thus capable of responding to
endogenously produced NTN and/or GDNF.
Additional survival or growth promoting effects may
be achieved by administering NTN and/or GDNF along with
the TrnR2 polypeptide. It is believed that treatment
with one or both of these growth factors together with a
TrnR co-receptor would increase the sensitivity of cells
normally responsive to the growth factor(s). In
addition, such treatment would be expected to promote the
survival or growth of other cell types that express Ret
but that are not normally responsive to NTN or GDNF.
Alternatively, expression of TrnR2 could be
increased in tissues defective in such expression by gene
therapy. Patients may be implanted with vectors or cells
capable of producing a biologically-active TrnR2
polypeptide. In one approach, cells that secrete soluble
TrnR2 may be encapsulated into semipermeable membranes
for implantation into a patient. The cells can be those
that normally express a TrnR2 protein or the cells can be
transformed to express a TrnR2 poiypeptide. When the
patient is human, it is preferred that the TrnR2 be human
TrnR2. However, the formulations and methods herein can
be used for veterinary as well as human applications and
the term "patient" as used herein is intended to include
human and veterinary patients.
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Cells can be grown ex vivo, for example, for use in
transplantation or engraftment into patients (Muench et
al., Leuk & Lymph 16:1-11, 1994 which is incorporated by
reference). TrnR2 in combination with NTN or GDNF can be
administered to such cells to elicit growth and
differentiation, provided the cells express Ret. Ret
expression has been observed during embryogenesis in many
cell lineages of the developing central and peripheral
nervous systems. Ret has also been detected outside the
nervous system as well, including gut and kidney. Thus,
in another embodiment of the present invention, a
composition comprising TrnR2 and NTN or GDNF is used to
promote the ex vivo expansion of Ret-expressing cells for
transplantation or engraftment.
These compositions and methods are useful for
treating a number of degenerative diseases. Where the
cellular degeneration involves neuronal degeneration, the
diseases include, but are not limited to peripheral
neuropathy, amyotrophic lateral sclerosis, Alzheimer's
disease, Parkinson's disease, Huntington's disease,
ischemic stroke, acute brain injury, acute spinal chord
injury, nervous system tumors, multiple sclerosis,
peripheral nerve trauma or injury, exposure to
neurotoxins, metabolic diseases such as diabetes or renal
dysfunctions and damage caused by infectious agents.
Where the cellular degeneration involves bone marrow cell
degeneration, the diseases include, but are not limited
to disorders of insufficient blood cells such as, for
example, leukopenias including eosinopenia and/or
basopenia, lymphopenia, monocytopenia, neutropenia,
anemias, thrombocytopenia as well as an insufficiency of
stem cells for any of the above. The above cells and
tissues can also be treated for depressed function.
The compositions and methods herein can also be
useful to prevent degeneration and/or promote survival in
other non-neuronal tissues as well. One skilled in the
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art can readily determine using a variety of assays known
in the art whether a particular cell type expresses Ret
and would thus likely be activated in the presence of
TrnR2 and a TRN such as NTN or GDNF.
In certain circumstances, it may be desirable to
modulate or decrease the trophic effect of endogenously
synthesized TRN growth factors, including NTN and/or
GDNF. This may be achieved by blocking binding of the
growth factor to its receptor in the target tissue or by
decreasing TrnR2 expression in the target tissue. Thus,
appropriate treatments, for example, may involve
administration of TrnR2 antibodies or other compounds
having TRN antagonist properties, or the use of antisense
polynucleotides to modulate TrnR2 expression.
Specific antibodies, either polyclonal or
monoclonal, may be capable of preventing binding of NTN
an/or GDNF to TrnR2 or, alternatively, may prevent the
formation of a functional TrnR2/Ret receptor complex.
Such antibodies can be produced by any suitable method
known in the art. For example, murine or human
monoclonal antibodies can be produced by hybridoma
technology or by combinatorial antibody library
technology, including panning a phage display library.
The antibody may be engineered using recombinant
techniques to produce an antibody with desirable
characteristics such as being "humanized" to be better
tolerated by the patient or having specificities for both
TrnR2 and Ret or both TrnR2 and a TRN growth factor.
Such antibody engineering techniques are known in the
art. See for example, Hoyden et al., Curr. Opin.
Immunol. 9(2):201-212, 1997, incorporated herein by
reference. Alternatively, the TrnR2 protein, or an
immunologically active fragment thereof, or an anti-
idiotypic antibody, or fragment thereof can be
administered to an animal to elicit the production of
antibodies capable of recognizing and binding to the
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TrnR2 protein. Such antibodies can be from any class of
- antibodies including, but not limited to IgG, IgA, IgM,
IgD, and IgE or in the case of avian species, IgY and
from any subclass of antibodies.
It is also envisioned that soluble TrnR2
polypeptides and fragments can also serve as TRN
antagonists. For example, it is believed that a soluble
TrnR2 administered in excess would GDNF, NTN, and
possibly other TRN growth factors, thereby sequestering
the TRN growth factor from the anchored TrnR2 receptors
on target cells. Similarly, if excessive levels of a TRN
growth factor, particularly GDNF or NTN, was circulating,
administration of soluble TrnR2 may act to sequester the
growth factor in the plasma and possibly facilitate their
excretion, thereby limiting the effects of the growth
factor in the body.
In another aspect of the present invention, TrnR2
antisense oligonucleotides can be made and a method
utilized for diminishing the level of expression of TrnR2
protein by a cell comprising administering one or more
TrnR2 antisense oligonucleotides. By TrnR2 antisense
oligonucleotides reference is made to oligonucleotides
that have a nucleotide sequence that interacts through
base pairing with a specific complementary nucleic acid
sequence involved in the expression of TrnR2 such that
the expression of TrnR2 is reduced. Preferably, the
specific nucleic acid sequence involved in the expression
of TrnR2 is contained within a genomic DNA molecule or
mRNA molecule that encodes TnrR2. A genomic DNA molecule
may comprise regulatory regions of the TrnR2 gene and/or
coding sequences for precursor or mature TrnR2 protein.
The term complementary to a nucleotide sequence in the
context of TrnR2 antisense oligonucleotides and methods
therefor means sufficiently complementary to such a
sequence as to allow hybridization to that sequence in a
cell, i.e., under physiological conditions. The TrnR2
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, 36
antisense oligonucleotides preferably comprise a sequence
containing from about 8 to about 100 nucleotides and more
preferably the TrnR2 antisense oligonucleotides comprise
from about 15 to about 30 nucleotides.
The TrnR2 antisense oligonucleotides can also
include derivatives which contain a variety of
modifications that confer resistance to nucleolytic
degradation such as, for example, modified
internucleoside linkages modified nucleic acid bases
and/or sugars and the like (Uhlmann and Peyman, Chemical
Reviews 90:543-584, 1990; Schneider and Banner,
Tetrahedron Lett 31:335, 1990; Milligan et al., J Med
Chem 36:1923-1937, 1993; Tseng et al., Cancer Gene Therap
1:65-71, 1994; Miller et al., Parasitology 10:92-97, 1994
which are incorporated by reference). Such derivatives
include but are not limited to backbone modifications
such as phosphotriester, phosphorothioate,
methylphosphonate, phosphoramidate, phosphorodithioate
and formacetal as well as morpholino, peptide nucleic
acid analogue and dithioate repeating units.
The TrnR2 antisense polynucleotides of the present
invention can be used in treating overexpression of TrnR2
or reduce sensitivity of cells to inappropriate
expression of NTN or GDNF. Such treatment can also
include the ex vivo treatment of cells.
The therapeutic or pharmaceutical compositions of
the present invention can be administered by any suitable
route known in the art including for example intravenous,
subcutaneous, intramuscular, transdermal, intrathecal or
intracerebral or administration to cells in ex vivo
treatment protocols. Administration can be either rapid
as by injection or over a period of time as by slow
infusion or administration of slow release formulation.
For treating tissues in the central nervous system,
administration can be by injection or infusion into the
cerebrospinal fluid (CSF). When it is intended that
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37
TrnR2 be administered to cells in the central nervous
system, administration can be by intravenous injection
with one or more agents capable of promoting penetration
of TrnR2 across the blood-brain barrier such as an
antibody to the transferrin receptor. Co-administration
may comprise physically coupling any known blood-brain
penetrating agent to TrnR2. (See for example, Friden et
al., Science 259:373-377, 1993 which is incorporated by
reference).
A TrnR2 polypeptide can also be linked or conjugated
with agents that provide other desirable pharmaceutical
or pharmacodynamic properties. For example, a TrnR2
polypeptide can be stably linked to a polymer such as
polyethylene glycol to obtain desirable properties of
solubility, stability, half-life and other
pharmaceutically advantageous properties. (See for
example Davis et al. Enzyme Eng 4:169-73, 1978; Burnham,
Am J Hosp Pharm 52:210-21B, 1994 which are incorporated
by reference).
The compositions are usually employed in the form of
pharmaceutical preparations. Such preparations are made
in a manner well known in the pharmaceutical art. One
preferred preparation utilizes a vehicle of physiological
saline solution, but it is contemplated that other
pharmaceutically acceptable carriers such as
physiological concentrations of other non-toxic salts,
five percent aqueous glucose solution, sterile water or
the like may also be used. It may also be desirable that
a suitable buffer be present in the composition. Such
solutions can, if desired, be lyophilized and stored in a
sterile ampoule ready for reconstitution by the addition
of sterile water for ready injection. The primary
solvent can be aqueous or alternatively non-aqueous.
TrnR2 can also be incorporated into a solid or semi-solid
biologically compatible matrix which can be implanted
into tissues requiring treatment.
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The carrier can also contain other pharmaceutically-
acceptable excipients for modifying or maintaining the
pH, osmolarity, viscosity, clarity, color, sterility,
stability, rate of dissolution, or odor of the
formulation. Similarly, the carrier may contain still
other pharmaceutically-acceptable excipients for
modifying or maintaining release or absorption or
penetration across the blood-brain barrier. Such
excipients are those substances usually and customarily
employed to formulate dosages for parenteral
administration in either unit dosage or multi-dose form
or for direct infusion into the cerebrospinal fluid by
continuous or periodic infusion.
Dose administration can be repeated depending upon
the pharmacokinetic parameters of the dosage formulation
and the route of administration used.
It is also contemplated that certain formulations
containing TrnR2 are to be administered orally. Such
formulations are preferably encapsulated and formulated
with suitable carriers in solid dosage forms. Some
examples of suitable carriers, excipients, and diluents
include lactose, dextrose, sucrose, sorbitol, mannitol,
starches, gum acacia, calcium phosphate, alginates,
calcium silicate, microcrystalline cellulose,
polyvinylpyrrolidone, cellulose, gelatin, syrup, methyl
cellulose, methyl- and propylhydroxybenzoates, talc,
magnesium, stearate, water, mineral oil, and the like.
The formulations can additionally include lubricating
agents, wetting agents, emulsifying and suspending
agents, preserving agents, sweetening agents or flavoring
agents. The compositions may be formulated so as to
provide rapid, sustained, or delayed release of the
active ingredients after administration to the patient by
employing procedures well known in the art. The
formulations can also contain substances that diminish
proteolytic degradation and promote absorption such as,
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for example, surface active agents.
The specific dose is calculated according to the
approximate body weight or body surface area of the
patient or the volume of body space to be occupied. The
dose will also be calculated dependent upon the
particular route of administration selected. Further
refinement of the calculations necessary to determine the
appropriate dosage for treatment is routinely made by
those of ordinary skill in the art. It is believed that
such calculations can be readily made by one skilled in
the art in light of the dose-response curves disclosed
herein for NTN- or GDNF-induced Ret activation in
TrnR2/Ret-expressing cells. Exact dosages are determined
in conjunction with standard dose-response studies. It
will be understood that the amount of the composition
actually administered will be determined by a
practitioner, in the light of the relevant circumstances
including the condition or conditions to be treated, the
choice of composition to be administered, the age,
weight, and response of the individual patient, the
severity of the patient's symptoms, and the chosen route
of administration.
The invention also provides the identification of a
novel receptor gene family for TRN neurotrophic factors.
The known members of this family, Trnl and TrnR2, share
approximately 48% percent amino acid sequence identity
and about 53% sequence homology. The inventors herein
believe that other unidentified genes may exist that
encode proteins that have substantial amino acid sequence
homology to TrnRl and TrnR2 and Which function as
receptors for growth factors selective for the same or
different tissues having the same or different biological
activities. A different spectrum of activity with
respect to tissues affected and/or response elicited
could result from preferential activation of different
receptors by different family members as is known to
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occur with members of the NGF family of neurotrophic
factors and Trk receptors (Tuszynski and Gage, supra).
As a consequence of members of a particular gene
family showing substantial conservation of amino acid
5 sequence among the protein products of the family
members, there is considerable conservation of sequences
at the DNA level. This forms the basis for a new
approach for identifying other members of the receptor
gene family to which TrnRl and TrnR2 belong. The method
10 used for such identification is cross-hybridization using
nucleic acid probes derived from one family member to
form stable hybrid duplex molecules with homologous
sequences from different members of the gene family or to
amplify nucleic acid sequences from different family
15 members. (See for example, Kaisho et al. FEES Letters
266:187-191, 1990 which is incorporated by reference).
The sequence from the different family member may not be
identical to the probe, but will, nevertheless be
sufficiently related to the probe sequence to hybridize
20 with the probe. Alternatively, PCR using primers from
one family member can be used to amplify homologous
sequences in additional family members.
The above approaches would not have heretofore been
successful in identifying other gene family members
25 because only one family member, TrnRl (GDNFR-a) was
known. With the identification of TrnR2 herein, however,
unique new probes and primers can be made that contain
sequences from the longer conserved regions of this gene
family (see boxed regions in Figure 1). The new probes
30 and primers made available from the present work make
possible this powerful new approach which can now
successfully identify other gene family members. Using
this new approach, one may screen for genes related to
TrnRl and TrnR2 in amino acid sequence homology by
35 preparing DNA or RNA probes based upon the conserved
regions in the TrnRl and TrnR2 proteins.
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Therefore, one embodiment of the present invention
comprises probes and primers that are unique to or
derived from a nucleotide sequence encoding such
conserved regions and a method for identifying further
members of the TrnR gene family. Examples of such
conserved region amino acid sequences include but are not
limited to Cys-Arg-Cys-Lys-Arg-Gly-Met-Lys-Lys-Glu (SEQ
ID N0:9); Cys-Asn-Arg-Arg-Lys-Cys-His-Lys-Ala-Lys-Arg
(SEQ ID NO:10), and Cys-Leu-Xaa-Asn-Ala-Ile-Glu-Ala-Phe-
Gly-Asn-Gly (SEQ ID NO:11) where Xaa is Lys or Arg.
Degenerate oligonucleotides containing all of the
possible nucleotide sequences which code for one or more
of the TrnR2 conserved amino acid sequences can be
synthesized for use as hybridization probes or
amplification primers. The nucleotide sequence may be
based on the above listed conserved sequences or chosen
from the other boxed conserved regions shown in Figure 1.
To reduce the number of different oligonucleotides in a
degenerate mix, an inosine base, or another "universal"
base, can be incorporated in the synthesis at positions
where all four nucleotides are possible. Univeral bases
such as inosine form base pairs with each of the four
normal DNA bases which are less stabilizing than AT and
GC base pairs but which are also less destabilizing than
mismatches between the normal bases (i.e. AG, AC, TG,
TC ) .
Sources of nucleic acid for screening would include
mammalian genomic DNA, cDNA reversed transcribed from
mRNA obtained from mammalian cells, or genomic or cDNA
libraries prepared from mammalian species cloned into any
suitable vector.
Hybridization using the new probes to conserved
regions of the nucleic acid sequences would be performed
under reduced stringency conditions. Factors involved in
determining stringency conditions are well known in the
art (for example, see Sambrook et al., Molecular Cloning,
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2rtd Ed., 1989 which is incorporated by reference).
Sources of nucleic acid for screening would include
genomic DNA libraries from mammalian species or cDNA
libraries constructed using RNA obtained from mammalian
cells cloned into any suitable vector.
PCR primers would be utilized under PCR conditions
of reduced annealing temperature which would allow
amplification of sequences from gene family members other
than TrnRl and TrnR2. To identify the sequences of these
products, they can be gel purified and ligated into any
suitable cloning vector and transformed into bacteria.
The resulting clones can be screened with an
oligonucleotide probe for either a unique TrnRl or a
unique TrnR2 sequence in the amplified region. Clones
not hybridizing to either unique probe can be sequenced
and if found to encode previously unisolated family
members, the sequence of that clone can be used to
isolate full length cDNA clones and genomic clones. A
similar method was used to isolate new gene members
(GDF-3 and GDF-9) of the TGF-Ci superfamily based on
homology between previously identified genes (McPherron J
Bio1 Chem 268: 3444-3449, 1993 which is incorporated by
reference).
Alternatively, other TrnR family members may be
identified and/or obtained by screening a cDNA expression
library for the presence of proteins cross-reacting with
an antibody capable of reacting with a polypeptide
containing a TrnR conserved region, e.g., an amino acid
sequence selected from the group consisting of SEQ ID
N0:9, SEQ ID NO:10 and SEQ ID N0:11. Preparation of cDNA
libraries in mammalian expression systems is known in the
art (see e.g., Jing et al., supra, Treanor et al., supra,
Gearing et al., EMBO J. 8:3667-3676, 1989, and Takebe, et
al., Mol. Cell. Hiol. 8:466-472, 1988, incorporated
herein by reference). A clone expressing a polypeptide
that binds to an anti-TrnR conserved region would be
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isolated and its cDNAs sequenced to determine whether it
_ encoded a TrnR family member based on comparing its
predicted amino acid sequences with those of TrnRl and
TrnR2.
Preferred embodiments of the invention are described
in the following examples. Other embodiments within the
scope of the claims herein will be apparent to one
skilled in the art from consideration of the specifica-
tion or practice of the invention as disclosed herein.
It is intended that the specification, together with the
examples, be considered exemplary only, with the scope
and spirit of the invention being indicated by the claims
which follow the examples.
Example 1
This example illustrates that either TrnRl or TrnR2
can mediate the signaling of NTN or GDNF through the Ret
protein tyrosine kinase.
Generation of fibroblasts expressing Ret and TrnRl or
TrnR2
To examine the possibility that TrnRl or TrnR2 can
form a functional receptor complex with Ret for NTN
and/or GDNF, NIH3T3 fibroblasts which stably express Ret
alone, both Ret and TrnRl, or both Ret and TrnR2 were
generated.
Briefly, full length human Ret cDNA (gift of Dr. H.
Donnis-Kelley, Washington University, St. Louis, MO) was
subcloned into the pCMV-Neo vector (Brewer, C.B., Meth.
Cell Hiol. 43:233-245, 1994, incorporated herein by
reference). NIH3T3 cells (subclone MG87; Zhan et al.,
1987) were transfected with the Ret-CMV-neo plasmid,
grown in DMEM plus 10% fetal bovine serum (Hyclone), and
stable transfectants expressing Ret were selected with 1
mg/ml 6418. Positive clones were screened for Ret
expression on immunoblots probed with an anti-Ret
antibody (Santa Cruz Biotechnology, Inc., Santa Cruz,
CA).
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A clonal Ret-expressing cell line was used as the
parent to generate TrnRl/Ret and TrnR2/Ret expressing
cells by transfection with TrnRl or TrnR2 expression
constructs. To prepare the TrnRl expression construct, a
TrnRl cDNA was obtained from a rat postnatal-day-1
library by Klentaq LA PCR with the primers: 5'-
GCGGTACCATGTTCCTAGCCACTCTGTACTTCGC-3' (SEQ ID N0:14) and
5'-GCTCTAGACTACGACGTTTCTGCCAACGATACAG-3' (SEQ ID N0:15).
The amplified product was cloned into the EcoRV site of
pHluescript KS (Stratagene, La Jolla, CA, sequenced and
subcloned into the HindIII and BamHI sites of pCMV-Neo
(Brewer, 1994). For the TrnR2 expression construct, the
coding region of the long form of human TrnR2 cDNA was
amplified from the same Marathon RACE human brain cDNA
library used to clone and sequence TrnR2. Amplification
primers were 5'-GCGGTACCATGATCTTGGCAAACGTCTGC-3' (SEQ ID
N0:16) and 5'-GCTCTAGAGTCAGGCGGCTGTTCTTGTCTGCG-3' (SEQ ID
N0:17). The product was cloned into pCMV-neo and the
insert confirmed by sequencing.
The TrnRl-CMV-Neo plasmid or the TrnR2 CMV-Neo
plasmid was co-transfected with SV2-HisD (gift of Dr.
Richard Mulligan, Massachusetts Institute of Technology)
into the Ret-expressing 3T3 cells and double
transfectants selected in 2 mM L-histidinol (Sigma, St.
Louis, MO). TrnRl- and TrnR2-expressing clones were
confirmed by western (Ret) and northern (TrnRl or TrnR2)
blotting.
Preparation of recombinant NTN and GDNF
A synthetic gene for the mature mouse NTN coding
sequence was prepared from four partially overlapping
oligonucleotides containing the Eschericia coli (E. coli)
codon preferences: 5'-GCA TAT GCC GGG TGC TCG TCC GTG CGG
CCT GCG TGC AAC TGG AAG TTC GTG TTT CTG AAC TGG GTC TGG
GTT ACA CTT CTG ACG AAA CTG T-3'(SEQ ID N0:18); 5'-GCT
GAC GCA GAC GAC GCA GAC CCA GGT CGT AGA TAC GGA TAG CAG
CTT CGC ATG CAC CAG CGC AGT AAC GGA ACA GAA CAG TTT CGT-
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3' (SEQ ID N0:19): 5'-CTG CGT CAG CGT CGT CGT GTT CGT CGT
GAA CGT GCT CGT GCT CAC CCG TGC TGC CGT CCG ACT GCT TAC
GAA GAC GAA GTT TCT TTC-3' (SEQ ID N0:20)~ 5'-CGG ATC CTT
AAA CGC AAG CGC ATT CAC GAG CAG ACA GTT CCT GCA GAG TGT
5 GGT AAC GAG AGT GAA CGT CCA GGA AAG AAA CTT CG-3' (SEQ ID
N0:21). These oligos were gel purified and then annealed
for 10 min at 68° C followed by 3O min at 22° C to form a
linear sequence. The annealed oligos were extended with
Klenow fragment, kinased and ligated into i:he Bluescript
10 KS plasmid. After verifying the authentic~.ty of the
Cloned NTN fragment by DNA sequencing, the fragment was
transferred to the Ndel and BamHI site of -the expression
vector pET30a(+) (Novagen, Madison, WI). a histidine tag
followed by a enterokinase site was placed at the amino
I5 terminus of the NTN sequence by inserting ~oligonucleotide
linkers A (5'-TAT GCA CCA TCA TCA TCA TCA TGA CGA CGA CGA
CAA GGC-3')(SEQ ID N0:22) and B (5'-TAG CCT TGT CGT CGT
CGT CAT GAT GAT GAT GAT GGT GCA-3')(SEQ ID N0:23) into
the Ndel site.
20 The mature rat GDNF coding sequence was obtained
from an embryonic-day-21 rat kidney cDNA library by PCR
using primers 5'-CAG CAT ATG TCA CCA GAT F,AA CAA GCG GCG
GCA CT-3' (SEQ ID N0:24) and 5'-CAG GGA TCC GGG TCA GAT
ACA TCC ACA CCG TTT AGC-3' (SEQ ID N0:25). The amplified
25 cDNA fragment was subcloned into the Ndel and SalI sites
of pET30a(+). A six-His tag and enterokinase site were
added to the amino terminus of the NTN sequence at the
Ndel site using linkers A and B as above.
The NTN and GDNF pET30a(+) constructs were sequenced
30 to confirm their authenticity and then transformed into
E. coli strain BL21/DE3. The transformed bacteria were
grown at 37° C in 2XYT medium (30 /~cg/ml kanamycin) with
vigorous shaking. For GDNF production, IPTG was added to
a final concentration of 1.0 mM to induce expression of
35 the protein after the culture reached an optical density
of A6oo ~ 0.7. Incubation was continued for an additional
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2 h. Bacteria containing the NTN expression construct
were grown for 24 h without IPTG.
Cells were harvested by centrifugation at 4000 X g
for 20 min, solubilized with Buffer A (6 M guanidine HC1,
0.1 M Na phosphate, 10 mM Tris HC1, pH 8.0) at 1/lOth
volume of the original culture volume and rocked
overnight. Lysate was centrifuged at 10,000 x g for 15
min at 4° C. The supernatant was exposed to 4 ml of
Nickel-NTA (nitrilo-tri-acetic acid) resin (Qiagen,
Chatsworth, CA) per 1 liter original culture and washed
with 10-20 column volumes of Buffer A, 10 column volumes
of Buffer B (8 M urea, 0.1 M Na phosphate, 10 mM Tris
HC1, pH 8.0), and 5-10 volumes of Buffer C (8 M urea, 0.1
M Na phosphate, 10 mM Tris HC1, pH 6.3) until the A280
was <0.01. The recombinant NTN or GDNF protein was
eluted with 10-20 ml Buffer E (8 M urea, 0.1 Na
phosphate, 10 mM Tris, pH 4.5). Fractions were collected
and analyzed by SDS-PAGE. The eluate was immediately
diluted to 50 ng/~C1 in Buffer B and dialyzed against 4 M
Renaturation Buffer (4 M urea, 5 mM cysteine, 0.02 Tween
20, 10~ glycerol, 10 mM Tris HCl, 150 mM NaCl, 100 mM Na
phosphate, pH 8.3, under argon) at 4° C overnight and
then against 2 M Renaturation Buffer (as above except
with 2 M urea) 2-3 days with changes every 24 h.
Ret phosphorylation assays PI-PLC treatment
TrnRl- and TrnR2-expressing fibroblasts were grown
to confluence in DMEM plus 10$ calf serum, treated with
50 ng/ml recombinant NTN or GDNF for 10 min, or left
untreated, and then lysed. A portion of the lysates was
removed and assayed for total Ret expression by western
blot analysis using an anti-Ret antibody. The remaining
lysates were immunoprecipitated with an anti-
phosphotyrosine antibody and analyzed by western blot
using the anti-Ret antibody. The results are shown in
Figure 3A.
Anti-Ret immunoblot analysis for total Ret shows
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that expression by the Ret and Ret/TrnR2 expressing
clones of the immature (150kD) intracellular Ret protein
and the glycosylated mature Ret protein (170 kD) was
essentially the same whether treated with NTN or GDNF or
left untreated (-) (Total, Fig. 3A).
However, when the lysates were immunoprecipitated
with anti-phosphotyrosine antibody before anti-Ret
western analysis (IP, Fig. 3A), no Ret protein was
observed in lysates from fibroblasts expressing only the
Ret tyrosine kinase (Ret, Fig. 3A) whether left untreated
or treated with NTN or GDNF at doses ranging from 50 to
3000 ng/ml (Fig. 3A and data not shown). A tyrosine-
phosphorylated band of approximately 170 kD was observed
only in cells which expressed both Ret and TrnR2 and
which were treated with GDNF or NTN, indicating that TRN-
induced activation of the mature Ret protein required the
presence of both Ret and TrnR2 (IP, Fig. 3A). Similar
results were seen for Ret/TrnRl expressing cells (data
not shown).
Further evidence that the effects of both GDNF and
NTN could be mediated by TrnR2 was obtained by treating
fibroblasts expressing Ret/TrnR2 with 1 U/ml PI-PLC for
45-60 min at 37°C and then washing the PI-PLC treated
cells prior to NTN or GDNF treatment. As shown in Fig.
2B, PI-PLC treatment, which specifically cleaves GPI-
linked proteins from the cell surface, significantly
depleted the NTN or GDNF induced phosphorylation of Ret
(compare +,- lanes with +,+ lanes). These data indicate
that TrnR2, a putative GPI-linked protein, can form a
functional receptor with Ret for NTN or GDNF. This is
analogous to the previously described requirement of
TrnRl as a co-receptor with Ret for GDNF signaling
(Treanor et al., supra; Jing et al., supra).
To further characterize the activities of these two
TRN co-receptors, the effects of 0 to 100 ng/ml of NTN or
GDNF on Ret phosphorylation was investigated. As shown
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in Fig. 3C, the Ret stimulating activity of both co-
receptors is dose dependent since more phosphorylation of
Ret in either construct was observed at higher levels of
GNDF and NTN. However, while the extent of Ret
phosphorylation in Ret/TrnRl expressing fibroblasts
treated with NTN or GDNF was approximately equivalent at
all doses tested, the TrnR2/Ret expressing fibroblasts
were more sensitive to NTN treatment than GDNF treatment.
In particular, Ret phosphorylation was clearly observed
in response to NTN treatment at 0.3 ng/ml; an equivalent
response to GDNF was observed at 10 ng/ml. Similar
results were obtained with multiple batches of
recombinant GDNF and NTN, and with another stable
TrnR2/Ret transfectant (data not shown).
The observed difference in the dose-response curves
of the Ret/TrnRl and Ret/TrnR2 fibroblasts to NTN and
GDNF suggests that there is a difference in the
functional affinity of the ligands for the two receptor
complexes, TrnRl/Ret and TrnR2/Ret. The TrnR2/Ret
complex may function preferentially as a NTN receptor,
whereas the TrnRl/Ret complex responds equivalently to
either factor.
Example 2
This example illustrates that the short splice
variant of TrnR2 (TrnR2-SV) can mediate signal
transduction through Ret like the long splice variant
(TrnR2-LV).
3T3 fibroblasts coexpressing Ret and either TrnR2-LV
or TrnR2-SV were generated by cotransfecting a clonal
Ret-expressing 3T3 cell line with a SV2-His plasmid and a
cDNA encoding TrnR2-LV and then selecting the desired
transfectants in 2 mM L-histidinol essentially as
described in Example 1.
The recombinant fibroblasts were stimulated with
either GDNF, neurturin or persephin at 100 ng/ml for 10
minutes and then lysed. To determine the amount of
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active phopshorylated Ret after stimulation with each
growth factor, the lysates were immunoprecipitated using
an anti-phosphotyrosine antibody, and then analyzed by
western blot using an anti-Ret antibody as described in
Example 1. The results are shown in Fig. 3D.
When stimulated with GDNF or neurturin (NTN), the
amount of tyrosine-phosphorylated Ret was approximately
equal in cells coexpressing TrnR2-SV as that in cells
coexpressing TrnR2-LV. In addition, neurturin
stimulation produced more Ret phosphorylation than GDNF
stimulation in both the TrnR2-SV-expressing and TrnR2-LV-
expressing cells, which is consistent with the hypothesis
that the TrnR2-LV/Ret complex has a preferential affinity
for neurturin over GDNF.
These data indicate that the short and long splice
variants of TrnR2 are essentially equivalent in mediating
GNDF and neurturin signaling through Ret activation.
Thus, it is believed the short splice variant contains
all the structural elements necessary for binding to GDNF
and neurturin and presenting these ligands to Ret in the
appropriate orientation such that Ret is phosphorylated.
Neither cell line responded to persephin (PSP),
indicating that this family member acts through a
differenct co-receptor complexed with Ret or through a
different receptor complex altoghether.
Example 2A
This example illustrates that a soluble TrnR2
polypeptide specifically binds to the GNDF and NTN
members of the TRN family.
A cDNA encoding a soluble TrnR2 receptor-
immunoglobulin fusion protein was prepared by fusing a
polynucleotide encoding the amino acid sequence from
methionine at position -21 through glycine at position
411 of SEQ ID N0:2, i.e., nucleotides 36-1331 of SEQ ID
NO: l, to a polynucleotide encoding the Fc region of human
IgGl using the plgPlus vector system (Invitrogen,
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Carlsbad, CA). The resulting construct was transfected
into COS cells and stable clones were selected using 1
mg/ml 6418. Stable COS clones were grown in conditioned
medium from which the secreted TrnR2-Fc fusion protein
5 was purified using protein-A chromotography.
This soluble TrnR2-Fc fusion protein was then used
in an ELISA binding assay to determine if soluble TrnR2-
LV is capable of binding to GDNF, NTN and PSP. Solutions
containing 250 ng/ml of GDNF, NTN or PSP were prepared
10 and 50 pl of each solution was applied to separate
Maxisorb ELISA plates (Nunc) (i.e., 12.5 ng growth factor
per well) and the growth factor was allowed to bind for 1
hr at room temperature. The wells of each plate were
then washed, blocked, and incubated with increasing
15 amounts of the soluble TrnR2-Fc fusion protein. The
wells were washed again and bound TrnR2-Fc protein was
detected using an anti-human IgG-HRP conjugated secondary
antibody (Jackson Immunoresearch), and TMB liquid
substrated detection reagent (Sigma). The amount of
20 luminescence was plotted against the amount of TrnR2-Fc
protein used in the assay and the results are shown in
Fig. 3(E).
In this immunoassay, approximately equal amounts of
soluble TrnR2-Fc bound to both GDNF and NTN when the
25 fusion protein was added at less than 1 nM. However, at
larger amounts of added fusion protein, much larger
amounts of soluble TrnR2 bound to immobilized NTN than to
immobilized GDNF, demonstrating that soluble TrnR2 also
has greater affinity for NTN than GDNF as was shown in
30 Example 1 for membrane bound TrnR2.
The specificity of TrnR2 for NTN and GDNF is
indicated by the lack of its binding to PSP at all
amounts of receptor tested.
Example 3
35 This example illustrates the expression of TrnRl and
TrnR2 in various tissues.
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TrnR2 expression in adult mouse is more limited than
TrnRl expression
TrnR2 expression in adult mouse was investigated by
Northern blot analysis of total RNA (25 ,ug) isolated from
various adult mouse tissues and electrophoresed in a 1$
agarose/formaldehyde gel and blotted onto a nylon
membrane (Zetaprobe) using standard procedures
(Chomczynski and Sacchi, Annal. Biochem 162: 156-159,
1987, incorporated herein by reference). To verify that
equal amounts of total RNA were present in each lane, the
28S ribosomal RNA band was visualized by staining with
ethidium bromide. RNA homologous to TrnR2 was detected
by probing the blot with a 3ZP-labeled fragment of TrnR2
cDNA. The results are shown in Figure 4.
RNA hybridizing to the TrnR2 probe was observed in
brain and testis. Two messages were observed in brain,
differing only slightly in size. These two bands likely
correspond to the two splice forms found in brain while
performing RACE PCR to amplify the 5' end of the TrnR2
cDNA, the shorter of which is missing 399 nucleotides
from the coding region (see Fig. 2). Two different bands
were also observed in testis, which were significantly
smaller (-1.5-1.8 kb) than either of the transcripts
detected in the brain (-4 kb). One of the smaller TrnR2
messages in testis may be analogous to a small TrnRl mRNA
reported which encodes a truncated protein of 158 amino
acids (Treanor et al., 1996). Low-level expression may
also be present in the spleen and in the adrenal.
These results indicate that the tissue distribution
of TrnR2 is more limited than TrnRl in the adult animal,
which has been detected in liver, kidney, and brain of
adult rat and mouse (Ding et al., supra).
Analysis of TrnRl TrnR2 and Ret expression in targets
of GDNF and NTN
Comparison of TrnR2 and TrnRl expression was also
investigated in known sites of GDNF and/or NTN action by
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in situ hybridization analysis. Mouse tissue samples
were obtained and prepared for in situ hybridization as
described previously Wanaka et al., Neruron 5: 267-281,
1990, which is incorporated herein by reference). The
results of in situ hybridization of these fresh frozen
tissue samples with antisense 33P-labeled RNA probes
transcribed from fragments of TrnRl, TrnR2 and Ret cDNAs
are shown in Figure 5.
In situ hybridization analysis showed only low-level
expression of TrnR2 in the substantia nigra in the adult
mouse, and in the ventral mesencephalon of an E14 mouse,
in contrast to high-level expression of TrnRl and Ret
(Fig. 5A and data not shown). Motor neurons in the
ventral horn (vh) of the adult spinal cord also express
TrnRl and Ret, but not TrnR2 (Fig. 5B). Ret is localized
predominately to motor neurons, whereas TrnRl shows
additional staining in the intermediate and dorsal horns
of the cord. TrnR2 is highly expressed in the developing
and adult dorsal root ganglia (drg), along with Ret and
TrnRl (Fig. 5C and data not shown). In addition, strong
expression of TrnRl and TrnR2, but not Ret, was observed
in the exiting nerve root (r) (Fig. 5D). In the
developing kidney (k) and gut (g), there is high level
expression of TrnRl and Ret, but not TrnR2 (Fig. 5c).
Finally, significant expression of both TrnR2 and Ret was
observed in the rat superior cervical ganglion (SCG),
with only low-level, diffuse staining of TrnRl (Fig. 5d).
These data indicate a partially overlapping
expression pattern for TrnRl and TrnR2 in embryonic and
adult central and peripheral nervous tissue. In several
areas of known GDNF action, including nigral and motor
neurons, high levels of TrnRl and Ret are expressed, with
only low or undetectable levels of TrnR2 expression.
Based on this initial survey, TrnR2 expression is largely
limited to neuronal tissue in both embryo and adult, with
highest levels of expression in sensory and sympathetic
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neuronal populations.
53
Example 4
This example illustrates that both NTN and GDNF
promote the survival of newborn rat SCG neurons in
' 5 culture through their activation of the Ret signaling
pathway and this activation is likely mediated by TrnR2,
not TrnRl.
Neuronal cultures were prepared from the SCG of
postnatal day-1 rats using known procedures (Martin et
al., J. Cell Hiol. 106:829-844, incorporated herein by
reference). The SCG cultures were plated on collagen-
coated dishes and maintained in NGF for seven days and
then deprived of NGF by switching to medium lacking NGF
and containing an anti-NGF antibody (Ruit et al., Neuron
8: 573-587, 1992, incorporated herein by references.
After 2-4 h, this medium was replaced with medium
containing 50 ng/ml NTN, GDNF, or NGF, After 10 min
incubation in these growth factors, lysates were prepared
and assayed for total Ret protein and tyrosine-
phosphorylated Ret as described in Example 1. Tyrosine
phosphorylated Ret protein was observed in cells treated
with NTN but not in NGF-treated cells (data not shown).
Thus, both NTN and GDNF can activate the Ret receptor.
It had been shown that the survival-promoting
ability of GDNF on several neuronal populations including
SCG neurons was significantly reduced by PI-PLC treatment
(Treanor et al., supra). To assess whether NTN-induced
Ret activation is similarly affected, SCG cultures were
grown in serum-free N2 medium to maximize the activity of
PI-PLC and then treated with 1 U/ml PI-PLC prior to the
addition of NTN (50 ng/ml). NTN-induced tyrosine
phosphorylation of Ret was significantly reduced in
cultures treated with PI-PLC (data not shown). Thus,
activation of the Ret PTK by NTN and GDNF appears to be
mediated by a GPI-linked protein.
As shown in Figure 5D, in situ hybridization
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analysis indicated that TrnR2 and Ret are expressed at
high levels in rat SCG neurons, whereas TrnRl is
expressed diffusely and does not appear to be localized
to neurons. To further assess the cellular localization
of TrnRl, TrnR2 and Ret mRNAs in this ganglion, their
expression in primary SCG cultures was analyzed. Because
the primary cultures contain a small contaminating
population of non-neuronal cells (Schwann cells and
fibroblasts), neuronal specific messages can be
identified by inducing apoptosis in the neuronal
population. Removal of NGF from the culture medium
results in near complete death of the neuronal population
within 48 hours (Martin et al., J. Cell Biol. 106:829-
844, 1988; Deckwerth et al., J. Cell Biol. 123:1207-1222,
1993; Edwards et al., J. Cell Biol. 124:537-546, 1994,
which are incorporated herein by reference). During this
period, neuronal messages decrease whereas messages from
non-neuronal cells remain constant (Freeman et al.,
Neuron 12:343-355, 1994; Estus et al., J. Cell Biol.
127:1717-1727, 1994, each incorporated herein by
reference).
The amount of TrnRl, TrnR2, Ret mRNA in neuronal
cultures was assessed using semiquantitative reverse
transcription-PCR (RT-PCR) as described in Freeman et
al., supra and Estus et al., supra. Five day SCG
cultures were switched to medium containing anti-NGF
antibodies for various times. Polyadenylated RNA was
isolated from the cultures at 0, 6, 12, 18, 24 and 36
hours after NGF removal using the QuickPrep Micro Kit
(Pharmacia, Piscataway NJ) according to the
manufacturer's instructions. Half of the poly-A RNA was
converted to cDNA by reverse transcription with Moloney
murine leukemia virus reverse transcriptase with random
hexamers (16 uM) as primers. cDNA from approximately 150
cells was used in a 50 ~ul PCR reaction using the
following primer sets: mouse Ret forward 5'
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TGGCACACCTCTGCTCTATG-3' (SEQ ID N0:26) and reverse 5'-
n TGTTCCCAGGAACTGTGGTC-3' (SEQ ID N0:27); TrnRl forward 5'-
GCACAGCTACGGGATGCTCTTCTG-3' (SEQ ID N0:28) and reverse
5'-GTAGTTGGGAGTCATGACTGTGCCAATC-3' (SEQ ID N0:29); TrnR2
5 forward 5'-AGCCGACGGTGTGGCTCTGCTGG-3' (SEQ ID N0:30) and
reverse 5'-CCAGTGTCATCACCACCTGCACG-3' (SEQ ID N0:31).
After amplification, the PCR products were separated by
electrophoresis on 10$ polyacrylamide gels, visualized by
autoradiography of the dried gels, and quantified with a
10 Phosphorlmager (Molecular Dynamics, Inc., Sunnyvale CA).
The results are shown in Figure 6.
Ret and TrnR2 messages decreased as the neurons
died, in a manner similar to neuron-specific enolase
(NSE). In contrast, TrnRl levels remained constant,
15 similar to the Schwann cell marker S-100.
These data indicate that Ret and TrnR2 expression is
largely limited to neurons in neonatal rat SCG cultures,
and likely mediates the functional response of these
neurons to NTN and GDNF. This is consistent with in situ
20 hybridization analysis (Fig. 5D) which also indicates
that the expressed receptor complex in SCG neurons
consists of TrnR2 and Ret. Interestingly, NTN is more
potent in promoting the survival of SCG neurons than GDNF
(Kotzbauer et al., supra), which is consistent with the
25 higher sensitivity of the TrnR2/Ret receptor complex to
NTN treatment (Fig. 3C). Although some low level
neuronal expression of TrnRl cannot be excluded by this
assay, these data indicate TrnRl is predominantly
expressed in the non-neuronal population, consistent with
30 its previously observed expression in Schwann cells
(Treanor et al., supra).
Example 5
This example illustrates that TrnRl, but not TrnR2,
is up-regulated in distal sciatic nerve after nerve
35 injury.
GDNF is a well characterized trophic factor for both
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56
embryonic and adult motor neurons (Henderson et al.,
Science 266:1062-1064, 1994; Yan et al., Nature 373:341-
344, 1995: Oppenheim et al., Nature 373:344-346, 1995; Li
et al., Proc.Natl.Acad.Sci.USA 92:9771-9775, 1995, all
are incorporated herein by reference). In the adult
animal, GDNF expression is up-regulated in the distal
segment of the sciatic nerve after transection, and in
denervated muscle (Trupp et al., J. Cell.Biol. 130:137-
148, 1995; Springer et al., Exp.Neurol. 131:47-52, 1995,
each is incorporated herein by reference). This is
similar to observations regarding NGF and the p75 low
affinity neurotrophin receptor (p75NTR), which are both
upregulated by Schwann cells in the distal segment of the
sciatic nerve after transection (Taniuchi et al.,
Proc.Natl.Acad.Sci.USA 83:4094-4098, 1986; Heumann et
al., J.CeII.Biol. 104:1623-1631, 1987, each is
incorporated herein by reference). Because GDNF is up-
regulated after injury, and because TrnRl is expressed by
Schwann cells (Treanor et al., supra), the expression of
TrnRl and TrnR2 in the distal segment of the rat sciatic
nerve before and after transection was examined to
determine if one or both of the TRN co-receptors is up-
regulated after transection.
Northern blot analysis was performed on total RNA
isolated from normal rat sciatic nerve and from the
distal segment of the sciatic nerve seven days post-
transection using 3~P-labeled TrnRl and TrnR2 cDNA
fragments as probes. Rat sciatic nerves were transected
and recovered as previously described (Araki et al.,
Neuron 17:353-361, 1996, incorporated herein by
reference). Brain total RNA was included on the blot as
a positive control for detection of TrnR2 mRNA. The
results are shown in Figure 7.
Seven days after nerve transection (7D), the distal
portion of the sciatic nerve showed a dramatic increase
in the level of TrnRl mRNA from the level observed before
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transection (N). In contrast, TrnR2 mRNA was not
detected in the nerve either before (N) or after
transection (7D). The 28S ribosomal RNA band, visualized
using ethidium bromide, is shown below to demonstrate
equal loading of total RNA samples.
Consistent with the observed differential expression
of TrnRl and TrnR2 in Schwann cells, RT-PCR analysis of
the JS-1 Schwann cell line also showed expression of
TrnRl, but not TrnR2 (data not shown). These results
indicate that TrnR2 is unlikely to play a major role in
Schwann cell mediated peripheral trophic support of the
regenerating nerve. However TrnRl, in conjunction with
GDNF produced by the distal sciatic nerve and muscle,
could potentially provide a potent trophic substrate for
growth of the regenerating nerve. _
Example 5
This example illustrates additional analysis by in
situ hybridization of the expression of neurturin, GDNF
and their receptors in the central nervous system of the
adult mouse.
MATERIALS AND METHODS
R_iboprobes
Riboprobes were synthesized from plasmids containing
mouse cDNA sequences of neurturin (nucleotides 293-598,
441-675 of GenBank accession number U78109), GDNF
(nucleotides 256-935 of GenBank accession number D88264),
GFRa-1 (TrnRl, nucleotides 574-1069 of GenBank accession
number U59486), GFRa-2 (TrnR2, nucleotides 1-569,
1002-1417 of GenBank accession number AF002701) and Ret
(nucleotides 207-611 of GenBank accession number X67812).
Both GFRa-2 (TrnR2) probes contained sequences that are
present in both splice variants of TrnR2 mRNA (Baloh et
al., Neuron 18:793-802, 1997). The Ret probe also
included sequence present in all of the known ret splice
variants. Plasmids were linearized with appropriate
restriction enzymes and transcribed in vitro with 50 pCi
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of [33P] UTP (NEN Dupont) by using T3, T7, or SP6 RNA
polymerise. All experiments were performed within 24
hours of probe synthesis. The probes were clearly
specific based on the distinct expression patterns
observed for the different mRNA. Two different sense
probes were also used to ensure specificity further.
Pret~aration of Tissue
Animals were housed and treated in accordance with
the guidelines of NIH and Washington University. Young
adult (6-8 weeks) female ICR mice that were at mid to
late gestation or recently delivered were anesthetized
with an overdose of xylazine (20 mg/ml): ketamine (100
mg/ml): acepromazine (10 mg/ml), (3:3:1). Brains (N=5)
were removed, frozen on dry ice, and sectioned at 14 ~m
in the sagittal or coronal plane on a cryostat. Frozen
sections were thawed then postfixed in 4$
paraformaldehyde in PHS, pH 7.4, for 20 minutes.
Sections were pretreated for hybridization as follows:
3x5 minutes in PBS; 3 minutes in 0.2$ glycine in PBS; 5
minutes in PBS; 10 minutes in O.1M triethanolamine, pH 8;
2x10 minutes in 0.025$ acetic anhydride/O.1M
triethanolamine; and, 5 minutes in PHS. Sections were
then dehydrated in a graded series of alcohol and
defatted with chloroform. 33P-labeled RNA probes were
diluted in hybridization buffer (50$ formamide, 50mM
Tris-HCL, pH 7.5, 5mM EDTA, lx Denhardt's solution, 10$
dextrin sulfate) to 1x106 counts per 75 ~1. Slides were
incubated overnight at 55°C in a humidified chamber with
75~C1 of hybridization solution per slide. After
hybridization, slides were washed as follows: 4x15
minutes in 4x SSC at room temperature; 20 minutes in
2xSSC at 55°C; 2x15 minutes in RNase buffer (0.5M NaCl,
lOmM Tris-HC1, pH 8, 1mM EDTA) at 37°C; 30 minutes in 20
ug/ml RNase A in RNase buffer at 37°C; 2x15 minutes in
RNase buffer at 37°C; 20 minutes in 2xSSC at 55°C; 20
minutes in lxSSC at 65°C; and, 30 minutes in O.lx SSC at
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65°C. Slides were then dehydrated and dipped in Kodak
NTB2 emulsion. After an exposure time of 14-18 days,
slides were developed and counter-stained with
hematoxylin and eosin. Neurons were identified based on
morphology. Cells with a pale-staining cytoplasm and
large-round, pale nuclei were identified as neurons.
Cells with small oval or irregularly shaped, dark-
staining nuclei were identified as glial cells. Slides
were photographed and figures were prepared from the
photographs with Adobe Photoshop 4Ø1. With the
exception of dust removal, and dadging/burning to produce
uniform tone photographs were not altered.
Labeling was considered specific if it was above the
level of background. Background label is defined as
labeling obtained with a sense probe or labeling that was
not specifically localized to cells. The intensity of
labeling for each probe was classified as follows:
high~he greatest intensity of labeling observed far a
particular probe; moderate-high-to-medium level of
labeling; low-easily detected but low level of intensity;
barely detected-very low level of labeling in few or
scattered cells; or, not detected-~o labeling above
background. Abbreviations are according to Paxinos and
Watson, The Rat Brain in Stereotaxic Coordinates,
Academic Press Inc., 1986: and Franklin and Paxinos, The
Mouse Brain in Stereotaxic Coordinates, Academic Press
Inc., 1997.
RESULTS
Forebrain
In the forebrain, GFRa-1 (TrnRl), GFRa-2 (TrnR2),
and Ret mRNAs were widely expressed in the septum and
olfactory system (Figures 8A-C; Table 2). GFRa-1
(TrnRl), GFRa-2 (TrnR2), and Ret mRNAs were expressed in
the medial and lateral septal nuclei, the nucleus of the
diagonal band of Hroca, and the piriform cortex (Figures
8A, 88). In each of these areas, the receptors were
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expressed in cells morphologically consistent with
neurons. All three receptor components were expressed at
higher levels in the lateral septal nucleus than in the
medial septal nucleus. Ret was expressed at much lower
5 levels than either GFRa-1 (TrnR1) or GFRa-2 (TrnR2) in
the lateral septal nucleus. In the nucleus of the
diagonal band, all three receptors were expressed at low
levels with GFRa-2 (TrnR2) expressed at the lowest level.
GFRa-1 (TrnRl) and GFRa-2 (TrnR2) mRNAs were expressed in
10 the absence of detectable Ret mRNA in the neocortex,
claustrum, endopiriform nucleus, the bed nucleus of the
stria terminalis, the basal nucleus of Meynert, the
ventral pallidum, and the olfactory tubercle (Figure 8,
Table 2). GFRa-1 (TrnRl) was expressed at high levels in
15 scattered clusters of neurons in the ventral pallidum.
GFRa-2 (TrnR2) was expressed in neurons in the ventral
pallidum at much lower levels and in fewer cells than
GFRa-1 (TrnRl).
NTN (Figures~8D, 9D) and GDNF (data not shown) mRNAs
20 were expressed in piriform cortex, in the hippocampus
and, at low levels, in neocortex (Figures 8D, 9D; Table
2). In addition, expression of GDNF was seen at moderate
levels in the olfactory tubercle and at very low levels
in the nucleus accumbens, the globus pallidus, the
25 ventral pallidum, the subiculum, and the striatum (Table
2).
O_lfactorv Bulb
In the olfactory bulb, mRNAs for all three receptors
were expressed in the glomerular layer and in the granule
30 layer (Table 2). In the glomerular layer, GFRa-1 (TrnRl)
and Ret were expressed at moderate levels and GFRa-2
(TrnR2) was expressed at a lower level. In the granule
layer, GFRa-1 (TrnRl) and GFRa-2 (TrnR2) were expressed
at higher levels than Ret. In the mitral cell layer,
35 both GFRa-1 (TrnRl) and GFRa-2 (TrnR2) were highly
expressed in the absence of ret. GFRa-1 (TrnRl) was
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expressed in the absence of Ret and GFRa-2 (TrnR2) in the
internal plexiform layer and the external plexiform
layer.
NTN and GDNF mRNA expression were not detected in
the olfactory bulb (Table 2).
Midbrain
Receptors for NTN and GDNF were widely expressed in
the midbrain. Most notably, mRNAs for all three receptor
components were detected in the pars compacts of the
substantia nigra, in which dopaminergic neurons,
responsive to both GDNF and NTN in adult mice are located
(Figure 10; Table 2) (Lin et al., 1993; Rosenblad et al.,
Soc. Neurosci. Abstr. 23:248.12, 1997). Receptor
expression in the substantia nigra was predominantly seen
in the pays compacts with much sparser labeling in the
pars reticulate. In particular, Ret and GFRa-1 (TrnRl)
were expressed at highest levels, while GFRa-2 (TrnR2)
was expressed at substantially lower levels. All three
receptor components were predominantly, but not
exclusively, expressed in neurons. Expression of all
three receptor components was also found in the ventral
tegmental area (VTA), the periaqueductal grey (PAG), the
rostral linear raphe (RLi), the interfascicular nucleus
(IF), and the Edinger Wesphal nucleus (EW). All three
receptor components were also expressed in the large
motor neurons of the oculomotor nucleus (3). GFRa-1
(TrnRl) and GFRcc-2 (TrnR2) were expressed in the absence
of Ret in the superficial layers of the superior
colliculus, in the interpeduncular nucleus, and in the
cerebral cortex (Figure 10). In addition, GFRa-1 (TrnRl)
and Ret, but not GFRa-2 (TrnR2), were expressed in the
red nucleus (Table 2).
NTN mRNA was expressed at a barely detectable level
in the oculomotor nucleus. NTN expression was not seen
in any other areas of the midbrain. GDNF mRNA was
expressed at very low levels in the substantia nigra pays
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compacta, in the superficial layers of the superior
colliculus and in the interfascicular nucleus.
Thalamus
Receptor components were expressed in several nuclei
in the thalamus including both relay and association
nuclei (Figures 9A-C: Table 2). The receptors were most
prominently expressed in the reticular nucleus (Rt), the
zona incerta, and the habenular nuclei (Figures 9A-C).
In the reticular nucleus, mRNAs for GFRa-1 (TrnRl) and
GFRa-2 (TrnR2) were expressed at high levels while Ret
mRNA was expressed at moderate-to-low levels. In the
habenular nuclei, GFRa-1 (TrnRl) was expressed at high
levels and the other two receptors were expressed at
lower levels, particularly in the lateral habenular
nucleus. All three receptor components were also
expressed at low levels in the medial geniculate nucleus
(Figure 10).
Several of the sensory and motor relay nuclei
expressed Ret mRNA at very low levels in the absence of
GFRa-1 or GFRa-2 (Figure 9; Table 2). These included the
two main sensory relay nuclei, the ventroposteromedial
(VPM) and the ventroposterolateral (VPL), and the primary
motor relays, the ventromedial (VM) and ventrolateral
(VL) nuclei. In the other major sensory relay, the
posterior nuclear group (Po), both Ret and GFRa-1 (TrnRl)
were expressed at low levels but GFRa-2 (TrnR2) was not
detected. The same pattern was observed in the
laterodorsal and lateroposterior nuclei, and the
mediodorsal nucleus in which Ret and GFRa-1 (TrnRl) were
expressed at low levels and GFRa-2 (TrnR2) was absent.
In addition, Ret and GFRa-2 (TrnR2) were expressed in the
absence of GFRa-1 (TrnRl) in the subthalamic nucleus
(Figures 9A-C).
NTN was expressed at moderate levels in the
anteromedial and anteroventral nuclei of the thalamus
(Figure 9D). GDNF mRNA was expressed at low levels in
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the anteromedial and anteroventral nuclei and, at very
low levels in the reticular, ventromedial, ventrolateral,
ventroposteromedial, and ventroposterolateral nuclei, and
in the posterior nuclear group and the zona incerta
(Table 2).
Hypothalamus
In the hypothalamus, GF (TRN) receptor components
were prominently expressed in many areas (Figure 9; Table
2). All three receptor components were detected in the
periventricular nucleus, the medial preoptic nuclei, both
central and median, in the medial and lateral preoptic
area, the ventromedial (VM) and dorsomedial (DM)
hypothalamic nuclei, the supramammillary nucleus, and the
posterior, anterior and lateral hypothalamic areas.
GFRa-1 (TrnRl) and GFRa-2 (TrnR2) were expressed, in the
absence of Ret, in the paraventricular nucleus and the
arcuate nucleus.
NTN was expressed in the supraoptic, and
paraventricular nuclei (Figure 11A). NTN expression in
the supraoptic and paraventricular nuclei was
particularly intense. In the supraoptic and
paraventricular nuclei, NTN expression was localized to
cells whose morphology is consistent with that of the
large secretory neurons that are found in these nuclei
(Figures 11H, 11C). GDNF mRNA was expressed at barely
detectable levels in the hypothalamus in the dorsomedial,
ventromedial, arcuate and medial mammillary nuclei (Table
2).
Hrainstem
GF (TRN) receptor components were expressed in a
number of brainstem nuclei including cranial nerve nuclei
(Figure 12; Table 2). All three receptor components were
expressed in the facial motor nucleus, in the region of
the nucleus ambiguous, the abducens nucleus, the
prepositus hypoglossal nucleus, the spinal trigeminal
sensory nuclei, the vagal dorsal motor nucleus, the
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64
lateral and medial vestibular nuclei, and the dorsal and
ventral cochlear nuclei. GFRa-1 (TrnRl) and Ret were
also expressed, in the absence of GFRa-2, in the region
of the inferior salvitory nucleus and nucleus
retroambiguous, and in the hypoglossal nucleus, and in
motor neurons in the trigeminal motor nucleus. In the
principal sensory trigeminal nucleus, Ret was expressed
in the absence of GFRa-1 TrnRl) and GFRa-2 (TrnR2). In
the cranial motor nuclei, the receptors were expressed in
large motor neurons.
Other areas of the brainstem in which expression of
all three receptors was detected included the raphe
nuclei, the inferior colliculus, the pontine reticular
nucleus, the gigantocellular reticular nucleus, the
I5 tegmental nuclei, and the locus coeruleus.
NTN was expressed at very low levels in the region
of the nucleus ambiguous. GDNF mRNA was expressed at a
low level in the facial motor nucleus and the ventral
cochlear nucleus (-Figure 12C).
Spinal Cord
Transverse sections of cervical, thoracic and lumbar
spinal cord were examined. The labeling pattern for each
of the receptors was the same at each level studied.
GFRa-1 (TrmRl) and Ret were expressed prominently in
motor neurons of the ventral horn (Figures 13A, 13C).
Low levels of GFRa-2 (TrnR2) were expressed diffusely in
the spinal cord gray matter with highest levels of
expression found in the superficial layers of the dorsal
horn (Figure 13B). In the ventral horn GFRa-2 (TrnR2)
mRNA was expressed in a few scattered motor neurons.
Cerebellum
In the cerebellum, Ret and GFRa-1 (TrnRl) were
expressed at a high level in the Purkinje cell layer
(Figures 14A, 14C). Both Ret and GRFa-1 (TrnRl) were
expressed in cells adjacent to Purkinje cells, but not in
Purkinje cells. GFRa-2 (TrnR2) was strongly expressed in
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Purkinje cells in a pattern complementary to Ret and
GFRa-1 (TrnRl) expression (Figure 14B). GFRa-2 was also
expressed at a low level in the granule cell layer and
Ret was expressed at a low level in the molecular layer.
5 In the deep cerebellar nuclei, all three receptor
components were detected, with Ret and GFRa-1 (TrnRl)
detected at higher levels than GFRa-2 (TrnR2) (Table 2).
Both NTN (Figure 14D) and GDNF (Table 2) were
expressed at low levels in the Purkinje cell and granular
10 layers of the cerebellum. NTN and GDNF were not detected
in the molecular layer. NTN, but not GDNF, was detected
in the deep cerebellar nuclei.
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TABLE I'-(Summary of GF T'actor and Receptor Expression
NTN GDNF GFRoc-1 GFRa-2 RET
FOREBRAIN
Olfactory Bulb OB
Glemerular Layer - - ++ + ++
Mitral Cell Layer - , - + ++ -
Internal Plexiform - - + - -
Layer
Extemal Plexiform Layer- - + - -
Granule Layer - - + +
Olfactory Tubercle - + ++ ++ - Tu
Piriform Cortex + +-~- ++ ++ + Pir
Claustrum - - ++ ++ - Cl
Dorsal Endopiriform - - ++ ++ - DEn
Nucleus
Medial Septal Nucleus - - + + ++ MS
Lateral Septal Nucleus- - ++ ++ + LS
Nucleus Diagonal Band - - + t + DB
Bed Nucleus Stria Terminalis- - + + - BST
Nucleus Accumbens - + - - - Acb
Amygdala - t + + t A
Basal Nucleus of Meynert- - f + - B
Ventral Pallidum - + ++ t - VP
Giobus Pallidus - t - - - GP
Striatum t - - - Cpu
MIDBRAIN
Substantia Nigra SN
Compacts - t +++ ++ +++ SNc
Reticulata - - + + + SNr
Ventral Tegmental Area- - +++ ++ +++ VTA
Periaqueductal Gray - - +++ ++ +++ PAG
Rostral Linear Raphe - - + + ' RLi
+
Dorsal Raphe - - ++ + + DR
Oculomotor Nucleus ++ ++ ++ 3
Edinger Westphal - - ++ + + EW
Superior Colliculus - ~ ++ ++ - SC
Red Nucleus - - t - + R
Interfascicular Nucleus- f ++ ++ ++ IF
Interpeduncular Nucleus- - ++ ++ - IP
CORTEX
Neocortex + t ++ +++ -
Cingulate Cortex + + + ++ - Cg
Hippocampus
Subiculum - + ++ + - S
CA 1 + ++ ++ ++ -
CA2 ++ ++ ++ ++ -
CA3 ++ ++ ++ -t-+- -
Dentate + ++ ++ ++ - DL
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TABLE 1'-Summary ur GF Factor and Receptor Expression
NTN GDNF GFRa-1 GFRa-2 ~T
,. CEREBELLUM
Granule Cell Layer + + - + - GR
Purkinje Cell Layer + + ++ ++ ++ P
Molecular Layer - - - - + Mol
Deep Nuclei + - ++ + ++
'
THALAMUS
Medial Habenula - - +++ + + MHb
Lateral Habenula - - ++ + + LHb
Reticular Nucleus - - +++ +++- ++ Rt
Mediodorsal Nucleus - - + - ++ MD
Anteromedial Nucleus ++ + - - - AM
Anteroventral Nucleus ~ ++ + - - - AV
Laterodorsal Nucleus - + + - t LD
Lateral Posterior Nucleus - f - t LP
Zona Incerta - ~ ++ ++ ++ ZI
Subthalamic Nucleus - - - ++ ++ STh
Ventromedial - t - - t VM
Ventrolateral - + - - t VL
Ventral Anterior t - - - VA
Posterior Nuclear Group - t - ~ Po
Ventroposterolateral - + - - t VPL
Ventroposteromedial - + - - t VPM
Lateral Geniculate - - + - + LGN
Medial Geniculate - - + + + MGN
HYPOTHALAMUS
Medial Preoptic Nucleus MP
Central - - ++ ++ ++
Median - - ++ ++ ~
++
Preoptic Area PA
Medial - - -+-+- ++ ~-+-
Laterai - - ++ ++ -i-+-
Periventricular Nucleus - - + + + Pe
Tubercinerium - - ++ + f T
Lateral Hypothalamic Area - - + +++ t LHA
Anterior Hypothalamic Area - - ++ + + AHA
Posterior Hypothalamic Area - - + + + PHA
Ventromedial Hypothalamic Nucleus - t + ++ t VM
Dorsomedial Nucleus - t + ++ + DM
Supraoptic Nucleus ++ - - - - SO
Arcuate Nucleus - t + ++ - Arc
Medial Mammillary Nucleus - t - - - MM
Supramammillary Nucleus - - t ++ + SuM
Paraventricular Nucleus ++ - ++ + - PV
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TABLE 1'-summary of GF Factor and Receptor Expression
NTN GDNF GFRa-1 GFRa-2 RET
BRAINSTEM
Median Raphe - - ++ - + MnR
Raphe Pallidus - - + + + Rpa
Raphe Magnus - - + + + RMg
Inferior Colliculus - - ++ +++ + IC
Preolivary Region - - + +
Trigeminal Principalis- - - -
+ Pr5
Spinal Trigeminal - - ++ ++
Motor Trigeminal - - +++ - ++~ Mo5
Nucleus Ambiguous - - ++ t ++
Nucleus Retroambiguous- - ++ - ++
Vagal Dorsal Motor - - + + +++
Nucleus
Hypoglossal - - +++ - +++ 12
Prepositus Hypoglossal- - + + ++
Abducens - - ++ ++ ++ 6
Facial Nucleus - + +++ + +++ 7
Dorsal Cochlear Nucleus- - ++ ++ + DC
Ventral Cochlear - + + ++ ++ VC
Lateral Vestibular - - + + ++
Medial Vestibular - - + + ++
Inferior Salvitory - - ++ - +
Pontine Reticular Nucleus- - + + +
Gigantocellular reticular- - + + ++ Gi
Tegmental Nuclei - - + +++ - Tg
Locus Coeruleus - - ++ ++ ++ LC
SPINAL CORD
Ventral Hom - - -~-++ + +i"E'
Dorsal Horn - - - ++ - DH
1 Level of expression: +++, high; ++, moderate; +, low; t, barely detected; -,
not detected.
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DISCUSSION
In the experiments described in this example, we
studied the expression of NTN and GDNF and their
receptors in the adult mouse brain. GF (TRN) receptors
are widely expressed throughout the adult brain. In many
areas of the brain, including all the areas in which GF-
responsive neurons are present, Ret and either GFRa-1
(TrnRl) or GFRa-2 (TrnR2) mRNA are present. In some
areas of the brain, most notably the cerebral cortex and
the hippocampus, co-receptors GFRa-1 (TrnRl) and GFRa-2
(TrnR2) were expressed without Ret.
NTN and GDNF mRNA were expressed at lower levels and
in fewer areas of the adult mouse brain than GF receptor
(TrnR) mRNA. NTN and GDNF were expressed in several
areas of the brain that receive projections from neurons
expressing receptor mRNA. This expression pattern
suggests that NTN and GDNF function through a classical
target-derived mechanism of trophic factor action to
maintain neuronal circuits in the mature CNS.
Neurons That Respond to NTN and/or GDNF in Adult Rodents
Express GF (TRN) Receptors.
Several central neuronal populations respond to GDNF
in the adult rodent including spinal motor neurons (Li et
al., Proc. Natl. Acad. Sci. 92:9771-9775, 1995), facial
motor neurons (Yan et al., Nature 373 (6512):341-344,
1995), dopaminergic neurons of the ventral midbrain
(Bowenkamp et al., J. Comp. Neurol. 355:479-489, 1995;
Kearns and Gash, Brain Res. 672:104-111, 1995; Tomac et
al., Nature 373:335-339, 1995; Cass, J. Neurosci.
16(24):8132-8139, 1996: Bowenkamp et al., Exp. Neurol.
145:104-117, 1997: Choi-Lunberg et al., Science 275:838-
841, 1997; Lapchak et al., Neurosci. 78(1):61-72, 1997),
noradrenergic neurons of the locus coeruleus (Arenas et
al., Neuron 15:1465-1473, 1995), and cholinergic neurons
of the basal forebrain (4Jilliams et al., J. Pharm. Exper.
Ther. 277:1140-1151, 1996). In addition, dopaminergic
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midbrain neurons are responsive to NTN in adult rats
(Rosenblad et al., supra).
In the experiments described in this example, we
observed expression of GF (TRN) receptors in areas of the
5 brain in which responsive neurons are located. For
example, in vivo, GDNF is a potent neurotrophic factor
for spinal (Li et al., supra) and facial (Yan et al.,
supra) motoneurons in adult rodents. Hoth facial motor
neurons and spinal motor neurons expressed Ret and GFRa-1
10 (TrnRl). In adult rodents, dopaminergic neurons in the
pars compacts of the substantia nigra and in the VTA are
protected from chemical and mechanical injury by NTN
(Rosenblad et al., supra) or GDNF (Kearns and Gash,
supra: Bowenkamp et al., 1997, supra). Ret, GFRa-1
15 (TrnRl) and GFRa-2 (TrnR2) were all expressed in neurons
in these areas of the ventral midbrain. All three
receptor components were also expressed in the
interfascicular nucleus and the rostral linear raphe. In
adult mice, dopaminergic neurons in these nuclei are
20 protected from MPTP (1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine) treatment by GDNF (Tomac et al.,
supra). GDNF treatment also protects cholinergic neurons
of the septal/diagonal band nuclei from axotomy by
fimbria/fornix section in adult rats (Williams et al.,
25 supra). In our experiments, we found GF (TRN) receptors
in both the medial septal nucleus and in the nucleus of
the diagonal band of Broca in neurons morphologically
consistent with large cholinergic neurons characteristic
of this area. In the locus coeruleus, GDNF treatment
30 protects noradrenergic neurons from death (Arenas et al.,
supra). Neurons in the locus coeruleus expressed all
three GF receptors. In summary, there is a strong
positive coorelation between GF (TRN) receptor expression
in mature neurons and responsiveness of these neurons to
35 NTN and GDNF.
GF (TRN) receptors were also expressed in areas of
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. 71
the adult brain in which responsiveness to NTN and GDNF
has not yet been directly evaluated. Areas of the brain
in which Ret and one or both of the co-receptors were
expressed include the oculomotor nucleus, the subthalamic
nucleus, and several nuclei in the thalamus and
hypothalamus. The strong correlation between receptor
expression and responsiveness to NTN and GDNF suggests
that neurons in these areas may respond to one or both
factors.
Our finding that GF (TRN) receptors were expressed
in areas of the hypothalamus involved in the control of
feeding behavior and regulation of body weight, including
the lateral hypothalamic area and the ventromedial and
dorsomedial nuclei of the hypothalamus, suggests that NTN
and/or GDNF may be involved in these processes. Previous
studies that show weight loss in rats (Hudson et al.,
Brain Res. Bull. 35;425-432, 1995) and monkeys (Gash et
al., J. Comp. Neurol. 363:345-358, 1995) treated with
GDNF support such a role for GDNF. Other neurons that
appear especially likely to benefit from the GF (TRN)
factors are the motoneurons in the oculomotor nucleus;
these neurons expressed all three GF (TRN) receptor
components and their loss would be consistent with the
ptosis observed in the NTN-deficient mice (Heuckeroth et
al., Soc. Neurosci. Abstr. 23:668.1, 1997).
NTN and GDNF Expression
Both NTN and GDNF were expressed in the targets of
neurons that respond to these factors. As shown
previously (Trupp et al., J. Neurosci. 17(10):3554-3567,
1997) and confirmed by the results reported here, GDNF
mRNA is expressed in targets of dopaminergic neurons of
the substantia nigra compacta (SNc); these targets
include the olfactory tubercle, the nucleus accumbens,
the striatum, and the globus pallidus. In addition, we
found low-level expression of GDNF mRNA in other SN
targets including the piriform cortex, and the arcuate,
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dorsomedial, and ventromedial nuclei of the hypothalamus.
We found GDNF mRNA in the compacta region of the
substantia nigra, where GDNF protein may support
dopaminergic neurons through an autocrine or paracrine
mechanism. The present study also showed NTN mRNA
expression in nigral targets including the arcuate and
paraventricular nuclei of the hypothalamus. This
distribution of NTN and GDNF mRNA is consistent with both
of these growth factors functioning as target-derived
trophic factors for nigral dopaminergic neurons in the
mature brain.
NTN and GDNF were expressed diffusely in all layers
of neocortex. The cerebral cortex may serve as a source
of NTN and GDNF for a number of cortical-projecting
neurons that express NTN and GDNF receptors; these
include neurons in the nuclei of the medial septum and
diagonal band of Broca that expressed Ret and GFRa-1
(TrnRl) and neurons in the locus coeruleus that express
Ret and GFRa-1 (TrnRl) and GFRa-2 (TrnR2). NTN and GDNF
mRNA were also expressed in a number of areas of the
hippocampal formation including all three fields of
Amnion's horn (CA1-3) within the hippocampus proper and in
the dentate gyrus. In addition, GDNF mRNA was expressed
fn the subiculum. Like the neocortex, the hippocampus is
a potential source of NTN and GDNF for neurons that
project to this region of the brain and express GF
receptors. Similarly, the medial septal nucleus and the
nucleus of diagonal band of Broca, which also expressed
GF (TRN) receptors, have large efferent projections to
all fields of the hippocampal formation. The
supramammillary nucleus of the hypothalamus in which all
three GF (TRN) receptors are expressed could obtain NTN
or GDNF by way of its efferents to the dentate gyrus and
the hippocampus proper. The dentate gyrus and the
hippocampus proper also receive a prominent projection
from the locus coeruleus, which also expressed GF (TRN)
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receptors.
The strongest and most circumscribed NTN expression
in the adult brain was seen in the lateral and medial
magnocellular paraventricular nucleus and in the
a
supraoptic nucleus of the hypothalamus. Within these
nuclei are large magnocellular secretory neurons; these
cells secrete oxytocin and vasopressin directly into the
general circulation via the posterior pituitary. What
function NTN may serve in these neurons is unclear.
Other growth factors, including fibroblast growth factor-
2 (Gonzales et al., Endocrinology 134(5):2289-2297, 1994)
and insulin-Like growth factor I (Aguado et al.,
Neuroendocrinol. 56:856-863, 1992) are found in
hypothalamic magnocellular neurons and are believed to
be
involved in neuroendocrine function; NTN may serve a
similar role. Alternatively, NTN in the supraoptic and
paraventricular nuclei may be a source of target-derived
trophic support for a number of neuronal populations that
express GF (TRN) receptors and have efferent projections
to these hypothalamic nuclei. For example, neurons in
the medial preoptic nucleus and the medial and lateral
septal nuclei project heavily to the supraoptic nucleus.
Cells in these nuclei expressed Ret mRNA as well as mRNA
for GFRa-1 (TrnRi) and GFRa-2 (TrnR2). Afferent
projections to the magnocellular neurons of the
paraventricular nucleus are less well defined but are
believed to include projections from the medial preoptic
nucleus and the median and dorsal raphe nuclei. We found
GF (TRN) receptor expression in each of these nuclei.
Another possibility is that NTN acts at some site distant
to the hypothalamus and is secreted directly into the
peripheral circulation like oxytocin and vasopressin.
Hrain Reaions That Do Not Contain Complete Receptor
Complexes
3 5 In our experiments as well as in previous studies (Nosrat
et al., Exp. Brain
Res. 115;410-422, 1997; Tomac et al., supra) of GF (TRN)
receptor expression,
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74
incomplete receptor complexes have been found in some areas of the brain. For
instance, in the thalamus and the trigeminal principal sensory nucleus, ret
was
expressed in the absence of GFRa-1 (TrnRl) and GFRa-2 (TrnR2). In other
regions, most strikingly neocortex and hippocampus, GFRa-I (TrnRl) or GFRa-2
(TrnR2) were expressed in the absence of Ret. These expression patterns raise
the
possibility that additional, as yet unidentified, receptor components for GDNF
and
NTN exist. Additional ligand-binding receptor components or other signaling
components may exist in addition to Ret. This latter possibility seems less
probable since all neurons that respond to NTN and GDNF express Ret.
An alternative possibility, which has been suggested previously (Trupp et
al., supra), is that, in some cases, responsive neurons express Ret only and
the
ligand-binding component is supplied in trans perhaps by the target of the
responsive neuron. Several patterns of NTN and GDNF receptor expression are
consistent with this possibility. For example, in the principle thalamic motor
(VL,
VM) and sensory (VPM, VPL) relay nuclei, Ret was expressed in the absence of
GFRa-1 (T'rnRl) or GFRa-2 (TrnR2). Cells in these nuclei send projections to
cerebral cortex where both GFRa-1 (TrnRl) and GFRa-2 (TrnR2) were expressed
at high levels. NTN and GDNF were also expressed in cerebral cortex.
Therefore,
thalamic neurons that express Ret may obtain NTN or GDNF as well as the
ligand-binding receptor component from their target cortical neurons.
Another example of incomplete receptor expression was found in the
Purkinje neurons of the cerebellum. In these neurons, only GFRa-2 (TrnR2)
mRNA was expressed. Ret mRNA was expressed in other cells in the Purkinje
layer, possibly basket cells or glial cells. The reason for this segregated
expression
in Purkinje neurons is unclear. Whether mature Purkinje neurons respond to NTN
or GDNF is unknown, although embryonic Purkinje neurons respond to GDNF in
vitro (Mount et al., Proc. Natl. Acad. Sci. 92:9092-9096, 1995).
Our findings show that GF (TRN) receptors are expressed in areas of the
adult brain in which neurons that respond to these factors are located. In
addition,
GF (TRN) receptor expression in other areas suggests that additional NTN- or
GDNF-responsive populations exist. In summary, the mRNA expression pattern
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- 75
for NTN, GDNF, and GF (TRN) receptors in the adult brain strongly suggests a
role for these proteins as target-derived trophic factors for mature neurons.
In view of the above, it will be seen that the several advantages of the
invention are achieved and other advantageous results attained.
As various changes could be made in the above methods and compositions
without departing from the scope of the invention, it is intended that all
matter
contained in the above description and shown in the accompanying drawings
shall
be interpreted as illustrative and not in a limiting sense.
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76
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: MILBRANDT, JEFFREY D
JOHNSON JR, EUGENE M
BALOH, ROBERT H
(ii) TITLE OF INVENTION: TrnR2, A Novel Receptor Which Mediates
Neurturin and GDNF Signaling Through Ret
(iii) NUMBER OF SEQUENCES: 31
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: HOWELL & HAFERKAMP, LC
(B) STREET: 7733 FORSYTH BLVD
(C) CITY: ST LOUIS
(D) STATE: MO
(E) COUNTRY: USA
(F) ZIP: 63105
(v) COMPUTER READABLE FORM:
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(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:
(C) CLASSIFICATION:
-(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: HOLLAND, DONALD R
(B) REGISTRATION NUMBER: 35197
(C) REFERENCE/DOCKET NUMBER: 976328
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 314-727-5188
(B) TELEFAX: 314-727-6092
(2) INFORMATION FOR SEQ ID NO:1:
(i1 SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1543 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 36..1427
(ix) FEATURE:
(A) NAME/FCEY: sig~eptide
(B) LOCATION: 36..98
CA 02291705 1999-11-22
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77
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: l:
GAGAAAGACA AAAAAAACGG TGGGATTTAT TTAAC ATG ATC TTG GCA AAC GTC 53
Met Ile Leu Ala Asn Val
-21 -20
TTC TGC CTC TTC TTC TTT CTA GAC GAG ACC CTC CGC TCT TTG GCC AGC 101
Phe Cys Leu Phe Phe Phe Leu Asp Glu Thr Leu Arg Ser Leu Ala Ser
-15 -10 -5 1
CCT TCC TCC CTG CAG GGC CCC GAG CTC CAC GGC TGG CGC CCC 149
CCA GTG
Pro Ser Ser Leu Gln Gly Pro Glu Leu His Gly Trp Arg Pro
Pro Val
10 15
GAC TGT GTC CGG GCC AAT GAG CTG TGT GCC GCC GAA TCC AAC 197
TGC AGC
Asp Cys Val Arg Ala Asn Glu Leu Cys Ala Ala Glu Ser Asn
Cys Ser
20 25 30
TCT CGC TAC CGC ACT CTG CGG CAG TGC CTG GCA GGC CGC GAC 245
CGC AAC
Ser Arg Tyr Arg Thr Leu Arg Gln Cys Leu Ala Gly Arg Asp
Arg Asn
35 40 45
ACC ATG CTG GCC AAC AAG GAG TGC CAG GCG GCC TTG GAG GTC 293
TTG CAG
Thr Met Leu Ala Asn Lys Glu Cys Gln Ala Ala Leu Glu Val
Leu Gln
50 55 60 65
GAG AGC CCG CTG TAC GAC TGC CGC TGC AAG CGG GGC ATG AAG 341
AAG GAG
Glu Ser Pro Leu Tyr Asp Cys Arg Cys Lys Arg Gly Met Lys
Lys Glu
70 75 80
CTG CAG TGT CTG CAG ATC TAC TGG AGC ATC CAC CTG GGG CTG 389
ACC GAG
Leu Gln Cys Leu Gln Ile Tyr Trp Ser Ile His Leu Gly Leu
Thr Glu
85 90 95
GGT GAG GAG TTC TAC GAA GCC TCC CCC TAT GAG CCG GTG ACC 437
TCC CGC
Gly Glu Glu Phe Tyr Glu Ala Ser Pro Tyr Glu Pro Val Thr
Ser Arg
100 105 110
Y
CTC TCG GAC ATC TTC AGG CTT GCT TCA ATC TTC TCA GGG ACA 485
GGG GCA
Leu Ser Asp Ile Phe Arg Leu Ala Ser Ile Phe Ser Gly Thr
Gly Ala
115 120 125
GAC CCG GTG GTC AGC GCC AAG AGC AAC CAT TGC CTG GAT GCT 533
GCC AAG
Asp Pro Val Val Ser Ala Lys Ser Asn His Cys Leu Asp Ala
Ala Lys
130 135 140 145
GCC TGC AAC CTG AAT GAC AAC TGC AAG AAG CTG CGC TCC TCC 581
TAC ATC
Ala Cys Asn Leu Asn Asp Asn Cys Lys Lys Leu Arg Ser Ser
Tyr Ile
150 155 160
TCC ATC TGC AAC CGC GAG ATC TCG CCC ACC GAG CGC TGC AAC 629
CGC CGC
Ser Ile Cys Asn Arg Glu Ile Ser Pro Thr Glu Arg Cys Asn
Arg Arg
165 170 175
AAG TGC CAC AAG GCC CTG CGC CAG TTC TTC GAC CGG GTG CCC 677
AGC GAG
Lys Cys Hie Lys Ala Leu Arg Gln Phe Phe Asp Arg Val Pro
Ser Glu
180 185 190
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TAC ACC TAC CGC ATG CTC TTC TGC TCC TGC CAA GAC CAG GCG 725
TGC GCT
Tyr Thr Tyr Arg Met Leu Phe Cys Ser Cys Gln Asp Gln Ala
Cys Ala
195 200 205
GAG CGC CGC CGG CAA ACC ATC CTG CCC AGC TGC TCC TAT GAG 773
GAC AAG
Glu Arg Arg Arg Gln Thr Ile Leu Pro Ser Cys Ser Tyr Glu
Asp Lys
210 215 220 225
GAG AAG CCC AAC TGC CTG GAC CTG CGT GGC GTG TGC CGG ACT 821
GAC CAC
Glu Lys Pro Asn Cys Leu Asp Leu Arg Gly Val Cys Arg Thr
Asp His
230 235 240
CTG TGT CGG TCC CGG CTG GCC GAC TTC CAT GCC AAT TGT CGA 869
GCC TCC
Leu Cys Arg Ser Arg Leu Ala Asp Phe His Ala Asn Cys Arg
Ala Ser
245 250 255
TAC CAG ACG GTC ACC AGC TGC CCT GCG GAC AAT TAC CAG GCG 917
TGT CTG
Tyr Gln Thr Val Thr Ser Cys Pro Ala Asp Asn Tyr Gln Ala
Cys Leu
260 265 270
GGC TCT TAT GCT GGC ATG ATT GGG TTT GAC ATG ACA CCT AAC 965
TAT GTG
Gly Ser Tyr Ala Gly Met Ile Gly Phe Asp Met Thr Pro Asn
Tyr Val
275 280 285
GAC TCC AGC CCC ACT GGC ATC GTG GTG TCC CCC TGG TGC AGC 1013
TGT CGT
Asp Ser Ser Pro Thr Gly Ile Val Val Ser Pro Trp Cys Ser
Cys Arg
290 295 300 305
GGC AGC GGG AAC ATG GAG GAG GAG TGT GAG AAG TTC CTC AGG 1061
GAC TTC
Gly Ser Gly Asn Met Glu Glu Glu Cys Glu Lys Phe Leu Arg
Asp Phe
310 315 320
ACC GAG AAC CCA TGC CTC CGG AAC GCC ATC CAG GCC TTT GGC 1109
AAC GGC
Thr Glu Asn Pro Cys Leu Arg Asn Ala Ile Gln Ala Phe Gly
Asn Gly
325 330 335
ACG GAC GTG AAC GTG TCC CCA AAA GGC CCC TCG TTC CAG GCC 1157
ACC CAG
Thr Asp Val Asn Val Ser Pro Lys Gly Pro Ser Phe Gln Ala
Thr Gln
340 345 350
GCC CCT CGG GTG GAG AAG ACG CCT TCT TTG CCA GAT GAC CTC 1205
AGT GAC
Ala Pro Arg Val Glu Lys Thr Pro Ser Leu Pro Asp Asp Leu
Ser Asp
355 360 365
AGT ACC AGC TTG GGG ACC AGT GTC ATC ACC ACC TGC ACG TCT 1253
GTC CAG
Ser Thr Ser Leu Gly Thr Ser Val Ile Thr Thr Cys Thr Ser
Val Gln
370 375 380 385
GAG CAG GGG CTG AAG GCC AAC AAC TCC AAA GAG TTA AGC ATG 1301
TGC TTC
Glu Gln Gly Leu Lys Ala Asn Asn Ser Lys Glu Leu Ser Met
Cys Phe
390 395 400
ACA GAG CTC ACG ACA AAT ATC ATC CCA GGG AGT AAC AAG GTG 1349
ATC AAA
Thr Glu Leu Thr Thr Asn Ile Ile Pro Gly Ser Asn Lys Val
Ile Lys
405 410 415
CCT AAC TCA GGC CCC AGC AGA GCC AGA CCG TCG GCT GCC TTG 1397
ACC GTG
Pro Asn Ser Gly Pro Ser Arg Ala Arg Pro Ser Ala Ala Leu
Thr Val
420 425 430
\, .y
CTG TCT GTC CTG ATG CTG AAA CAG GCC TTG~~TAGGCTGTGG GAACCGAGTC1447
Leu Ser Val Leu Met Leu Lys Gln Ala Leu
435 440
AGAAGATTTT TGAAAGCTAC GCAGACAAGA ACAGCCGCCT GACGAAATGG 1507
AAACACACAC
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AGACACACAC ACACCTTGCA AAAAAAAAAA AAAAAA 1543
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 464 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Ile Leu Ala Asn Val Phe Cys Leu Phe Phe Phe Leu Asp Glu Thr
-21 -20 -15 -10
Leu Arg Ser Leu Ala Ser Pro Ser Ser Leu Gln Gly Pro Glu Leu His
-5 1 5 10
Gly Trp Arg Pro Pro Val Asp Cys Val Arg Ala Asn Glu Leu Cys Ala
15 20 2S
Ala Glu Ser Asn Cys Ser Ser Arg Tyr Arg Thr Leu Arg Gln Cys L_eu
30 35 40
Ala Gly Arg Asp Arg Asn Thr Met Leu Ala Asn Lys Glu Cys Gln Ala
45 50 55
Ala Leu Glu Val Leu Gln Glu Ser Pro Leu Tyr Asp Cys Arg Cys Lys
60 65 70 75
Arg Gly Met Lys Lys Glu Leu Gln Cys Leu Gln Ile Tyr Trp Ser Ile
80 85 90
His Leu Gly Leu Thr Glu Gly Glu Glu Phe Tyr Glu Ala Ser Pro Tyr
95 100 105
Glu Pro Val Thr Ser Arg Leu Ser Asp Ile Phe Arg Leu Ala Ser Ile
110 115 120
Phe Ser Gly Thr Gly Ala Asp Pro Vai Val Ser Ala Lys Ser Asn His
125 130 135
Cys Leu Asp Ala Ala Lys Ala Cys Asn Leu Asn Asp Asn Cys Lys Lys
140 145 150 155
Leu Arg Ser Ser Tyr Ile Ser Ile Cys Asn Arg Glu Ile Ser Pro Thr
160 165 170
Glu Arg Cys Asn Arg Arg Lys Cys His Lys Ala Leu Arg Gln Phe Phe
17S 180 185
Asp Arg Val Pro Ser Glu Tyr Thr Tyr Arg Met Leu Phe Cys Ser Cys
190 195 200
Gln Asp Gln Ala Cys Ala Glu Arg Arg Arg Gln Thr Ile Leu Pro Ser
205 210 215
Cys Ser Tyr Glu Asp Lys Glu Lys Pro Asn Cys Leu Asp Leu Arg Gly
220 225 230 235
Val Cys Arg Thr Asp His Leu Cys Arg Ser Arg Leu Ala Asp Phe His
240 245 250
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Ala Asn Cys Arg Ala Ser Tyr Gln Thr Val Thr Ser Cys Pro Ala Asp
255 260 265
Asn Tyr Gln Ala Cys Leu Gly Ser Tyr Ala Gly Met Ile Gly Phe Asp
270 275 280
Met Thr Pro Asn Tyr Val Asp Ser Ser Pro Thr Gly Ile Val Val Ser
285 290 295
Pro Trp Cys Ser Cys Arg Gly Ser Gly Asn Met Glu Glu Glu Cys Glu
340 305 310 315
Lys Phe Leu Arg Asp Phe Thr Glu Asn Pro Cys Leu Arg Asn Ala Ile
320 325 330
Gln Ala Phe Gly Asn Gly Thr Asp Val Asn Val Ser Pro Lys Gly Pro
335 340 345
Ser Phe Gln Ala Thr Gln Ala Pro Arg Val Glu Lys Thr Pro Ser Leu
350 355 360
Pro Asp Asp Leu Ser Asp Ser Thr Ser Leu Gly Thr Ser Val Ile Thr
365 370 375
Thr Cys Thr Ser Val Gln Glu Gln Gly Leu Lys Ala Asn Asn Ser Lys
380 385 390 395
Glu Leu Ser Met Cys Phe Thr Glu Leu Thr Thr Asn Ile Ile Pro Gly
400 405 410
Ser Asn Lys Val Ile Lys Pro Asn Ser Gly Pro Ser Arg Ala Arg Pro
415 420 425
Ser Ala Ala Leu Thr Val Leu Ser Val Leu Met Leu Lys Gln Ala Leu
430 435 440
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 411 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
( HYPOTFiETI
i CAL
i :
i YES
)
(xi) SEQUENCE 3:
DESCRIPTION:
SEQ
ID
N0:
Ser ProSer Leu Gln Gly Glu LeuHis Trp Pro Pro
Ser Pro Gly Arg
1 5 10 15
Val AspCys Arg Ala Asn Leu CysAla Glu Asn Cys
Val Glu Ala Ser
20 25 30
Ser SerArg Arg Thr Leu Gln CysLeu Gly Asp Arg
Tyr Arg Ala Arg
35 40 45
Asn ThrMet Ala Asn Lys Cys GlnAla Leu Val Leu
Leu Glu Ala Glu
50 55 60
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Gln Glu Ser Pro Leu Tyr Asp Cys Arg Cys Lys Arg Gly Met Lys Lys
65 70 75 80
Glu Leu Gln Cys Leu Gln Ile Tyr Trp Ser Ile His Leu Gly Leu Thr
85 90 95
Glu Gly Glu Glu Phe Tyr Glu Ala Ser Pro Tyr Glu Pro Val Thr Ser
100 105 110
Arg Leu Ser Asp Ile Phe Arg Leu Ala Ser Ile Phe Ser Gly Thr Gly
115 120 125
Ala Asp Pro Val Val Ser Ala Lys Ser Asn His Cys Leu Asp Ala Ala
130 135 140
Lys Ala Cys Asn Leu Asn Asp Asn Cys Lys Lys Leu Arg Ser Ser Tyr
145 150 155 160
Ile Ser Ile Cys Asn Arg Glu Ile Ser Pro Thr Glu Arg Cys Asn Arg
165 170 175
Arg Lys Cys His Lys Ala Leu Arg Gln Phe Phe Asp Arg Val Pro Ser
180 185 190
Glu Tyr Thr Tyr Arg Met Leu Phe Cys Ser Cys Gln Asp Gln Ala Cys
195 200 205
Ala Glu Arg Arg Arg Gln Thr Ile Leu Pro Ser Cys Ser Tyr Glu Asp
210 215 220
Lys Glu Lys Pro Asn Cys Leu Asp Leu Arg Gly Val Cys Arg Thr Asp
225 230 235 240
His Leu Cys Arg Ser Arg Leu Ala Asp Phe His Ala Asn Cys Arg Ala
245 250 255
Ser Tyr Gln Thr Val Thr Ser Cys Pro Ala Asp Asn Tyr Gln Ala Cys
260 265 270
Leu Gly Ser Tyr Ala Gly Met Ile Gly Phe Asp Met Thr Pro Asn Tyr
275 280 285
Val Asp Ser Ser Pro Thr Gly Ile Val Val Ser Pro Trp Cys Ser Cys
290 295 300
Arg Gly Ser Gly Asn Met Glu Glu Glu Cys Glu Lys Phe Leu Arg Asp
305 310 315 320
Phe Thr Glu Asn Pro Cys Leu Arg Asn Ala Ile Gln Ala Phe Gly Asn
325 330 335
Gly Thr Asp Val Asn Val Ser Pro Lys Gly Pro Ser Phe Gln Ala Thr
340 345 350
Gln Ala Pro Arg Val Glu Lys Thr Pro Ser Leu Pro Asp Asp Leu Ser
355 360 365
Asp Ser Thr Ser Leu Gly Thr Ser Val Ile Thr Thr Cys Thr Ser Val
370 375 380
Gln Glu Gln Gly Leu Lys Ala Asn Asn Ser Lys Glu Leu Ser Met Cys
385 390 395 400
Phe Thr Glu Leu Thr Thr Asn Ile Ile Pro Gly
405 410
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(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1392 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1389
(ix) FEATURE:
(A) NAME/KEY: sig~eptide
(B) LOCATION: 1..63
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 64..1389
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
ATG ATC TTG GCA AAC GCC TTC TGC CTC TTC TTC 48
TTT TTA GAC GAA ACC
Met Ile Leu Ala Asn Ala Phe Cys Leu Phe Phe
Phe Leu Asp Glu Thr
-21 -20 -15 -10
CTC CGC TCT TTG GCC AGC CCT TCC TCT CCG CAG CTC CAC 96
GGC TCT GAG
Leu Arg Ser Leu Ala Ser Pro Ser Ser Pro Gln Leu His
Gly Ser Glu
-5 1 5 10
GGC TGG CGC CCC CAA GTG GAC TGT GTC CGG GCC TGT GCG 144
AAT GAG CTG
Gly Trp Arg Pro Gln Val Asp Cys Val Arg Ala Cys Ala
Asn Glu Leu
15 20 25
GCT GAA TCC AAC TGC AGC TCC AGG TAC CGC ACC TGC CTG 192
CTT CGG CAG
Ala Glu Ser Asn Cys Ser Ser Arg Tyr Arg Thr Cys Leu
Leu Arg Gln
30 35 40
GCC GGC CGG GAT CGC AAT ACC ATG CTG GCC AAT CAG GCG 240
AAG GAG TGC
Ala Gly Arg Asp Arg Asn Thr Met Leu Ala Asn Gln Ala
Lys Glu Cys
45 50 55
GCC CTG GAG GTC TTG CAG GAA AGC CCA TTG TAT TGC AAG 288
GAC TGC CGC
Ala Leu Glu Val Leu Gln Glu Ser Pro Leu Tyr Cys Lys
Asp Cys Arg
60 65 70 75
CGG GGC ATG AAG AAG GAG CTG CAG TGT CTG CAG AGC ATC 336
ATC TAT TGG
Arg Gly Met Lys Lys Glu Leu Gln Cys Leu Gln Ser Ile
Ile Tyr Trp
BO 85 90
CAT CTG GGG CTG ACG GAG GGT GAG GAG TTC TAC CCC TAT 384
GAA GCT TCG
His Leu Gly Leu Thr Glu Gly Glu Glu Phe Tyr Pro Tyr
Glu Ala Ser
95 100 105
GAG CCT GTG ACC TCC CGC CTC TCG GAC ATC TTC TCA ATC 432
AGG CTC GCT
Glu Pro Val Thr Ser Arg Leu Ser Asp Ile Phe Ser Ile
Arg Leu Ala
110 115 120
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TTC TCA GGG ACA GGG GCA GAC CCG GTG GTC AGT GCC AAG AGC AAC CAC 480
Phe Ser Gly Thr Gly Ala Asp Pro Val Val Ser Ala Lys Ser Asn His
125 130 135
TGC CTG GAT GCC GCC AAG GCC TGC AAC CTG AAC GAC AAC TGC AAG AAG 528
Cys Leu Asp Ala Ala Lys Ala Cys Asn Leu Asn Asp Asn Cys Lys Lys
140 145 150 155
CTC CGC TCC TCC TAC ATC TCC ATC TGC AAC CGC GAG ATC TCT CCC ACT 576
Leu Arg Ser Ser Tyr Ile Ser Ile Cys Asn Arg Glu Ile Ser Pro Thr
160 165 170
GAA CGC TGC AAC CGC CGC AAG TGC CAC AAG GCC CTG CGC CAG TTC TTC 624
Glu Arg Cys Asn Arg Arg Lys Cys His Lys Ala Leu Arg Gln Phe Phe
175 180 185
GAC CGT GTG CCC AGC GAG TAT ACC TAC CGC ATG CTC TTC TGC TCC TGT 672
Asp Arg Val Pro Ser Glu Tyr Thr Tyr Arg Met Leu Phe Cys Ser Cys
190 195 200
CAG GAC CAG GCA TGC GCC GAG CGT CGC CGG CAA ACC ATC CTG CCC AGC 720
Gln Asp Gln Ala Cys Ala Glu Arg Arg Arg Gln Thr Ile Leu Pro Ser
205 210 215
TGT TCC TAT GAG GAC AAG GAG AAG CCC AAC TGC TTG GAC CTG CGC AGC 768
Cys Ser Tyr Glu Asp Lys Glu Lys Pro Asn Cys Leu Asp Leu Arg Ser
220 225 230 235
CTG TGT CGT ACA GAC CAC TTG TGC CGG TCC CGC CTG GCA GAC TTC CAC 816
Leu Cys Arg Thr Asp His Leu Cys Arg Ser Arg Leu Ala Asp Phe His
240 245 250
GCC AAC TGT CGA GCC TCC TAC CGG ACA ATC ACC AGC TGC CCT GCG GAC 864
Ala Asn Cys Arg Ala Ser Tyr Arg Thr Ile Thr Ser Cys Pro Ala Asp
255 260 265
AAC TAC CAG GCA TGT CTG GGC TCC TAT GCT GGC ATG ATT GGG TTT GAT 912
Asn Tyr Gln Ala Cys Leu Gly Ser Tyr Ala Gly Met Ile Gly Phe Asp
270 275 280
ATG ACA CCG AAC TAT GTG GAC TCC AAC CCC ACG GGC ATC GTG GTG TCT 960
Met Thr Pro Asn Tyr Val Asp Ser Asn Pro Thr Gly Ile Val Val Ser
285 290 295
CCC TGG TGC AAT TGT CGT GGC AGT GGG AAC ATG GAA GAA GAG TGT GAG 1008
Pro Trp Cys Asn Cys Arg Gly Ser Gly Asn Met Glu Glu Glu Cys Glu
300 305 310 315
AAG TTC CTC AAG GAC TTC ACA GAA AAC CCA TGC CTC CGG AAT GCC ATT 1056
Lys Phe Leu Lys Asp Phe Thr Glu Asn Pro Cys Leu Arg Asn Ala Ile
320 325 330
CAA GCC TTT GGC AAT GGC ACA GAT GTG AAC ATG TCT CCC AAA GGC CCC 1104
Gln Ala Phe Gly Asn Giy Thr Asp Val Asn Met Ser Pro Lys Gly Pro
335 340 345
ACA TTT TCA GCT ACC CAG GCC CCT CGG GTA GAG AAA ACT CCT TCA CTG 1152
Thr Phe Ser Ala Thr Gln Ala Pro Arg Val Glu Lys Thr Pro Ser Leu
350 355 360
CCA GAT GAC CTC AGT GAT AGC ACC AGC CTG GGG ACC AGT GTC ATC ACC 1200
Pro Asp Asp Leu Ser Asp Ser Thr Ser Leu Gly Thr Ser Val Ile Thr
365 370 375
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ACCTGCACA TCTATC CAGGAGCAA GGGCTGAAG GCCAACAAC TCC 1248
AAA
ThrCysThr SerIle GlnGluGln GlyLeuLys AlaAsnAsn SerLys
380 385 390 395
GAGTTAAGC ATGTGT TTCACAGAG CTCACGACA AATATCAGC CCAGGG 1296
GluLeuSer MetCys PheThrGlu LeuThrThr AsnIleSer ProGly
400 405 410
AGTAAAAAG GTGATC AAACTTTAC TCAGGCTCC TGCAGAGCC AGACTG 1344
SerLysLys ValIle LysLeuTyr SerGlySer CysArgAla ArgLeu
415 420 425
TCGACTGCC TTGACT GCCCTCCCA CTCCTGATG GTGACCTTG GCC 1389
SerThrAla LeuThr AlaLeuPro LeuLeuMet ValThrLeu Ala
430 435 440
TAG 1392
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 463 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
Met Ile Leu Ala Asn Ala Phe Cys Leu Phe Phe Phe Leu Asp Glu Thr
-21 -20 -15 -10
Leu Arg Ser Leu Ala Ser Pro Ser Ser Pro Gln Gly Ser Glu Leu His
-S 1 5 10
Gly Trp Arg Pro Glri Val Asp Cys Val Arg Ala Asn Glu Leu Cys Ala
15 20 25
Ala Glu Ser Asn Cys Ser Ser Arg Tyr Arg Thr Leu Arg Gln Cys Leu
30 35 40
Ala Gly Arg Asp Arg Asn Thr Met Leu Ala Asn Lys Glu Cys Gln Ala
45 50 55
Ala Leu Glu Val Leu Gln Glu Ser Pro Leu Tyr Asp Cys Arg Cys Lys
60 65 70 75
Arg Gly Met Lys Lys Glu Leu Gln Cys Leu Gln Ile Tyr Trp Ser Ile
80 85 90
His Leu Gly Leu Thr Glu Gly Glu Glu Phe Tyr Glu Ala Ser Pro Tyr
95 100 105
Glu Pro Val Thr Ser Arg Leu Ser Asp Ile Phe Arg Leu Ala Ser Ile
110 115 120
Phe Ser Gly Thr Gly Ala Asp Pro Val Val Ser Ala Lys Ser Asn His
125 130 135
Cys Leu Asp Ala Ala Lys Ala Cys Asn Leu Asn Asp Asn Cys Lys Lys
140 145 150 155
Leu Arg Ser Ser Tyr Ile Ser Ile Cys Asn Arg Glu Ile Ser Pro Thr
160 165 170
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Glu Arg Cys Asn Arg Arg Lys Cys His Lys Ala Leu Arg Gln Phe Phe
175 180 185
Asp Arg Val Pro Ser Glu Tyr Thr Tyr Arg Met Leu Phe Cys Ser Cys
190 195 200
Gln Asp Gln Ala Cys Ala Glu Arg Arg Arg Gln Thr Ile Leu Pro Ser
. 205 210 21.5
Cys Ser Tyr Glu Asp Lys Glu Lys Pro Asn Cys Leu Asp Leu Arg Ser
220 225 230 235
Leu Cys Arg Thr Asp His Leu Cys Arg Ser Arg Leu Ala Asp Phe His
240 245 250
Ala Asn Cys Arg Ala Ser Tyr Arg Thr Ile Thr Ser Cys Pro Ala Asp
255 260 265
Asn Tyr Gln Ala Cys Leu Gly Ser Tyr Ala Gly Met Ile Gly Phe Asp
270 275 280
Met Thr Pro Asn Tyr Val Asp Ser Asn Pro Thr Gly Ile Val Val Ser
285 290 295
Pro Trp Cys Asn Cys Arg Gly Ser Gly Asn Met Glu Glu Glu Cys Glu
300 305 310 315
Lys Phe Leu Lys Asp Phe Thr Glu Asn Pro Cys Leu Arg Asn Ala Ile
320 325 330
Gln Ala Phe Gly Asn Gly Thr Asp Val Asn Met Ser Pro Lys Gly Pro
335 340 345
Thr Phe Ser Ala Thr Gln Ala Pro Arg Val Glu Lys Thr Pro Ser Leu
350 355 360
Pro Asp Asp Leu Ser Asp Ser Thr Ser Leu Gly Thr Ser Val Ile Thr
365 370 375
Thr Cys Thr Ser Ile Gln Glu Gln Gly Leu Lys Ala Asn Asn Ser Lys
380 385 390 395
Glu Leu Ser Met Cys Phe Thr Glu Leu Thr Thr Asn Ile Ser Pro Gly
400 405 410
Ser Lys Lys Val Ile Lys Leu Tyr Ser Gly Ser Cys Arg Ala Arg Leu
415 420 425
Ser Thr Ala Leu Thr Ala Leu Pro Leu Leu Met Val Thr Leu Ala
430 435 440
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 411 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Ser Pro Ser Ser Pro Gln Gly Ser Glu Leu His Gly Trp Arg Pro Gln
1 5 10 15
Val Asp Cys Val Arg Ala Asn Glu Leu Cys Ala Ala Glu Ser Asn Cys
20 25 30
Ser Ser Arg Tyr Arg Thr Leu Arg Gln Cys Leu Ala Gly Arg Asp Arg
35 40 45
Asn Thr Met Leu Ala Asn Lys Glu Cys Gln Ala Ala Leu Glu Val Leu
50 55 60
Gln Glu Ser Pro Leu Tyr Asp Cys Arg Cys Lys Arg Gly Met Lys Lys
65 70 75 80
Glu Leu Gln Cys Leu Gln Ile Tyr Trp Ser Ile His Leu Gly Leu Thr
85 90 95
Glu Gly Glu Glu Phe Tyr Glu Ala Ser Pro Tyr Glu Pro Val Thr Ser
100 105 110
Arg Leu Ser Asp Ile Phe Arg Leu Ala Ser Ile Phe Ser Gly Thr Gly
115 120 125
Ala Asp Pro Val Val Ser Ala Lys Ser Asn His Cys Leu Asp Ala Ala
130 135 140
Lys Ala Cys Asn Leu Asn Asp Asn Cys Lys Lys Leu Arg Ser Ser Tyr
145 150 155 160
Ile Ser Ile Cys Pan Arg Glu Ile Ser Pro Thr Glu Arg Cys Asn Arg
165 170 175
Arg Lys Cys His Lys Ala Leu Arg Gln Phe Phe Asp Arg Val Pro Ser
180 185 190
Glu Tyr Thr Tyr Arg Met Leu Phe Cys Ser Cys Gln Asp Gln Ala Cys
195 200 205
Ala Glu Arg Arg Arg Gln Thr Ile Leu Pro Ser Cys Ser Tyr Glu Asp
210 215 220
Lys Glu Lys Pro Asn Cys Leu Asp Leu Arg Ser Leu Cys Arg Thr Asp
225 230 235 240
His Leu Cys Arg Ser Arg Leu Ala Asp Phe His Ala Asn Cys Arg Ala
245 250 255
Ser Tyr Arg Thr Ile Thr Ser Cys Pro Ala Asp Asn Tyr Gln Ala Cys
260 265 270
Leu Gly Ser Tyr Ala Gly Met Ile Gly Phe Asp Met Thr Pro Asn Tyr
275 280 285
Val Asp Ser Asn Pro Thr Gly Ile Val Val Ser Pro Trp Cys Asn Cys
290 295 300
Arg Gly Ser Gly Asn Met Glu Glu Glu Cys Glu Lys Phe Leu Lys Asp
305 310 315 320
Phe Thr Glu Asn Pro Cys Leu Arg Asn Ala Ile Gln Ala Phe Gly Asn
325 330 335
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Gly Thr Asp Val Asn Met Ser Pro Lys Gly Pro Thr Phe Ser Ala Thr
340 345 350
Gln Ala Pro Arg Val Glu Lys Thr Pro Ser Leu Pro Asp Asp Leu Ser
355 360 365
' Asp Ser Thr Ser Leu Gly Thr Ser Val Ile Thr Thr Cys Thr Ser Ile
370 375 380
Gln Glu Gln Gly Leu Lys Ala Asn Asn Ser Lys Glu Leu Ser Met Cys
385 390 395 400
Phe Thr Glu Leu Thr Thr Asn Ile Ser Pro Gly
405 410
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 331 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
Met Ile Leu Ala Asn Val Phe Cys Leu Phe Phe Phe Leu Gly Thr Gly
1 5 10 15
Ala Asp Pro Val Val Ser Ala Lys Ser Asn His Cys Leu Asp Ala Ala
20 25 30
Lys Ala Cys Asn Leu Asn Asp Asn Cys Lys Lys Leu Arg Ser Ser Tyr
35 40 45
Ile Ser Ile Cys Asn Arg Glu Ile Ser Pro Thr Glu Arg Cys Asn Arg
50 55 60
Arg Lys Cys His Lys Ala Leu Arg Gln Phe Phe Asp Arg Val Pro Ser
65 70 75 80
Glu Tyr Thr Tyr Arg Met Leu Phe Cys Ser Cys Gln Asp Gln Ala Cys
85 90 95
Ala Glu Arg Arg Arg Gln Thr Ile Leu Pro Ser Cys Ser Tyr Glu Asp
100 105 110
Lys Glu Lys Pro Asn Cys Leu Asp Leu Arg Gly Val Cys Arg Thr Asp
115 120 125
His Leu Cys Arg Ser Arg heu Ala Asp Phe His Ala Asn Cys Arg Ala
130 135 140
Ser Tyr Gln Thr Val Thr Ser Cys Pro Ala Asp Asn Tyr Gln Ala Cys
145 150 155 160
Leu Gly Ser Tyr Ala Gly Met Ile Gly Phe Asp Met Thr Pro Asn Tyr
165 170 175
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Val Asp Ser Ser Pro Thr Gly Ile Val Val Ser Pro Trp Cys Ser Cys
180 185 190
Arg Gly Ser Gly Asn Met Glu Glu Glu Cys Glu Lys Phe Leu Arg Asp
195 200 205
Phe Thr Glu Asn Pro Cys Leu Arg Asn Ala Ile Gln Ala Phe Gly Asn
210 215 220
Gly Thr Asp Val Asn Val Ser Pro Lys Gly Pro Ser Phe Gln Ala Thr
225 230 235 240
Gln Ala Pro Arg Val Glu Lys Thr Pro Ser Leu Pro Asp Asp Leu Ser
245 250 255
Asp Ser Thr Ser Leu Gly Thr Ser Val Ile Thr Thr Cys Thr Ser Val
260 265 270
Gln Glu Gln Gly Leu Lys Ala Asn Asn Ser Lys Glu Leu Ser Met Cys
275 280 285
Phe Thr Glu Leu Thr Thr Asn Ile Ile Pro Gly Ser Asn Lys Val Ile
290 295 300
Lys Pro Asn Ser Gly Pro Ser Arg Ala Arg Pro Ser Ala Ala Leu Thr
305 310 315 320
Val Leu Ser Val Leu Met Leu Lys Gln Ala Leu
325 330
(2} INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 330 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: B:
Met Ile Leu Ala Asn Ala Phe Cys Leu Phe Phe Phe Leu Gly Thr Gly
1 5 10 15
Ala Asp Pro Val Val Ser Ala Lys Ser Asn His Cys Leu Asp Ala Ala
20 25 30
Lys Ala Cys Asn Leu Asn Asp Asn Cys Lys Lys Leu Arg Ser Ser Tyr
35 40 45
Ile Ser Ile Cys Asn Arg Glu Ile Ser Pro Thr Glu Arg Cys Asn Arg
50 55 60
Arg Lys Cys His Lys Ala Leu Arg Gln Phe Phe Asp Arg Val Pro Ser
65 70 75 80
Glu Tyr Thr Tyr Arg Met Leu Phe Cys Ser Cys Gln Asp Gln Ala Cys
85 90 95
Ala Glu Arg Arg Arg Gln Thr Ile Leu Pro Ser Cys Ser Tyr Glu Asp
100 105 110
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Lys Glu Lys Pro Asn Cys Leu Asp Leu Arg Ser Leu Cys Arg Thr Asp
115 120 125
His Leu Cys Arg Ser Arg Leu Ala Asp Phe His Ala Asn Cys Arg Ala
130 135 140
Ser Tyr Arg Thr Ile Thr Ser Cys Pro Ala Asp Asn Tyr Gln Ala Cys
145 150 155 160
Leu Gly Ser Tyr Ala Gly Met Ile Gly Phe Asp Met Thr Pro Asn Tyr
165 170 175
Val Asp Ser Asn Pro Thr Gly Ile Val Val Ser Pro Trp Cys Asn Cys
180 185 190
Arg Gly Ser Gly Asn Met Glu Glu Glu Cys Glu Lys Phe Leu Lys Asp
195 200 205
Phe Thr Glu Asn Pro Cys Leu Arg Asn Ala Ile Gln Ala Phe Gly Asn
210 215 220
Gly Thr Asp Val Asn Met Ser Pro Lys Gly Pro Thr Phe Ser Ala Thr
225 230 235 240
Gln Ala Pro Arg Val Glu Lys Thr Pro Ser Leu Pro Asp Asp Leu Ser
245 250 255
Asp Ser Thr Ser Leu Gly Thr Ser Val Ile Thr Thr Cys Thr Ser Ile
260 265 270
Gln Glu Gln Gly Leu Lys Ala Asn Asn Ser Lys Glu Leu Ser Met Cys
275 280 285
Phe Thr Glu Leu Thr Thr Asn Ile Ser Pro Gly Ser Lys Lys Val Ile
290 295 300
Lys Leu Tyr Ser Gly Ser Cys Arg Ala Arg Leu Ser Thr Ala Leu Thr
305 310 315 320
Ala Leu Pro Leu Leu Met Val Thr Leu Ala
325 330
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
Cys Arg Cys Lys Arg Gly Met Lys Lys Glu
1 5 10
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(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(xi} SEQUENCE DESCRIPTION: SEQ ID NO:10:
Cys Asn Arg Arg Lys Cys His Lys Aia Lys Arg
1 5 10
(2) INFORMATION FOR SEQ ID N0:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C} STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:peptide
(iii}HYPOTHETICAL:
NO
(v) FRAGMENT TYPE:internal
(ix) FEATURE:
(AI NAME/KEY:Modified-site
(B) LOCATION:3
(D) OTHER
INFORMATION:
/product=
"OTHER"
/note="Xaa is Lys Arg"
or
{xi) SEQ~NCE DESCRIPTION: SEQ ID N0:11:
Cys Leu Xaa Asn Ala Ile Glu Ala Phe Gly Asn Gly
1 5 10
(2} INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 465 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
Met Phe Leu Ala Thr Leu Tyr Phe Ala Leu Pro Leu Leu Asp Leu Leu
1 S 10 15
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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 60
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 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 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 29S 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 Lys Leu Phe Phe Asp Asn
Leu Phe Aen Lys Thr
325 330 335
Cys Leu Lys Asn Ala Gln Phe Asn Gly Asp Val
Ile Ala Gly Ser Thr
340 345 350
Val Trp Gln Pro Ala Pro Gln Thr Thr Thr Thr
Pro Val Thr Ala Thr
355 360 365
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Thr AlaLeuArg ValLysAsn LysProLeu GlyProAla GlySerGlu
370 375 380
Asn GluIlePro ThrHisVal LeuProPro CysAlaAsn LeuGlnAla
385 390 395 400
Gln LysLeuLys SerAsnVal SerGlyAsn ThrHisLeu CysIleSer
405 410 415
Asn GlyAsnTyr GluLysGlu GlyLeuGly AlaSerSer HisIleThr
420 425 430
Thr LysSerMet AlaAlaPro ProSerCys GlyLeuSer ProLeuLeu
435 440 445
Val LeuValVal ThrAlaLeu SerThrLeu LeuSerLeu ThrGluThr
450 455 460
Ser
465
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 468 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
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
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
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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 Sex 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
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
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(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "TrnRl primer 1"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
GCGGTACCAT GTTCCTAGCC ACTCTGTACT TCGC 34
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "TrnRl primer 2"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
GCTCTAGACT ACGACGTTTC TGCCAACGAT ACAG 34
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Trn2 primer 1"
( i i i ) HYPOTIiET I CAL : NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
GCGGTACCAT GATCTTGGCA AACGTCTGC 29
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(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "TrnR2 primer 2"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
GCTCTAGAGT CAGGCGGCTG TTCTTGTCTG CG 32
(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 90 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "NTN Primer 1"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ SID N0:18:
GCATATGCCG GGTGCTCGTC CGTGCGGCCT GCGTGCAACT GGAAGTTCGT GTTTCTGAAC 60
TGGGTCTGGG TTACACTTCT GACGAAACTG T 90
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 87 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "NTN Primer 2"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
GCTGACGCAG ACGACGCAGA CCCAGGTCGT AGATACGGAT AGCAGCTTCG CATGCACCAG 60
CGCAGTAACG GAACAGAACA GTTTCGT
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(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 87 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "NTN Primer 3"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
CTGCGTCAGC GTCGTCGTGT TCGTCGTGAA CGTGCTCGTG CTCACCCGTG CTGCCGTCCG 60
ACTGCTTACG AAGACGAAGT TTCTTTC 87
(2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 86 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "NTN Primer 4"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
CGGATCCTTA AACGCAAGCG CATTCACGAG CAGACAGTTC CTGCAGAGTG TGGTAACGAG 60
AGTGAACGTC CAGGAAAGAA ACTTCG 86
(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "OLIGOLINKER A"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
TATGCACCAT CATCATCATC ATGACGACGA CGACAAGGC 39
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(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "OLIGOLINKER B"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
TAGCCTTGTC GTCGTCGTCA TGATGATGAT GATGGTGCA 39
(2) INFORMATION FOR SEQ ID N0:24:
W ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "GDNF PRIMER"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
CAGCATATGT CACCAGATAA ACAAGCGGCG GCACT 35
(2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "GDNF PRIMER"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQ~CE DESCRIPTION: SEQ ID N0:25:
CAGGGATCCG GGTCAGATAC ATCCACACCG TTTAGC 36
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(2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "MOUSE RET FORWARD PCR
PRIMER"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:
TGGCACACCT CTGCTCTATG 20
(2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "MOUSE RET REVERSE PCR
PRIMER
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
TGTTCCCAGG AACTGTGGTC 20
(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "TrnRl FORWARD PRIMER"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
GCACAGCTAC GGGATGCTCT TCTG 24
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(2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(Ay LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "TrnRl REVERSE PRIMER"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
GTAGTTGGGA GTCATGACTG TGCCAATC 28
(2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "TrnR2 FORWARD PRIMER"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
AGCCGACGGT GTGGCTCTGC TGG 23
(2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base gairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "TrnR2 REVERSE PRIMER"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
CCAGTGTCAT CACCACCTGC ACG 23
