Note: Descriptions are shown in the official language in which they were submitted.
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GALANIN RECEPTOR GalR2
This application claims priority from U.S. Provisional Application No.
60/019946,
filed June 5, 1996.
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a novel neuropeptide galanin receptor and its
nucleic acid
sequence.
2. Description of the Related Art
Galanin is a 29 amino acid peptide hormone (30 amino acids in human) which is
present in a wide range of central and peripheral tissues. Skofitsch, Peptides
6, 509 (1985);
Merchenthaler, Prog. Neurobiol. 40, 711 (1993). Galanin is involved in many
diverse
physiological functions. Galanin is known to regulate the secretion of both
endocrine and
exocrine hormones. Galanin inhibits insulin secretion from pancreatic beta
cells, and can
inhibit pancreatic amylase secretion; in the stomach, galanin inhibits gastrin
and somatostatin
secretion. Galanin stimulates VIP (vasoactive intestinal protein) release from
the
hypothalamus, prolactin and growth hormone release from the pituitary; and
inhibits the
secretion of ACTH in the hypothalamus. Furthermore, the secretion of
neurotransmitters can
be modulated. For example, galanin can inhibit the release of histamine and
norepinephrine
in the hypothalamus. Other secondary messenger systems are also regulated:
galanin can
either stimulate or inhibit intracellular cAMP accumulation; is involved in
the closure of N-
and L-type voltage-sensitive calcium channels, and in the opening of ATP-
sensitive and -
insensitive potassium channels; and has been shown to stimulate the release of
calcium from
intracellular stores. Moreover, galanin is involved in the inhibition of
acetylcholine release
and the inhibition of muscarinic receptor-mediated phosphoinositide turnover.
Bartfai, Crit.
Rev. Neurobiol. 7, 229 ( 1993)
Galanin is implicated in the modulation of many cognitive and sensory
functions.
Galanin has potent antinociceptive effects, and can impair performance in one-
trial learning,
t-maze, and swim maze learning and memory paradigms. Its inhibition of the
anoxic release
of glutamate, as well as its inhibitory actions on cholinergic function
suggest a role in
neuroprotection, and in the development of Alzheimer's Disease. Crawley,
Regulatory
Peptides 59, 1 (1995). Galanin is known to induce feeding in rodents and, in
contrast with
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the effects of Neuropeptide Y on feeding, galanin increases preference for fat
intake.
Akabayashi, Proc. Natl. Acad. Sci. USA 9I, 10375 (1994). Galanin is also
involved in the
regulation of gastrointestinal smooth muscle contraction. Because of the
important role of
galanin in these many physiological processes, there is a strong need to
further develop
materials and methods for investigating the mechanistic behavior of the
receptors and for
treating diseased and other abnormal states associated with these
physiological processes.
Pharmacological data suggest the existence of several galanin receptor
subtypes.
Wynick, Proc. Natl. Acad. Sci. USA 90, 423 I ( 1990); Zen-Fa, J. Pharmacol.
Exp. Ther. 272,
371 (1995). Galanin receptors are known to be linked to the G; proteins, and
there is some
evidence that certain galanin receptor subtypes may be linked to cholera toxin-
sensitive GS
proteins. Gillison, Diabetes 43, 24 ( 1994); Chen, Am. J. Physiol. 266, G 113
( 1994). One
galanin receptor has been cloned and it is a member of the seven transmembrane
(7TMD)
class of G protein-linked receptors. It has been designated as GaIRI. Habert-
Ortoli, Proc.
Natl. Acad. Sci. USA 91, 9780; W095/22608. In addition to this human GalRl
receptor, the
GalRl receptor has been obtained from rat. Burgevin, J. Mol. Neurosci. b, 33
(1995). The in
vivo functions mediated through this cloned GaIR 1 receptor have not yet been
elucidated.
EP-0711830-A2 disclose a closely-related GalR1 sequence, differing in that
CyslS--~Trp is
varied.
SUMMARY OF THE INVENTION
The present invention provides a novel galanin receptor protein, the GalR2
receptor.
Also provided are the nucleic acid sequences encoding this novel receptor
protein as well as
methods for using this protein and its nucleic acid sequence, and methods
useful for
developing and identifying compounds for the treatment of diseases anc~
;aisorders in which
galanin is implicated. The importance of this discovery is manifested txe
effects of
galanin, which include antinociceptive activity, smooth muscle contrac~;r~n,
cardiovascular
activity, pituitary hormone release, cognition, and increased food intake.
Thus, this receptor
protein is useful for screening for galanin agonist and antagonist activity
for controlling these
conditions.
In one aspect of the present invention, we provide isolated nucleic acid
sequences for
a novel galanin receptor, the GalR2 receptor. In particular, we provide the
cDNA sequences
encoding the complete rat receptor and partial sequences of the human
receptor. These
nucleic acid sequences have a variety of uses. For example, they are useful
for making
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vectors and for transforming cells, both of which are ultimately useful for
production of the
GalR2 receptor protein. They are also useful as scientific research tools for
developing
nucleic acid probes for determining receptor expression levels, e.g., to
identify diseased or
otherwise abnormal states. They are useful for developing analytical tools
such as antisense
oligonucleotides for selectively inhibiting expression of the receptor gene to
determine
physiological responses. The present invention can also be used to isolate the
homologous
nucleic acid sequence of other species, such as human, primate, dog, mouse,
etc.
In another aspect of the present invention, we provide a homogeneous
composition
comprising the receptor GalR2 protein. The protein is useful for screening
drugs for agonist
and antagonist activity, and, therefore, for screening for drugs useful in
regulating
physiological responses associated with the GaIR2 receptor. Specifically,
antagonists to the
GalR2 receptor could be used to treat obesity and diabetes by reducing
appetite and food
consumption, whereas agonists could be used for the treatment of anorexic
conditions.
Furthermore, drugs could be used to treat Alzheimer's disease, stroke,
neuropathic pain,
and/or endocrine disorders. The proteins are also useful for developing
antibodies for
detection of the protein.
Flowing from the foregoing are a number of other aspects of the invention,
including
(a) vectors, such as plasmids, comprising the receptor GaIR2 nucleic acid
sequence that may
further comprise additional regulatory elements, e.g., promotors, (b)
transformed cells that
express the GalR2 receptor, (c) nucleic acid probes, (d) antisense
oligonucleotides, (e)
agonists, (f) antagonists, and (g) transgenic mammals. Further aspects of the
invention
comprise methods for making and using the foregoing compounds and
compositions.
The invention includes polynucleotide molecules coding for a rat or human
GalR2, or
a galanin binding fragment thereof. A polynucleotide molecule comprising SEQ
ID NO:1 or
a degenerate variant thereof. A polynucleotide molecule comprising the full-
length cDNA, or
a degenerate variant thereof, corresponding to the partial sequence shown in
SEQ ID NO:S.
A purified and isolated rat or human GalR2 protein. The GaIR2 protein
comprising SEQ ID
N0:2. The full-length GaIR2 protein, the partial sequence of which is
indicated in SEQ ID
N0:6. A purified and isolated rat or human GalR2 protein or fragment thereof
having galanin
binding activity. A polynucleotide molecule coding for a variant of rat GalR2
comprising
SEQ ID N0:3, or a galanin binding fragment thereof. A polynucleotide molecule
comprising
SEQ ID N0:3 or a degenerate variant thereof. A purified and isolated variant
of a rat or
human GalR2 protein or fragment thereof having galanin binding activity. A
purified and
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isolated variant of rat GalR2 protein comprising SEQ ID N0:4.
The foregoing merely summarize certain aspects of the present invention and is
not
intended, nor should it be construed, to limit the invention in any manner.
All patents and
other publications recited herein are hereby incorporated by reference in
their entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is the polynucleotide sequence of rat GalR2 of the invention.
Figure 2 is the amino acid sequence of rat GalR2.
Figures 3-4 are the polynucleotide sequence of the Y 107 variant of rat GalR2.
Figure S is the amino acid sequence of Y107(omitting putative intron).
Figure 6 is the partial polynucleotide sequence of human GalR2.
Figure 7 is the partial amino acid sequence of human GalR2.
Figure 8 is the representative saturation isotherm of ['ZSI]hGalanin binding
to rat GalR2
(clone BMB77) transiently expressed in COS-7 cells. The inset shows the
corresponding
linear Rosenthal plot. B/F axis on the Rosenthal plot indicates the ratio of
Bound to Free
radioligand.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention comprises, in part, a novel galanin receptor protein,
the GalR2
receptor. Particularly preferred embodiments of the GalR2 receptor are those
having an
amino acid sequence substantially the same as SEQ ID NO: 2, 4 or 6. As used
herein,
reference to the GalR2 receptor is meant as a reference to any protein having
an amino acid
sequence substantially the same as SEQ ID NO: 2, 4 or 6. The present invention
also
comprises the nucleic acid sequence encoding the GalR2 protein, which nucleic
acid
sequence is substantially the same as SEQ ID NO: 1, 3 or 5. Receptors SEQ ID
NO: 1 and
SEQ ID NO: 3 are nucleic acid sequences of rat GalR2 receptors but SEQ ID NO:
3 appears
to contain an intronic region; therefore, SEQ ID NO: 1 is the preferred
embodiment of the rat
GaIR2 receptor of this invention. Receptor SEQ ID NO: 5 is the partial nucleic
acid sequence
of human GalR2. Receptors SEQ ID NO: 2 and 4 are rat GalR2 receptors and
appear to be
allelic variants. Receptor SEQ ID NO: 6 is the partial amino acid sequence of
human GalR2.
As used herein, a protein "having an amino acid sequence substantially the
same as
SEQ ID NO: x" (where "x" is the number of one of the protein sequences recited
herein)
means a protein whose amino acid sequence is the same as SEQ ID NO: x or
differs only in a
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way such that ICso[galanin] as determined according to the method detailed in
Example 2,
infra, are less than or equal to 1 nM. Those skilled in the art will
appreciate that conservative
substitutions of amino acids can be made without significantly diminishing the
protein's
affinity for galanin and fragments and analogs thereof. Other substitutions
may be made that
5 increase the protein's affinity for these compounds. Making and identifying
such proteins is
a routine matter given the teachings herein, and can be accomplished, for
example, by altering
the nucleic acid sequence encoding the protein (as disclosed herein),
inserting it into a vector,
transforming a cell, expressing the nucleic acid sequence, and measuring the
binding amity
of the resulting protein, all as taught herein.
As used herein the term "a molecule having a nucleotide sequence substantially
the
same as SEQ ID NO: y" (wherein "y" is the number of one of the protein-
encoding
nucleotide sequences listed in the Sequence Listing) means a nucleic acid
encoding a protein
"having an amino acid sequence substantially the same as SEQ ID NO: y*"
(wherein "y*" is
the number of the amino acid sequence for which nucleotide sequence "y" codes)
as defined
above. This definition is intended to encompass natural allelic variations in
the GaIR2
sequence. Cloned nucleic acid provided by the present invention may encode
GalR2 protein
of any species of origin, including (but not limited to), for example, mouse,
rat, rabbbit, cat,
dog, primate, and human. Preferably the nucleic acid provided by the invention
encodes
GalR2 receptors of mammalian, and most preferably, rat or human origin.
The invention also includes nucleotide sequences encoding chimeric proteins
comprised of parts of the GalR2 receptor and parts of other related seven-
transmembrane
receptors.
The BMB77 clone (SEQ ID NO: 1) (see Example 1, infra) has a 1.7-kb cDNA insert
with a open reading frame from nucleotides 279 to 1394 that encodes a 372
amino acid
protein (SEQ ID NO: 2). Hydrophobicity plot analysis using the PEPPLOT
function of GCG
(Genetics Computer Group, Madison, WI) shows that the GaIR2 receptor has seven
transmembrane-like domains, indicating it might be a G-protein-coupled
receptor. GaIR2 is
26 amino acids longer in length than GaIRI, the only other published galanin
receptor. This
extra length of amino acids within GalR2 is due to an extended C-terminal tail
sequence.
However, the putative N-terminal extracellular domain of GaIR2 is about 7
amino acids
shorter than the corresponding region in GaIRl. It is also important to note
that the GalR2
sequence shows only 38% amino acid sequence identity to the GaIRI receptor.
The Y107 clone (SEQ ID NO: 3) (see Example 1, infra) has a 1.9-kb cDNA insert
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with an open reading frame from nucleotides 17-384, and from nucleotides 874-
1621. The
sequence between nucleotides 384 and 874 contains multiple STOP codons in all
three
reading frames. Furthermore, the dinucleotides GT and AG at positions 385-386
and 872-
873, respectively, fulfill the criteria for being a splice donor and acceptor
site, respectively.
Moreover, when the nucleotide sequences 17-384 and 874-1621 are joined
together, an open
reading frame is formed which has a cognate translation product nearly
identical to that of
clone BMB77 (SEQ ID NO: 4). Therefore, it is likely that the region between
these two open
reading frames is an intron.
There is a single nucleotide change between positions 963 in clone BMB77 (SEQ
ID
NO: 1 ) and 1190 in clone Y 107 (SEQ ID NO: 3). This adenine to cytosine
transversion
results in a change from Serz29 in clone BMB77 (SEQ ID NO: 2) to Arg229 in
clone Y107
(SEQ ID NO: 4). Amino acid 229 is located in the third intracellular loop of
GalIZ2. The
third intracellular loop of other seven transmembrane domain G protein-coupled
receptors is
an important domain for effecting coupling of the receptor to its G protein.
Bockaert, Curr.
Op. Neurobiol. 1, 32-42 (1991). This polymorphic amino acid position could
change the G
protein binding characterisitics of the GaIR2 receptor variants. Gillison,
Diabetes 43, 24
( 1994); Chen, Am. J. Physiol. 266, G 113 ( 1994).
The partial human GalR2 nucleic acid sequence (SEQ ID NO: 5) contains a 337
amino acid opening reading frame. The initial leucine residue of this partial
human GalR2
protein (SEQ ID NO: 6) corresponds to amino acid Leus' in rat GalR2 (SEQ ID
NO: 2 and 4).
Nucleic acid hybridization probes provided by the invention are DNAs
consisting
essentially of the nucleotide sequences complementary to any sequence depicted
in SEQ ID
NOa 1 and 3 that is effective in nucleic acid hybridization. Nucleic acid
probes are useful for
detecting GalR2 gene expression in cells and tissues using techniques well-
known in the art,
including, but not limited to, Northern blot hybridization, in situ
hybridization, and Southern
hybridization to reverse transcriptase - polymerase chain reaction product
DNAs. The probes
provided by the present invention, including oligonucleotide probes derived
therefrom, are
also useful for Southern hybridization of mammalian, preferably human, genomic
DNA for
screening for restriction fragment length polymorphism (RFLP) associated with
certain
genetic disorders. As used herein, the term complementary means a nucleic acid
having a
sequence that is sufficiently complementary in the Watson-Crick sense to a
target nucleic
acid to bind to the target under physiological conditions or experimental
conditions which
those skilled in the art routinely use when employing probes.
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Receptor GalR2 binds various fragments and analogs of galanin with affinities
different from that of the known receptors. The rank order of binding affinity
of receptor
GalR2 was found to be:
galanin = (2-29)galanin > ( I - I S)galanin » (3-29)galanin
Table 1, infra, presents a more detailed affinity profile of the GalR2
receptor for galanin and
various fragments thereof. As used herein, a protein having substantially the
same affinity
profile as the GalR2 receptor means a protein in which the ICso of each of the
peptides listed
in Table I , infra, is no more than an order of magnitude greater than those
listed in Table I
for each of the respective peptides as measured according to the methods
described in
Example 2.
The production of proteins such as receptor GalR2 from cloned genes by genetic
engineering means is well known in this art. The discussion which follows is
accordingly
intended as an overview of this field, and is not intended to reflect the full
state of the art.
DNA which encodes receptor GalR2 may be obtained, in view of the instant
disclosure, by chemical synthesis, by screening reverse transcripts of mRNA
from
appropriate cells or cell line cultures, by screening genomic libraries from
appropriate cells,
or by combinations of these procedures, as illustrated below. Screening of
mRNA or
genomic DNA may be carried out with oligonucleotide probes generated from the
GalR2
gene sequence information provided herein. Probes may be labeled with a
detectable group
such as a fluorescent group, a radioactive atom or a chemiluminescent group in
accordance
with known procedures and used in conventional hybridization assays, as
described in greater
detail in the Examples below. In the alternative, the GalR2 gene sequence may
be obtained
by use of the polymerase chain reaction (PCR) procedure, with the PCR
oligonucleotide
primers being produced from the GalR2 gene sequence provided herein. See U.S.
Patent Nos.
4,683,195 to MuIlis et al. and 4,683,202 to Mullis.
Receptor GalR2 may be synthesized in host cells transformed with a :ecombinant
expression construct comprising a nucleic acid encoding the receptor GalR2.
Such a
recombinant expression construct can also be comprised of a vector that is a
replicable DNA
construct. Vectors are used herein either to amplify DNA encoding GalR2 and/or
to express
DNA which encodes GaIR2. For the purposes of this invention, a recombinant
expression
construct is a replicable DNA construct in which a DNA sequence encoding GalR2
is
operably linked to suitable control sequences capable of effecting the
expression of GalR2 in
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a suitable host. The need for such control sequences will vary depending upon
the host
selected and the transformation method chosen. Generally, control sequences
include a
transcriptional promoter, an optional operator sequence to control
transcription, a sequence
encoding suitable mRNA ribosomal binding sites, and sequences which control
the
termination of transcription and translation. Amplification vectors do not
require expression
control domains. All that is needed is the ability to replicate in a host,
usually conferred by
an origin of replication, and a selection gene to facilitate recognition of
transformants. See,
Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Edition, Cold
Spring Harbor
Press, New York, 1989).
Vectors useful for practicing the present invention include plasmids, viruses
(including phage), retroviruses, and integratable DNA fragments (i.e.,
fragments integratable
into the host genome by homologous recombination). The vector replicates and
functions
independently of the host genome, or may, in some instances, integrate into
the genome itself.
Suitable vectors will contain replicon and control sequences which are derived
from species
compatible with the intended expression host. The vectors may be self
replicating. Suitable
vectors for the purposes of the present invention include pBluescript, pcDNA3,
pSV-SPORT,
and, for insect cells, baculovirus. A preferred vector is the plasmid pcDNA3
(Invitrogen, San
Diego, CA).
Construction of suitable vectors containing the desired coding and control
sequences
employs standard ligation and restriction techniques that are well understood
in the art.
Isolated plasmids, DNA sequences, or synthesized oligonucleotides are cleaved,
tailored, and
relegated in the form desired.
Site-specific DNA cleavage is performed by treating with the suitable
restriction
enzyme (or enzymes) under conditions that are generally understood in the art,
and the
particulars of which are specified by the manufacturer of these commercially
available
restriction enzymes. See, e.g., New England Biolabs, Product Catalog. In
general, about 1
,ug of plasmid or DNA sequence is cleaved by one unit of enzyme in about 20
,ul of buffer
solution. Often excess of restriction enzyme is used to ensure complete
digestion of the DNA
substrate. Incubation times of about one hour to two hours at about
37°C are workable,
although variations are tolerable. After each incubation, protein is removed
by extraction
with phenoUchloroform, and may be followed by ether extraction. The nucleic
acid may be
recovered from aqueous fractions by precipitation with ethanol. If desired,
size separation of
the cleaved fragments may be performed by polyacrylamide gel or agarose gel
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electrophoresis using standard techniques. A general description of size
separations is found
in Methods in Enrymology 65, 499-560 ( 1980).
Transformed host cells are cells which have been transformed or transfected
with
recombinant expression constructs made using recombinant DNA techniques and
comprising
mammalian GalR2-encoding sequences. Preferred host cells for transient
transfection are
COS-7 cells. Transformed host cells may ordinarily express GalR2, but host
cells
transformed for purposes of cloning or amplifying nucleic acid hybridization
probe DNA
need not express the receptor. When expressed, the mammalian GalR2 protein
will typically
be located in the host cell membrane. See, Sambrook et al., ibid.
Cultures of cells derived from multicellular organisms are desirable hosts for
recombinant GalR2 protein synthesis. In principal, any higher eukaryotic cell
culture is
workable, whether from vertebrate or invertebrate culture. However, mammalian
cells are
preferred, as illustrated in the Examples. Propagation of such cells in cell
culture has become
a routine procedure. See Tissue Culture (Academic Press, Kruse & Patterson,
Eds., 1973).
Examples of useful host cell lines are bacteria cells, insect cells, yeast
cells, human 293 cells,
VERO and HeLa cells, LMTK' cells, and WI138, BHK, CHO, COS-7, CV, and MDCK
cell
lines (American Type Culture Collection, Rockville, MD). CHO cells are
preferred.
The invention provides homogeneous compositions of mammalian GalR2 produced
by transformed eukaryotic cells as provided herein. Such homogeneous
compositions are
intended to be comprised of mammalian GalR2 protein that comprises at least
90% of the
protein in such homogenous composition. The invention also provides membrane
preparation from cells expressing GalR2 as the result of transformation with a
recombinant
expression construct, as described here.
Mammalian GalR2 protein made from cloned genes in accordance with the present
invention may be used for screening compounds for GalR2 agonist or antagonist
activity, or
for determining the amount of a GalR2 agonist or antagonist drug in a solution
(e.g., blood
plasma or serum). For example, host cells may be transformed with a
recombinant
expression construct of the present invention, GalR2 protein expressed in
those host cells, the
cells lysed, and the membranes from those cells used to screen compounds for
GalR2 binding
activity. Competitive binding assays in which such procedures may be carried
out are well
known in the art. By selection of host cells which do not ordinarily express
GalR2, pure or
crude preparations of membranes containing GalR2 can be obtained. Further,
GalR2 agonists
and antagonists can be identified by transforming host cells with a
recombinant expression
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construct as provided by the present invention. Membranes obtained from such
cells (and
membranes of intact cells) can be used in binding studies wherein the drug
dissociation
activity is monitored.
It is known that the neurotransmitter galanin is a regulator of appetite,
cognition,
5 endocrine function, pain, and smooth muscle control. As shown herein, the
various galanin
analogs/fragments that induce these physiological responses bind with a high
affinity to the
GalR2 receptor. It is therefore evident that by modulating the activity of the
GalR2 receptor,
various physiological activities can be regulated. Specifically, antagonists
to the GaIIR2
receptor, identified by the methods described herein, could be used to treat
obesity, diabetes,
10 hyperlipidemia, stroke, neuropathic pain, Alzheimer's disease, and/or
endocrine disorders.
This invention provides a pharmaceutical composition comprising an effective
amount of a drug identified by the method described herein and a
pharmaceutically
acceptable carrier. Such drugs and carrier can be administered by various
routes, for example
oral, subcutaneous, intramuscular, intravenous or intracerebral. The preferred
route of
administration would be oral at daily doses of about 0.01-100 mglkg.
This invention provides a method of treating obesity, diabetes,
hyperlipidemia, stroke,
neuropathic pain, Alzheimer's disease, or endocrine disorders wherein the
abnormality is
improved by reducing the activity of GalR2 receptor or blocking the binding of
ligands to a
GalR2 receptor which comprises administering an effective amount of the
pharmaceutical
composition described above.
The recombinant expression constructs of the present invention are useful in
molecular biology to transform cells which do not ordinarily express GaIR2 to
thereafter
express this receptor. Such cells are useful as intermediates for making cell
membrane
preparations useful for receptor binding assays, which are in turn useful for
drug screening.
Drugs identified from such receptor assays can be used for the treatment of
obesity, diabetes,
anorexia, hyperlipidemia, stroke, neuropathic pain, Alzheimer's disease, or
endocrine
disorders.
The recombinant expression constructs of the present invention are also useful
in gene
therapy. Cloned genes of the present invention, or fragments thereof, may also
be used in
gene therapy carried out by homologous recombination or site-directed
mutagenesis. See
generally Thomas & Capecchi, Cell 51, 503-512 (1987); Bertling, Bioscience
Reports 7, 107-
112 (1987); Smithies et al., Nature 3I7, 230-234 (1985).
. Oligonucleotides of the present invention are useful as diagnostic tools for
probing
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GallZ2 gene expression in tissues. For example, tissues are probed in situ
with
oligonucleotide probes carrying detectable groups by conventional
autoradiographic
techniques, as explained in greater detail in the Examples below, to
investigate native
' expression of this receptor or pathological conditions relating thereto.
Further, chromosomes
can be probed to investigate the presence or absence of the GaIR2 gene, and
potential
pathological conditions related thereto, as also illustrated by the Examples
below. Probes
according to the invention should generally be at least about 15 nucleotides
in length to
prevent binding to random sequences, but, under the appropriate circumstances
may be
smaller.
The invention also provides antibodies that are immunologically reactive to a
mammalian GalR2, preferably rat or human GaIR2. The antibodies provided by the
invention
are raised in animals by inoculation with cells that express a mammalian
GalIZ2 or epitopes
thereof, using methods well known in the art. Animals that are used for such
inoculations
include individuals from species comprising cows, sheep, pigs, mice, rats,
rabbits, hamsters,
goats and primates. Preferred animals for inoculation are rodents (including
mice, rats,
hamsters) and rabbits. The most preferred animal is the mouse.
Cells that can be used for such inoculations, or for any of the other means
used in the
invention, include any cell line which naturally expresses a mammalian GalR2,
or any cell or
cell line that expresses a mammalian GalR2 or any epitope thereof as a result
of molecular or
genetic engineering, or that has been treated to increase the expression of a
mammalian
GaIR2 by physical, biochemical or genetic means. Preferred cells are human
cells, most
preferably HEK 293 cells that have been transformed with a recombinant
expression
construct comprising a nucleic acid encoding a mammalian GalR2, preferably a
rat or human
GaIR2, and that express the mammalian GalR2 gene product.
The present invention provides monoclonal antibodies that are immunologically
reactive with an epitope of mammalian GalR2 or fragment thereof and that is
present on the
surface of mammalian cells, preferably human or mouse cells. These antibodies
are made
using methods and techniques well known to those of skill in the art.
Monoclonal antibodies provided by the present invention are produced by
hybridoma
cell lines, that are also provided by the invention and that are made by
methods well known
in the art. Hybridoma cell lines are made by fusing individual cells of a
myeloma cell line
with spleen cells derived from animals immunized with cells expressing the
GaIR2 receptor,
preferably rat or human cells, as described above. The myeloma cell lines used
in the
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invention include lines derived from myelomas of mice, rats, hamsters,
primates and humans.
Preferred myeloma cell lines are from mouse. The animals from whom spleens are
obtained
after immunization are rats, mice and hamsters, preferably mice, most
preferably Balb/c mice.
Spleen cells and myeloma cells are fused using a number of methods well known
in the art,
S including but not limited to incubation with inactivated Sendai virus and
incubation in the
presence of polyethylene glycol (PEG). The most preferred method for cell
fusion is
incubation in the presence of a solution of 45% (w/v) PEG-1450. Monoclonal
antibodies
produced by hybridoma cell lines can be harvested from cell culture
supernatant fluids from
in vitro cell growth; alternatively, hybridoma cells can be injected
subcutaneously and/or into
the peritoneal cavity of an animal, most preferably a mouse, and the
monoclonal antibodies
obtained from blood and/or ascites fluid.
Monoclonal antibodies provided by the present invention are also produced by
recombinant genetic methods well known to those of skill in the art, and the
present invention
encompasses antibodies made by such methods that are immunologically reactive
with an
epitope of a mammalian GalR2.
The present invention encompasses fragments of the antibody that are
immunologically reactive with an epitope of a mammalian GalR2. Such fragments
are
produced by any number of methods, including but not limited to proteolytic
cleavage,
chemical synthesis or preparation of such fragments by means of genetic
engineering
technology. The present invention also encompasses single-chain antibodies
that are
immunologically reactive with an epitope of a mammalian GalR2 made by methods
known to
those of skill in the art.
The present invention also encompasses an epitope of a mammalian GalR2 that is
comprised of sequences and/or a conformation of sequences present in the
mammalian GalR2
molecule. This epitope may be naturally occurring, or may be the result of
proteolytic
cleavage of the mammalian GaIR2 molecule and isolation of an epitope-
containing peptide or
may be obtained by synthesis of an epitope-containing peptide using methods
well known to
those skilled in the art. The present invention also encompasses epitope
peptides produced as
a result of genetic engineering technology and synthesized by genetically
engineered
prokaryotic or eukaryotic cells.
The invention also includes chimeric antibodies, comprised of Iight chain and
heavy
chain peptides immunologically reactive to an epitope that is a mammalian
GalR2. The
chimeric antibodies embodied in the present invention include those that are
derived from
CA 02256523 1998-11-19
WO 97/46681 PCT/US97/09787
13
naturally occurring antibodies as well as chimeric antibodies made by means of
genetic
engineering technology well known to those of skill in the art.
Also provided by the present invention are non-human transgenic animals grown
from
germ cells transformed with the GaIR2 nucleic acid sequence according to the
invention and
that express the GaLR2 receptor according to the invention and offspring and
descendants
thereof. Also provided are transgenic non-human mammals comprising a
homologous
recombination knockout of the native GalR2 receptor, as well as transgenic non-
human
mammals grown from germ cells transformed with nucleic acid antisense to the
GalR2
nucleic acid of the invention and offspring and descendants thereof. Further
included as part
of the present invention are transgenic animals which the native GalR2
receptor has been
replaced with the human homolog. Of course, offspring and descendants of all
of the
foregoing transgenic animals are also encompassed by the invention.
Transgenic animals according to the invention can be made using well known
techniques with the nucleic acids disclosed herein. E.g., Leder et al., U.S.
Patent Nos.
4,736,866 and 5,175,383; Hogan et al., Manipulating the Mouse Embryo, A
Laboratory
Manual (Cold Spring Harbor Laboratory (19$6)); Capecchi, Science 244, 1288
(1989); and
Zimmer and Gruss, Nature 338, 150 (1989). Such transgenic animals are useful
for screening
for and determining the physiological effects of GalR2 receptor agonists and
antogonist.
Consequently, such transgenic animals are useful for developing drugs to
regulate
physiological activities in which galanin participates.
. The following Examples are provided for illustrative purposes only and are
not
intended, nor should they be construed, as limiting the invention in any
manner.
EXAMPLES
Example Z: Isolation and Sequencing of Rat GalR2 Receptor
An expression cloning strategy was used to clone the novel galanin receptor
from a rat
hypothalamus cDNA Library. RNA was obtained from 9 frozen rat hypothalami
weighing a
total of 0.87 grams. Poly(A) RNA was isolated directly from the tissue using
the Promega
PolyATtract System 1000 kit (Promega (Madison, WI) Z5420). The hypothalami
were
homogenized in 4 mL of 4M guanidine thiocyanate-25mM sodium citrate, pH 7.1-2%
13-
mercaptoethanol using a Polytron at full-speed for approximately 1 minute. To
the
. homogenized tissue 8 mL of 4M guanidine thiocyanate-25mM sodium citrate, pH
7.1-1% 13-
CA 02256523 1998-11-19
WO 97146681 PCT/US97/09787
14
mercaptoethanol which had been preheated to 70°C was added. After
mixing thoroughly, 870
pmol biotinylated oligo(dT) was added; the mixture was incubated at
70°C for 5 minutes.
The homogenate was subjected to centrifugation at 12000 x g for 10 minutes at
room
temperatwe; the homogenate was transferred to a clean tube and 10.44 mL
Streptavidin
MAGNESPHERE~ Paramagnetic Particles (SA-PMPs) which had been prepared as per
the
published protocol was added. (Promega Corporation (Madison, WI) published
protocol TM
228). The homogenate and SA-PMPs were incubated together for 2 minutes at room
temperature after which the homogenate was decanted while the SA-PMP-
biotinylated
oligo(dT)-hypothalamic poly(A) RNA complex was retained in the tube by a
magnetic stand.
The complex was washed as per the protocol, after which the RNA was
precipitated and
resuspended in water. 25 micrograms of this poly(A) RNA was used by Invitrogen
(Invitrogen Corporation, San Diego, CA) to prepare a cDNA expression library.
The
protocols used by Invitrogen to prepare the cDNA library are essentially based
upon the
procedures of Okayama and Berg (Molec. Cell. Biol. 2, 161 (1982)) and Gubler
and Hoffman
(Gene 25, 263 (1983)) (Invitrogen Corporation (San Diego, CA) publications
130813sa and
130928sa). An oligo(dT) anchor primer was used for reverse transcription, and
the library
was cloned unidirectionally into pcDNA3 vector which contains a CMV
(cytomegalovirus)
promoter for eukaryotic expression. The cDNA library had 5.3 x 105 primary
recombinants
with an average insert size of 2.59 kb.
Isolation o a nove~,galanin recewtor cDNA clone
1. Homology cloning strategy
In order to isolate novel receptors) for gal4... an, approximately 2 million
phage
plaques of a rat small intestine library (Stratagene (La Jolla, CA) 936'08)
were screened with
rat GalRl coding sequence DNA as probe under low stringency conditions (30%
formamide,
6X SSC (0.9M NaCU0.09M NaCitrate), 0.1% N-lauroyl sarcosine, 0.2% SDS, 3%
blocking
reagent.) The probe was prepared by digesting the parent GaIRI plasmid with
SacI and AccI,
separating the fragments by agarose gel electrophoresis, and purifying the 1-
kb SacI-AccI
fragment from the gel. The probe was labeled with digoxigenin dUTP according
to the
manufacturer's instructions (GENIUS Kit, Boehringer Mannheim, Indianapolis,
IN, PN 1093
657). The filter lifts, denaturation, neutralization, hybridization, and
washing were done
according to the manufacturer's instructions except that hybridization was
done at 30°C and
the washes were performed twice for 40 minutes each: once at room temperature;
the second
CA 02256523 1998-11-19
WO 97/46681 PCT/US97/09787
at 37°C.
One plaque containing DNA homologous to the probe was purified and subcloned
into pBluescript vector (Stratagene (La Jolla, California) 212206) by standard
molecular
' biological techniques. This clone, designated SI2I 12, was subjected to
sequence analysis and
S was found to contain a sequence which had characteristics of a novel, but
truncated member
of the G-protein-coupled 7TMD receptor family. This clone was later used as
probe to
detenmine the identity of a novel galanin-binding clone (Y107) found in the
expression
cloning strategy (see below.)
The partial human GalR2 cDNA was obtained by screening approximately two
10 million phage of a human small intestine library (Clontech (Palo Alto, CA)
HL 1133a) were
screened with a human GalR2 probe obtained by PCR amplification from human
genomic
DNA. The conditions used were essentially as described for the rat GalR2
isolation.
2. Expression cloning strategy
15 The rat hypothalamus cDNA library was plated on the Luria Broth/Ampicillin
(GIBCO) plates in pools of 1,OOO~independent colonies. The plates were
incubated at 37°C
for about 20 hours and the bacteria from each plate were scraped in 4-5 ml
LB/Ampicillin
media. Two ml of the bacteria samples were used for plasmid preparation and
one ml of each
pool was stored at -80°C in 15% glycerol.
COS-7 cells were grown in Dulbecco's Modified Eagle Medium (DMEM, GIBCO
{Gaithersburg, MD) 11965-092), IO% fetal bovine serum (GIBCO {Gaithersburg,
MD)
16000-028), and IX antibiotic/antimycotic solution (GIBCO (Gaithersburg, MD)
15240-
039). Cells were maintained by trypsinizing and splitting at 50 to 70%
confluency.
Twenty-four hours before transfection, cells were plated into flaskette
chambers
(Nunc, Inc. (Naperville, IL) 177453) at 3x105 cells/flaskette (equivalent to
3x104 cells/cmz).
Two ~g of plasmid DNA from each pool was transfected into the cells using 10
~cl of
Lipofectamine (GIBCO (Gaithersburg, MD) 18324-012) according to the
manufacturer's
protocol.
Forty-eight hours after transfection, the ['Z5I]galanin binding assay was
performed in
the flaskette chamber. The cells were washed once with 25 mM Tris-HCI, 10 mM
MgCl2, pH
7.4, and blocked for 1 S minutes with 1 ml total binding buffer (25 mM Tris-
HCI, 10 mM
MgCl2, 1 % bovine serum albumin, pH 7.4) at room temperature. After aspirating
off the
blocking solution, 1 ml of binding buffer containing 100 pM 'Z5I-hGalanin (NEN
DuPont
CA 02256523 1998-11-19
WO 97/46681 PCT/US97/09787
16
(Boston, MA) NEX-333) was added and flasks were incubated at room temperature
for 90
minutes. Following the incubation, the labeling buffer was removed and the
flaskettes were
rinsed (approximately 2 mL per flaskette) four times with ice-cold binding
buffer. After a
final rinse with ice-cold phosphate buffered saline (PBS)(GIBCO (Gaithersburg,
MD) 14190-
136), the cells were fixed with ice-cold PBS/1 % glutaraldehyde (Sigma (St.
Louis, MO)
G5882). The solution was removed and residual glutaraldehyde rinsed away with
one wash
of ice-cold PBS/0.5 M Ttis (pH 7.5) followed by one wash of ice-cold PBS.
After separating
the slide bases from their tops, the slides were dipped in 0.5% gelatin at
42°C and dried under
vacuum. The dried slides were dipped in photographic emulsion (NTB-2) (Kodak
(Rochester, NY) 165 4433) diluted 1:1 in 0.02% Aerosol-OT (Sigma (St. Louis,
MO) A6627)
at 42 °C, dried at room temperature for 1 hour, and exposed in the
darkbox for four or five
days at 4°C. After sufficient exposure time, the darkbox was brought to
room temperature
for 1 hour after which the slides were developed in D-19 developer (Kodak
(Rochester, NY)
146 4593) for three minutes at 15°C, placed in fixer (Kodak (Rochester,
NY) 197 1746) for
three minutes at I 5 °C, washed in water, and air dried. Cells were
stained with Diff Quik
stain set (Baxter (McGaw Park, IL) B4132-I) and air dried. Slides were dipped
into xylenes
and mounted with DPX mountant (Electron Microscopy Sciences (Fort Washington,
PA)
13510). Positive cells were identified using dark field microscopy.
Two positive pools were identified. Since the hypothalamus could express
different
subtypes of galanin receptor, we analyzed the positive pools for GaIRI
receptors by PCR and
homology. Of the 2 positive pools tested as described above, 1 contained GalRl
as
detenmined by PCR and homology analyses. However, the other pool (Y107) was
negative
for GalRl by PCR, but homologous to SI2112 probe (see Homology strategy,
above).
Because: 1) SI2112 sequence indicated it was likely a novel, albeit truncated
and
unexpressible, G-protein-coupled receptor; 2) pool YI07 DNA showed homology to
the
SI2112 probe; and, 3) DNA from pool Y107, when used to transiently transfect
COS-7 cells,
conferred the ability upon the cells to bind galanin, it was deduced that the
clone within pool
Y107, which conferred gaIanin-binding ability when expressed, was likely to be
an
expressible version of the SI2112 clone. Therefore, clones from pool Y107 were
probed with
radiolabeled DNA prepared from SI2112, and a single clone hybridizing to the
SI2112 probe
was purified from non-homologous clones. This clone was called Y107.
Sequence analysis of clone Y107 (SEQ ID NO: 3) revealed the presence of one
intron
of 489-by length, beginning after nucleotide 384 (the second nucleotide of the
codon for
CA 02256523 1998-11-19
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17
amino acid 133, an arginine residue). Using Y107 DNA as probe, an intronless
version of the
Y107 cDNA was obtained from a PCI2 cell cDNA library by homology cloning. The
first
intronless version of the Y107 was in the pSV-SPORT vector (GIBCO
(Gaithersburg, MD)
' 15386-014); the complete cDNA insert of this clone was subcloned into the
pcDNA3 vector
(Invitrogen (San Diego, CA) V790-20) in order to maintain, for subsequent
pharamacological
analyses, common vector and promoter (CMV) backgrounds amongst our clones. The
intronless clone in pSV-SPORT was designated BMB77.sv40; the intronless clone
contained
within pcDNA3 is named BMB77. Clones BMB77 and Y107 differ by one amino acid
in
sequence. Pharmacological analyses have been performed with both the Y107 (SEQ
ID NO:
3 and 4) and BMB77 {SEQ ID NO: 1 and 2) clones.
DNA and DBDtIdB SPo»y»roc ns~ajy~ls
Plasmid DNA was sequenced by Lark Technologies Inc. (Houston, Texas) and
Biotechnology Resource Laboratory of Yale University (New Haven, CT) using
Sequenase
Kit (LJS Biochemical (Cleveland, OH) 70770) or Applied Biosystems' automatic
sequencer
system (Model 373A). The peptide sequence was deduced from the long open-
reading-frame
of the nucleotide sequence. DNA and peptide sequences were analyzed using the
GCG
program (Genetics Computer Group, Madison, WI). The results are embodied in
SEQ ID
NO: 1 (the nucleic acid sequence of'clone BMB77), SEQ ID NO: 2 (the amino acid
sequence
of clone BMB77), SEQ ID NO: 3 (the nucleic acid sequence of clone Y107), SEQ
ID NO: 4
(the amino acid sequence, omitting the putative intronic region, of clone
Y107), SEQ ID NO:
5 (the partial nucleic acid sequence of human GaIR2), and SEQ ID NO: 6 (the
partial amino
acid sequence of human GalR2).
Example 2: Pharmacological Characterization of the Novel Rat Galanin Receptor
Iransient TrancfP~i:~H
Monkey kidney cells (COS-7) were maintained in T-175 cm2 flasks (Nunc, Inc.
(Naperville, IL) 171226) at 37°C with 5% COZ in a humidified
atmosphere. Cells were
grown in Dulbecco's Modified Eagle Medium (DMEM) (GIBCO (Gaithersburg, MD)
11965-
092) supplemented with 2 mM glutamine, 10% fetal bovine serum, 1 mM sodium
pyruvate
and a antibiotic/antimycotic comprised of
pennicillin/streptomycin/amphotericinB (GIBCO,
Gaithersburg, MD, PN 15240-013). Cells at 70% confluency were transfected with
rat GalR2
DNA using the Lipofectamine reagent (GIBCO (Gaithersburg, MD) 18324-012). I S
pg of
CA 02256523 1998-11-19
WO 97/46681 PCT/LTS97/09787
18
DNA and 90 pl of lipofectamine were added to each flask. Media was replaced 24
hours post
transfection, and membranes were harvested 24 hours later.
Stable EYbre ctn'ofthe Rat mlR7 Ror ~~lelone BMB77)
293 cells (human embryonic kidney, ATCC) were plated onto a T-25 flask one day
prior to transfection, such that they were 50-70% confluent at the time of
transfection. 15 ug
of rat GalR2 (BMB77) DNA were added to 0.3 ml of Optimem I culture media
(GIBCO Life
Sciences), and 25 ul of lipofectamine were added to 0.3 ml of Optimem I. The
DNA and
lipofectamine solutions were combined and incubated at room temperature for 20
minutes.
An additional 2.4 ml of Optimem I was added to the DNA/lipofectamine mixture.
The media
was aspirated from T-25 flask containing 293 cells, washed with Optimem I, and
the
DNA/lipofectamine mixture was added to the 293 cells. After a S-6 hour
incubation in a
37°C incubator (5% COZ) for 5-6 hours, the DNA/lipofectamine mixture
was replaced with
culture media (DMEM, 10% fetal bovine serum, 2 mM glutamine and
I S antibiotic/antimycotic.)
The day following transfection, the media was replaced with selecting media
(culture
media with the addition of 350 uglml of Geneticin G-418), and the flask was
returned to the
37°C / 5% COZ incubator. When discrete colonies became apparent, cells
were pooled.
Growth was monitored, followed by cloning by limited dilution, such that an
average of one
cell was seeded in each well of a 96-well microtiter culture plate. After
about 21 days in
culture under selection conditions, those wells containing single colonies
were selected and
transferred to 24-well culture plates pretreated with poly-1-lysine, following
trypsinization.
Each of these clones was propagated until sufficient quantities were available
for testing in
the ['ZS I]galanin binding assay, from which one particular clone designated
293-rGalR2-1
was selected on the basis of its high level of receptor expression.
Membrane Prey ar '
The media was removed from each flask of transfected cells, and the cells were
washed twice with 20 ml ice-cold phosphate buffered saline. The cells were
scraped from the
flask in S ml of Tris buffer (20 mM Tris-HCI, 5 mM EDTA, pH 7.7), and then
transferred to
a centrifuge tube. Each flask was rinsed with an additional 5 ml of Tris
buffer, and combined
in the centrifuge tube. The cells were homogenized in a Polytron PT-3000
(Brinkman
Instruments, Mill Valley, New York) for 2 x 10 seconds (12 mM probe, 7000-8000
rpm) and
CA 02256523 1998-11-19
WO 97/46681 PCT/US97/09787
19
centrifuged at 20,000 x g for 30 minutes at 4°C. The pellet was
resuspended in fresh Tris
buffer, and centrifuged again at 20,000 x g for 30 minutes at 4°C.
Protein concentration was
measured using the Bio-Rad kit according to the standard manufacturer's
protocol (Bio-Rad
Laboratories (Hercules, CA) S00-0001 ) using bovine IgG as the standard.
LflIGalanin Bindin A av for rat GalRl ar~d.~g~IR2 rln~oc
The binding assays were performed on 96-well plates (GF/C Millipore Corp.,
Bedford, MA PN MAFC NOB 50) pretreated with 0.3% polyethylenimine (PEI) for at
least 3
hours prior to use. The PEI was aspirated from the plates on a vacuum
manifold, and the
wells were rinsed with 200 pl of ice-cold binding buffer (25 mM Tris, 10 mM
MgCl2, 0.1%
BSA, pH 7.4) immediately before samples were added to the wells. For
competition assays,
increasing concentrations of peptide were incubated with ['ZSI)hGalanin (NEN
DuPont
(Boston, MA) NEX333) and membrane. In a final volume of 200 pl, samples
consisted of
1.25 pg of protein, 50 pM ['Z3IJhGalanin, and peptide dilution or binding
buffer.
Nonspecific binding was defined by 100 nM rat galanin. For saturation
experiments,
increasing concentrations of ('ZSIjhGalanin were incubated with membrane and
100 nM rat
galanin. Samples were incubated for 90 minutes at room temperature with
constant shaking.
To terminate the reaction, samples were aspirated on a vacuum manifold and
rinsed with 3 x
200 pl ice-cold buffer. Samples were then counted on a gamma counter to
quantitate the
amount of radioactivity. Rat galanin, fragment peptides (1-15}galanin, (1-
12)galanin, (1-10
)galanin, chimeric peptide M40, (2-29)rat galanin, (3-29)rat galanin, (5-
29)rat galanin, (9-
29)rat galanin, (10-29)rat galanin, (2-30)human galanin, and (3-30)human
galanin were
synthesized at Bayer Corp. (West Haven, CT}. All other peptides were purchased
from either
Peninsula (Belmont, CA) or Bachem (Torrance, CA).
In Vitro Pharma oloQv
Saturation analysis for rat GalR2 transiently expressed in COS-7 cells yielded
a one-
component model with an average ICd value of 0.28 nM (n=2) and a receptor
density (Bm",~ of
770 fmol/mg protein (Fig. 6). This is very similar to the rat GalR2 receptor
stably expressed
in 293 cells, which yielded an average IC,~ value of 0.20 nM (n=2) and a
receptor density
(Bm"~ of 2289 fmoUmg protein (data not shown}.
Table 1 summarizes the ICso values (50% inhibition of specific binding, as
determined
using nonlinear regression analysis) of various standard peptides, fragments
and chimeras for
CA 02256523 1998-11-19
WO 97/46681 PCT/US97/09787
[~ZSI)hGalanin binding to membranes expressing the rGalR2 receptor.
Transiently expressed
rat GaIR 1 is included in Table I for comparison of its pharmacological
profile to this novel
rat GalR2 receptor. The preliminary pharmacological binding profile for rat
GalR2 differs
from rat GalR1 such that rat galanin itself has about 10-fold lower affinity
for GalR2, no
5 matter how it is expressed. When comparing transiently expressed GalRl and
GalR2, the
chimeric peptide C7 also has about 10-fold lower affinity for GalR2; this
difference,
however, is not observed with rat GalR2 stably expressed in 293 cells. Rat(I-
16)galanin has
about S- to 10-fold lower affinity for GalR2 than GaIR 1 (Table 1 ). In
addition, the ratio of
affinities of ( I - I 2) and ( I - I S)galanin are markedly different for GaIR
I (22-fold difference),
10 while these two peptides have more equivalent binding affinities for GalR2.
The binding profiles for intron-containing (Y107) and intronless (BMB77 clone)
GalR2 receptors transiently expressed are very similar, with the possible
exceptions of MI5
and (1-12)galanin, which have approximately S- and 3-fold higher amities,
respectively, for
clone Y107.
CA 02256523 1998-11-19
WO 97!46681 PCT/US97/09787
21
Table 1
M40 0.78 (0.75,0.810.77 f 0.03 1.2 t 0.07
) 0.32 0.10
r(1-16)gaI0.86 (0.97,0.75)1.2 t 0.16 1.1 t 0.15 0.18 0.02
h(2-30)gal0.94 0.65 (0.8,0.5)ND I .9 0.26
(I-IS)gal I.0 (1.1,0.99)1.2 f 0.21 2.4 f 0.98 1.1 0.27
( 1-12)galI .6 ( I .8, 5.7 f 0.49 4. I t 1.0 24 (22,25)
I .4)
C7 2.6 3.2 f 0.56 0.4410.06 0.26 0.02
MI5 7.0 38 t 9.8 ND 2.0 (1.5,2.4)
(1-10)gal 240 (284,195) 393 t 37 560 t 89 ND
r(3-29)gal> 1000 > I 000 > 1000 > 1000
h(3-30)gal> 1000 ND ND > 1000
r(5-29)gal> 1000 > I 000 ND > 1000
r{9-29)gal> 1000 > I 000 ND > 1000
r( I 0-29)gal> 1000 > I 000 ND > I 000
Table I summarizes the ICso values for various standard peptides for
['2'I)hGalanin binding to
rat GalRl and GalR2 clones. The averages _+ standard error of the mean (SEM)
represent
values from at least three independent experiments. Two independent
experiments are
represented by the average, followed by the individual values in parentheses.
Remaining
values without SEM are from a single experiment. Peptide species are indicated
with the
following prefixes: r = rat, h = human. ND = not determined
CA 02256523 1998-11-19
WO 97/46681 PCT/US97/09787
22
Abbreviations of Chimers:
MI5 = (1-13)Galanin + (5-I 1)Substance P = Galantide (see Bartfai, TIPS 13,
312-317 (1992)
M35 = (I-13)Galanin + (2-9)Bradykinin (Bartfai, infra)
M40 = (I-I3)Galanin + proPro(AIaLeu)ZAIa amide (Bartfai, infra)
C7 = (I-13)Galanin + Sp~tide (see Crawley, Brain Research 600, 268-27? (1993)
Having now fully described this invention, it will be appreciated by those
skilled in
the art that the same can be performed with any wide range of equivalent
parameters of
composition, conditions, and methods of preparing such recombinant molecules,
vectors,
transformed hosts and proteins without departing from the spirit or scope of
the invention or
any embodiment thereof.
CA 02256523 1999-09-29
23
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Bloomquist, Brian T. ; McCaleb, Michael L.;
Cornfield, Linda J.; Yoo-Warren, Heeja
(ii) TITLE OF INVENTION: GALANIN RECEPTOR GALR2
(iii) NUMBER OF SEQUENCES: 6
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Borden Elliot Scott & Aylen
(B) STREET: 1000-60 Queen Street
(C) CITY: Ottawa
(D) STATE: Ontario
(E) COUNTRY: Canada
(F) ZIP: K1P 5Y7
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette, 3.5 inch, 1.44 Mb storage
(B) COMPUTER: IBM PC Compatible
(C) OPERATING SYSTEM: Windows 95
(D) SOFTWARE: WordPerfect for Windows version 6.1
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 2,256,523
(B) FILING DATE: 5-JUN-1997
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 60/019,946
(B) FILING DATE: 5-JUN-1996
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Collard, Christine J.
(B) REGISTRATION NUMBER: 10030
(C) REFERENCE/DOCKET NUMBER: PAT 43573W-1
(ix)TELECOMUNICATION
INFORMATION:
(A) TELEPHONE: (613) 237-5160
(B) TELEFAX: (613) 787-3558
(2) INFORMATION
FOR
SEQUENCE
ID
NO.:
l:
(i) SEQUENCE
CHARACTERISTICS:
(A) LENGTH: 1690
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(vii) IMMEDIATE SOURCE:
CA 02256523 1999-09-29
24
(B) CLONE: Clone BMB77 nucleic acid sequence
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
TCGACCCACGCGTCCGCTCA AGAGCGAGTCCCAGGACTTGAGCGCGGGAA 60
AGTCTAAAGC
GCGAATGGAGTCAGGGTCATTCGATTGCACCTCTCTCGGCTGCGGGCCGGAGCGGGGTAC 120
CATCCTACACTCTGGGTGCTCCCTCCTCCTCCCGTCCCCCGCGCACCCCTGCCCTGGCTC 180
CTGGAGCTCGGCAGTCTCGCTGGGGCGCTGCAGCGAGGGAGCAGCGTGCTCACCAAGACC 240
CGGACAGCTGCGGGAGCGGCGTCCACTTTGGTGATACCATGAATGGCTCCGGCAGCCAGG 300
GCGCGGAGAACACGAGCCAGGAAGGCGGTAGCGGCGGCTGGCAGCCTGAGGCGGTCCTTG 360
TACCCCTATTTTTCGCGCTCATCTTCCTCGTGGGCACCGTGGGCAACGCGCTGGTGCTGG 420
CGGTGCTGCTGCGCGGCGGCCAGGCGGTCAGCACCACCAACCTGTTCATCCTCAACCTGG 480
GCGTGGCCGACCTGTGTTTCATCCTGTGCTGCGTGCCTTTCCAGGCCACCATCTACACCC 540
TGGACGACTGGGTGTTCGGCTCGCTGCTCTGCAAGGCTGTTCATTTCCTCATCTTTCTCA 600
CTATGCACGCCAGCAGCTTCACGCTGGCCGCCGTCTCCCTGGACAGGTATCTGGCCATCC 660
GCTACCCGCTGCACTCCCGAGAGTTGCGCACACCTCGAAACGCGCTGGCCGCCATCGGGC 720
TCATCTGGGGGCTAGCACTGCTCTTCTCCGGGCCCTACCTGAGCTACTACCGTCAGTCGC 780
AGCTGGCCAACCTGACAGTATGCCACCCAGCATGGAGCGCACCTCGACGTCGAGCCATGG 840
ACCTCTGCACCTTCGTCTTTAGCTACCTGCTGCCAGTGCTAGTCCTCAGTCTGACCTATG 900
CGCGTACCCTGCGCTACCTCTGGCGCACAGTCGACCCGGTGACTGCAGGCTCAGGTTCCC 960
AGAGCGCCAAACGCAAGGTGACACGGATGATCATCATCGTGGCGGTGCTTTTCTGCCTCT 1020
GTTGGATGCCCCACCACGCGCTTATCCTCTGCGTGTGGTTTGGTCGCTTCCCGCTCACGC 1080
GTGCCACTTACGCGTTGCGCATCCTTTCACACCTAGTTTCCTATGCCAACTCCTGTGTCA 1140
ACCCCATCGTTTACGCTCTGGTCTCCAAGCATTTCCGTA,AAGGTTTCCGCAAAATCTGCG 1200
CGGGCCTGCTGCGCCCTGCCCCGAGGCGAGCTTCGGGCCGAGTGAGCATCCTGGCGCCTG 1260
GGAACCATAGTGGCAGCATGCTGGAACAGGAATCCACAGACCTGACACAGGTGAGCGAGG 1320
CAGCCGGGCCCCTTGTCCCACCACCCGCACTTCCCAACTGCACAGCCTCGAGTAGAACCC 1380
CA 02256523 1999-09-29
TGGATCCGGC TTGTTAAAGG ACCAAAGGGC ATCTAACAGC TTCTAGACAG TGTGGCCCGA 1440
GGATCCCTGG GGGTTATGCT TGAACGTTAC AGGGTTGAGG CTAAAGACTG AGGATTGATT 1500
GTAGGGAACC TCCAGTTATT AAACGGTGCG GATTGCTAGA GGGTGGCATA GTCCTTCAAT 1560
CCTGGCACCC GAAAAGCAGA TGCAGGAGCA GGAGCAGGAG CAAAGCCAGC CATGGAGTTT 1620
GAGGCCTGCT TGAACTACCT GAGATCCAAT AATAAAACAT TTCATATGCT GTGAAAAAAA 1680
1690
(2) INFORMATION FOR SEQUENCE ID NO. 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 372
(B) TYPE: Amino Acid Sequence
(C) STR.ANDEDNESS: Single
(D) TOPOLOGY: Linear
(vii) IMMEDIATE SOURCE:
(B) CLONE: Clone BMB77 amino acid sequence
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Met Asn Gly Ser Gly Ser Gln Gly Ala Glu Asn Thr Ser Gln Glu
1 5 10 15
Gly Gly Ser Gly Gly Trp Gln Pro Glu Ala Val Leu Val Pro Leu
20 25 30
Phe Phe Ala Leu Ile Phe Leu Val Gly Thr Val Gly Asn Ala Leu
40 45
Val Leu Ala Val Leu Leu Arg Gly Gly Gln Ala Val Ser Thr Thr
50 55 60
Asn Leu Phe Ile Leu Asn Leu Gly Val Ala Asp Leu Cys Phe Ile
65 70 75
Leu Cys Cys Val Pro Phe Gln Ala Thr Ile Tyr Thr Leu Asp Asp
80 85 90
Trp Val Phe Gly Ser Leu Leu Cys Lys Ala Val His Phe Leu Ile
95 100 105
Phe Leu Thr Met His Ala Ser Ser Phe Thr Leu Ala Ala Val Ser
110 115 120
Leu Asp Arg Tyr Leu Ala Ile Arg Tyr Pro Leu His Ser Arg Glu
125 130 135
Leu Arg Thr Pro Arg Asn Ala Leu Ala Ala Ile Gly Leu Ile Trp
140 145 150
Gly Leu Ala Leu Leu Phe Ser Gly Pro Tyr Leu Ser Tyr Tyr Arg
155 160 165
Gln Ser Gln Leu Ala Asn Leu Thr Val Cys His Pro Ala Trp Ser
170 175 180
Ala Pro Arg Arg Arg Ala Met Asp Leu Cys Thr Phe Val Phe Ser
185 190 195
CA 02256523 1999-09-29
26
Tyr Leu Leu Pro Val Leu Val Leu Ser Leu Thr Tyr Ala Arg Thr
200 205 210
Leu Arg Tyr Leu Trp Arg Thr Val Asp Pro Val Thr Ala Gly Ser
215 220 225
Gly Ser Gln Ser Ala Lys Arg Lys Val Thr Arg Met Ile Ile Ile
230 235 240
Val Ala Val Leu Phe Cys Leu Cys Trp Met Pro His His Ala Leu
245 250 255
Ile Leu Cys Val Trp Phe Gly Arg Phe Pro Leu Thr Arg Ala Thr
260 265 270
Tyr Ala Leu Arg Ile Leu Ser His Leu Val Ser Tyr Ala Asn Ser
275 280 285
Cys Val Asn Pro Ile Val Tyr Ala Leu Val Ser Lys His Phe Arg
290 295 300
Lys Gly Phe Arg Lys Ile Cys Ala Gly Leu Leu Arg Pro Ala Pro
305 310 315
Arg Arg Ala Ser Gly Arg Val Ser Ile Leu Ala Pro Gly Asn His
320 325 330
Ser Gly Ser Met Leu Glu Gln Glu Ser Thr Asp Leu Thr Gln Val
335 340 345
Ser Glu Ala Ala Gly Pro Leu Val Pro Pro Pro Ala Leu Pro Asn
350 355 360
Cys Thr Ala Ser Ser Arg Thr Leu Asp Pro Ala Cys
365 370
(2) INFORMATION FOR SEQUENCE ID NO. 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1958
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(vii) IMMEDIATE SOURCE:
(B) CLONE: Clone Y107 nucleic acid sequence
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 3:
CCACTTTGGT GATACCATGA ATGGCTCCGG CAGCCAGGGC GCGGAGAACA CGAGCCAGGA 60
AGGCGGTAGC GGCGGCTGGC AGCCTGAGGC GGTCCTTGTA CCCCTATTTT TCGCGCTCAT 120
CTTCCTCGTG GGCACCGTGG GCAACGCGCT GGTGCTGGCG GTGCTGCTGC GCGGCGGCCA 180
GGCGGTCAGC ACCACCAACC TGTTCATCCT CAACCTGGGC GTGGCCGACC TGTGTTTCAT 240
CCTGTGCTGC GTGCCTTTCC AGGCCACCAT CTACACCCTG GACGACTGGG TGTTCGGCTC 300
GCTGCTCTGC AAGGCTGTTC ATTTCCTCAT CTTTCTCACT ATGCACGCCA GCAGCTTCAC 360
GCTGGCCGCC GTCTCCCTGG ACAGGTAAAG GACCCAGAAA GAAACATCCA GTATGCCCGG 420
CA 02256523 1999-09-29
27
AGGGATCTTGACTGGAAAAGACTGAATCCTGGTCTGGTGA CCTTAGTTCCCTGCCCTTTC 480
ACATCACTTGGACATTCCCACAGAAGAGCGGTGAAGAGGC GGTGGTCCTTATTCTCCTCT 540
GGTTTCCACTGAGTGCAACATGTGCGTCCTGAGTACGCTG GAGGGACTCACAAAATTTCA 600
GCTTTCTTTAGGAGTTTCCTTGCTGTAGTTTGACCCAAGT CTTCTCCAGGTTTCTGTCAG 660
AACTCAGGCATGAGGGATCTGCCTCCCCTGGTTGTCACCA GAGGATAACAATCACTGCCC 720
CCAGAAATCCAGACAGATTCTACAACTTTTAGTCTTCGGT GTTTTGGGGGTGCCCCTTCA 780
CGTGGAGTAGGTCGGTGGCCACATTCCCAGGAGTGACAAT AGCCTAGCAGTGAATCCTCT 840
CGCTTAGCTGATGCCCCCCCACTGTCCCCACAGGTATCTG GCCATCCGCTACCCGCTGCA 900
CTCCCGAGAGTTGCGCACACCTCGAAACGCGCTGGCCGCC ATCGGGCTCATCTGGGGGCT 960
AGCACTGCTCTTCTCCGGGCCCTACCTGAGCTACTACCGT CAGTCGCAGCTGGCCAACCT 1020
GACAGTATGCCACCCAGCATGGAGCGCACCTCGACGTCGA GCCATGGACCTCTGCACCTT 1080
CGTCTTTAGCTACCTGCTGCCAGTGCTAGTCCTCAGTCTG ACCTATGCGCGTACCCTGCG 1140
CTACCTCTGGCGCACAGTCGACCCGGTGACTGCAGGCTCA GGTTCCCAGCGCGCCAAACG 1200
CAAGGTGACACGGATGATCATCATCGTGGCGGTGCTTTTC TGCCTCTGTTGGATGCCCCA 1260
CCACGCGCTTATCCTCTGCGTGTGGTTTGGTCGCTTCCCG CTCACGCGTGCCACTTACGC 1320
GTTGCGCATCCTTTCACACCTAGTTTCCTATGCCAACTCC TGTGTCAACCCCATCGTTTA 1380
CGCTCTGGTCTCCAAGCATTTCCGTAAAGGTTTCCGCAAA ATCTGCGCGGGCCTGCTGCG 1440
CCCTGCCCCGAGGCGAGCTTCGGGCCGAGTGAGCATCCTG GCGCCTGGGAACCATAGTGG 1500
CAGCATGCTGGAACAGGAATCCACAGACCTGACACAGGTG AGCGAGGCAGCCGGGCCCCT 1560
TGTCCCACCACCCGCACTTCCCAACTGCACAGCCTCGAGT AGAACCCTGGATCCGGCTTG 1620
TTAAAGGACCAAAGGGCATCTAACAGCTTCTAGACAGTGT GGCCCGAGGATCCCTGGGGG 1680
TTATGCTTGAACGTTACAGGGTTGAGGCTAAAGACTGAGG ATTGATTGTAGGGAACCTCC 1740
AGTTATTAAACGGTGCGGATTGCTAGAGGGTGGCATAGTC CTTCAATCCTGGCACCCGAA 1800
AAGCAGATGCAGGAGCAGGAGCAGGAGCAAAGCCAGCCAT GGAGTTTGAGGCCTGCTTGA 1860
ACTACCTGAGATCCAATAATAAAACATTTCATATGCTGTG AAAAAAAAAAAAAAAAAAAA 1920
CA 02256523 1999-09-29
28
AAAAAAAAAPr AAAAAAAAA.A AAAi~AAAA 19 5 8
(2) INFORMATION FOR SEQUENCE ID N0. 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 372
(B) TYPE: Amino Acid Sequence
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(vii) IMMEDIATE SOURCE:
(B) CLONE: Clone Y107 amino acid sequence (omitting putative
intron)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Met Asn Gly Ser Gly Ser Gln Gly Ala Glu Asn Thr Ser Gln Glu
1 5 10 15
Gly Gly Ser Gly Gly Trp Gln Pro Glu Ala val Leu Val Pro Leu
20 25 30
Phe Phe Ala Leu Ile Phe Leu val Gly Thr Val Gly Asn Ala Leu
35 40 45
Val Leu Ala Val Leu Leu Arg Gly Gly Gln Ala Val Ser Thr Thr
50 55 60
Asn Leu Phe Ile Leu Asn Leu Gly Val Ala Asp Leu Cys Phe Ile
65 70 75
Leu Cys Cys Val Pro Phe Gln Ala Thr Ile Tyr Thr Leu Asp Asp
80 85 90
Trp Val Phe Gly Ser Leu Leu Cys Lys Ala Val His Phe Leu Ile
95 100 105
Phe Leu Thr Met His Ala Ser Ser Phe Thr Leu Ala Ala Val Ser
110 115 120
Leu Asp Arg Tyr Leu Ala Ile Arg Tyr Pro Leu His Ser Arg Glu
125 130 135
Leu Arg Thr Pro Arg Asn Ala Leu Ala Ala Ile Gly Leu Ile Trp
140 145 150
Gly Leu Ala Leu Leu Phe Ser Gly Pro Tyr Leu Ser Tyr Tyr Arg
155 160 165
Gln Ser Gln Leu Ala Asn Leu Thr Val Cys His Pro Ala Trp Ser
170 175 180
Ala Pro Arg Arg Arg Ala Met Asp Leu Cys Thr Phe Val Phe Ser
185 190 195
Tyr Leu Leu Pro Val Leu Val Leu Ser Leu Thr Tyr Ala Arg Thr
200 205 210
Leu Arg Tyr Leu Trp Arg Thr Val Asp Pro Val Thr Ala Gly Ser
215 220 225
Gly Ser Gln Arg Ala Lys Arg Lys Val Thr Arg Met Ile Ile Ile
230 235 240
Val Ala Val Leu Phe Cys Leu Cys Trp Met Pro His His Ala Leu
245 250 255
CA 02256523 1999-09-29
29
Ile Leu Cys Val Trp Phe Gly Arg Phe Pro Leu Thr Arg Ala Thr
260 265 270
Tyr Ala Leu Arg Ile Leu Ser His Leu Val Ser Tyr Ala Asn Ser
275 280 285
Cys Val Asn Pro Ile Val Tyr Ala Leu Val Ser Lys His Phe Arg
290 295 300
Lys Gly Phe Arg Lys Ile Cys Ala Gly Leu Leu Arg Pro Ala Pro
305 310 315
Arg Arg Ala Ser Gly Arg Val Ser Ile Leu Ala Pro Gly Asn His
320 325 330
Ser Gly Ser Met Leu Glu Gln Glu Ser Thr Asp Leu Thr Gln Val
335 340 345
Ser Glu Ala Ala Gly Pro Leu Val Pro Pro Pro Ala Leu Pro Asn
350 355 360
Cys Thr Ala Ser Ser Arg Thr Leu Asp Pro Ala Cys
365 370
(2) INFORMATION FOR SEQUENCE ID NO. 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1083
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(vii) IMMEDIATE SOURCE:
(B) CLONE: Human GalR2 partial nucleic acid sequence
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
CTGCGCGGCG GCCAGGCGGT CAGCACTACC AACCTGCTCA TCCTTAACCT GGGCGTGGCC 60
GACCTGTGTT TCATCCTGTG CTGCGTGCCC TTCCAGGCCA CCATCTACAC CCTGGACGGC 120
TGGGTGTTCG GCTCGCTGCT GTGCAAGGCG GTGCACTTCC TCATCTTCCT CACCATGCAC 180
GCCAGCAGCT TCACGCTGGC CGCCGTCTCC CTGGACAGGT ATCTGGCCAT CCGCTACCCG 240
CTGCACTCCC GCGAGCTGCG CACGCCTCGA AACGCGCTGG CAGCCATCGG GCTCATCTGG 300
GGGCTGTCGC TGCTCTTCTC CGGGCCCTAC CTGAGCTACT ACCGCCAGTC GCAGCTGGCC 360
AACCTGACCG TGTGCCATCC CGCGTGGAGC GCCCCTCGCC GCCGCGCCAT GGACATCTGC 420
ACCTTCGTCT TCAGCTACCT GCTTCCTGTG CTGGTTCTCG GCCTGACCTA CGCGCGCACC 480
TTGCGCTACC TCTGGCGCGC CGTCGACCCG GTGGCCGCGG GCTCGGGTGC CCGGCGCGCC 540
AAGCGCAAGG TGACACGCAT GATCCTCATC GTGGCCGCGC TCTTCTGCCT CTGCTGGATG 600
CCCCACCACG CGCTCATCCT CTGCGTGTGG TTCGGCCAGT TCCCGCTCAC GCGCGCCACT 660
CA 02256523 1999-09-29
TATGCGCTTC GCATCCTCTC GCACCTGGTC TCCTACGCCA ACTCCTGCGT CAACCCCATC 720
GTTTACGCGC TGGTCTCCAA GCACTTCCGC AAAGGCTTCC GCACGATCTG CGCGGGCCTG 780
CTGGGCCGTG CCCCAGGCCG AGCCTCGGGC CGTGTGTGCG CTGCCGCGCG GGGCACCCAC 840
AGTGGCAGCG TGTTGGAGCG CGAGTCCAGC GACCTGTTGC ACATGAGCGA GGCGGCGGGG 900
GCCCTTCGTC CCTGCCCCGG CGCTTCCCAG CCATGCATCC TCGAGCCCTG TCCTGGCCCG 960
TCCTGGCAGG GCCCAAAGGC AGGCGACAGC ATCCTGACGG TTGATGTGGC CTGAAAGCAC 1020
TTAGCGGGCG CGCTGGGATG TCACAGAGTT GGAGTCATTG TTGGGGGACC GTGGGCCGGA 1080
AZ'I' 10 8 3
(2) INFORMATION FOR SEQUENCE ID N0. 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 337
(B) TYPE: Amino Acid sequence
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(vii) IMMEDIATE SOURCE:
(B) CLONE: Human GalR2 partial amino acid sequence
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Leu Arg Gly Gly Gln Ala Val Ser Thr Thr Asn Leu Leu Ile Leu
1 5 10 15
Asn Leu Gly Val Ala Asp Leu Cys Phe Ile Leu Cys Cys Val Pro
20 25 30
Phe Gln Ala Thr Ile Tyr Thr Leu Asp Gly Trp Val Phe Gly Ser
40 45
Leu Leu Cys Lys Ala Val His Phe Leu Ile Phe Leu Thr Met His
50 55 60
Ala Ser Ser Phe Thr Leu Ala Ala Val Ser Leu Asp Arg Tyr Leu
65 70 75
Ala Ile Arg Tyr Pro Leu His Ser Arg Glu Leu Arg Thr Pro Arg
80 85 90
Asn Ala Leu Ala Ala Ile Gly Leu Ile Trp Gly Leu Ser Leu Leu
95 100 105
Phe Ser Gly Pro Tyr Leu Ser Tyr Tyr Arg Gln Ser Gln Leu Ala
110 115 120
Asn Leu Thr Val Cys His Pro Ala Trp Ser Ala Pro Arg Arg Arg
125 130 135
Ala Met Asp Ile Cys Thr Phe Val Phe Ser Tyr Leu Leu Pro Val
140 145 150
Leu Val Leu Gly Leu Thr Tyr Ala Arg Thr Leu Arg Tyr Leu Trp
155 160 165
CA 02256523 1999-09-29
31
Arg Ala Val Asp Pro Val Ala Ala Gly Ser Gly Ala Arg Arg Ala
170 175 180
Lys Arg Lys Val Thr Arg Met Ile Leu Ile Val Ala Ala Leu Phe
185 190 195
Cys Leu Cys Trp Met Pro His His Ala Leu Ile Leu Cys Val Trp
200 205 210
Phe Gly Gln Phe Pro Leu Thr Arg Ala Thr Tyr Ala Leu Arg Ile
215 220 225
Leu Ser His Leu Val Ser Tyr Ala Asn Ser Cys Val Asn Pro Ile
230 235 240
Val Tyr Ala Leu Val Ser Lys His Phe Arg Lys Gly Phe Arg Thr
245 250 255
Ile Cys Ala Gly Leu Leu Gly Arg Ala Pro Gly Arg Ala Ser Gly
260 265 270
Arg Val Cys Ala Ala Ala Arg Gly Thr His Ser Gly Ser Val Leu
275 280 285
Glu Arg Glu Ser Ser Asp Leu Leu His Met Ser Glu Ala Ala Gly
290 295 300
Ala Leu Arg Pro Cys Pro Gly Ala Ser Gln Pro Cys Ile Leu Glu
305 310 315
Pro Cys Pro Gly Pro Ser Trp Gln Gly Pro Lys Ala Gly Asp Ser
320 325 330
Ile Leu Thr Val Asp Val Ala
335
