U.S. patent application number 10/212980 was filed with the patent office on 2003-01-30 for galanin receptor galr3 and nucleotides encoding same.
This patent application is currently assigned to Merck & Co., Inc.. Invention is credited to Cascieri, Margaret A., Howard, Andrew D., Lynch, Kevin R., Smith, Roy G., Sullivan, Kathleen A., Tan, Carina, Van Der Ploeg, Leonardus H.T..
Application Number | 20030023074 10/212980 |
Document ID | / |
Family ID | 22090825 |
Filed Date | 2003-01-30 |
United States Patent
Application |
20030023074 |
Kind Code |
A1 |
Howard, Andrew D. ; et
al. |
January 30, 2003 |
Galanin receptor GALR3 and nucleotides encoding same
Abstract
A new galanin receptor, GALR3, is described. Also provided are
nucleic acids encoding same and various assays to identify ligands
particular to said receptor. Ligands so identified are useful for
the treatment of obesity, treatment of pain, and treatment of
cognitive disorders.
Inventors: |
Howard, Andrew D.; (Park
Ridge, NJ) ; Cascieri, Margaret A.; (East Windsor,
NJ) ; Smith, Roy G.; (Houston, TX) ; Sullivan,
Kathleen A.; (Springfield, NJ) ; Tan, Carina;
(Metuchen, NJ) ; Van Der Ploeg, Leonardus H.T.;
(Scotch Plains, NJ) ; Lynch, Kevin R.;
(Charlottesville, VA) |
Correspondence
Address: |
MERCK AND CO INC
P O BOX 2000
RAHWAY
NJ
070650907
|
Assignee: |
Merck & Co., Inc.
|
Family ID: |
22090825 |
Appl. No.: |
10/212980 |
Filed: |
August 6, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10212980 |
Aug 6, 2002 |
|
|
|
09595549 |
Jun 16, 2000 |
|
|
|
09595549 |
Jun 16, 2000 |
|
|
|
PCT/US98/26812 |
Dec 17, 1998 |
|
|
|
60069725 |
Dec 17, 1997 |
|
|
|
Current U.S.
Class: |
536/23.5 ;
435/320.1; 435/325; 435/69.1; 530/350 |
Current CPC
Class: |
C07K 14/72 20130101;
A61K 38/00 20130101 |
Class at
Publication: |
536/23.5 ;
530/350; 435/69.1; 435/320.1; 435/325 |
International
Class: |
C12P 021/02; C12N
005/06; C07K 014/705; C07H 021/04 |
Claims
What is claimed:
1. Galanin receptor 3 (GALR3), substantially free from associated
proteins, or a GALR3-like receptor, wherein the GALR3-like receptor
shares at least about 40% homology to GALR3 and has substantially
the same biological activity.
2. A GALR3-like receptor according to claim 1, which shares at
least about 50% homology to a GALR3.
3. A GALR3-like receptor according to claim 1, which shares at
least about 75% homology to a GALR3.
4. A GALR3-like receptor according to claim 1, which shares at
least about 85% homology to a GALR3.
5. A GALR3 in accordance with claim 1 which is human.
6. A GALR3 in accordance with claim 1 which is rat.
7. GALR3 according to claim 1 which is shown in FIG. 1.
8. GALR3 according to claim 1 which is shown in FIG. 3.
9. GALR3 according to claim 1 which is shown in FIG. 8.
10. A nucleic acid, substantially free from associated nucleic
acids, which encodes a GALR3 or a GALR3-like receptor which is at
least about 40% homologous to GALR3 and which has substantially the
same biological activity.
11. A nucleic acid encoding a GALR3-like receptor according to
claim 10, wherein the GALR3-like receptor shares at least about 50%
homology to a GALR3.
12. A nucleic acid encoding a GALR3-like receptor according to
claim 10, wherein the GALR3-like receptor shares at least about 75%
homology to a GALR3.
13. A nucleic acid encoding a GALR3-like receptor according to
claim 10, wherein the GALR3-like receptor shares at least about 85%
homology to human GALR3.
14. A nucleic acid according to claim 10 which is DNA.
15. A vector comprising the nucleic acid of claim 13.
16. A host cell comprising the nucleic acid of claim 10.
17. A method of determining if a compound is a GALR3 ligand
comprising contacting the compound and GALR3 and determining if
binding occurs.
18. A method of identifying a compound that modulates GALR3
receptor activity, comprising: (a) culturing cells expressing GALR3
receptor in the presence of the compound; and (b) measuring GALR3
receptor activity or second messenger activity.
19. A method according to claim 18 wherein the cells are
transformed to express a GALR3 receptor.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] Not applicable
STATEMENT REGARDING FEDERALLY-SPONSORED R&D
[0002] Not applicable
REFERENCE TO MICROFICHE APPENDIX
[0003] Not applicable
FIELD OF THE INVENTION
[0004] This invention relates to a novel galanin receptor,
designated GALR3, to nucleotides encoding same, and to assays
making use thereof.
BACKGROUND OF THE INVENTION
[0005] Although first isolated from porcine intestine, galanin is
widely distributed in the central and peripheral nervous system.
Galanin in most species is a 29 amino acid peptide with an amidated
carboxyl terminus. Human galanin is unique in that it is longer, 30
amino acids, and is not amidated. There is strong conservation of
the galanin sequence with the amino terminal fifteen residues being
absolutely conserved in all species. Galanin immunoreactivity and
binding is abundant in the hypothalamus, the locus coeruleus, the
hippocampus and the anterior pituitary, as well as regions of the
spinal cord, the pancreas and the gastrointestinal tract.
[0006] Like neuropeptide Y (NPY), injection of galanin into the
paraventricular nucleus (PVN) of the hypothalamus produces a
dose-dependent increase in feeding in satiated rats. While galanin,
like norepinephrine, enhances carbohydrate ingestion, some studies
have shown that it profoundly increases fat intake. It has been
suggested that galanin shifts macronutrient preference from
carbohydrate to fat. The same injections that increase feeding
reduce energy expenditure and inhibit insulin secretion. There is
enhanced galanin expression in the hypothalamus of genetically
obese rats compared with their lean littermate controls. Injection
of peptide receptor antagonists into the PVN blocks the
galanin-specific induction of increased fat intake. Specific
galanin antisense oligonucleotides when injected into the PVN
produce a specific decrease in galanin expression associated with a
decrease in fat ingestion and total caloric intake while hardly
affecting either protein or carbohydrate intake. Thus galanin
appears to be one potential neurochemical marker related to the
behavior of fat ingestion.
[0007] Galanin inhibits cholinergic function and impairs working
memory in rats. Lesions that destroy cholinergic neurons result in
deficits in spatial learning tasks. While locally administered
acetylcholine (ACh) reverses some of this deficit, galanin blocks
this ACh-mediated improvement. Evidence from autopsy samples from
Alzheimer's disease-afflicted brains suggests an increased
galinergic innervation of the nucleus basilis. Thus, if galinergic
overactivity contributes to the decline in cognitive performance in
Alzheimer's disease, galanin antagonists may be therapeutically
useful in alleviating cognitive impairment.
[0008] In the rat, administration of galanin
intracerebroventricu-larly, subcutaneously or intravenously
increases plasma growth hormone. Infusion of human galanin into
healthy subjects also increases plasma growth hormone and potently
enhances the growth hormone response to GHRH.
[0009] Galanin levels are particularly high in dorsal root ganglia.
Sciatic nerve resection dramatically up-regulates galanin peptide
and mRNA levels. Chronic administration of galanin receptor
antagonists (M35, M15) after axotomy results in a marked increase
in self mutilation behavior in rats, generally considered to be a
response to pain. Application of antisense oligonucleotides
specific for galanin to the proximal end of a transected sciatic
nerve suppressed the increase in galanin peptide levels with a
parallel increase in autotomy. Galanin injected intrathecally acts
synergistically with morphine to produce analgesia, this
antinociceptive effect of morphine is blocked by galanin receptor
antagonists. Thus, galanin agonists may have some utility in
relieving neural pain.
[0010] The actions of galanin are mediated by high affinity galanin
receptors that are coupled by pertussis toxin sensitive
G.sub.i/G.sub.o proteins to inhibition of adenylate cyclase
activity, closure of L-type Ca.sup.++ channels and opening of
AT?-sensitive K.sup.+ channels. Specific binding of
.sup.125I-galanin (Kd approximately 1 nM) has been demonstrated in
areas paralleling localization of galanin immunoreactivity:
hypothalamus, ventral hippocampus, basal forebrain, spinal cord,
pancreas and pituitary. In most tissues the amino terminus (GAL
1-15) is sufficient for high affinity binding and agonist
activity.
[0011] Recently, a galanin receptor cDNA was isolated by expression
cloning from a human Bowes melanoma cell line. (Habert-Ortoli, et
al. 1994. Proc. Nat. Acad. Sci., USA 91: 9780-9783). This receptor,
GALR1, is expressed in human fetal brain and small intestine, but
little else is known of its distribution. Gal(1-16) is at least
1000 times more active than pGAL(3-29) as an inhibitor of
.sup.125I-porcine galanin binding to this receptor transiently
expressed in COS cells. It remains to be determined whether this
receptor subtype represents the hypothalamic receptor that mediates
the galanin specific feeding behavior.
[0012] It would be desirable to identify further galanin receptors
so that they can be used to further characterize this biological
system and to identify galanin receptor subtype selective agonists
and antagonists.
SUMMARY OF THE INVENTION
[0013] This invention relates to a novel galanin receptor,
designated GALR3, substantially free from associated proteins, and
to GALR3-like receptors which are at least about 40% homologous and
which have substantially the same biological activity.
[0014] In preferred embodiments of this invention, the GALR3-like
receptors are at least about 60%, and more preferably at least
about 75%, and even more preferably at least about 85% homologous
to a GALR3 receptor. This invention also relates specifically to
rat, human and mouse GALR3, substantially free from associated
proteins, and to receptors which are at least about 50% homologous
and which have substantially the same biological activity.
[0015] Another aspect of this invention are primate and non-primate
GALR3 proteins which are encoded by substantially the same nucleic
acid sequences, but which have undergone changes in splicing or
other RNA processing-derived modifications or mutagenesis-induced
changes, so that the expressed protein has a homologous, but
different amino acid sequence from the native forms. These variant
forms may have different and/or additional functions in human and
animal physiology or in vitro in cell based assays.
[0016] A further aspect of this invention are nucleic acids which
encode a GALR3 receptor, a GALR3-like receptor or a functional
equivalent of a GALR3 receptor from rat, human, mouse, swine, or
other species. These nucleic acids may be free from associated
nucleic acids, or they may be isolated or purified. The nucleic
acids which encode a receptor of this invention may be any type of
nucleic acid. Preferred forms are DNAs, including genomic and cDNA,
although this invention specifically includes RNAs as well. Nucleic
acid constructs may also contain regions which control
transcription and translation such as one or more promoter regions,
termination regions, and if desired enhancer regions. The nucleic
acids may be inserted into any known vector including plasmids, and
used to transfect suitable host cells using techniques generally
available to one of ordinary skill in the art.
[0017] Another aspect of this invention are vectors comprising
nucleic acids which encode GALR3, and host cells which contain
these vectors. Still another aspect of this invention is a method
of making GALR3 comprising introducing a vector comprising nucleic
acids encoding GALR3 into a host cell under culturing
conditions.
[0018] Yet another aspect of this invention are assays for GALR3
ligands which utilize the receptors and/or nucleic acids of this
invention. Preferred assays of this embodiment compare the binding
of the putative GALR3 ligand to the binding of galanin to
GALR3.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 is the DNA sequence of human GALR3 gene, clone
GALR3-3 (SEQ ID NO:1).
[0020] FIG. 2 is the deduced amino acid sequence of human GALR3,
clone GALR3-3 (SEQ ID NO:2).
[0021] FIG. 3 is the DNA sequence (open reading frame only) of
human GALR3, clone GALR3-2 (SEQ ID NO:3).
[0022] FIG. 4 is the deduced amino acid sequence of GALR3, clone
GALR3-2 (SEQ ID NO:4)
[0023] FIG. 5 is a comparison of the open reading frame protein
sequences of human and rat GALR3 with the corresponding sequences
of GALR1 (mouse--SEQ ID NO:5, rat--SEQ ID NO:6, and human--SEQ ID
NO:7) and GALR2 (mouse--SEQ ID NO:8, rat--SEQ ID NO:9, and
human--SEQ ID NO:10).
[0024] FIG. 6 is a phylogenetic analysis of the putative GALR3
protein sequence.
[0025] FIG. 7 illustrates the competition curves for
.sup.125I-porcine galanin against human and porcine galanin.
[0026] FIG. 8 is the DNA sequence of rat GALR3 from region TM4 to
region TM7 (SEQ ID NO:11).
[0027] FIG. 9 is the deduced amino acid sequence of rat GALR3 from
region TM4 to region TM7 (SEQ ID NO:12).
DETAILED DESCRIPTION OF THE INVENTION
[0028] As used throughout the specification and claims, the
following definitions apply:
[0029] "Substantially free from associated proteins" means that the
receptor is at least about 90%, and preferably at least about 95%
free from other cell membrane proteins which are normally found in
a living mammalian cell which expresses a galanin receptor.
[0030] "Substantially free from associated nucleic acids" means
that the nucleic acid is at least about 90%, and preferably at
least about 95%, free from other nucleic acids which are normally
found in a living mammalian cell which naturally expresses a
galanin receptor gene.
[0031] "Substantially the same biological activity" means that the
receptor-galanin binding constant is within 5-fold of the binding
constant of GALR3 and galanin, and preferably within 2-fold of the
binding constant of GALR3 and galanin.
[0032] "Stringent post-hybridizational washing conditions" means
0.1.times. standard saline citrate (SSC) at 65.degree. C.
[0033] "Standard post-hybridizational washing conditions" means
6.times. SSC at 55.degree. C.
[0034] "Relaxed post-hybridizational washing conditions" means
6.times. SSC at 30.degree. C., or 1 to 2.times. SSC at 55.degree.
C.
[0035] "Functional equivalent" means that a receptor which does not
have the exact same amino acid sequence of a naturally occurring
GALR3 protein due to alternative splicing, deletions, mutations, or
additions, but retains at least 1%, preferably 10%, and more
preferably 25% of the biological activity of the naturally
occurring receptor. Such derivatives will have a significant
homology with a natural GALR3 and can be detected by reduced
stringency hybridization with a DNA sequence obtained from a GALR3.
The nucleic acid coding a functional equivalent has at least about
60% homology at the nucleotide level to a naturally occurring
receptor nucleic acid.
[0036] It has been found, in accordance with this invention, that
there is a third galanin receptor, which is designated GALR3. The
human (clone 3-3 and 3-2) and rat GALR3 sequences are given in
FIGS. 1, 3 and 8, respectively, and are referenced in the Examples;
however it is to be understood that this invention specifically
includes GALR3 without regard to the species and, in particular,
specifically includes rodent (including rat and mouse), rhesus,
swine, chicken, cow and human. The galanin 3 receptors are highly
conserved throughout species, and one of ordinary skill in the art,
given the rat, human and/or mouse sequences presented herein, can
easily design probes to obtain the GALR3 from other species.
[0037] GALR3 proteins contain various functional domains, including
one or more domains which anchor the receptor in the cell membrane,
and at least one ligand binding domain. As with many receptor
proteins, it is possible to modify many of the amino acids,
particularly those which are not found in the ligand binding
domain, and still retain at least a percentage of the biological
activity of the original receptor. Thus this invention specifically
includes modified functionally equivalent GALR3s which have
deleted, truncated, or mutated N-terminal portions. This invention
also specifically includes modified functionally equivalent GALR3s
which contain modifications and/or deletions in other domains,
which are not accompanied by a loss of functional activity.
[0038] Additionally, it is possible to modify other functional
domains such as those that interact with second messenger effector
systems, by altering binding specificity and/or selectivity. Such
functionally equivalent mutant receptors are also within the scope
of this invention.
[0039] The proteins of this invention were found to have structural
features which are typical of the 7-transmembrane domain (TM)
containing G-protein linked receptor superfamily (GPC-R's or 7-TM
receptors). Thus GALR3 proteins make up new members of the GPC-R
family of receptors. The intact GALR3 of this invention was found
to have the general features of GPC-R's, including seven
transmembrane regions, three intra- and extracellular loops, and
the GPC-R protein signature sequence. The TM domains and GPC-R
protein signature sequence are noted in the protein sequences of
the GALR3. Not all regions are required for functioning, and
therefore this invention also comprises functional receptors which
lack one or more non-essential domains.
[0040] Determination of the nucleotide sequence indicated that the
GALR3 belongs to the intron-containing class of GPC-R's.
[0041] The DNA sequence encoding the putative GALR3 is shown in
FIGS. 1 and 3. The human putative GALR3 gene is organized similarly
to human GALR2 with a single intron (-1 kb) dividing the open
reading into two exons with Exon 1 consisting of -350 bp, and Exon
2-700 bp. Based on database searching, the open reading frame
protein sequence for this novel gene (FIGS. 2 and 4) is most
closely related to GALR2 and GALR1 with 58, 75% identity and
similarity to human GALR2, and 37, 61% identity and similarity to
rat GALR1 (FIG. 5). Differences in open reading frame DNA sequence
and the resulting deduced amino acid sequence between clone GALR3-2
and GALR3-3 may be allelic in nature. Phylogenetic analysis of the
putative GALR3 protein sequence supports the notion that this gene
encodes a receptor for galanin (FIG. 6).
[0042] The human GALR3 protein bears strong sequence identity and
similarity to the rat GALR3 ortholog.
[0043] This invention also relates to truncated forms of GALR3,
particularly those which encompass the extracellular portion of the
receptor, but lack the intracellular signaling portion of the
receptor, and to nucleic acids encoding these truncated forms. Such
truncated receptors are useful in various binding assays. Thus this
invention specifically includes modified functionally equivalent
GALR3s which have deleted, truncated, or mutated N-terminal
portions. This invention also specifically includes modified
functionally equivalent GALR3s including receptor chimeras which
contain modifications and/or deletions in other domains, which are
not accompanied by a loss of functional activity.
[0044] Additionally, it is possible to modify other functional
domains such as those that interact with second messenger effector
systems, by altering binding specificity and/or selectivity. Such
functionally equivalent mutant receptors are also within the scope
of this invention.
[0045] Assays which make up further aspects of this invention
include binding assays (competition for .sup.125I-galanin binding),
coupling assays (including galanin-mediated inhibition of
forskolin-stimulated adenylate cyclase in cells expressing galanin
receptors), measurement of galanin-stimulated calcium release in
cells expressing galanin receptors (such as aequorin assays),
stimulation of inward rectifying potassium channels (GIRK channels,
measured by voltage changes) in cells expressing galanin receptors,
and measurement of pH changes upon galanin stimulation of cells
expressing galanin receptors as measured with a
microphysiometer.
[0046] Host cells may be cultured under suitable conditions to
produce GALR3. An expression vector containing DNA encoding the
receptor may be used for expression of receptor in a recombinant
host cell. Recombinant host cells may be prokaryotic or eukaryotic,
including but not limited to bacteria such as E. coli, fungal cells
such as yeast, mammalian cells including but not limited to cell
lines of human, bovine, porcine, monkey and rodent origin, and
insect cells including but not limited to Drosophila, Spodoptera,
and silkworm derived cell lines. Cell lines derived from mammalian
species which are suitable and which are commercially available
include, but are not limited to, L cells L-M(TK.sup.-) (ATCC CCL
1.3), L cells L-M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC
CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL
1651), CHO-KI (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL
1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL
26) and MRC-5 (ATCC CCL 171).
[0047] The specificity of binding of compounds showing affinity for
the receptor is shown by measuring the affinity of the compounds
for cells transfected with the cloned receptor or for membranes
from these cells. Expression of the cloned receptor and screening
for compounds that inhibit the binding of radiolabeled ligand to
these cells provides a rational way for rapid selection of
compounds with high affinity for the receptor. These compounds
identified by the above assays may be agonists or antagonists of
the receptor and may be peptides, proteins, or non-proteinaceous
organic molecules. Alternatively, functional assays of the receptor
may be used to screen for compounds which affect the activity of
the receptor. Such functional assays range from ex vivo muscle
contraction assays to assays which determine second messenger
levels in cells expressing the receptor. The second messenger
assays include, but are not limited to, assays to measure cyclic
AMP or calcium levels or assays to measure adenyl cyclase activity.
These compounds identified by the above assays may be agonists,
antagonists, suppressors, or inducers of the receptor. The
functional activity of these compounds is best assessed by using
the receptor either natively expressed in tissues or cloned and
exogenously expressed.
[0048] Using the assays of this invention, galanin agonists and
antagonists may be identified. A galanin agonist is a compound
which binds to the GALR3, such as a galanin mimetic, and produces a
cellular response which is at least about equivalent to that of
galanin, and which may be greater than that of galanin. Such
compounds would be useful in situations where galanin insufficiency
causes anorexia, or for treatment of pain.
[0049] Also using this embodiment of the assay, galanin antagonists
may be identified. A galanin antagonist is a compound which can
bind to the GALR3, but produces a lesser response than that of
native galanin. Such compounds would be useful in the treatment of
obesity.
[0050] One assay of this invention is a method of identifying a
compound which modulates GALR3 receptor comprising: a) culturing
cells expressing the GALR3 receptor in the presence of the compound
and b) measuring GALR3 receptor activity or second messenger
activity. If desired, the determined activity can be compared to a
standard, such as that measured using galanin as the compound. In
preferred embodiments, the cells are transformed and express the
GALR3 receptor.
[0051] The consultant cDNA clone (or shorter portions of, for
instance, only 15 nucleotides long) may be used to probe libraries
under hybridization conditions to find other receptors which are
similar enough so that the nucleic acids can hybridize, and is
particularly useful for screening libraries from other species. In
this step, one of ordinary skill in the art will appreciate that
the hybridization conditions can vary from very stringent to
relaxed. Proper temperature, salt concentrations, and buffers are
well known.
[0052] The following non-limiting Examples are presented to better
illustrate the invention.
EXAMPLE 1
[0053] Human GALR3:
[0054] Identification and Cloning of Human GalR3 Gene, Sequence and
Gene Structure
[0055] Automated searching of sequence data from GenBank (National
Center For Biotechnology Information, Bethesda, Md.) were queried
using sequences from known receptor clones. Using a list of 50-60
rhodopsin family amino acid sequences, the NEW division of GenBank
was searched. The query algorithm is TFASTX and the output is
placed in a file where alignments are sorted by query sequence and
scored (cut-off based on the expectation value, set for example, at
0.01). A DNA sequence alignment of 300 bp to a portion of a large
BAC clone (-100,000 bp) with accession number Z97630 was identified
from the high through-put genomic sequence (HTGS database,
GenBank). The complete open reading frame (ORF) for the putative
gene encoding GALR3 was then identified using sequence from BAC
Z97630 and an additional BAC clone, with assession number Z82241,
from the HTGS database. The Genbank assession numbers corresponded
to the following HTGS BAC clones (HS entries): Z97630, HS466N1;
Z82241, HS8112.
[0056] DNA sequences derived from these BACs were used to choose
PCR primers. PCR primers beginning at the predicted initiating Met
and ending more 3' than the predicted stop codon were utilized to
PCR from human genomic DNA a fragment containing the predicted Exon
I, the intervening intron, and predicted Exon II. This PCR product
was subcloned and sequenced, resulting in expression plasmid
GALR3-3.
[0057] In a parallel approach, a human genomic DNA library
(Stratagene, La Jolla, Calif.) was screened to isolate the putative
GALR3 gene. Primary screening under medium stringency resulted in 6
positive plaques using an Exon 2 probe. One hybridizing phage
plaque was obtained upon secondary screening. A 13 kb EcoR1/EcoRV
fragment was identified from the genomic clone by Southern
blotting, transferred into pBluescript vector (Stratagene, La
Jolla, Calif.), and confirmed to be GALR3 by sequencing. A
intronless GALR3 expression construct was assembled in a similar
manner to that described above using Pfu DNA polymerase
(Stratagene, La Jolla, Calif.) resulting in expression plasmid
GALR3-2.
EXAMPLE 2
[0058] Chromosomal Location
[0059] The BAC clones which were identified by the searches of the
HTGS dataset have been mapped by the The Sanger Centre (Cambridge,
UK) genome research laboratory to human chromosome 22.
[0060] FISH analysis conducted herein has confirmed this assignment
and refined it to 22q12.2-13.1.
EXAMPLE 3
[0061] Receptor Expression:
[0062] Construction of Human GalR3 Expression Plasmid
[0063] The human GalR3 cDNA expression construct was assembled
stepwise from PCR products amplified from human genomic DNA. Each
exon was PCR amplified using standard conditions. The primers in
for exon I were: Forward Exon I (5'-gcg aat tcg gta cca tgg ctg atg
ccc aga aca t-3'; SEQ ID NO:13) and Reverse Exon 1 (5'-cgc ctg tcg
aca gat aca gca gc-3'; SEQ-ID NO:14). The primers for exon U were:
Forward Exon II (5'-tgt atc tgt cga cag gta acc tgg ccg tgc ggc acc
c-3'; SEQ ID NO: 15) and Reverse Exon II (5'-gcg cgg ccg ctt att
ccg gtc ctc ggg c-3'; SEQ ID NO: 16). PCR products were subcloned
into pCRII and sequenced. For expression in mammalian cells, the
putative GALR3 ORF was subcloned into pcDNA-1/amp (Invitrogen)
resulting in plasmid GALR3-3; and pcDNA-3 (Invitrogen), resulting
in plasmid GALR3-2.
EXAMPLE 4
[0064] RNA Expression Profile
[0065] Using RNase protection analysis, the relative levels of
human GALR3 mRNA was assessed. As shown below GALR3 is expressed in
numerous brain regions and peripheral tissues, as observed for
GALR1 and GALR2.
1 Expression Tissue Level Amygdala + Cerebellum + Frontal Cortex +
Hippocampus + Hypothalamus ++ Pituitary + Brain stem + Lung ++
Heart + Spleen + Liver + Pancreas + Duodenum + Colon + Straited
muscle ++
EXAMPLE 5
[0066] Radioligand Binding:
[0067] Pharmacology of Human GALR3
[0068] Mammalian COS-7 cells were transfected by electroporation.
COS-7 cells (1.times.10.sup.7) were suspended in 0.85 ml of
Ringers' buffer and 15 mg of the GALR3-2 or GALR3-3 expression
plasmid was added to a 0.4 mm electroporation cuvette (Bio-Rad,
Hercules, Calif.). Current was applied (960 mF, 260 V) using a
Bio-Rad Electroporator device and the cells were transferred to a
T-180 flask (Corning). Expression was allowed to proceed for 72
hrs.
[0069] Membranes were prepared from transfected cells following
dissociation in enzyme-free dissociation solution (Specialty Media,
Lavallette, N.J.) by disruption in a Dounce homogenizer in ice-cold
membrane buffer (10 mM Tris, pH 7.4, 10 mM PMSF, 10 mM
phosphoramidon, and 40 mg/ml bacitracin). After a low speed
(1100.times. g for 10 min. at 4.degree. C.) and a high speed
centrifugation (38,700.times. g for 15 min. at 4.degree. C.),
membranes were resuspended in buffer and protein concentration
determined (Bio-Rad assay kit). Binding of .sup.125I-human or
porcine galanin (specific activity of 2200 Ci/mmol, DuPont NEN) was
measured in membranes using a buffer of 25 mM Tris pH 7.4, 0.5%
BSA, 2 mM MgCl.sub.2, 40 .mu.g/ml bacitracin, 4 mg/ml
phosphoramidon, and 10 .mu.M leupeptin in a total volume of 250 ml.
70 pM .sup.125I-human or porcine galanin was used. Transfected
cells expressing plasmid GALR3-3 were bound with .sup.125I-human
galanin whereas cells expressing plasmid GALR3-2 were bound with
.sup.125I-porcine galanin. Reactions were initiated by the addition
of membranes and the incubation was allowed to proceed at room
temperature for 1 hour. Non-specific binding was defined as the
amount of radioactivity remaining bound in the presence of 1 mM
respectively unlabeled galanin and was generally not above 200 cpm
(<10% of total radioactivity bound). Titration of membrane
protein from 1 to 50 .mu.g was conducted. In competition studies
various concentrations of unlabeled human or porcine galanin were
included along with .sup.125I-porcine galanin (70 pM) in cells
expressing the GALR3-2 plasmid. Incubations were terminated by
rapid filtration through GF/C filters which had been presoaked with
0.1% polyethylamine using a TOMTEC (Orange, Conn.) cell harvester.
The results were analyzed using the Prism software package
(GraphPad, San Diego, Calif.). The table below illustrates that
both clones confer specific binding to COS-7 cells for both human
and porcine galanin radioligands as a function of protein
concentration. In COS-7 cells mock transfected with expression
vector only (no GALR3 gene), no specific binding of either
radioligand was observed.
2 Clone GALR3-3 Clone GALR3-2: Membrane Protein .sup.125I-human
galanin .sup.125I-porcine galanin (.mu.g) (cpm) (cpm) 1 ND ND 5 211
695 10 407 1134 20 886 1763 50 2061 3728
[0070] Competition curves for .sup.125I-porcine galanin against
human and porcine galanin were generated to to determine the IC50
for both unlabeled peptides (clone GALR3-2), as shown in FIG. 7.
The IC50 values for porcine and human galanin were 16 nM and 93 nM,
respectively.
EXAMPLE 6
[0071] Rat GALR3:
[0072] Identification and Cloning of Rat GalR3 Gene
[0073] Primers based on the intronless human GALR3 sequence from
TM4 and TM7 were designed and used to PCR amplify with Pfu DNA
polymerase the rat GALR3 ortholog from rat genomic DNA. A PCR
product of the appropriate size (approximately 400 bp) that
hybridized with an Exon 2 probe from the human GALR3 gene was
subcloned into pBluescript vector (Stratagene, La Jolla, Calif.).
The DNA sequence is shown is FIG. 8 and the deduced amino acid
sequence is shown in FIG. 9. DNA sequence analysis revealed
significant homology with human GALR3: approximately 95% protein
sequence identity for 129 amino acids spanning TM4 through
TM-7.
[0074] RNA Expression
[0075] Northern blot analysis using a probe dervived from the rat
GALR3 ORF has revealed expression of rat GALR3 mRNA in
hypothalamus, mid-brain, pons, and whole fetal brain.
Sequence CWU 1
1
16 1 2263 DNA human 1 ccaggtcggg ggagttagat cccggggtca agcaaccaga
actgggggct cttgcctgag 60 gattccagct tctcttccca ggtgcccgtc
tgatggggag atggctgatg cccagaacat 120 ttcactggac agcccaggga
gtgtgggggc cgtggcagtg cctgtggtct ttgccctaat 180 cttcctgctg
ggcacagtgg gcaatgggct ggtgctggca gtgctcctgc agcctggccc 240
gagtgcctgg caggagcctc gcagcaccac ggacctgttc atcctcaacc tggcggtggc
300 tgacctctgc ttcatcctgt gctgcgtgcc cttccaggcc accatataca
cgctggatgc 360 ctggctcttt ggggccctcg tttgcaaggc cgtgcacctg
ttcatctacc tcaccatgaa 420 cgccagcagc tttacgctgg ctgctgtatc
tgtggacagg tgcgctgtgc ctggggcctg 480 gctgggcagg gctgtggggg
cgggggttgg gggaggagtc ctgaacagat cctcactggc 540 cttaggaagg
agagagtggg ggaccagaaa gggaggtggg tgggaggaaa caaaagctcc 600
ctgacccctc gcaagcagcc tctgggcacc tgcagggcgt gcttgagggg actgtcctgc
660 ccttcccctc ctccactgtg aacttccaga ggacgcctct gagtctcaag
tggcagcaca 720 gggtctggca catagtaagt gctctgtaag cgcgaaatga
atcgcaaaag aagctcacga 780 atgcgttcat cagttttttt gttttgtttt
gttttgttgt tttttttttt ttggatcttg 840 gctcactgca acctctgcct
cctgggttcc agcgattctc ctgccacagc ctcctgagta 900 gctgggatta
caggccacca cacctggcta attttttgta tttttagtag aaacggggtt 960
ttgccatgtt ggacaggctg gtctcgaacc cctgacctca agtgatccgc ccgcctcggc
1020 ctcccaagtg ctgggattac aggcgtgagc caccgcgccc agcccagcta
ttttctaact 1080 gcccacacct ggccaagctg tgcacacatc tgcttccaca
gcttgaaact tggggtcaaa 1140 tccaggctca ctccagctga tgaccctggg
caagtcactt ctctctggac ctcatctgac 1200 gcatccataa aataatccta
gaaataacaa gtcaccggga tcgggccctt gctaggtgca 1260 agggcctaag
caccttgcgc gttcacaccc ttaatccccg ccacgtcccc cacggttcac 1320
aggaggcgca ctgggccgca gggcccgggc gcgggacgtg gcgcgggccc ctgcgggagg
1380 gcacctgccc gccccgctga ccasgcgccc tccgcaggta cctggccgtg
cggcacccgc 1440 tgcgctcgcg cgccctgcgc acgccgcgta acgcccgcgc
cgcagtgggg ctggtgtggc 1500 tgctggcggc gctcttctcg gcgccctacc
tcagctacta cggcaccgtg cgctacggcg 1560 cgctggagct ctgcgtgccc
gcctgggagg acgcgcgccg ccgcgccctg gacgtggcca 1620 ccttcgctgc
cggctacctg ctgcccgtgg cygtggtgag cctggcctac gggcgcacgc 1680
tgcgcttcct gtgggccgcc gtgggtcccg cgggcgcggc ggcrgccaar gcgcggcgga
1740 gggcgackgg ccgcgcgggg cgcgccatgc tggcggtggc cgcgctctac
gcgctmtgct 1800 ggggtccgca ccacgcgctc atcctgtgct tctggtacgg
ccgmttcgcc ttcagcccgg 1860 ccacctacgc mtgccgcctg gcctcacact
gcctggccta cgccaactcm tgcctcaacc 1920 cgctcgtmta cgcgctcgcc
tcgcgccact tccgcgcgcg cttccgccgc ctgtggccgt 1980 gcggycgccg
acgccgccac cgtgcccgcc gcgccttgcg tcgcgtccgc cccgcgtcct 2040
cgggcccacc cggctgcccc ggagacgccc ggcctagcgg gacgctgctg gctggtggcg
2100 gccagggccc sgagcccagg gagggacccg tccacggcgg agaggctgcc
cgaggaccgg 2160 aataaaccct gccgcctgga ctccgcctgt gtccgtctgt
ctcactcccg ttctccgaag 2220 gcgggacgcc accgggccag ggatggggca
atgccacgag ctc 2263 2 368 PRT human 2 Met Ala Asp Ala Gln Asn Ile
Ser Leu Asp Ser Pro Gly Ser Val Gly 1 5 10 15 Ala Val Ala Val Pro
Val Val Phe Ala Leu Ile Phe Leu Leu Gly Thr 20 25 30 Val Gly Asn
Gly Leu Val Leu Ala Val Leu Leu Gln Pro Gly Pro Ser 35 40 45 Ala
Trp Gln Glu Pro Arg Ser Thr Thr Asp Leu Phe Ile Leu Asn Leu 50 55
60 Ala Val Ala Asp Leu Cys Phe Ile Leu Cys Cys Val Pro Phe Gln Ala
65 70 75 80 Thr Ile Tyr Thr Leu Asp Ala Trp Leu Phe Gly Ala Leu Val
Cys Lys 85 90 95 Ala Val His Leu Phe Ile Tyr Leu Thr Met Asn Ala
Ser Ser Phe Thr 100 105 110 Leu Ala Ala Val Ser Val Asp Arg Tyr Leu
Ala Val Arg His Pro Leu 115 120 125 Arg Ser Arg Ala Leu Arg Thr Pro
Arg Asn Ala Arg Ala Ala Val Gly 130 135 140 Leu Val Trp Leu Leu Ala
Ala Leu Phe Ser Ala Pro Tyr Leu Ser Tyr 145 150 155 160 Tyr Gly Thr
Val Arg Tyr Gly Ala Leu Glu Leu Cys Val Pro Ala Trp 165 170 175 Glu
Asp Ala Arg Arg Arg Ala Leu Asp Val Ala Thr Phe Ala Ala Gly 180 185
190 Tyr Leu Leu Pro Val Ala Val Val Ser Leu Ala Tyr Gly Arg Thr Leu
195 200 205 Arg Phe Leu Trp Ala Ala Val Gly Pro Ala Gly Ala Ala Ala
Ala Lys 210 215 220 Ala Arg Arg Arg Ala Thr Gly Arg Ala Gly Arg Ala
Met Leu Ala Val 225 230 235 240 Ala Ala Leu Tyr Ala Leu Cys Trp Gly
Pro His His Ala Leu Ile Leu 245 250 255 Cys Phe Trp Tyr Gly Arg Phe
Ala Phe Ser Pro Ala Thr Tyr Ala Cys 260 265 270 Arg Leu Ala Ser His
Cys Leu Ala Tyr Ala Asn Ser Cys Leu Asn Pro 275 280 285 Leu Val Tyr
Ala Leu Ala Ser Arg His Phe Arg Ala Arg Phe Arg Arg 290 295 300 Leu
Trp Pro Cys Gly Arg Arg Arg Arg His Arg Ala Arg Arg Ala Leu 305 310
315 320 Arg Arg Val Arg Pro Ala Ser Ser Gly Pro Pro Gly Cys Pro Gly
Asp 325 330 335 Ala Arg Pro Ser Gly Thr Leu Leu Ala Gly Gly Gly Gln
Gly Pro Glu 340 345 350 Pro Arg Glu Gly Pro Val His Gly Gly Glu Ala
Ala Arg Gly Pro Glu 355 360 365 3 1107 DNA human 3 atggctgatg
cccagaacat ttcactggac agcccaggga gtgtgggggc cgtggcagtg 60
cctgtggtct ttgccctaat cttcctgctg ggcacagtgg gcaatgggct ggtgctggca
120 gtgctcctgc agcctggccc gagtgcctgg caggagcctg gcagcaccac
ggacctgttc 180 atcctcaacc tggcggtggc tgacctctgc ttcatcctgt
gctgcgtgcc cttccaggcc 240 accatctaca cgctggatgc ctggctcttt
ggggccctcg tctgcaaggc cgtgcacctg 300 ctcatctacc tcaccatgta
cgccagcagc tttacgctgg ctgctgtctc cgtggacagg 360 tacctggccg
tgcggcaccc gctgcgctcg cgcgccctgc gcacgccgcg taacgcccgc 420
gccgcagtgg ggctggtgtg gctgctggcg gcgctcttct cggcgcccta cctcagctac
480 tacggcaccg tgcgctacgg cgcgctggag ctctgcgtgc ccgcctggga
ggacgcgcgc 540 cgccgcgccc tggacgtggc caccttcgct gccggctacc
tgctgcccgt ggctgtggtg 600 agcctggcct acgggcgcac gctgcgcttc
ctgtgggccg ccgtgggtcc cgcgggcgcg 660 gcggcggccg aggcgcggcg
gagggcgacg ggccgcgcgg ggcgcgccat gctggcggtg 720 gccgcgctct
acgcgctctg ctggggtccg caccacgcgc tcatcctgtg cttctggtac 780
ggccgcttcg ccttcagccc ggccacctac gcctgccgcc tggcctcaca ctgcctggcc
840 tacgccaact cctgcctcaa cccgctcgtc tacgcgctcg cctcgcgcca
cttccgcgcg 900 cgcttccgcc gcctgtggcc gtgcggccgc cgacgccgcc
accgtgcccg ccgcgccttg 960 cgtcgcgtcc gccccgcgtc ctcgggccca
cccggctgcc ccggagacgc ccggcctagc 1020 gggaggctgc tggctggtgg
cggccagggc ccggagccca gggagggacc cgtccacggc 1080 ggagaggctg
cccgaggacc ggaataa 1107 4 368 PRT human 4 Met Ala Asp Ala Gln Asn
Ile Ser Leu Asp Ser Pro Gly Ser Val Gly 1 5 10 15 Ala Val Ala Val
Pro Val Val Phe Ala Leu Ile Phe Leu Leu Gly Thr 20 25 30 Val Gly
Asn Gly Leu Val Leu Ala Val Leu Leu Gln Pro Gly Pro Ser 35 40 45
Ala Trp Gln Glu Pro Gly Ser Thr Thr Asp Leu Phe Ile Leu Asn Leu 50
55 60 Ala Val Ala Asp Leu Cys Phe Ile Leu Cys Cys Val Pro Phe Gln
Ala 65 70 75 80 Thr Ile Tyr Thr Leu Asp Ala Trp Leu Phe Gly Ala Leu
Val Cys Lys 85 90 95 Ala Val His Leu Leu Ile Tyr Leu Thr Met Tyr
Ala Ser Ser Phe Thr 100 105 110 Leu Ala Ala Val Ser Val Asp Arg Tyr
Leu Ala Val Arg His Pro Leu 115 120 125 Arg Ser Arg Ala Leu Arg Thr
Pro Arg Asn Ala Arg Ala Ala Val Gly 130 135 140 Leu Val Trp Leu Leu
Ala Ala Leu Phe Ser Ala Pro Tyr Leu Ser Tyr 145 150 155 160 Tyr Gly
Thr Val Arg Tyr Gly Ala Leu Glu Leu Cys Val Pro Ala Trp 165 170 175
Glu Asp Ala Arg Arg Arg Ala Leu Asp Val Ala Thr Phe Ala Ala Gly 180
185 190 Tyr Leu Leu Pro Val Ala Val Val Ser Leu Ala Tyr Gly Arg Thr
Leu 195 200 205 Arg Phe Leu Trp Ala Ala Val Gly Pro Ala Gly Ala Ala
Ala Ala Glu 210 215 220 Ala Arg Arg Arg Ala Thr Gly Arg Ala Gly Arg
Ala Met Leu Ala Val 225 230 235 240 Ala Ala Leu Tyr Ala Leu Cys Trp
Gly Pro His His Ala Leu Ile Leu 245 250 255 Cys Phe Trp Tyr Gly Arg
Phe Ala Phe Ser Pro Ala Thr Tyr Ala Cys 260 265 270 Arg Leu Ala Ser
His Cys Leu Ala Tyr Ala Asn Ser Cys Leu Asn Pro 275 280 285 Leu Val
Tyr Ala Leu Ala Ser Arg His Phe Arg Ala Arg Phe Arg Arg 290 295 300
Leu Trp Pro Cys Gly Arg Arg Arg Arg His Arg Ala Arg Arg Ala Leu 305
310 315 320 Arg Arg Val Arg Pro Ala Ser Ser Gly Pro Pro Gly Cys Pro
Gly Asp 325 330 335 Ala Arg Pro Ser Gly Arg Leu Leu Ala Gly Gly Gly
Gln Gly Pro Glu 340 345 350 Pro Arg Glu Gly Pro Val His Gly Gly Glu
Ala Ala Arg Gly Pro Glu 355 360 365 5 348 PRT mouse 5 Met Glu Leu
Ala Met Val Asn Leu Ser Glu Gly Asn Gly Ser Asp Pro 1 5 10 15 Glu
Pro Pro Ala Pro Glu Ser Arg Pro Leu Phe Gly Ile Gly Val Glu 20 25
30 Asn Phe Ile Thr Leu Val Val Phe Gly Leu Ile Phe Ala Met Gly Val
35 40 45 Leu Gly Asn Ser Leu Val Ile Thr Val Leu Ala Arg Ser Lys
Pro Gly 50 55 60 Lys Pro Arg Ser Thr Thr Asn Leu Phe Ile Leu Asn
Leu Ser Ile Ala 65 70 75 80 Asp Leu Ala Tyr Leu Leu Phe Cys Ile Pro
Phe Gln Ala Thr Val Tyr 85 90 95 Ala Leu Pro Thr Trp Val Leu Gly
Ala Phe Ile Cys Lys Phe Ile His 100 105 110 Tyr Phe Phe Thr Val Ser
Met Leu Val Ser Ile Phe Thr Leu Ala Ala 115 120 125 Met Ser Val Asp
Arg Tyr Val Ala Ile Val His Ser Arg Arg Ser Ser 130 135 140 Ser Leu
Arg Val Ser Arg Asn Ala Leu Leu Gly Val Gly Phe Ile Trp 145 150 155
160 Ala Leu Ser Ile Ala Met Ala Ser Pro Val Ala Tyr His Gln Arg Leu
165 170 175 Phe His Arg Asp Ser Asn Gln Thr Phe Cys Trp Glu Gln Trp
Pro Asn 180 185 190 Lys Leu His Lys Lys Ala Tyr Val Val Cys Thr Phe
Val Phe Gly Tyr 195 200 205 Leu Leu Pro Leu Leu Leu Ile Cys Phe Cys
Tyr Ala Lys Val Leu Asn 210 215 220 His Leu His Lys Lys Leu Lys Asn
Met Ser Lys Lys Ser Glu Ala Ser 225 230 235 240 Lys Lys Lys Thr Ala
Gln Thr Val Leu Val Val Val Val Val Phe Gly 245 250 255 Ile Ser Trp
Leu Pro His His Val Val His Leu Trp Ala Glu Phe Gly 260 265 270 Ala
Phe Pro Leu Thr Pro Ala Ser Phe Phe Phe Arg Ile Thr Ala His 275 280
285 Cys Leu Ala Tyr Ser Asn Ser Ser Val Asn Pro Ile Ile Tyr Ala Phe
290 295 300 Leu Ser Glu Asn Phe Arg Lys Ala Tyr Lys Gln Val Phe Lys
Cys His 305 310 315 320 Val Cys Asp Glu Ser Pro Arg Ser Glu Thr Lys
Glu Asn Lys Ser Arg 325 330 335 Met Asp Thr Pro Pro Ser Thr Asn Cys
Thr His Val 340 345 6 346 PRT rat 6 Met Glu Leu Ala Pro Val Asn Leu
Ser Glu Gly Asn Gly Ser Asp Pro 1 5 10 15 Glu Pro Pro Ala Glu Pro
Arg Pro Leu Phe Gly Ile Gly Val Glu Asn 20 25 30 Phe Ile Thr Leu
Val Val Phe Gly Leu Ile Phe Ala Met Gly Val Leu 35 40 45 Gly Asn
Ser Leu Val Ile Thr Val Leu Ala Arg Ser Lys Pro Gly Lys 50 55 60
Pro Arg Ser Thr Thr Asn Leu Phe Ile Leu Asn Leu Ser Ile Ala Asp 65
70 75 80 Leu Ala Tyr Leu Leu Phe Cys Ile Pro Phe Gln Ala Thr Val
Tyr Ala 85 90 95 Leu Pro Thr Trp Val Leu Gly Ala Phe Ile Cys Lys
Phe Ile His Tyr 100 105 110 Phe Phe Thr Val Ser Met Leu Val Ser Ile
Phe Thr Leu Ala Ala Met 115 120 125 Ser Val Asp Arg Tyr Val Ala Ile
Val His Ser Arg Arg Ser Ser Ser 130 135 140 Leu Arg Val Ser Arg Asn
Ala Leu Leu Gly Val Gly Phe Ile Trp Ala 145 150 155 160 Leu Ser Ile
Ala Met Ala Ser Pro Val Ala Tyr Tyr Gln Arg Leu Phe 165 170 175 His
Arg Asp Ser Asn Gln Thr Phe Cys Trp Glu His Trp Pro Asn Gln 180 185
190 Leu His Lys Lys Ala Tyr Val Val Cys Thr Phe Val Phe Gly Tyr Leu
195 200 205 Leu Pro Leu Leu Leu Ile Cys Phe Cys Tyr Ala Lys Val Leu
Asn His 210 215 220 Leu His Lys Lys Leu Lys Asn Met Ser Lys Lys Ser
Glu Ala Ser Lys 225 230 235 240 Lys Lys Thr Ala Gln Thr Val Leu Val
Val Val Val Val Phe Gly Ile 245 250 255 Ser Trp Leu Pro His His Val
Ile His Leu Trp Ala Glu Phe Gly Ala 260 265 270 Phe Pro Leu Thr Pro
Ala Ser Phe Phe Phe Arg Ile Thr Ala His Cys 275 280 285 Leu Ala Tyr
Ser Asn Ser Ser Val Asn Pro Ile Ile Tyr Ala Phe Leu 290 295 300 Ser
Glu Asn Phe Arg Lys Ala Tyr Lys Gln Val Phe Lys Cys Arg Val 305 310
315 320 Cys Asn Glu Ser Pro His Gly Asp Ala Lys Glu Lys Asn Arg Ile
Asp 325 330 335 Thr Pro Pro Ser Thr Asn Cys Thr His Val 340 345 7
349 PRT human 7 Met Glu Leu Ala Val Gly Asn Leu Ser Glu Gly Asn Ala
Ser Cys Pro 1 5 10 15 Glu Pro Pro Ala Pro Glu Pro Gly Pro Leu Phe
Gly Ile Gly Val Glu 20 25 30 Asn Phe Val Thr Leu Val Val Phe Gly
Leu Ile Phe Ala Leu Gly Val 35 40 45 Leu Gly Asn Ser Leu Val Ile
Thr Val Leu Ala Arg Ser Lys Pro Gly 50 55 60 Lys Pro Arg Ser Thr
Thr Asn Leu Phe Ile Leu Asn Leu Ser Ile Ala 65 70 75 80 Asp Leu Ala
Tyr Leu Leu Phe Cys Ile Pro Phe Gln Ala Thr Val Tyr 85 90 95 Ala
Leu Pro Thr Trp Val Leu Gly Ala Phe Ile Cys Lys Phe Ile His 100 105
110 Tyr Phe Phe Thr Val Ser Met Leu Val Ser Ile Phe Thr Leu Ala Ala
115 120 125 Met Ser Val Asp Arg Tyr Val Ala Ile Val His Ser Arg Arg
Ser Ser 130 135 140 Ser Leu Arg Val Ser Arg Asn Ala Leu Leu Gly Val
Gly Cys Ile Trp 145 150 155 160 Ala Leu Ser Ile Ala Met Ala Ser Pro
Val Ala Tyr His Gln Gly Leu 165 170 175 Phe His Pro Arg Ala Ser Asn
Gln Thr Phe Cys Trp Glu Gln Trp Pro 180 185 190 Asp Pro Arg His Lys
Lys Ala Tyr Val Val Cys Thr Phe Val Phe Gly 195 200 205 Tyr Leu Leu
Pro Leu Leu Leu Ile Cys Phe Cys Tyr Ala Lys Val Leu 210 215 220 Asn
His Leu His Lys Lys Leu Lys Asn Met Ser Lys Lys Ser Glu Ala 225 230
235 240 Ser Lys Lys Lys Thr Ala Gln Thr Val Leu Val Val Val Val Val
Phe 245 250 255 Gly Ile Ser Trp Leu Pro His His Ile Ile His Leu Trp
Ala Glu Phe 260 265 270 Gly Val Phe Pro Leu Thr Pro Ala Ser Phe Leu
Phe Arg Ile Thr Ala 275 280 285 His Cys Leu Ala Tyr Ser Asn Ser Ser
Val Asn Pro Ile Ile Tyr Ala 290 295 300 Phe Leu Ser Glu Asn Phe Arg
Lys Ala Tyr Lys Gln Val Phe Lys Cys 305 310 315 320 His Ile Arg Lys
Asp Ser His Leu Ser Asp Thr Lys Glu Asn Lys Ser 325 330 335 Arg Ile
Asp Thr Pro Pro Ser Thr Asn Cys Thr His Val 340 345 8 371 PRT mouse
8 Met Asn Gly Ser Asp Ser Gln Gly Ala Glu Asp Ser Ser Gln Glu Gly 1
5 10 15 Gly Gly Gly Trp Gln Pro Glu Ala Val Leu Val Pro Leu Phe Phe
Ala 20 25 30 Leu Ile Phe Leu Val Gly Ala Val Gly Asn Ala Leu Val
Leu Ala Val 35 40 45 Leu Leu Arg Gly Gly Gln Ala Val Ser Thr Thr
Asn Leu Phe Ile Leu 50 55 60 Asn Leu Gly Val Ala Asp Leu Cys Phe
Ile Leu Cys Cys Val Pro Phe 65 70 75 80 Gln Ala Thr Ile Tyr Thr Leu
Asp Asp Trp Val Phe Gly Ser Leu Leu 85 90
95 Cys Lys Ala Val His Phe Leu Ile Phe Leu Thr Met His Ala Ser Ser
100 105 110 Phe Thr Leu Ala Ala Val Ser Leu Asp Arg Tyr Leu Ala Ile
Arg Tyr 115 120 125 Pro Met His Ser Arg Glu Leu Arg Thr Pro Arg Asn
Ala Leu Ala Ala 130 135 140 Ile Gly Leu Ile Trp Gly Leu Ala Leu Leu
Phe Ser Gly Pro Tyr Leu 145 150 155 160 Ser Tyr Tyr Ser Gln Ser Gln
Leu Ala Asn Leu Thr Val Cys His Pro 165 170 175 Ala Trp Ser Ala Pro
Arg Arg Arg Ala Met Asp Leu Cys Thr Phe Val 180 185 190 Phe Ser Tyr
Leu Leu Pro Val Leu Val Leu Ser Leu Thr Tyr Ala Arg 195 200 205 Thr
Leu His Tyr Leu Trp Arg Thr Val Asp Pro Val Ala Ala Gly Ser 210 215
220 Gly Ser Gln Arg Ala Lys Arg Lys Val Thr Arg Met Ile Val Ile Val
225 230 235 240 Ala Val Leu Phe Cys Leu Cys Trp Met Pro His His Ala
Leu Ile Leu 245 250 255 Cys Val Trp Phe Gly Arg Phe Pro Leu Thr Arg
Ala Thr Tyr Ala Leu 260 265 270 Arg Ile Leu Ser His Leu Val Ser Tyr
Ala Asn Ser Cys Val Asn Pro 275 280 285 Ile Val Tyr Ala Leu Val Ser
Lys His Phe Arg Lys Gly Phe Arg Lys 290 295 300 Ile Cys Ala Gly Leu
Leu Arg Arg Ala Pro Arg Arg Ala Ser Gly Arg 305 310 315 320 Val Cys
Ile Leu Ala Pro Gly Asn His Ser Gly Gly Met Leu Glu Pro 325 330 335
Glu Ser Thr Asp Leu Thr Gln Val Ser Glu Ala Ala Gly Pro Leu Val 340
345 350 Pro Ala Pro Ala Leu Pro Asn Cys Thr Thr Leu Ser Arg Thr Leu
Asp 355 360 365 Pro Ala Cys 370 9 372 PRT rat 9 Met Asn Gly Ser Gly
Ser Gln Gly Ala Glu Asn Thr Ser Gln Glu Gly 1 5 10 15 Gly Ser Gly
Gly Trp Gln Pro Glu Ala Val Leu Val Pro Leu Phe Phe 20 25 30 Ala
Leu Ile Phe Leu Val Gly Thr Val Gly Asn Ala Leu Val Leu Ala 35 40
45 Val Leu Leu Arg Gly Gly Gln Ala Val Ser Thr Thr Asn Leu Phe Ile
50 55 60 Leu Asn Leu Gly Val Ala Asp Leu Cys Phe Ile Leu Cys Cys
Val Pro 65 70 75 80 Phe Gln Ala Thr Ile Tyr Thr Leu Asp Asp Trp Val
Phe Gly Ser Leu 85 90 95 Leu Cys Lys Ala Val His Phe Leu Ile Phe
Leu Thr Met His Ala Ser 100 105 110 Ser Phe Thr Leu Ala Ala Val Ser
Leu Asp Arg Tyr Leu Ala Ile Arg 115 120 125 Tyr Pro Leu His Ser Arg
Glu Leu Arg Thr Pro Arg Asn Ala Leu Ala 130 135 140 Ala Ile Gly Leu
Ile Trp Gly Leu Ala Leu Leu Phe Ser Gly Pro Tyr 145 150 155 160 Leu
Ser Tyr Tyr Arg Gln Ser Gln Leu Ala Asn Leu Thr Val Cys His 165 170
175 Pro Ala Trp Ser Ala Pro Arg Arg Arg Ala Met Asp Leu Cys Thr Phe
180 185 190 Val Phe Ser Tyr Leu Leu Pro Val Leu Val Leu Ser Leu Thr
Tyr Ala 195 200 205 Arg Thr Leu Arg Tyr Leu Trp Arg Thr Val Asp Pro
Val Thr Ala Gly 210 215 220 Ser Gly Ser Gln Arg Ala Lys Arg Lys Val
Thr Arg Met Ile Ile Ile 225 230 235 240 Val Ala Val Leu Phe Cys Leu
Cys Trp Met Pro His His Ala Leu Ile 245 250 255 Leu Cys Val Trp Phe
Gly Arg Phe Pro Leu Thr Arg Ala Thr Tyr Ala 260 265 270 Leu Arg Ile
Leu Ser His Leu Val Ser Tyr Ala Asn Ser Cys Val Asn 275 280 285 Pro
Ile Val Tyr Ala Leu Val Ser Lys His Phe Arg Lys Gly Phe Arg 290 295
300 Lys Ile Cys Ala Gly Leu Leu Arg Pro Ala Pro Arg Arg Ala Ser Gly
305 310 315 320 Arg Val Ser Ile Leu Ala Pro Gly Asn His Ser Gly Ser
Met Leu Glu 325 330 335 Gln Glu Ser Thr Asp Leu Thr Gln Val Ser Glu
Ala Ala Gly Pro Leu 340 345 350 Val Pro Pro Pro Ala Leu Pro Asn Cys
Thr Ala Ser Ser Arg Thr Leu 355 360 365 Asp Pro Ala Cys 370 10 387
PRT human 10 Met Asn Val Ser Gly Cys Pro Gly Ala Gly Asn Ala Ser
Gln Ala Gly 1 5 10 15 Gly Gly Gly Gly Trp His Pro Glu Ala Val Ile
Val Pro Leu Leu Phe 20 25 30 Ala Leu Ile Phe Leu Val Gly Thr Val
Gly Asn Thr Leu Val Leu Ala 35 40 45 Val Leu Leu Arg Gly Gly Gln
Ala Val Ser Thr Thr Asn Leu Phe Ile 50 55 60 Leu Asn Leu Gly Val
Ala Asp Leu Cys Phe Ile Leu Cys Cys Val Pro 65 70 75 80 Phe Gln Ala
Thr Ile Tyr Thr Leu Asp Gly Trp Val Phe Gly Ser Leu 85 90 95 Leu
Cys Lys Ala Val His Phe Leu Ile Phe Leu Thr Met His Ala Ser 100 105
110 Ser Phe Thr Leu Ala Ala Val Ser Leu Asp Arg Tyr Leu Ala Ile Arg
115 120 125 Tyr Pro Leu His Ser Arg Glu Leu Arg Thr Pro Arg Asn Ala
Leu Ala 130 135 140 Ala Ile Gly Leu Ile Trp Gly Leu Ser Leu Leu Phe
Ser Gly Pro Tyr 145 150 155 160 Leu Ser Tyr Tyr Arg Gln Ser Gln Leu
Ala Asn Leu Thr Val Cys His 165 170 175 Pro Ala Trp Ser Ala Pro Arg
Arg Arg Ala Met Asp Ile Cys Thr Phe 180 185 190 Val Phe Ser Tyr Leu
Leu Pro Val Leu Val Leu Gly Leu Thr Tyr Ala 195 200 205 Arg Thr Leu
Arg Tyr Leu Trp Arg Ala Val Asp Pro Val Ala Ala Gly 210 215 220 Ser
Gly Ala Arg Arg Ala Lys Arg Lys Val Thr Arg Met Ile Leu Ile 225 230
235 240 Val Ala Ala Leu Phe Cys Leu Cys Trp Met Pro His His Ala Leu
Ile 245 250 255 Leu Cys Val Trp Phe Gly Gln Phe Pro Leu Thr Arg Ala
Thr Tyr Ala 260 265 270 Leu Arg Ile Leu Ser His Leu Val Ser Tyr Ala
Asn Ser Cys Val Asn 275 280 285 Pro Ile Val Tyr Ala Leu Val Ser Lys
His Phe Arg Lys Gly Phe Arg 290 295 300 Thr Ile Cys Ala Gly Leu Leu
Gly Arg Ala Pro Gly Arg Ala Ser Gly 305 310 315 320 Arg Val Cys Ala
Ala Ala Arg Gly Thr His Ser Gly Ser Val Leu Glu 325 330 335 Arg Glu
Ser Ser Asp Leu Leu His Met Ser Glu Ala Ala Gly Ala Leu 340 345 350
Arg Pro Cys Pro Gly Ala Ser Gln Pro Cys Ile Leu Glu Pro Cys Pro 355
360 365 Gly Pro Ser Trp Gln Gly Pro Lys Ala Gly Asp Ser Ile Leu Thr
Val 370 375 380 Asp Val Ala 385 11 390 DNA rat 11 ccctacctca
gctactacgg cacggtgcgc tacggccggc tcgagctctg cgtgcccgct 60
tgggaggagg acgcgcggcg gcgcgcgctg gacgtggcca ccttcgccgc gggctacctg
120 ctgccggtgg ccgtggtgag cctggcctac ggacgcacgc tatgtttcct
atgggccgcc 180 gtgggtcccg cgggcagcgc ggcagcagag gcgcgcagac
gggcgaccgg ccgggcggga 240 cgccgcatgc tggcagtggc gctctacgcg
ctttgctggg gcccgcacca cgcgctcatc 300 ctctgcttct ggtacggtcc
gttcgccttc agcccggcca cctacgcctg tcgcctggcc 360 tcacactgcc
tcgcctacgc caactcctgc 390 12 129 PRT rat 12 Pro Tyr Leu Ser Tyr Tyr
Gly Thr Val Arg Tyr Gly Arg Leu Glu Leu 1 5 10 15 Cys Val Pro Ala
Trp Glu Asp Ala Arg Arg Arg Ala Leu Asp Val Ala 20 25 30 Thr Phe
Ala Ala Gly Tyr Leu Leu Pro Val Ala Val Val Ser Leu Ala 35 40 45
Tyr Gly Arg Thr Leu Cys Phe Leu Trp Ala Ala Val Gly Pro Ala Gly 50
55 60 Ser Ala Ala Ala Glu Ala Arg Arg Arg Ala Thr Gly Arg Ala Gly
Arg 65 70 75 80 Arg Met Leu Ala Val Ala Leu Tyr Ala Leu Cys Trp Gly
Pro His His 85 90 95 Ala Leu Ile Leu Cys Phe Trp Tyr Gly Pro Phe
Ala Phe Ser Pro Ala 100 105 110 Thr Tyr Ala Cys Arg Leu Ala Ser His
Cys Leu Ala Tyr Ala Asn Ser 115 120 125 Cys 13 34 DNA Artificial
Sequence PCR primer 13 gcgaattcgg taccatggct gatgcccaga acat 34 14
23 DNA Artificial Sequence PCR primer 14 cgcctgtcga cagatacagc agc
23 15 37 DNA Artificial Sequence PCR primer 15 tgtatctgtc
gacaggtaac ctggccgtgc ggcaccc 37 16 28 DNA Artificial Sequence PCR
primer 16 gcgcggccgc ttattccggt cctcgggc 28
* * * * *