U.S. patent application number 10/668035 was filed with the patent office on 2004-07-29 for endogenous, constitutively activated g protein-coupled orphan receptors.
This patent application is currently assigned to Arena Pharmaceuticals, Inc.. Invention is credited to Behan, Dominic P., Chalmers, Derek T., Chen, Ruoping, Liaw, Chen W., Lin, I-Lin, Lowitz, Kevin P..
Application Number | 20040147429 10/668035 |
Document ID | / |
Family ID | 29587896 |
Filed Date | 2004-07-29 |
United States Patent
Application |
20040147429 |
Kind Code |
A1 |
Behan, Dominic P. ; et
al. |
July 29, 2004 |
Endogenous, constitutively activated G protein-coupled orphan
receptors
Abstract
Disclosed herein are techniques for directly identifying
candidate compounds as agonists, partial agonists and/or, most
preferably, inverse agonists, to endogenous, constitutively
activated orphan G protein-coupled receptors. Such directly
identified compounds can be utilized, most preferably, in
pharmaceutical compositions.
Inventors: |
Behan, Dominic P.; (San
Diego, CA) ; Chalmers, Derek T.; (Cardiff, CA)
; Liaw, Chen W.; (San Diego, CA) ; Lin, I-Lin;
(San Diego, CA) ; Lowitz, Kevin P.; (Sunnyvale,
CA) ; Chen, Ruoping; (San Diego, CA) |
Correspondence
Address: |
COZEN O'CONNOR, P.C.
1900 MARKET STREET
PHILADELPHIA
PA
19103-3508
US
|
Assignee: |
Arena Pharmaceuticals, Inc.
San Diego
CA
|
Family ID: |
29587896 |
Appl. No.: |
10/668035 |
Filed: |
September 22, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10668035 |
Sep 22, 2003 |
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09364425 |
Jul 30, 1999 |
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6653086 |
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09364425 |
Jul 30, 1999 |
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09060188 |
Apr 14, 1998 |
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60094879 |
Jul 31, 1998 |
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60106300 |
Oct 30, 1998 |
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60110906 |
Dec 4, 1998 |
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60121851 |
Feb 26, 1999 |
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Current U.S.
Class: |
435/7.1 ;
435/7.2; 514/20.6 |
Current CPC
Class: |
C07K 14/70571 20130101;
C07D 403/12 20130101; C07D 231/12 20130101; C07D 513/04 20130101;
G01N 2500/00 20130101; C07D 231/16 20130101; G01N 33/68 20130101;
C07D 413/12 20130101; C07D 233/42 20130101; C07D 405/12 20130101;
C07D 409/12 20130101 |
Class at
Publication: |
514/002 ;
435/007.2 |
International
Class: |
A61K 038/00; G01N
033/53; G01N 033/567 |
Claims
What is claimed is:
1. A method for directly identifying a candidate compound as a
compound selected from the group consisting of an inverse agonist,
a partial agonist and an agonist, to an endogenous, constitutively
active G protein coupled orphan receptor, comprising the steps of:
(a) contacting a candidate compound with GPCR Fusion Protein, said
GPCR Fusion Protein comprising an endogenous, constitutively active
G protein coupled orphan receptor and a G protein; and (b)
determining, by measurement of the compound efficacy at said
contacted receptor, whether said compound is an inverse agonist, a
partial agonist or an agonist of said receptor.
2. The method of claim 1 wherein the compound is directly
identified as an inverse agonist to said orphan receptor.
3. The method of claim 1 wherein the compound is directly
identified as an agonist to said orphan receptor.
4. The method of claim 1 wherein the compound is directly
identified as partial agonist to said orphan receptor.
5. A composition comprising a compound identified by the method of
claim 2.
6. A composition comprising a compound identified by the method of
claim 3.
7. A composition comprising a compound identified by the method of
claim 4.
8. The method of claim 1 wherein said orphan receptor is selected
from the group consisting of: GPR3, GPR4, GPR6, GPR12, GPR21, OGR1,
GHSR, RE2 and ALO22171.
9. The method of claim 1 wherein said orphan receptor is GPR6.
10. The method of claim 1 wherein said G protein is selected from
the group consisting of: Gs, Gi, Gq and Go.
11. The method of claim 1 wherein said G protein is Gs.alpha..
12. A method for directly identifying a candidate compound as a
compound selected from the group consisting of an inverse agonist,
a partial agonist and an agonist, to an endogenous, constitutively
active G protein coupled orphan receptor, comprising the steps of.
(a) contacting a candidate compound with GPCR Fusion Protein, said
GPCR Fusion Protein comprising an endogenous, constitutively active
G protein coupled orphan receptor and a Gs.alpha. protein; and (b)
determining, by measurement of the compound efficacy at said
contacted receptor, whether said compound is an inverse agonist, a
partial agonist or an agonist of said receptor.
13. The method of claim 12 wherein said orphan receptor is selected
from the group consisting of: GPR3, GPR4, GPR6, GPR12, GPR21, OGR1,
GHSR, RE2 and ALO22171.
14. The method of claim 12 wherein said orphan receptor is
GPR6.
15. The method of claim 14 wherein said compound is directly
identified as a compound selected from the group consisting of an
inverse agonist and an agonist.
16. The method of claim 15 wherein said compound is an inverse
agonist.
17. A composition comprising the compound of claim 16.
18. A method for modulating a G protein coupled oprhan receptor
comprising the step of contacting said receptor with a compound
identified by the method of claim 1.
19. A method for modulating a G protein coupled oprhan receptor
comprising contacting said receptor with a compound identified by
the method of claim 12.
Description
[0001] The benefit of commonly owned: (1) Provisional Patent
Application Serial No. 60/094,879, filed Jul. 31, 1998; (2)
Provisional Patent Application Serial No. 60/106,300, filed Oct.
30, 1998; (3) Provisional Patent Application Serial No. 60/110,906,
filed Dec. 4, 1998, and (4) Provisional Application Serial No.
60/121,851, filed Feb. 26, 1999 is hereby claimed. This patent
document is related to U.S. Ser. No. 09/060,188, filed Apr. 14,
1998. The entire disclosures of each of the foregoing patent
applications are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention disclosed in this patent document relates to
transmembrane receptors, more particularly to endogenous,
constitutively active G protein-coupled receptors for which the
endogenous ligand is unknown, and most particularly to the use of
such receptors for the direct identification of candidate compounds
via screening as agonists, partial agonists or inverse agonists to
such receptors.
BACKGROUND OF THE INVENTION
[0003] A. G Protein-Coupled Receptors
[0004] G protein-coupled receptors share a common structural motif.
All these receptors have seven sequences of between 22 to 24
hydrophobic amino acids that form seven alpha helices, each of
which spans the membrane. The transmembrane helices are joined by
strands of amino acids having a larger loop between the fourth and
fifth transmembrane helix on the extracellular side of the
membrane. Another larger loop, composed primarily of hydrophilic
amino acids, joins transmembrane helices five and six on the
intracellular side of the membrane. The carboxy terminus of the
receptor lies intacellularly with the amino terminus in the
extracellular space. It is thought that the loop joining helices
five and six, as well as the carboxy terminus, interact with the G
protein. Currently, Gq, Gs, Gi, and Go are G proteins that have
been identified. The general structure of G protein-coupled
receptors is shown in FIG. 1.
[0005] Under physiological conditions, G protein-coupled receptors
exist in the cell membrane in equilibrium between two different
states or conformations: an "inactive" state and an "active" state.
As shown schematically in FIG. 2, a receptor in an inactive state
is unable to link to the intracellular transduction pathway to
produce a biological response. Changing the receptor conformation
to the active state allows linage to the transduction pathway and
produces a biological response.
[0006] A receptor may be stabilized in an active state by an
endogenous ligand or an exogenous agonist ligand. Recent
discoveries such as, including but not exclusively limited to,
modifications to the amino acid sequence of the receptor provide
means other than ligands to stabilize the active state
conformation. These means effectively stabilize the receptor in an
active state by simulating the effect of a ligand binding to the
receptor. Stabilization by such ligand-independent means is termed
"constitutive receptor activation." A receptor for which the
endogenous ligand is unknown or not identified is referred to as an
"orphan receptor."
[0007] B. Traditional Compound Screening
[0008] Generally, the use of an orphan receptor for screening
purposes to identify compounds that modulate a biological response
associated with such receptor has not been possible. This is
because the traditional "dogma" regarding screening of compounds
mandates that the ligand for the receptor be known, whereby
compounds that competitively bind with the receptor, i.e., by
interfering or blocking the binding of the natural ligand with the
receptor, are selected. By definition, then, this approach has no
applicability with respect to orphan receptors. Thus, by adhering
to this dogmatic approach to the discovery of therapeutics, the
art, in essence, has taught and has been taught to forsake the use
of orphan receptors unless and until the natural ligand for the
receptor is discovered. The pursuit of an endogenous ligand for an
orphan receptor can take several years and cost millions of
dollars.
[0009] Furthermore, and given that there are an estimated 2,000 G
protein-coupled receptors in the human genome, the majority of
which being orphan receptors, the traditional dogma castigates a
creative approach to the discovery of therapeutics to these
receptors.
[0010] C. Exemplary Orphan Receptors: GPR3, GPR4, GPR6, GPR12,
GPR21, GHSR, OGR1 and AL022171
[0011] GPR3 is a 330 amino acid G protein coupled receptor for
which the endogenous ligand is unknown. (Marchese, A. et al. (1994)
Genomics 23:609; see also, Iismaa, T. P. et al (1994) Genomics
24:391; see FIG. 1 for reported nucleic acid and amino acid
sequence.) GPR3 is constitutively active in its endogenous form.
(Eggerick, D. et al. (1995) Biochem. J. 389:837). GPR12 is a 334
amino acid homolog of GPR3; the endogenous ligand for GPR12 is
unknown (Song, Z.-H., et al (1995) Genomics, 28:347; see FIG. 1 for
reported amino acid sequence). GPR6 is a 362 amino acid homolog of
GPR3; the endogenous ligand for GPR6 is unknown (Song, Z.-H. et al,
supra.; see FIG. 1 for reported amino acid sequence). GPR6
transcripts are reported to be abundant in the human putamen and to
a lesser extent in the frontal cortex, hippocampus, and
hypothalamus (Heiber, M. et al. DNA and Cell Biology (1995) 14(1):
25; see FIG. 1 for reported nucleic acid and amino acid sequences
for GPR6). GPR4 has also been identified as an orphan GPCR (Heiber,
M. et al, 14 DNA Cell Biol. 25 (1995)). OGR1, an orphan GPCR, is
reported to have a high level of homology with GPR4 (Xu, Y. and
Casey, G., 35 Genomics 397 (1996)). GPR21 is a 349 amino acid G
protein coupled receptor for which the endogenous ligand is unknown
(see GenBank Accession # U66580 for nucleic acid and deduced amino
acid sequence). GPR21 has been reported to be located at chromosome
9q33. O'Dowd B. et al., 187 Gene 75 (1997). AL022171 is a human DNA
sequence from clone 384F21 on chromosome 1q24. AL022171 has been
identified to contain an open reading frame of 1,086 bp encoding
for a 361 amino acid protein. (see GenBank Accession number
AL022171). AL022171 is 68% homologous to GPR21 (see FIG. 5B). GHSR
is also identified as an orphan GPCR (Howard, A. D. et al, 273
Science 974 (1996)).
SUMMARY OF THE INVENTION
[0012] Disclosed herein are methods for screening of candidate
compounds against endogenous, constitutively activated G
protein-coupled orphan receptors (GPCRs) for the direct
identification of candidate compounds as agonists, inverse agonists
or partial agonists to such receptors. For such screening purposes,
it is preferred that an endogenous, constitutively activated orphan
GPCR:G protein--fusion protein be utilized.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 shows a generalized structure of a G protein-coupled
receptor with the numbers assigned to the transmembrane helixes,
the intracellular loops, and the extracellular loops.
[0014] FIG. 2 schematically shows the two states, active and
inactive, for a typical G protein coupled receptor and the linkage
of the active state to the second messenger transuction
pathway.
[0015] FIG. 3 is computerized representation of a "dot-blot"
showing the distribution of the orphan receptor GPR4 across a
variety of human tissues (see Appendix A for grid-code).
[0016] FIG. 4 is a diagram showing enhanced binding of
[.sup.35S]GTP.gamma.S to membranes prepared from 293T cells
transfected with the orphan receptor GPR3 compared to those
transfected with control vector alone at 75% g/well membrane
protein. The radiolabeled concentration of [.sup.35S]GTP.gamma.S
was held constant at 1.2 nM and the GDP concentration was held
constant at 1 .mu.M. The assay was performed on 96-well format in
Wallac scintistrips.
[0017] FIG. 5A shows the amino acid alignment of orphan receptors
GPR3, GPR6, and GPR12. FIG. 5B shows the amino acid alignment of
orphan receptors GPR21 and A1022171 (Consensus #1 indicates
matching residues).
[0018] FIG. 6A is a diagram showing that the orphan receptors GPR3,
GPR6, and GPR 12 are confirmed to be constitutively active by their
enhanced ability to induce expression of .beta.-galactosidase from
a CRE driven reporter system in VIP cells. FIGS. 6B and 6C are
diagrams of orphan receptors GPR21 and AL022171, respectively, that
have also been confirmed to be constitutively active by their
enhanced ability to induce expression of the luciferase gene from a
CRE driven reporter system in both 293 and 293T cells.
[0019] FIGS. 7A, 7B and 7C show the relative distribution of the
expression of the GPR3 (A), GPR6 (B), and GPR12 (C) orphan
receptors across several normal human tissues as determined by
RT-PCR Abbreviations: Ocx=occipital cortex; Hypoth=hypothalamus;
Tex=temporal cortex; Fcx=frontal cortex.
[0020] FIGS. 8A and 8B show GPR3 receptor expression in normal (A)
and epileptic (B) human brain tissue as examined by RT-PCP
[0021] FIG. 9A is a copy of an autoradiograph evidencing the
results from in situ hybridization (normal rat) using GPR6 probe;
FIG. 9B is a reference map of the corresponding region of the rat
brain.
[0022] FIG. 10A is a copy of an autoradiograph evidencing the
results from in situ hybridization (Zucker rat--lean) using GPR6
probe; FIG. 10B is a copy of an autoradiograph evidencing the
results from in situ hybridization (Zucker rat--obese) using GPR6
probe; FIG. 10C is a reference map of the corresponding region of
the rat brain.
[0023] FIGS. 11A-F are copies of autoradiographs evidencing the
results from in situ hybridization (normal rat) using GPR12
probe.
[0024] FIG. 12 is a copy of an autoradiograph evidencing the
results from in situ hybridization (normal rat) using GPR6 probe
(12A), and orexin 1 receptor probe (12B) with overlays for
determination of co-localization of the two receptors (12C and
12D).
[0025] FIG. 13 is a copy of an autoradiograph evidencing the
results from in situ hybridization (normal rat) using GPR6 probe
(13A), and melanocortin-3 receptor probe (13B) with overlays for
determination of co-localization of the two receptors (13C and
13D).
[0026] FIG. 14 provides results from co-localization experiment,
evidencing that GPR6 and AGRP are localized within the arcuate. The
arrow directs attention to to a specific cell within the arcuate,
with the circle surrounding the cell; the "dots" are radiolabeled
GPR6, and beneath those, in a darker shade, is AGRP.
[0027] FIG. 15 provides graphic results of body weight over time
from animals (n=5) receiving antisense oligonucleotides to GPR6
(star symbol at Day 5 indicates day on which animals received
d-amphetamine sulfate injection; see FIG. 16).
[0028] FIG. 16 provides bar graph results from baseline locomotor
activity and from amphetamine-induced locomotive behavior in the
animals of FIG. 15.
[0029] FIG. 17 provides bar-graph results from the direct
identification of candidate compounds screened against GPR3 Fusion
Protein (FIG. 17A) and GPR6 Fusion Protein (FIG. 17B).
[0030] FIG. 18A-L is a sequence diagram of the preferred vector
pCMV, including restriction enzyme site locations.
DETAILED DESCRIPTION
[0031] The scientific literature that has evolved around receptors
has adopted a number of terms to refer to ligands having various
effects on receptors. For clarity and consistency, the following
definitions will be used throughout this patent document. To the
extent that these definitions conflict with other definitions for
these terms, the following definitions shall control:
[0032] AGONISTS shall mean materials (e.g., ligands, candidate
compounds) that activate the intracellular response when they bind
to the receptor, or enhance GTP binding to membranes.
[0033] AMINO ACID ABBREVIATIONS used herein are set out in Table
1:
1 TABLE 1 ALANINE ALA A ARGININE ARG R ASPARAGINE ASN N ASPARTIC
ACID ASP D CYSTEINE CYS C GLUTAMIC ACID GLU E GLUTAMINE GLN Q
GLYCINE GLY G HISTIDINE HIS H ISOLEUCINE ILE I LEUCINE LEU L LYSINE
LYS K METHIONINE MET M PHENYLALANINE PHE F PROLINE PRO P SERINE SER
S THREONINE THR T TRYPTOPHAN TRP W TYROSINE TYR Y VALINE VAL V
[0034] PARTIAL AGONISTS shall mean materials (e.g., ligands,
candidate compounds) which activate the intracellular response when
they bind to the receptor to a lesser degree/extent than do
agonists, or enhance GTP binding to membranes to a lesser
degree/extent than do agonists
[0035] ANTAGONIST shall mean materials (e.g., ligands, candidate
compounds) that competitively bind to the receptor at the same site
as the agonists but which do not activate the intracellular
response initiated by the active form of the receptor, and can
thereby inhibit the intracellular responses by agonists or partial
agonists. ANTAGONISTS do not diminish the baseline intracellular
response in the absence of an agonist or partial agonist.
[0036] CANDIDATE COMPOUND shall mean a molecule (for example, and
not limitation, a chemical compound) which is amenable to a
screening technique. Preferably, the phrase "candidate compound"
does not include compounds which were publicly known to be
compounds selected from the group consisting of inverse agonist,
agonist or antagonist to a receptor, as previously determined by an
indirect identification process ("indirectly identified compound");
more preferably, not including an indirectly identified compound
which has previously been determined to have therapeutic efficacy
in at least one mammal; and, most preferably, not including an
indirectly identified compound which has previously been determined
to have therapeutic utility in humans.
[0037] COMPOSITION means a material comprising at least one
component; a "pharmaceutical composition" is an example of a
composition.
[0038] COMPOUND EFFICACY shall mean a measurement of the ability of
a compound to inhibit or stimulate receptor functionality, as
opposed to receptor binding affinity. A most preferred means of
detecting compound efficacy is via measurement of GTP (via
[.sup.35S]GTP.gamma.S) or cAMP, as further disclosed in the Example
section of this patent document.
[0039] CONSTITUTIVELY ACTIVATED RECEPTOR (Constitutivey Active
Receptor) shall mean a receptor subject to constitutive receptor
activation. A constitutively activated receptor can be endogenous
or non-endogenous.
[0040] CONSTITUTIVE RECEPTOR ACTIVATION shall mean stabilization of
a receptor in the active state by means other than binding of the
receptor with its endogenous ligand or a chemical equivalent
thereof.
[0041] CONTACT or CONTACTING shall mean bringing at least two
moieties together, whether in an in vitro system or an in vivo
system.
[0042] DIRECTLY IDENTIFYING or DIRECTLY IDENTIFIED, in relationship
to the phrase "candidate compound", shall mean the screening of a
candidate compound against a constitutively activated receptor,
preferably a constitutively activated orphan receptor, and most
preferably against a constitutively activated G protein-coupled
cell surface orphan receptor, and assessing the compound efficacy
of such compound. This phrase is, under no circumstances, to be
interpreted or understood to be encompassed by or to encompass the
phrase "indirectly identifing" or "indirectly identified."
[0043] ENDOGENOUS shall mean a material that a mammal naturally
produces. ENDOGENOUS in reference to, for example and not
limitation, the term "receptor," shall mean that which is naturally
produced by a mammal (for example, and not limitation, a human) or
a virus. By contrast, the term NON-ENDOGENOUS in this context shall
mean that which is not naturally produced by a mammal (for example,
and not limitation, a human) or a virus. For example, and not
limitation, a receptor which is not constitutively active in its
endogenous form, but when manipulated becomes constitutively
active, is most preferably referred to herein as a "non-endogenous,
constitutively activated receptor." Both terms can be utilized to
describe both "in vivo" and "in vitro"-systems. For example, and
not limitation, in a screening approach, the endogenous or
non-endogenous receptor may be in reference to an in vitro
screening system. As a further example and not limitation, where
the genome of a mammal has been manipulated to include a
nonendogenous constitutively activated receptor, screening of a
candidate compound by means of an in vivo system is viable.
[0044] G PROTEIN COUPLED RECEPTOR FUSION PROTEIN and GPCR FUSION
PROTEIN, in the context of the invention disclosed herein, each
mean a non-endogenous protein comprising an endogenous,
constitutively activated orphan GPCR fused to at least one G
protein, most preferably, the alpha (a) subunit of such G protein
(this being the subunit that binds GTP), with the G protein
preferably being of the same type as the G protein that naturally
couples with endogenous orphan GPCR For example, and not
limitation, in an endogenous state, the G protein "Gs.alpha." is
the predominate G protein that couples with GPR6 such that a GPCR
Fusion Protein based upon GPR6 would be a non-endogenous protein
comprising GPR6 fused to Gs.alpha.. The G protein can be fused
directly to the c-terminus of the endogenous, constitutively active
orphan GPCR, or there may be spacers between the two.
[0045] INDIRECTLY IDENTIFYING or INDIRECTLY IDENTIFIED means the
traditional approach to the drug discovery process involving
identification of an endogenous ligand specific for an endogenous
receptor, screening of candidate compounds against the receptor for
determination of those which interfere and/or compete with the
ligand-receptor interaction, and assessing the efficacy of the
compound for affecting at least one second messenger pathway
associated with the activated receptor.
[0046] INHIBIT or INHIBITING, in relationship to the term
"response" shall mean that a response is decreased or prevented in
the presence of a compound as opposed to in the absence of the
compound.
[0047] INVERSE AGONISTS shall mean materials (e.g., ligand,
candidate compound) which bind to either the endogenous form of the
receptor or to the constitutively activated form of the receptor,
and which inhibit the baseline intracellular response initiated by
the active form of the receptor below the normal base level of
activity which is observed in the absence of agonists or partial
agonists, or decrease GTP binding to membranes. Preferably, the
baseline intracellular response is inhibited in the presence of the
inverse agonist by at least 30%, more preferably by at least 50%,
and most preferably by at least 75%, as compared with the baseline
response in the absence of the inverse agonist.
[0048] LIGAND shall mean an endogenous, naturally occurring
molecule specific for an endogenous, naturally occurring
receptor.
[0049] ORPHAN RECEPTOR shall mean an endogenous receptor for which
the endogenous ligand specific for that receptor has not been
identified or is not known.
[0050] PHARMACEUTICAL COMPOSITION shall mean a composition
comprising at least one active ingredient, whereby the composition
is amenable to investigation for a specified, efficacious outcome
in a mammal (for example, and not limitation, a human). Those of
ordinary skill in the art will understand and appreciate the
techniques appropriate for determining whether an active ingredient
has a desired efficacious outcome is based upon the needs of the
artisan.
[0051] NON-ORPHAN RECEPTOR shall mean an endogenous naturally
occurring molecule specific for an endogenous naturally occurring
ligand wherein the binding of a ligand to a receptor activates an
intracellular signaling pathway.
[0052] STIMULATE or STIMULATING, in relationship to the term
"response" shall mean that a response is increased in the presence
of a compound as opposed to in the absence of the compound.
[0053] The order of the following sections is set forth for
presentational efficiency and is not intended, nor should be
construed, as a limitation on the disclosure or the claims to
follow.
[0054] A. Introduction
[0055] The traditional study of receptors has always proceeded from
the a priori assumption (historically based) that the endogenous
ligand must first be identified before discovery could proceed to
find antagonists and other molecules that could affect the
receptor. Even in cases where an antagonist might have been known
first, the search immediately extended to looking for the
endogenous ligand. This mode of thinking has persisted in receptor
research even after the discovery of constitutively activated
receptors. What has not been heretofore recognized is that it is
the active state of the receptor that is most useful for
discovering agonists, partial agonists, and inverse agonists of the
receptor. For those diseases which result from an overly active
receptor, what is desired in a therapeutic drug is a compound which
acts to diminish the active state of a receptor, not necessarily a
drug which is an antagonist to the endogenous ligand. This is
because a compound (drug) which reduces the activity of the active
receptor state need not bind at the same site as the endogenous
ligand. Thus, as taught by a method of this invention, any search
for therapeutic compounds should start by screening compounds
against the ligand-independent active state. The search, then, is
for an inverse agonist to the active state receptor.
[0056] Screening candidate compounds against the endogenous,
constitutively activated orphan receptors, for example, and not
limited to, the endogenous, constitutively active GPCRs set forth
herein, GPR3, GPR4, GPR6, GPR12, GPR21, GHSR, OGR1, RE2 and
AL022171, allows for the direct identification of candidate
compounds which act at these orphan cell surface receptors, without
requiring any prior knowledge or use of the receptor's endogenous
ligand. By determining areas within the body where such receptors
are expressed and/or over-expressed, it is possible to determine
related disease/disorder states which are associated with the
expression and/or over-expression of these receptors; such an
approach is disclosed in this patent document.
[0057] B. Disease/Disorder Identification and/or Selection
[0058] As will be set forth in greater detail below, most
preferably inverse agonists to endogenous, constitutively activated
orphan receptors, e.g, such as those set forth herein (GPR3, GPR4,
GPR6, GPR12, GPR21, GHSR, OGR1, RE2 and AL022171) can be identified
by the methodologies of this invention. Such inverse agonists are
ideal candidates as lead compounds in drug discovery programs for
treating diseases related to these receptors. Indeed, an antagonist
to such a receptor (even if the ligand were known) may be
ineffective given that the receptor is activated even in the
absence of ligand-receptor binding. Because of the ability to
directly identify inverse agonists to these receptors, thereby
allowing for the development of pharmaceutical compositions, a
search, for diseases and disorders associated with these receptors
is possible. For example, scanning both diseased and normal tissue
samples for the presence of these orphan receptors now becomes more
than an academic exercise or one which might be pursued along the
path of identifying an endogenous ligand. Tissue scans can be
conducted across a broad range of healthy and diseased tissues.
Such tissue scans provide a preferred first step in associating a
specific receptor with a disease and/or a disorder.
[0059] Preferably, the DNA sequence of the endogenous,
constitutively activated GPCR is used to make a probe for RT-PCR
identification of the expression of the receptor in tissue samples.
The presence of a receptor in a diseased tissue, or the presence of
the receptor at elevated concentrations in diseased tissue compared
to normal tissue, can be utilized to identify a correlation with
that disease. Receptors can equally well be localized to regions of
organs by this technique. Based on the known functions of the
specific tissues to which the receptor is localized, the putative
functional role of the receptor can be deduced.
[0060] C. Homology Identification
[0061] The identification and association of an orphan receptor
with diseases and/or disorders can be beneficially enhanced via
identification of additional receptors having homology with the
original orphan receptor. This approach was utilized in the
identification of both GPR6 and GPR12, based upon their sequence
homology with GPR3, and in the identification of AL022171, having
sequence homology to GPR21. GPR3 was previously identified as a
constitutively activated orphan receptor (see Eggerick, supra).
What was not known, prior to this invention, was that GPR6, GPR12,
GPR21 and AL022171 are also constitutively active in their
endogenous states. Using known computerized databases (e.g.,
dbEST), GPR6, GPR12, GPR21 and AL022171 were identified.
[0062] This highlights certain unique benefits of the invention
disclosed herein: because the dogma in drug screening relies upon
knowledge and identification of a receptor's endogenous ligand, the
art had no motivation to explore whether or not GPR3 homologs were
constitutively active in their endogenous forms (other than for, at
best, academic curiosity). However, with the power of the present
invention to directly identify inverse agonists to such receptors,
coupled with the ability to locate the distribution of such
receptors in tissue samples, the present invention dramatically
transcends such idle curiosity and provides a means for alleviating
diseases and disorders which impact the human condition.
[0063] D. Screening of Candidate Compounds
[0064] 1. Generic GPCR Screening Assay Techniques
[0065] When a G protein receptor becomes constitutively active, it
binds to a G protein (eg., Gq, Gs, Gi, Go) and stimulates the
binding of GTP to the G protein. The G protein then acts as a
GTPase and slowly hydrolyzes the GTP to GDP, whereby the receptor,
under normal conditions, becomes deactivated. However,
constitutively activated receptors continue to exchange GDP to GTP.
A non-hydrolyzable analog of GTP, [.sup.35S]GTP.gamma.S, can be
used to monitor enhanced binding to membranes which express
constitutively activated receptors. It is reported that
[.sup.35S]GTP.gamma.S can be used to monitor G protein coupling to
membranes in the absence and presence of ligand. An example of this
monitoring, among other examples well-known and available to those
in the art, was reported by Traynor and Nahorski in 1995. The
preferred use of this assay system is for initial screening of
candidate compounds because the system is generically applicable to
all G protein-coupled receptors regardless of the particular G
protein that interacts with the intracellular domain of the
receptor. It is in the context of the use of a GTh assay system
that a GPCR Fusion Protein is preferably utilized.
[0066] B 2. Specific GPCR Screening Assay Techniques
[0067] Once candidate compounds are identified using the "generic"
G protein-coupled receptor assay (i.e. an assay to select compounds
that are agonists, partial agonists, or inverse agonists), further
screening to confirm that the compounds have interacted at the
receptor site is preferred. For example, a compound identified by
the "generic" assay may not bind to the receptor, but may instead
merely "uncouple" the G protein from the intracellular domain. In
the case of GPR3, GPR4, GPR6, GPR12, GPR21, GHSR, OGR1, RE2 and
AL022171, it has been determined that these receptors couple the G
protein Gs. Gs stimulates the enzyme adenylyl cyclase (Gi, on the
other hand, inhibits this enzyme). Adenylyl cyclase catalyzes the
conversion of ATP to cAMP; thus, because these receptors are
activated in their endogenous forms, increased levels of cAMP are
associated therewith (on the other hand, endogenously activated
receptors which couple the Gi protein are associated with decreased
levels of cAMP). See, generally, "Indirect Mechanisms of Synaptic
Transmission," Chpt. 8, From Neuron To Brain (3.sup.rd Ed.)
Nichols, J. G. et al eds. Sinauer Associates, Inc. (1992). Thus,
assays that detect cAMP can be utilized to determine if a candidate
compound is an inverse agonist to the receptor (i.e., such a
compound which contacts the receptor would decrease the levels of
cAMP relative to the uncontacted receptor). A variety of approaches
known in the art for measuring cAMP can be utilized; a most
preferred approach relies upon the use of anti-cAMP antibodies.
Another type of assay that can be utilized is a whole cell second
messenger reporter system assay. Promoters on genes drive the
expression of the proteins that a particular gene encodes. Cyclic
AMP drives gene expression by promoting the binding of a
cAMP-responsive DNA binding protein or transcription factor (CREB)
which then binds to the promoter at specific sites called cAMP
response elements and drives the expression of the gene. Reporter
systems can be constructed which have a promoter containing
multiple cAMP response elements before the reporter gene, e.g.,
.beta.-galactosidase or luciferase. Thus, an activated Gs receptor
such as GPR3 causes the accumulation of cAMP which then activates
the gene and expression of the reporter protein. The reporter
protein such as .beta.-galactosidase or luciferase can then be
detected using standard biochemical assays (see, for example, Chen
et al. 1995). A cAMP assay is particularly preferred.
[0068] The foregoing specific assay approach can, of course, be
utilized to initially directly identify candidate compounds, rather
than by using the generic assay approach. Such a selection is
primarily a matter of choice of the artisan. With respect to GPR6,
use of a modified, commercially available cAMP assay was initially
utilized for the direct identification of inverse agonists.
[0069] C3. GPCR Fusion Protein
[0070] The use of an endogenous, constitutively activated orphan
GPCR for use in screening of candidate compounds for the direct
identification of inverse agonists, agonists and partial agonists
provides a unique challenge in that, by definition, the endogenous
receptor is active even in the absence of an endogenous ligand
bound thereto. Thus, in order to differentiate between, e.g, the
endogenous receptor in the presence of a candidate compound and the
endogenous receptor in the absence of that compound, with an aim of
such a differentiation to allow for an understanding as to whether
such compound may be an inverse agonist, agonist, partial agonist
or have no affect on such a receptor, it is preferred that an
approach be utilized that can enhance such differentiation. A
preferred approach is the use of a GPCR Fusion Protein.
[0071] Generally, once it is determined that an endogenous orphan
GPCR is constitutively active, using the assay techniques set forth
above (as well as others), it is possible to determine the
predominant G protein that couples with the endogenous GPCR.
Coupling of the G protein to the GPCR provides a signaling pathway
that can be assessed. Because it is most preferred that screening
take place by use of a mammalian expression system, such a system
will be expected to have endogenous G protein therein. Thus, by
definition, in such a system, the endogenous, constitutively active
orphan GPCR will continuously signal. In this regard, it is
preferred that this signal be enhanced such that in the presence
of, e.g., an inverse agonist to the receptor, it is more likely
that one will be able to more readily differentiate, particularly
in the context of screening, between the receptor when it is or is
not contacted with the inverse agonist.
[0072] The GPCR Fusion Protein is intended to enhance the efficacy
of G protein coupling with the endogenous GPCR The GPCR Fusion
Protein appears to be important for screening with an endogenous,
constitutively activated GPCR because such an approach increases
the signal that is most preferably utilized in such screening
techniques. Facilitating a significant "signal to noise" ratio is
important for the screening of candidate compounds as disclosed
herein.
[0073] The construction of a construct useful for expression of a
GPCR Fusion Protein is within the purview of those having ordinary
skill in the arL Commercially available expression vectors and
systems offer a variety of approaches that can fit the particular
needs of an investigator. One important criterion for such a GPCR
Fusion Protein construct is that the endogenous GPCR sequence and
the G protein sequence both be in-frame (preferably, the sequence
for the endogenous GPCR is upstream of the G protein sequence) and
that the "stop" codon of the GPCR must be deleted or replaced such
that upon expression of the GPCR, the G protein can also be
expressed. The GPCR can be linked directly to the G protein, or
there can be spacer residues between the two (preferably no more
than about 12, although this number can be readily ascertained by
one of ordinary skill in the art). We have evaluated both
approaches, and in terms of measurement of the activity of the
GPCR, the results are substantially the same; however, there is a
preference (based upon convenience) of use of a spacer in that some
restriction sites that are not used will, effectively, upon
expression, become a spacer. Most preferably, the G protein that
couples to the endogenous GPCR will have been identified prior to
the creation of the GPCR Fusion Protein construct. Because there
are only a few G proteins that have been identified, it is
preferred that a construct comprising the sequence of the G protein
(i.e., a universal G protein construct) be available for insertion
of an endogenous GPCR sequence therein; this provides for
efficiency in the context of large-scale screening of a variety of
different endogenous GPCRs having different sequences.
[0074] E. Medicinal Chemistry
[0075] Generally, but not always, direct identification of
candidate compounds is preferably conducted in conjunction with
compounds generated via combinatorial chemistry techniques, whereby
thousands of compounds are randomly prepared for such analysis.
Generally, the results of such screening will be compounds having
unique core structures; thereafter, these compounds are preferably
subjected to additional chemical modification around a preferred
core structure(s) to further enhance the medicinal properties
thereof. In this way, inverse agonists, agonists and/or partial
agonists that are directly identified can be beneficially improved
upon prior to development of pharmaceutical compositions comprising
such compounds. Generally, it is preferred that the binding
affinity of a directly identified compound selected for further
refinement into a pharmaceutical composition have a binding
affinity for the receptor of less than 100 nM, although this is
generally a preference selection based upon the particular needs of
the artisan. Such techniques are known to those in the art and will
not be addressed in detail in this patent document.
[0076] F. Pharmaceutical Compositions
[0077] Candidate compounds selected for further development can be
formulated into pharmaceutical compositions using techniques well
known to those in the art. Suitable pharmaceutically-acceptable
carriers are available to those in the art; for example, see
Remington's Pharmaceutical Sciences, 16.sup.th Edition, 1980, Mack
Publishing Co., (Oslo et al., eds.).
EXAMPLES
[0078] The following examples are presented for purposes of
elucidation, and not limitation, of the present invention. While
specific nucleic acid and amino acid sequences are disclosed
herein, those of ordinary skill in the art are credited with the
ability to make minor modifications to these sequences while
achieving the same or substantially similar results reported below.
It is intended that equivalent, endogenous, constitutively
activated human orphan receptor sequences having eighty-five
percent (85%) homology, more preferably having ninety percent (90%)
homology, and most preferably having grater than ninety-five
percent (95%) homology to GPR3, GPR4, GPR6, GPR12, GPR21, GHSR,
OGR1, RE2 and AL022171 fall within the scope of any claims appended
hereto.
Example 1
[0079] Preparation of In Situ Probes
[0080] In situ probes for GPR3, GPR6, and GPR12 were prepared. The
following PCR protocol was utilized for all three probes: the
reaction condition utilized was 1.times.rTth DNA polymerase buffer
II, 1.5 mM Mg(OAc).sub.2, 0.2 mM each of the 4 nucleotides, 0.228
.mu.g rat genomic DNA, 0.25 .mu.M of each primer (see below) and 1
unit of rTth DNA polymerase (Perkin Elmer) in 50 .mu.l reaction
volume. The cycle condition was 30 cycles of 94.degree. C. for 1
min, 55.degree. C. for 1 min and 72.degree. C. for 45 sec with a
Perkin Elmer Cetus 2400 thermal cycler.
[0081] 1. Rat GPR3 In Situ Probe
[0082] Because the full length cDNA sequence for rat GPR3 is not
data-base available, the DNA fragment for the in situ probe was
obtained by PCR using a 3' degenerate oligonucleotide based on the
published human and mouse GPR3 sequences in the middle of the
transmembrane domain 3, and a 5' degenerate oligonucleotide near
the beginning of the 5' extracellular domain. The sequences of the
oligonucleotides utilized were as follows:
2 5'-GGAGGATCCATGGCCTGGTTCTCAGC-3' (SEQ. ID. NO.: 1; 5' oligo)
5'-CACAAGCTTAGRCCRTCC MG RCA RTTCCA-3' (SEQ. ID. NO.: 2; 3'
oligo)
[0083] where R=A or G, and M=A or C.
[0084] A 537 bp PCR fragment containing nucleotide 24 through to
the middle of transmembrane 3 was digested with Bam HI and Hind III
and was subcloned into a Bam HI-Hind III site of pBluescript.
[0085] 2. Rat GPR 6 In Situ Probe
[0086] The in situ probe DNA fragment of rat GPR6 was obtained by
PCR based on the published rat GPR6 cDNA sequences. The sequences
of the oligonucleotides utilized were as follows:
3 5'-GGAGAAGCTTCTGGCGGCGATGAACGCTAG-3' (SEQ. ID. NO.: 3; 5' oligo)
5'-ACAGGATCCAGGTGGCTGCTAGCAAGAG-3' (SEQ. ID. NO.: 4; 3' oligo)
[0087] A 608 bp PCR fragment containing nucleotide -10 through to
the middle of transmembrane domain 4 was digested with Bam HI and
Hind III and was subcloned into Bam HI-Hind III site of
pBluescript.
[0088] 3. Rat GPR12 In Situ Probe
[0089] The in situ probe DNA fragment of rat GPR12 was obtained by
PCR based on the published rat GPR12cDNA sequences. The sequences
of the oligonucleotides utilized were as follows:
4 5'-CTTAAGCTTAAAATGAACGAAGACCCGAAG-3' (SEQ. ID. NO.: 5; 5' oligo)
5'-GGAGGATCCCCAGAGCATCACTAGCAT-3' (SEQ. ID. NO.: 6; 3' oligo)
[0090] A 516 bp PCR fragment containing nucleotide -5 through to
the middle of transmembrane domain 4 was digested with Bam HI and
Hind III and subcloned into a Bam HI-Hind III site of
pBluescript.
[0091] In situ probe sequences generated were as follows:
5 Rat GPR3 probe: GGAGGATCCATGGCCTGGTTCTCAGCCGGCTCAGGCAGTG-
TGAATGTGAGCAT (SEQ. ID. NO.: 7) AGACCCAGCAGAGGAACCTACAGGCC-
CAGCTACACTGCTGCCCTCTCCCAGGG CCTGGGATGTGGTGCTGTGCATCTCAGGCA-
CCCTGGTGTCCTGCGAGAATGCT CTGGTGATGGCCATCATTGTGGGCACGCCTGCCT-
TCCGCGCCCCCATGTTCCTG CTGGTGGGCAGCTTGGCCGTAGCAGACCTGCTGGCAG-
GCCTGGGCCTGGTCCT GCACTTCGCTGCTGACTTCTGTATTGGCTCACCAGAGATGA-
GCTTGGTGCTGGT TGGCGTGCTAGCAACGGCCTTTACTGCCAGCATCGGCAGCCTGC-
TGGCCATCA CCGTTGACCGCTACCTTTCCCTGTACAACGCCCTCACCTACTACTCAG- AGACAA
CAGTAACTCGAACCTACGTGATGCTGGCCTTGGTGTGGGTGGGTGCCCTGG- GC
CTGGGGCTGGTTCCCGTGCTGGCCTGGAACTGCCGGGACGGTCTAAGCTT Rat GPR6 probe:
AAGCTTCTGGCGGCGATGAACGCTAGCGCCGCCGCGCTC-
AACGAGTCCCAGGTGGTGGCAGTAGCG (SEQ. ID. NO.: 8)
GCCGAGGGAGCGGCAGCTGCGGCTACAGCAGCAGGGACACCGGACACCAGCGAATGGGGACCTCCG
GCAGCATCCGCGGCGCTGGGAGGCGGCGGAGGACCTAACGGGTCACTGGAGCTGTCTTCGCAGC-
TG CCCGCAGGACCCTCAGGACTTCTGCTTTCGGCAGTGAATCCCTGGGATGTGCTGC-
TGTGCGTGTCGGG GACTGTGATCGCAGGCGAAAATGCGCTGGTGGTGGCGCTCATCG-
CATCCACTCCCGCGCTGCGCACG CCCATGTTTGTGCTCGTGGGTAGTCTGGCCACTG-
CTGACCTGCTGGCGGGCTGTGGCCTCATCCTACA CTTCGTGTTCCAGTACGTGGTGC-
CCTCGGAGACTGTGAGCCTGCTCATGGTGGGCTTCCTGGTGGCGT
CCTTCGCCGCCTCAGTCAGCAGCCTGCTCGCTATCACAGTGGACCGTTACCTGTCCCTTTACAACGCG
CTCACCTACTACTCGCGCCGGACCCTGTTGGGCGTGCACCTCTTGCTAGCAGCCACCTGGAT- CC
Rat GPR12 probe: AAGCTTAAAATGAACGAAGACCCGAAGGTCA-
ATTTAAGCGGGCTGCCTCGGGACTGTATAGAAGCT (SEQ. ID. NO.: 9)
GGTACTCCGGAGAACATCTCAGCCGCTGTCCCCTCCCAGGGCTCTGTTGTGGAGTCAGAACCCGAGC
TCGTTGTCAACCCCTGGGACATTGTCTTGTGCAGCTCAGGAACCCTCATCTGCTGTGAAAATG-
CCGTC GTGGTCCTTATCATCTTCCACAGCCCCAGCCTGCGAGCACCCATGTTCCTGC-
TGATAGGCAGCCTGGC TCTTGCAGACCTGCTGGCTGGTCTGGGACTCATCATCAATT-
TTGTTTTTGCCTACCTGCTTCAGTCAGA AGCCACCAAGCTGGTCACAATTGGACTCA-
TTGTCGCCTCTTTCTCTGCCTCTGTCTGCAGTTTGCTGG
CTATCACTGTGGACCGCTACCTCTCGCTGTATTACGCCCTGACGTACCACTCCGAGAGGACCGTCACC
TTTACCTATGTCATGCTAGTGATGCTCTGGGGATCC
Example 2
[0092] Receptor Expression
[0093] 1. cDNA and Vectors
[0094] With respect to GPR3 and GPR6, expression vectors comprising
cDNA were generously supplied by Brian O'Dowd (University of
Toronto). The vector for GPR3 cDNA was pcDNA3; the vector for GPR6
was pRcCMV (the coding region for GPR6 was subcloned into pCMV
vector at a Hind III-XbaI site). GPR12 cDNA was prepared using the
following protocol: Human GPR12 cDNA was obtained by PCR using
human genomic DNA and a 5' primer from the 5' untranslated region
with a Hind III restriction site, and a 3' primer from the 3'
untranslated region containing a Bam HI site'. Primers had the
following sequences:
6 5'-CTTAAGCTTGTGGCATTTGGTACT-3' (SEQ. ID. NO.: 10; 5' oligo)
5'-TCTGGATCCTTGGCCAGGCAGTGGAAGT-3 (SEQ. ID. NO.: 11; 3' oligo)
[0095] PCR was performed using rTth polymerase (Perkin Elmer) with
the buffer system provided by the manufacturers, 0.25 .mu.M of each
primer, 0.2 .mu.M of each of the four nucleotides and 0.2 .mu.g of
genomic DNA as template. The cycle condition was 30 cycles of
94.degree. C. for 1 min, 57.degree. C. for min and 72.degree. C.
for 1.5 min. The 1.2 kb PCR fragment was digested with Hind III and
Bam HI, and subcloned into Hind III-Bam HI site of pCMV expression
vector. The resulting cDNA clones were fully sequenced and
consistent with published sequences.
[0096] With respect to GPR21, PCR was performed using genomic DNA
as template and rTth polymerase (Perkin Elmer) with the buffer
system provided by the manufacturer, 0.25 .mu.M of each primer, and
0.2 mM of each of the four nucleotides. The cycle condition was 30
cycles of 94.degree. C. for 1 min, 62.degree. C. for min and
72.degree. C. for 1 min and 20 sec. The 5' PCR primer was kinased
with the sequence:
7 5'-GAGAATTCACTCCTGAGCTCAAGATGAACT-3' (SEQ. ID. NO.: 12)
[0097] and the 3' primer contained a BamHI site with the
sequence:
8 5'-CGGGATCCCCGTAACTGAGCCACTTCAGAT-3' (SEQ. ID. NO.: 13).
[0098] The resulting 1.1 kb PCR fragment was digested with BamHI
and cloned into EcoRV-BamHI site of pCMV expression vector. Nucleic
acid (SEQ.ID.NO.:14) and amino acid (SEQ.ID.NO.: 15) sequences for
human GPR21 were thereafter determined.
[0099] With respect to AL022171, PCR was performed using genomic
DNA as template and rTth polymerase (Perkin Elmer) with the buffer
system provided by the manufacturer, 0.25 .mu.M of each primer, and
0.2 mM of each of the four nucleotides. The cycle condition was 30
cycles of 94.degree. C. for 1 min, 54.degree. C. for 1 min and
72.degree. C. for 1 min and 20 sec. The 5' primer contains an
HindIII site with the following sequence:
9 5'-AGGAAGCTTTAAATTTCCAAGCCATGAATG-3' (SEQ. ID. NO.: 16)
[0100] and the 3' primer contained a EcoRI site with the following
sequence:
[0101] 5'-ACCGAATTCAGATTACATTTGATTTACTATG-3' (SEQ.ID.NO.: 17). The
resulting 1.15 kb PCR fragment was digested with HindIII and EcoRI
and cloned into HindIII-EcoRI site of pCMV expression vector.
Nucleic acid (SEQ.ID.NO.: 18) and amino acid (SEQ.ID.NO.: 19)
sequences for human AL022171 were thereafter determined and
verified.
[0102] With respect to GPR4 (GenBank accession number L36148),
expression vectors comprising the cDNA was generously supplied by
Brian O'Dowd (University of Toronto). The vector for GPR4 cDNA was
pcDNA3 and this subcloned into pCMV vector at a Hind III-XbaI site
(the 5' untranslated region between HindIII and an ApaI site was
trimmed by conducting digestion/self ligation).
[0103] With respect to RE2 (GenBank accession number AF091890), PCR
was performed using human brain cDNA as template and rTth
polymerase (Perkin Elmer) with the buffer system provided by the
manufacturer, 0.25 .mu.M of each primer, and 0.2 mM of each of the
four nucleotides. The cycle condition was 30 cycles of 94.degree.
C. for 1 min, 62.degree. C. for 1 min and 72.degree. C. for 1 min
and 30 sec. The 5' PCR primer contained an EcoRI site with the
sequence
10 5'-AGCGAATTCTGCCCACCCCACGCCGAGGTGCT-3' (SEQ. ID. No. 20)
[0104] and the 3' primer contained a BamHI site with the
sequence
[0105] 5'-TGCGGATCCGCCAGCTCTTGAGCCTGCACA-3' (SEQ. ID.NO.: 21). The
1.36 kb PCR fragment that resulted after two rounds of PCR was then
digested with EcoRI and BamHI and cloned into EcoRI-BamHI site of
pCMV. Nucleic acid (SEQ. ID. NO. 22) and amino acid sequence (SEQ.
ID. NO. 23) was thereafter determined.
[0106] With respect to OGR1 (GenBank accession number U48405), PCR
was performed using human genomic DNA as template and rTth
polymerase (Perkin Elmer) with the buffer system provided by the
manufacturer, 0.25 .mu.M of each primer, and 0.2 mM of each of the
four nucleotides. The cycle condition was 30 cycles of 94.degree.
C. for 1 min, 62.degree. C. for 1 min and 72.degree. C. for 1 min
and 20 sec. The 5' PCR primer contained a HindIII site with the
sequence
11 5'-GGAAGCTTCAGGCCCAAAGATGGGGAACAT-3' (SEQ. ID. No. 24)
[0107] and the 3' primer contain a BamHI site with the sequence
[0108] 5'-GTGGATCCACCCGCGGAGGACCCAGGCTAG-3' (SEQ. ID. NO.25). The
resulting 1.14 kb PCR fragment was digested with HindIII and BamHI
and cloned into HindIII-BamHI site pCMV. Nucleic acid (SEQ. ID. NO.
26) and amino acid sequence (SEQ. ID. NO. 27) was thereafter
determined.
[0109] With respect to GHSR, PCR was performed using hippocampus
cDNA as template and TaqPlus Precision polymerase (Stratagene) with
the buffer system provided by the manufacturer, 0.25 .mu.M of each
primer, and 0.2 mM of each 4 nucleotides. The cycle condition was
30 cycles of 94.degree. C. for 1 min, 68.degree. C. for 1 min and
72.degree. C. for 1 min and 10 sec. For first round PCR, the 5' PCR
primer sequence:
12 5'-ATGTGGAACGCGACGCCCAGCG-3' (SEQ. ID. NO. 40)
[0110] and the 3' primer sequence:
13 5'-TCATGTATTAATACTAGATTCT-3'. (SEQ. ID. NO. 41)
[0111] Two microliters of the first round PCR was used as a
template for the second round PCR where the 5' primer was kinased
with sequence:
[0112] 5'-TACCATGTGGAACGCGACGCCCAGCGAAGAGCCGGGGT-3' (SEQ.ID.NO.:42)
and the 3' primer contains an EcoRI site with the sequence:
[0113] 5'-CGGAATTCATGTATTAATACTAGATTCTGTCCAGGCCCG-3'
(SEQ.ID.NO.:43). The 1.1 kb PCR fragment was digested with EcoRI
and cloned into blunt-EcoRI site of CMVp expression vector. Nucleic
acid (SEQ.ID.NO.:44) and amino acid (SEQ.ID.NO.:45) sequences for
human GHSR were thereafter determined.
[0114] 2. Transfection Procedure
[0115] On day one, 1.times.10.sup.7 293 or 293T cells per 150 mm
plate were plated out. On day two, two reaction tubes were prepared
(the proportions to follow for each tube are per plate): tube A was
prepared by nixing between 8-20 .mu.g DNA (e.g., pCMV vector; pCMV
vector with receptor cDNA; pCMV with GPCR Fusion Protein, supra) in
1-2 ml serum free DMEM (Irvine Scientific, Irvine, Calif.); tube B
was prepared by mixing 50-120 .mu.l lipofectamine (Gibco BRL) in
1-2 ml serum free DMEM. Tubes A and B were then admixed by
inversions (several times), followed by incubation at room
temperature for 30-45 min. The admixture is referred to as the
"transfection mixture". Plated cells were washed with 1.times.PBS,
followed by addition of 10-12 ml serum free DMEM. 2.4 ml of the
transfection mixture was then added to the cells, followed by
incubation for 4 hrs at 37.degree. C./5% CO.sub.2. The transfection
mixture was then removed by aspiration, followed by the addition of
25 ml of DMEMN10% Fetal Bovine Serum. Cells were then incubated at
37.degree. C./5% CO.sub.2.
[0116] For GPCR Fusion Protein, preferred amounts to the above are
as follows: 12 .mu.g DNA; 2 ml serum free DMEM; 60 .mu.l
lipofectamine; 293 cells 9 and an addition of 12 ml serum free
DMEM).
Example 3
[0117] Tissue Distribution of GPCR
[0118] For some orphan receptors, it will be apparent to those in
the art that there is an understanding of the distribution of such
receptors within, e.g., a human, or associated with a disease
state. However, for many orphan receptors, such information is not
known, or will not be known. It is therefore preferred that some
understanding of where such receptors may be distributed be
understood; this allows for the ability to gain a predictive
opportunity to associate a particular receptor with a disease state
or disorder associated with the particular tissue where the
receptor may be preferentially expressed. Using a commercially
available mRNA dot-blot format, the distribution of endogenous,
constitutively active GPCRs in various tissue types was
assessed.
[0119] Preferably, the entire coding region of the receptor is used
to generate a radiolabeled probe using a Prime-It II.TM. Random
Primer Labeling Kit (Stratagene, #300385), according to the
manufacturer's instructions. As an example, this approach was
utilized for GPR4.
[0120] Human RNA Master Blot.TM. kit (Clontech, #7770-1) was
hybridized with this probe and washed under stringent conditions,
in accordance with manufacturer instructions. The blot was exposed
to Kodak BioMax.TM. Autoradiography film overnight, at -80.degree.
C. Results are presented in FIG. 3. Based upon these results, it is
noted that GPR4 appears to be expressed throughout a variety of
fetal tissue types (row G), as well as non-fetal heart (C1), and
non-fetal lung (F1). This approach can be readily utilized for
other receptors.
Example 4
[0121] GTP Membrane Binding Scintillation Proximity Assay
[0122] When a G protein-coupled receptor is in its active state,
either as a result of ligand binding or constitutive activation,
the receptor binds to a G protein (in the case of GPR3, GPR4, GPR6,
GPR12, GPR21, GHSR, OGR1, RE2 and AL022171, Gs) and stimulates the
binding of GTP to the G protein. The trimeric G protein-receptor
complex acts as a GTPase and slowly hydrolyzes the GTP to GDP, at
which point the receptor normally is deactivated. Constitutively
activated receptors continue to exchange GDP for GTP. The
non-hydrolyzable GTP analog, [.sup.35S]GTP.gamma.S, can be utilized
to demonstrate enhanced binding of [.sup.35S]GTP.gamma.S to
membranes expressing constitutively activated receptors. The
advantage of using [.sup.35S]GTP.gamma.S binding to measure
constitutive activation is that: (a) it is generically applicable
to all G protein-coupled receptors; (b) it is proximal at the
membrane surface making it less likely to pick-up molecules which
affect the intracellular cascade.
[0123] The assay utilizes the ability of G protein coupled
receptors to stimulate [.sup.35S]GTP.gamma.S binding to membranes
expressing the relevant receptors. The assay can, therefore, be
used in the direct identification method to screen candidate
compounds to known, orphan and constitutively activated G protein
coupled receptors. The assay is generic and has application to drug
discovery at all G protein coupled receptors.
[0124] The [.sup.35S]GTP.gamma.S assay was incubated in 20 mM
HEPES, pH 7.4, binding buffer with 12 nM [.sup.35S]GTP.gamma.S and
75 .mu.g membrane protein [e.g., 293T cells expressing GPR3] and 1
.mu.M GDP for 1 hour. Wheatgerm agglutinin beads (25 .mu.l;
Amersham) were then added and the mixture was incubated for another
30 minutes at room temperature. The tubes were then centrifuged at
1500.times.g for 5 minutes at room temperature and then counted in
a scintillation counter.
[0125] Referring to FIG. 4, GPR3 receptor was determined to have
increased activity as compared to control; this heightened activity
is not the result of autocrine stimulation in that the data were
obtained from membrane preparations, as opposed to whole cell
preparations.
Example 5
[0126] Receptor Homology Determination
[0127] Following confirmation that GPR3 is a constitutively
activated receptor, a homology search of the available G
protein-coupled data banks (GeneBank), using the commercially
available program, DNA Star, identified two highly homologous
receptors, GPR6 and GPR12 (see FIG. 5A); both of these receptors
are orphan receptors. While the sequence of these receptors was
previously "known" (i.e., they were available on the databases), it
was not known that these two receptors are constitutively activated
in their endogenous forms (see Example 6, FIG. 7). Furthermore,
heretofore there would be no reason to search for such receptors
for use in a drug discovery program in that the ligands therefore
are not known or have not been identified. As such, the dogma
approach to drug discovery would at best find the homology between
GPR3, GPR6 and GPR12 of minor interest or, more likely,
irrelevant.
Example 6
[0128] Analysis of Homologous Receptors For Constitutive
Activation
[0129] Although a variety of cells are available to the art for the
expression of proteins, it is most preferred that mammalian cells
be utilized. The primary reason for this is predicated upon
practicalities, ie., utilization of, e.g., yeast cells for the
expression of a GPCR, while possible, introduces into the protocol
a non-mammalian cell which may not (indeed, in the case of yeast,
does not) include the receptor-coupling, genetic-mechanism and
secretary pathways that have evolved for mammalian systems--thus,
results obtained in non-mammalian cells, while of potential use,
are not as preferred as that obtained from mammalian cells. Of the
mammalian cells, COS-7,293 and 293T cells are particularly
preferred, although the specific mammalian cell utilized can be
predicated upon the particular needs of the artisan.
[0130] 1. Analysis of GPR3, GPR6 and GPR12
[0131] To generate a .beta.-galactosidase reporter containing
multiple Gal4 binding sites, a Bgl II/HindIII fragment was removed
from the somatostatin promoter-containing plasmid
1.4(5.times.Gal)CAT (Leonard, J. et al (1992) PNAS USA
89:6247-6251) and cloned into p B gal-Basic (Promega). The Bgl II
HindIII fragment contains a variant of the minimal somatostatin
promoter (from -71 bp to +50 bp relative to the transcription start
site) in which the core 4 bp of the cAMP Response Element (-46 to
-43) were replaced with 5 copies of the recognition sequence for
the yeast transcription factor Gal4. When this reporter is
co-trarsfected with an expression plasmid encoding a Gal4CREB
fusion protein, it is highly responsive to agents that increase the
cAMP signaling pathway.
[0132] VIP2.0Zc is a cell line that has been stably transfected
with the reporter gene .beta.-galactosidase under the control of a
cAMP responsive VIP promoter (Konig et al. Mol. Cell. Neuro. 1991,
2, 331-337). The cell line was used here to indirectly measure the
accumulation of intracellular cAMP. Approximately 2 million cells
were plated in 6 cm plate the day before trnsfection. DNA (5
.mu.g), for each receptor, was mixed with 2.5 ml serum-free DMEM
containing 200 .mu.g/ml DEAE dextran and 100 .mu.M chloroquine, and
added to a rinsed cell monolayer. After incubation for 90 min in a
CO.sub.2 incubator, the transfection medium was removed. The cells
were washed with serum-free medium and supplemented with flesh
complete medium. Twenty four hours after transfection, the cells
were replated into 96-well plate at a density of 50-100 K per well
and the .beta.-galactosidase activity was assayed 48 to 72 hours
after transfection.
[0133] The assay buffer contained 100 mM sodium phosphate, 2 mM
MgSO.sub.4, 0.1 mM MnCl.sub.2, pH 8.0. The cells were washed with
PBS, and 25 .mu.l/well of hypotonic lysis buffer consisting of
0.1.times. assay buffer was added. Ten minutes later, 100 .mu.l of
assay buffer containing 0.5% Triton X-100 and 40 mM
.beta.-mercaptoehanol was added to each well and incubation at room
temperature continued for 10 minutes. The substrate solution
containing 5 mg/ml chlorophenol red-.beta.D-galactopyranoside
(CPRG) in assay buffer was added at 25 .mu.l/well and the plate was
incubated at 37.degree. C. for 30 minutes before absorbance at 595
nm was measured with a plate reader.
[0134] GPR3, GPR6 and GPR12 were assayed using the foregoing
system, and it was determined that both GPR6 and GPR12 are
constitutively active. See FIG. 6A.
[0135] 2. Analysis of GPR21 and AL022171
[0136] 293 and 293T cells were plated-out on 96 well plates at a
density of 2.times.10.sup.4 cells per well and were transfected,
using Lipofectamine Reagent (BRL), the following day according to
manufacturer instructions. A DNA/lipid mixture was prepared for
each 6-well transfection as follows: 260 ng of plasmid DNA in 100
.mu.l of DMEM were gently mixed with 2 .mu.l of lipid in 100 .mu.l
of DMEM (the 260 ng of plasmid DNA consisted of 200 ng of a
8xCRE-Luc reporter plasmid, 50 ng of pCMV comprising endogenous
receptor or non-endogenous receptor or pCMV alone, and 10 ng of a
GPRS expression plasmid (GPRS in pcDNA3 (Invitrogen)). The
8XCRE-Luc reporter plasmid was prepared as follows: vector
SRIF-.beta.-gal was obtained by cloning the rat somatostatin
promoter (-71/+51) at BglV-HindIII site in the p.beta.gal-Basic
Vector (Clontech). Eight (8) copies of cAMP response element were
obtained by PCR from an adenovirus template AdpCF126CCRE8 (see 7
Human Gene Therapy 1883 (1996)) and cloned into the SRIF-.beta.-gal
vector at the Kpn-BglV site, resulting in the 8xCRE-O-gal reporter
vector. The 8xCRE-Luc reporter plasmid was generated by replacing
the beta-galactosidase gene in the 8xCRE-.beta.-gal reporter vector
with the luciferase gene obtained from the pGL3-basic vector
(Promega) at the HindIII-BamHI site. Following 30 min. incubation
at room temperature, the DNA/lipid mixture was diluted with 400
.mu.l of DMEM and 100 .mu.l of the diluted mixture was added to
each well. 100 .mu.l of DMEM with 10% FCS were added to each well
after a 4 hr incubation in a cell culture incubator. The following
day the transfected cells were changed with 200 .mu.l/well of DMEM
with 10% FCS. Eight (8) hours later, the wells were changed to 100
.mu.l/well of DMEM without phenol red, after one wash with PBS.
Luciferase activity were measured the next day using the
LucLite.TM. reporter gene assay kit (Packard) following
manufacturer instructions and read on a 1450 MicroBeta.TM.
scintillation and luminescence counter (Wallac). Results are
summarized in FIGS. 6B and 6C.
[0137] GPR21 and AL022171 were assayed using the foregoing system,
and based upon these results, it was determined that both GPR21 and
AL022171 are constitutively active in their endogenous forms. See
FIGS. 6B and 6C.
[0138] 3. Analysis of GPR4, RE2, OGRL and GHSR
[0139] Using the protocols defined herein, GPR4, RE2, OGR1 and GHSR
were analyzed and determined to be constitutively active in their
endogenous forms (data not shown).
Example 7
[0140] Tissue Distribution of GPR3, GPR6 and GPR12
[0141] Tissue samples were examined for expression of these orphan
receptors by comparative RT-PCR, using the following primers:
14 GPR3: 5'-CTGGTCCTGCACTTTGCTGC-3' (SEQ. ID. NO.: 28)
5'-AGCATCACATAGGTCCGTGTCAC-3' (SEQ. ID. NO.: 29)
[0142] These primers amplify a 194 bp fragment.
15 GPR6: 5'-ACCAGAAAGGGTGTGGGTACACTG-3' (SEQ. ID. NO.: 30)
5'-GGAACGAAAGGGCACTTTGG-3' (SEQ. ID. NO.: 31)
[0143] These primers amplify a 249 bp fragment
16 GPR12: 5'-GCTGCCTCGGGATTATTTAG-3' (SEQ. ID. NO.: 32)
5'-GCCTATTAGCAGGAACATGGGTG-3' (SEQ. ID. NO.: 33)
[0144] These primers amplify a 220 bp fragment.
[0145] These amplicons were designed to be non-overlapping, i.e.,
there is no sequence similarity between them, and to have similar
Tm's, such that each primer pair amplifies its respective target at
the same optimal annealing temperare. This diminishes the chance
that an amplicon from one primer pair will act as an annealing
target for the other primers in the multiplex reaction, therefore
reducing the chance of interference with other primer pairs.
[0146] Total RNA was extracted from tissue samples (human) using
TRIzol.TM. Reagent (Gibco/BRL), following manufacture instructions.
cDNA was generated using 2 mg total RNA and a First-Strand.TM. cDNA
synthesis kit (Phamracia). The cDNA samples were then diluted 1:3
in H.sub.2O and comparative PCR was performed as described (Jensen,
J. et al. (1996) J. Biol. Chem. 271:187490) in the presence of
[.sup.32P]dCTP. All reactions included the SP1-specific primers,
which amplify a 300 bp fragment, to serve as an internal control.
Using the primers outlined above, under defined PCR conditions (1
cycle: 95.degree. C., 5 min; 23 cycles: 95.degree. C., 30 sec,
58.degree. C., 30 sec, 72.degree. C., 1 min; 1 cycle: 72.degree.
C., 10 min) gave consistently reliable and quantitatively accurate
results. It was further determined that the selected primer pairs
did not interfere with each other when multiplexed. PCR products
were visualized by denaturing gel electrophoresis (7M urea, 5%
polyacrylamide (Long Ranger.TM. Solution, AT Biochemical,
0.6.times.TBE) and subsequent autoradiography.
[0147] FIGS. 7A, 7B, and 7C show the distribution of GPR3, GPR6 and
GPR12 across human tissues. This information allows for assessing
disease states that are associated with such tissue, as well as
determining specific regions within such tissue where such
expression predominates, thus allowing for correlating such
receptor expression with particular disease states. This, in turn,
then allows for direct identification of compounds that impact such
receptors, without the need to understand or know the endogenous
ligand for such receptor. Further screening reveals that GPR3 is
expressed in much higher levels in human epilepsy tissue samples
(tissue source: temporal cortex), as compared with controls, as
evidenced by RT-PCR analysis (FIG. 8).
Example 8A
[0148] Functional Analysis--GPR6 (In Situ Analysis)
[0149] The distribution of GPR6 in the hypothalamus suggested
possible involvement in feeding behavior. Accordingly, a functional
analysis of this receptor was undertaken. In situ analysis was
conducted as follows:
[0150] 1. Probe Design
[0151] GPR6 probe was produced from a 450 bp HindIII-ScaI fragment
of the GPR6 receptor cloned into the HindIII-SmaI site of
pBluescriptSK+. Riboprobes were produced using a T7 transcription
system in a standard labeling reaction consisting of: 1 .mu.g of
linearized plasmid, 2 .mu.l of 5.times. transcription buffer, 125
.mu.Ci of .sup.35S-UTP, 150 .mu.M of GTP, CTP and ATP, 12.5 mM
dithiothreitol, 20U of RNase inhibitor and 6U of appropriate
polymerase. The reaction was incubated at 37.degree. C. for 90
min., labeled probe being separated from free nucleotides over
Sephadex G-50 spin columns.
[0152] 2. Tissue Preparation
[0153] Dissected tissue was frozen in isopentane cooled to
-42.degree. C. and susequently stored at -80.degree. C. prior to
sectioning on a cryostat maintained at -20.degree. C. Slide-mounted
tissue sections were then stored at -80.degree. C.
[0154] 3. In Situ Hybridization Protocol
[0155] Tissue sections were removed from the -80.degree. C. freezer
and incubated with a 1 .mu.g/ml solution of proteinase-K to
permeabilize the tissue and inactivate endogenous RNase. After this
treatment, sections were incubated in succession in water (1 min),
0.1 M triethanolamine (pH 8.0; 1 min), and 0.25% acetic anhydride
in 0.1 M trietianolamine (10 min). The tissue was then washed in
2.times.SSC (0.3 mM NaCl, 0.03 nM Na citrate, pH 7.2; 5 min) and
dehydrated through graded concentrations of ethanol. Sections were
then hybridized with 1.5.times.10.sup.6 dpm of
[.sup.35S]UTP-labeled CRNA probes in 20 .mu.l of a hybridization
buffer containing 75% formamide, 10% dextran sulfate, 3.times.SSC,
50 mM sodium phosphate buffer (pH 7.4), 1.times. Denhart's
solution, 0.1 mg/ml yeast tRNA, and 0.1 mg/ml sheared salmon sperm
DNA. Tissue sections were covered with coverslips that were sealed
with rubber cement. The slides were incubated overnight at
50.degree. C. On the following day, the rubber cement was removed,
the coverslips were soaked-off with 2.times.SSC, and the tissue
sections were washed for 10 min in fresh 2.times.SSC solution.
Single stranded probe not hybridized with endogenous mRNAs was
removed by incubating the sections for 30 min in 200 .mu.g/ml
solution of RNase-A at 37.degree. C. The tissue was then washed in
increasingly stringent SSC solutions (2, 1 and 0.5.times.SSC; 10
min each), followed by a 1 hr wash in 0.5.times.SSC at 60.degree.
C. After this final wash, the tissue sections were dehydrated using
graded concentrations of ethanol, air dried and prepared for
detection by x-ray autoradiography on Kodak XAR-5 film.
[0156] 4. Analysis
[0157] Utilizing the above protocol on normal male rats
(Sprague-Dawley, Charles River), it was determined that GPR6 is
expressed in the following areas of the brain: hypothalamus,
hippocampus, nucleus accumbens, caudate and cerebral cortex. See
FIG. 9A for a representative tissue section (GPR6 receptor is
presented in the dark areas; FIG. 9B provides a reference map of
the rat brain.)
[0158] Given the high levels of expression of GPR6 in areas of the
brain associated with feeding, an in situ analysis was conducted
using the above protocol on both lean and obese male Zucker rats
(Charles River). As those in the art appreciate, the Zucker animals
are genetically bred to result in animals that exhibit a lean or
obese phenotype. FIG. 10A provides a representative tissue section
of GPR6 receptor expression in the lean Zucker animals; FIG. 10B
provides a representative tissue section of GPR6 receptor
expression in the obese Zucker animals; FIG. 10C is a reference map
of this section of the rat brain. These results support the
position that the endogenous, constitutively activated orphan
receptor GPR6 is relatively overexpressed in a model of
obesity.
Example 8B
[0159] Functional Analysis--GPR12 (In Situ Analysis)
[0160] In situ analysis for the GPR12 receptor was conducted as
follows:
[0161] 1. Probe Design
[0162] GPR12 probe was produced from a 515 bp (NT5-NT520)
HindIII-BamHI fragment of the rat GPR12 receptorcloned into the
HindIII-BamHI site ofpBluescriptSK+. Riboprobes were produced using
a T3/T7 transcription system in a standard labeling reaction
consisting of: 1 .mu.g of linearized plasmid, 2 .mu.l of 5.times.
transcription buffer, 125 .mu.Ci of .sup.35S-UTP, 150 .mu.M of GTP,
CTP and ATP, 12.5 mM dithiothreitol, 20U of Rnase inhibitor and 6U
of appropriate polymerase. The reaction was incubated at 37.degree.
C. for 90 min., labeled probe being separated from free nucleotides
over Sephadex G-50 spin columns.
[0163] 2. Tissue Preparation
[0164] Dissected tissue was frozen in isopentane cooled to
42.degree. C. and subsequently stored at -80.degree. C. prior to
sectioning on a cryostat maintained at -20.degree. C. Slide-mounted
tissue sections were then stored at -80.degree. C.
[0165] 3. In Situ Hybridization Protocol
[0166] Tissue sections were removed from the -80.degree. C. freezer
and incubated with a 1 .mu.g/ml solution of proteinase-K to
permeabilize the tissue and inactivate endogenous RNase. After this
treatment, sections are incubated in succession in water (1 min),
0.1 M triethanolamine (pH 8.0; 1 min), and 0.25% acetic anhydride
in 0.1 M triethanolamine (10 min). The tissue was then washed in
2.times.SSC (0.3 mM NaCl, 0.03 nM Na citrate, pH 7.2; 5 min) and
dehydrated through graded concentrations of ethanol. Sections were
then hybridized with 1.5.times.10.sup.6 dpm of
[.sup.35S]UTP-labeled cRNA probes in 20 .mu.l of a hybridization
buffer containing 75% formamide, 10% dextran sulfate, 3.times.SSC,
50 mM sodium phosphate buffer (pH 7.4), 1.times. Denhart's
solution, 0.1 mg/ml yeast tRNA, and 0.1 mg/ml sheared salmon sperm
DNA. Tissue sections were covered with coverslips that were sealed
with rubber cement. The slides were incubated overnight at
50.degree. C. On the following day, the rubber cement was removed,
the coverslips were soaked-off with 2.times.SSC, and the tissue
sections were washed for 10 min in fresh 2.times.SSC solution.
Single stranded probe not hybridized with endogenous mRNAs was
removed by incubating the sections for 30 min in 200 .mu.g/ml
solution of RNase-A at 37.degree. C. The tissue was then washed in
increasingly stringent SSC solutions (2, 1 and 0.5.times.SSC; 10
min each), followed by a 1 hr wash in 0.5.times.SSC at 60.degree.
C. After this final wash, the tissue sections were dehydrated using
graded concentrations of ethanol, air dried and prepared for
detection by x-ray autoradiography on Kodak XAR-5 film.
[0167] 4. Analysis
[0168] Utilizing the above protocol on normal male rats
(Sprague-Dawley, Charles River), it was determined that GPR12 is
expressed in the following areas of the brain: hippocampus
(particularly in regions CA.sub.3, CA.sub.4 and the dentate gyrus;
outer layers of the cerebral cortex; and the amygdala--all of these
regions are well known in the art as associated with regions
important for learning and memory); and thalamic relay nuclei,
including the lateral geniculate nucleus, the medial geniculate
nucleus and the lateral thelamic nucleus (regions related to
lateral relay functions, e.g., vision and hearing). See FIGS. 1A-F
for representative tissue sections (GPR12 receptor is presented in
the dark areas).
Example 8C
[0169] Functional Analysis--Co-Localization of GPR6 with
Feeding-Behavior Receptors (In Situ Analysis)
[0170] The human orexin receptor OX.sub.1R, previously an orphan
GPCR (ak.a., "HFGAN72"), has been localized in the lateral
hypothalamic region of the brain and has been hypothesized to be
involved in regulation of feeding behavior. Sakurai, T. et al 92(4)
Cell 573 (1998). As noted in Sakurai, "pharmacological intervention
directed at the orexin receptors may prove to be an attractive
avenue toward the discovery of novel therapeutics for diseases
involving disregulation of energy homeostasis, such as obesity and
diabetes mellitus." Id at 582. The melanocortin-3 receptor (MC-3)
has also been identified, Gantz, I. Et al, 268(11) J. Biol. Chem.
8246 (1993), and is similarly associated with energy
homeostasis.
[0171] An understanding of the neural pathways involved in the
regulation and disregulation of energy homeostasis is important for
appreciation of hierarchical nuances that are critical for rational
drug design. Merely affecting one receptor, particularly a receptor
that is "downsteam" of a more relevant receptor-pathway, may lead
to a substantial expenditure of time and resources that ultimately
results in the development of a pharmaceutical compound that may
have little, if any, substantive impact on a particular disease
state. For example, leptin, while clearly involved in some fashion
with energy homeostasis, has not, to date, evidenced an opportunity
for the development of a pharmaceutical product in the area of
obesity. And while both the OX.sub.1 and MC-3 receptors (as well as
other melanocortin receptors) are also, in some manner, involved
with energy homeostasis, development of pharmaceuticals based upon
the traditional receptor "antagonist" approach may prove to be more
frustrating than fruitful if, for example, these receptors are not
constitutively active in their endogenous forms, and, within the
energy homeostasis pathway, there is a receptor that is
constitutively active in its endogenous state. Indeed, the
endogenous, constitutively active receptor would, by definition,
continually signal whereas the endogenous, non-constitutively
active receptors would require ligand-binding for such signaling.
Thus, in the case of GPR6, which is not only constitutively active
in its endogenous form, but also appears to be significantly
up-regulated in an animal model of obesity, GPR6 would, in essence,
"trump" other energy homeostasis related receptors in that even
with complete blockage via receptor antagonists to these receptors,
GPR6 would continue signaling. Thus, a determination of whether
these receptors (and others within the energy homeostasis pathway)
are co-localized within discrete, neuronal regions, is useful in
providing a more refined receptor target for drug development.
[0172] In situ hybridization studies were performed as described
above for GPR6, OX.sub.1R and MC-3 receptors. For GPR6, the in situ
probe utilized was as set forth in Example 7A. For OX.sub.1R and
MC-3, the probes were based upon the published rat sequences and
were approximately 950 bp and 441 bp, respectively. Tissue
preparation (normal rats) and in situ hybridization were
substantially the same as set forth in Example 8A.
[0173] Results are presented in FIG. 12 (GPR6 and OX.sub.1R) and
FIG. 13 (GPR6 and MC-3), where a red filter was used for GPR6
hybridization and a green filter was used for OX.sub.1R (FIG. 12B)
and MC-3 (FIG. 13B). FIGS. 12C and 12D (a magnified version of 12A)
are generated by overlay of FIGS. 12A and 12B; co-localization is
evidenced by areas having an orange color (from the combination of
red and green). Thus, in FIG. 12C, it can be seen that GPR6 and
OX.sub.1R are co-localized in a sub-set of cells in the lateral
arcuate and in the ventromedial hypothalamic nucleus, both of these
regions being involved in the energy homeostasis pathways. A
similar overlay-procedure for FIGS. 13A (GPR6) and 13B (MC-3)
provides evidence that these receptors are co-localized primarily
in the lateral arcuate.
[0174] Information continues to develop within the art as to the
neural pathways associated with feeding behavior. An important
component of this pathway is the neuropeptide agouti-related
peptide (AGRP) sometimes referred to as agouti-related transcript
(ART). The expression of AGRP is largely restricted to the arcuate
nucleus (see Flier, J. S. and Maratos-Flier, E., and FIG. 1
therein). The cells that produce AGRP also produce neuropeptide Y
(NPY). Animal studies have evidenced that administration of AGRP
and administration of NPY leads to increases in feeding behavior
and obesity. AGRP has also been shown to be an antagonist to the
melanocortin 4 (MC4) receptor, and antagonism of the MCA receptor
is also known to increase feeding behavior an obesity. Thus, AGRP
appears to be involved in at least two pathways associated with
feeding behavior. As set forth below, it has been discovered that
the GPR6 receptor is co-localized within cells that produce AGRP,
and based upon the results set forth below in Example 8, coupled
with the fact that GPR6 is an endogenous, constitutively activated
GPCR, it is apparent that GPR6 is in some manner a potential
"regulator" of the system--when expression of the GPR6 receptor is
reduced via the use of antssense protocols (Example 9) there was a
excengly rapid loss in body weight of the animals tested,
suggesting that GPR6 may regulate the expression of AGRP.
[0175] Unlike the "overlay" approach above, the protocol set forth
in Marks, D. L. et al. 3 Mol. & Cell. Neuro. 395 (1992) was
utilized for assessment of co-localization. AGRP (the AGRP cRNA
probe was synthesized from a 382 bp fragment of AGRP cDNA cloned
into Bluescript SK vector) was analyzed in conjunction with
radiolabeled GPR6 and both were found to be co-localized in the
arcuate (see FIG. 14). Given the role that AGRP plays with respect
to homeostasis, and further given hat GPR6 is constitutively active
in its endogenous state, the results obtained from Example 9,
infra, would be consistent with these data in that the almost
immediate, significant loss of weight can be understood in the
context of GPR6 influencing AGRP.
Example 9
[0176] Functional Analysis--GPR6 (In Vivo Analysis: GPR6
Antisense)
[0177] Based upon the results developed from Example 7, and while
not wishing to be bound by any particular theory, it was
hypothesized that reduction in the expression of the GPR6 receptor
would lead to a reduction in, inter alia, feeding behavior,
metabolism, body weight, etc.; thus, by decreasing expression of
this receptor via use of an antisense oligonucleotide, it was
hypothesized that such animals would evidence changes in functional
feeding behavior and/or feeding-related metabolism. Examination of
this hypothesis was considered analogous to utilization of an
inverse agonist to the receptor in that an inverse agonist would
reduce the constitutive activity of the GPR6 receptor, akin to
reducing the expression of the receptor itself It is noted that
such an approach results in "knock-down", as opposed to
"knock-out", of the receptor, ie., in general, it is accepted that
an antisense approach reduces expression of the target protein by
approximately 30%.
[0178] Sixteen adult male Sprague-Dawley rats (Harlan, San Diego)
were used for this study. Animals were vivarium-acclimated for at
least one week prior to use. Animals were housed (groups of two) in
hanging plastic cages with food and water available ad lib. Animals
were weighed and handled for at least one day prior to surgery (to
establish baseline weight) and throughout the study (to assess the
effects of the treatment). Daily food intake for pairs of animals
in a cage was assessed by weighing the food in the feeding trough
each morning before and after refilling. Groups included antisense
(n=5), missense (n=4) and sterile water (n=5).
[0179] Surgeries were performed under sodium pentobarbital
anesthesia (60 mg/kg), supplemented with halothane as necessary.
Animals were stereotaxically implanted with a single cannula (brain
infusion kit, Alza Pharmaceuticals) aimed at the lateral ventricle
(bregma, AP -1.0, Lat -1.5, DV -3.8 from the surface of the brain).
The inlet of the cannula was connected via flexible tubing to the
outlet of an osmotic minipump (Model 2001, Alza Pharmaceuticals),
that was implanted subcutaneously between the shoulder blades
according to instructions provided by the manufacturer.
[0180] Pumps contained antisense oligonucleotide
5'-GsCTAGCGTTCATCGCCGsC-3- ' (SEQ.ID.NO.:34; antisense) (wherein
the small "s" denotes a phosphorothioate linkage) or missense
oligonucleotide 5'-CsTGGACTGTATCGCCCCsG-3' (SEQ.ID.NO.: 35;
missense), or sterile water vehicle. Oligonucleotides were
synthesized by Genset Corp and diluted to 2 .mu.g/.mu.l in sterile
water. Because the pumps utilized deliver 1 .mu.l/hour, animals
received 48% g/day of antisense or missense oligonucleotides, or 24
.mu.l/day of sterile water. Pumps were primed prior to implant by
incubation in sterile saline at 37.degree. C. for at least four
hours prior to implant.
[0181] Five days after surgery, animals were treated with
d-amphetamine sulfate; six days after surgery, baseline and
amphetamine-stimulated locomotor behavior were examined; seven days
after surgery, animals were euthanized and brains rapidly removed
and frozen for histological analysis.
[0182] Animals were taken from the vivarium to the testing room,
placed into an open field enclosure (see below), and baseline
activity assessed for 30 minutes. At the end of 30 minutes, animals
were briefly removed from the enclosure, injected with
d-amphetamine sulfate (1.0 mg/kg s.c., diluted in sterile saline;
National Institute on Drug Abuse Drug Supply Program), and
immediately returned to the enclosure for 150 minutes. Locomotor
behavior was quantified at 10 minute intervals in order to follow
the time-course of baseline and amphetamine-stimulated
activity.
[0183] Baseline and amphetamine-stimulated locomotor behavior were
assessed in a San Diego Instruments Flex Field System, consisting
of 16".times.16".times.15" clear plexiglas open field enclosures.
Photocell arrays (16 in each dimension) which surrounded the open
fields were interfaced with a personal computer for collection of
data. One array at 2" above the floor of an enclosure detected
locomotor activity, and a second at 5" detected rearing behavior.
The computer provided a variety of measures of locomotor activity,
including total photocell beam breaks, time active, time resting,
distance traveled, total number of rears, and time spent rearing
(data not shown). During testing, the testing room was dimly lit by
an overhead incandescent bulb, with white noise to mask outside
sounds.
[0184] Results are presented in FIGS. 15 and 16. In FIG. 15, it is
noted that animals receiving the antisense oligonucleotide (GPR6
"knock-down" animals) had significantly greater loss of weight as
compared with either the missense oligonucleotide-treated animals,
or the control-treated animals. With respect to locomotor activity,
the results of FIG. 16 support the position that the base-line and
amphetamine-treatment locomotor activities were substantially the
same across all three groups.
Example 10
[0185] GPCR Fusion Protein Preparation
[0186] The design of the endogenous, constitutively activated
GPCR-G protein fusion construct was accomplished as follows: both
the 5' and 3' ends of the rat G protein Gs.alpha. (long form; Itoh,
H. et al., 83 PNAS 3776 (1986)) were engineered to include a
HindIII (5'-AAGCTT-3') sequence thereon. Following confirmation of
the correct sequence (including the flanking HindIII sequences),
the entire sequence was shuttled into pcDNA3.1(-) (Invitrogen, cat.
no. V795-20) by subcloning using the HindIII restriction site of
that vector. The correct orientation for the Gs.alpha. sequence was
determined after subcloning into pcDNA3.1(-). The modified
pcDNA3.1(-) containing the rat Gs.alpha. gene at HindIII sequence
was then verified; this vector was now available as a "universal"
Gs.alpha. protein vector. The pcDNA3.1 (-) vector contains a
variety of well-known restriction sites upstream of the HindIII
site, thus beneficially providing the ability to insert, upstream
of the Gs protein, the coding sequence of an endogenous,
constitutively active GPCR. This same approach can be utilized to
create other "universal" G protein vectors, and, of course, other
commercially available or proprietary vectors known to the artisan
can be utilized--the important criteria is that the sequence for
the GPCR be upstream and in-frame with that of the G protein.
[0187] Both GPR3-Gs.alpha. Fusion Protein construct and
GPR6-Gs.alpha. Fusion Protein construct were then made as follows:
primers were designed for both the GPR3 and GPR6. For GPR3, the
primers were as follows:
17 5'-gatcTCTAGAATGATGTGGGGTGCAGGCAGCC-3' (SEQ. ID. NO. 36; sense)
5'-ctagGGTACCCGGACATCACTGGGGGAGCGGGATC-3' (SEQ. ID. NO. 37,
antisense)
[0188] The sense and anti-sense primers included the restriction
sites for XbaI and KpnI, respectively. For GPR6, the primers were
as follows:
18 5'-gatcTCTAGAATGCAGGGTGCAAATCCGGCC-3' (SEQ. ID. NO. 38, sense)
5'-ctagGGTACCCGGACCTCGCTGGGAGACCTGGAAC-3'. (SEQ. ID. NO. 39,
antisense)
[0189] The sense and anti-sense primers also contained restriction
sites for XbaI and KpnI, respectively. These restriction sites are
available upstrem of the HindIII site in the pcDNA3.1(-)
vector.
[0190] PCR was then utilized to secure the respective receptor
sequences for fusion within the Gs.alpha. universal vector
disclosed above, using the following protocol for each: 100 ng cDNA
for GPR3 and GPR6 was added to separate tubes containing 2 .mu.l of
each primer (sense and anti-sense), 3 uL of 10 mM dNTPs, 10 uL of
10.times.TaqPlus.TM. Precision buffer, 1 uL of TaqPlus.TM.
Precision polymerase (Stratagene: #600211), and 80 uL of water.
Reaction temperatures and cycle times for GPR3 were as follows: the
initial denaturing step was done it 94.degree. C. for five minutes,
and a cycle of 94.degree. C. for 30 seconds; 55.degree. C. for 30
seconds; 72.degree. C. for two minutes (repeated 30 times for
GPR3). A final extension time was done at 72.degree. C. for ten
minutes. For GPR6, the initial denaturing step was done at
96.degree. C. for seven minutes, and a cycle of 96.degree. C. for
30 seconds, 55.degree. C. for 30 seconds, and 72.degree. C. for two
minutes was repeated 30 times. A final extension time of ten
minutes at 72.degree. C. was done for GPR6. Both PCR products for
GPR3 and GPR6 were ran on a 1% agarose gel and then purified (data
not shown). Each purified product was digested with XbaI and KpnI
(New England Biolabs) and the desired inserts were isolated,
purified and ligated into the Gs universal vector at the respective
restriction site. The positive clones were isolated following
transformation and determined by restriction enzyme digest;
expression using 293 cells was accomplished following the protocol
set forth infra. Each positive clone for GPR3:Gs--Fusion Protein
and GPR6:Gs--Fusion Protein was sequenced and made available for
the direct identification of candidate compounds.
[0191] GPCR Fusion Proteins were analyzed as above and verified to
be constitutively active (data not shown).
Example 11
[0192] Protocol: Direct Identification of Inverse Agonists and
Agonists Using [.sup.35S]GTP.gamma.S
[0193] Although we have utilized endogenous, constitutively active
GPCRs for the direct identification of candidate compounds as,
e.g., inverse agonists, for reasons that are not altogether
understood, intra-assay variation can become exacerbated.
Preferably, then, a GPCR Fusion Protein, as disclosed above, is
utilized. We have determined that when such a protein is used,
intra-assay variation appears to be substantially stabilized,
whereby an effective signal-to-noise ratio is obtained. This has
the beneficial result of allowing for a more robust identification
of candidate compounds.
[0194] It is important to note that the following results have been
obtained using an orphan receptor; as that data support, it is
possible, using the techniques disclosed herein, to directly
identify candidate compounds that modulate the orphan receptor as
inverse agonists, agonists and partial agonists, directly from a
primary screen; indeed, the methods disclosed herein are sensitive
enough to allow for direct identification of both inverse agonist
and agonist modulators on the same assay plate.
[0195] 1. Membrane Preparation
[0196] Membranes comprising the endogenous, constitutively active
orphan GPCR fusion protein of interest (see Examples 2 and 10) and
for use in the direct identification of candidate compounds as
inverse agonists, agonists or partial agonists were prepared as
follows:
[0197] (a) Materials
[0198] Membrane Scrape Buffer was comprised of 20 mM HEPES and 10
mM EDTA, pH 7.4; Membrane Wash Buffer was comprised of 20 mM HEPES
and 0.1 mM EDTA, pH 7.4; Binding Buffer was comprised of 20 mM
HEPES, 100 mM NaCl, and 10 mM MgCl.sub.2, pH 7.4
[0199] (b) Procedure
[0200] All materials were kept on ice throughout the procedure.
Firstly, the media was aspirated from a confluent monolayer of
cells, followed by rinse with 10 ml cold PBS, followed by
aspiration. Thereafter, 5 ml of membrane Scrape Buffer was added to
scrape cells; this was followed by transfer of cellular extract
into 50 ml centrifuge tubes (centrifuged at 20,000 rpm for 17
minutes at 4.degree. C.). Thereafter, the supernatant was aspirated
and the pellet was resuspended in 30 ml Membrane Wash Buffer
followed by centrifuge at 20,000 rpm for 17 minutes at 4.degree. C.
The supernatant was then aspirated and the pellet resuspended in
Binding Buffer. This was then homogenized using a Brinkman
polytrontm homogenizer (15-20 second bursts until the all material
was in suspension). This is referred to herein as "Membrane
Protein".
[0201] 2. Bradford Protein Assay
[0202] Following the homogenization, protein concentration of the
membranes was determined using the Bradford Protein Assay (protein
can be diluted to about 1.5 mg/ml, aliquoted and frozen
(-80.degree. C.) for later use; when frozen, protocol for use is as
follows: on the day of the assay, frozen Membrane Protein is thawed
at room temperature, followed by vortex and then homogenized with a
polytron at about 12.times.1,000 rpm for about 5-10 seconds; it is
noted that for multiple preparations, the homogenizer should be
thoroughly cleaned between homoginezation of different
preparations).
[0203] (a) Materials
[0204] Binding Buffer (as per above); Bradford Dye Reagent;
Bradford Protein Standard were utilized, following manufacturer
instructions (Biorad, cat. no. 500-0006).
[0205] (b) Procedure
[0206] Duplicate tubes were prepared, one including the membrane,
and one as a control "blank". Each contained 800 ul Binding Buffer.
Thereafter, 10 ul of Bradford Protein Standard (1 mg/ml) was added
to each tube, and 10 ul of membrane Protein was then added to just
one tube (not the blank). Thereafter, 200 ul of Bradford Dye
Reagent was added to each tube, followed by vortex of each. After
five (5) minutes, the tubes were re-vortexed and the material
therein was transferred to cuvettes. The cuvettes were then read
using a CECIL 3041 spectrophotometer, at wavelength 595.
[0207] 3. Direct Identification Assay
[0208] (a) Materials
[0209] GDP Buffer consisted of 37.5 ml Binding Buffer and 2 mg GDP
(Sigma, cat. no. G-7127), followed by a series of dilutions in
Binding Buffer to obtain 0.2 uM GDP (final concentration of GDP in
each well was 0.1 uM GDP); each well comprising a candidate
compound, had a final volume of 200 ul consisting of 100 oul GDP
Buffer (final concentration, 0.1 uM GDP), 50 ul Membrane Protein in
Binding Buffer, and 50 ul [.sup.35S]GTP.gamma.S (0.6 mM) in Binding
Buffer (2.5 ul [.sup.35S]GTP.gamma.S per 10 ml Binding Buffer).
[0210] (b) Procedure
[0211] Candidate compounds (Tripos, Inc., St. Louis, Mo.) were
received in 96-well plates (these can be frozen at -80.degree. C.).
Membrane Protein (or membranes with expression vector excluding the
GPCR Fusion Protein, as control), were homogenized briefly until in
suspension. Protein concentration was then determined using the
Bradford Protein Assay set forth above. Membrane Protein (and
control) was then diluted to 0.25 mg/ml in Binding Buffer (final
assay concentration, 12.5 ug/well). Thereafter, 100 ul GDP Buffer
was added to each well of a Wallac Scintistrip.TM. (Wallac). A 5 ul
pin-tool was then used to transfer 5 ul of a candidate compound
into such well (i.e., 5 ul in total assay volume of 200 ul is a
1:40 ratio such that the final screening concentration of the
candidate compound is 10 uM). Again, to avoid contamination, after
each transfer step the pin tool was rinsed in three reservoirs
comprising water (1.times.), ethanol (1.times.) and water
(2.times.)--excess liquid should be shaken from the tool after each
rinse and dried with paper and kimwipes. Thereafter, 50 ul of
Membrane Protein is added to each well (a control well comprising
membranes without the GPCR Fusion Protein is also utilized), and
pre-incubated for 5-10 minutes at room temperature (the plates were
covered with foil in that the candidate compounds obtained from
Tripos are light sensitive). Thereafter, 50 ul of
[.sup.35S]GTP.gamma.S (0.6 nM) in Binding Buffer was added to each
well, followed by incubation on a shaker for 60 minutes at room
temperature (again, in this example, plates were covered with
foil). The assay was then stopped by spinning of the plates at 4000
RPM for 15 minutes at 22.degree. C. The plates were then aspirated
with an 8 channel manifold and sealed with plate covers. The plates
were then read on a Wallacc 1450 using setting "Prot. #37" (as per
manufacturer instructions).
[0212] Exemplary results are presented in FIG. 17A (GPR3:Gs Fusion
Protein) and FIG. 17B (GPR6:Gs Fusion Protein) where each
designation is a well comprising a different candidate compound,
standard deviations based upon the mean results of each plate are
in dashed lines and the vertical lines are the percent response.
Note in FIG. 17A well designation C4--this compound was directly
identified as an inverse agonist to the GPR3 receptor. Note in FIG.
17B wells designated G7 and H9--these compounds were directly
identified as an inverse agonist and a agonist, respectively, to
the GPR6 receptor. In both cases, these are orphan receptors.
[0213] It is preferred that following such direct identification,
IC.sub.50 (inverse agonist) or EC.sub.50 (agonist) values be
determined; those having ordinary skill in the art are credited
with utilizing IC.sub.50 and EC.sub.50 assay protocols of
choice.
Example 12
[0214] Protocol: Confirmation Assay
[0215] After using an independent assay approach to provide a
directly identified candidate compound as set forth above, it is
preferred that a confirmation assay then be utilized. In this case,
the preferred confirmation assay is a cyclase-based assay.
[0216] A modified Flash Plate.TM. Adenylyl Cyclase kit (New England
Nuclear; Cat. No. SMP004A) was utilized for confirmation of
candidate compounds directly identified as inverse agonists and
agonists to endogenous, constitutively activated orphan GPCRs in
accordance with the following protocol.
[0217] Transfected cells were harvested approximately three days
after transfection. Membranes were prepared by homogenization of
suspended cells in buffer containing 20 mM HEPES, pH 7.4 and 10 mM
MgCl.sub.2. Homogenization was performed on ice using a Brinkman
Polytron.TM. for approximately 10 seconds. The resulting homogenate
was centrifuged at 49,000.times.g for 15 minutes at 4.degree. C.
The resulting pellet was then resuspended in buffer containing 20
mM HEPES, pH 7.4 and 0.1 mM EDTA, homogenized for 10 seconds,
followed by centrifugation at 49,000.times.g for 15 minutes at
4.degree. C. The resulting pellet can be stored at -80.degree. C.
until utilized. On the day of direct identification screening, the
membrane pellet is slowly thawed at room temperature, resuspended
in buffer containing 20 mM HEPES, pH 7.4 and 10 mM MgCL2, to yield
a final protein concentration of 0.60 mg/ml (the resuspended
membranes are placed on ice until use).
[0218] cAMP standards and Detection Buffer (comprising 2 .mu.Ci of
tacer [.sup.125I cAMP (100 .mu.l] to 11 ml Detection Buffer) were
prepared and maintained in accordance with the manufacturer's
instructions. Assay Buffer was prepared fresh for screening and
contained 20 mM HEPES, pH 7.4, 10 mM MgCl.sub.2, 20 mM
phospocreatine (Sigma), 0.1 units/ml creatine phosphokinase
(Sigma), 50 .mu.M GTP (Sigma), and 0.2 mM ATP (Sigma); Assay Buffer
can be stored on ice until utilized.
[0219] Candidate compounds identified as per above (if frozen,
thawed at room temperature) were added to plate wells (3
.mu.l/well; 12 .mu.M final assay concentration), together with 40
.mu.l Membrane Protein (30 .mu.g/well) and 50 .mu.l of Assay
Buffer. This admixture was then incubated for 30 minutes at room
temperature, with gentle shaking.
[0220] Following the incubation, 100 .mu.l of Detection Buffer was
added to each well, followed by incubation for 2-24 hours. Plates
were then counted in a Wallac MicroBetam plate reader using "Prot.
#31" (as per manufacturer instructions).
[0221] Although a variety of expression vectors are available to
those in the art, it is most preferred that the vector utilized be
pCMV. This vector has been deposited with the American Type Culture
Collection (ATCC) on Oct. 13, 1998 (10801 University Blvd.,
Manassas, Va. 20110-2209 USA) under the provisions of the Budapest
Treaty for the International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedure. The vector was
tested by the ATCC and determined to be viable. The ATCC has
assigned the following deposit number to pCMV: ATCC #203351. A
diagram of pCMV (including restriction sites) is set forth in FIG.
18.
[0222] It is intended that each of the patents, applications, and
printed publications mentioned in this patent document be hereby
incorporated by reference in their entirety. As those skilled in
the art will appreciate, numerous changes and modifications may be
made to the preferred embodiments of the invention without
departing from the spirit of the invention. It is intended that all
such variations fall within the scope of the invention.
Appendix A
FIG. 3 Grid Code
[0223] A2--amygdala; A3--caudate nucleus; A4--cerebellum;
A5--cerebral cortex; A6--frontal cortex; A7--hippocampus;
A8--medulla oblongata
[0224] B1--occipital cortex; B2--putamen; B3--substantia nigra;
B4--temporal cortex; B5--thalamus; B6--sub-thalamic nucleus;
B7--spinal cord
[0225] C1 --heart; C2--aorta; C3--skeletal muscle; C4--colon;
C5--bladder; C6--uterus; C7-prostate; C8--stomach
[0226] D1 --testis; D2--ovary; D3--pancreas; D4--pituitary gland;
D5--adrenal gland; D6--thyroid; D7--salivary gland; D8--mammary
gland
[0227] E1--kidney; E2--liver; E3--small intestine; E4--spleen;
E5--thymus; E6-peripheral leukocyte; E-8 lymph node; E9--bone
marrow
[0228] F1--tonsil; F2--lung; F3--trachea; F4--placenta
[0229] G1 --fetal brain; G2--fetal heart; G3--fetal kidney;
G4--fetal liver; G5--fetal spleen; G6--fetal thymus; G8--fetal
lung
[0230]
Sequence CWU 1
1
60 1 26 DNA Artificial Sequence Oligonucleotide 1 ggaggatcca
tggcctggtt ctcagc 26 2 29 DNA Artificial Sequence Oligonucleotide 2
cacaagctta grccrtccmg rcarttcca 29 3 30 DNA Artificial Sequence
Oligonucleotide 3 ggagaagctt ctggcggcga tgaacgctag 30 4 28 DNA
Artificial Sequence Oligonucleotide 4 acaggatcca ggtggctgct
agcaagag 28 5 30 DNA Artificial Sequence Oligonucleotide 5
cttaagctta aaatgaacga agacccgaag 30 6 27 DNA Artificial Sequence
Oligonucleotide 6 ggaggatccc cagagcatca ctagcat 27 7 530 DNA Rattus
rattus 7 ggaggatcca tggcctggtt ctcagccggc tcaggcagtg tgaatgtgag
catagaccca 60 gcagaggaac ctacaggccc agctacactg ctgccctctc
ccagggcctg ggatgtggtg 120 ctgtgcatct caggcaccct ggtgtcctgc
gagaatgctc tggtgatggc catcattgtg 180 ggcacgcctg ccttccgcgc
ccccatgttc ctgctggtgg gcagcttggc cgtagcagac 240 ctgctggcag
gcctgggcct ggtcctgcac ttcgctgctg acttctgtat tggctcacca 300
gagatgagct tggtgctggt tggcgtgcta gcaacggcct ttactgccag catcggcagc
360 ctgctggcca tcaccgttga ccgctacctt tccctgtaca acgccctcac
ctactactca 420 gagacaacag taactcgaac ctacgtgatg ctggccttgg
tgtgggtggg tgccctgggc 480 ctggggctgg ttcccgtgct ggcctggaac
tgccgggacg gtctaagctt 530 8 601 DNA Rattus rattus 8 aagcttctgg
cggcgatgaa cgctagcgcc gccgcgctca acgagtccca ggtggtggca 60
gtagcggccg agggagcggc agctgcggct acagcagcag ggacaccgga caccagcgaa
120 tggggacctc cggcagcatc cgcggcgctg ggaggcggcg gaggacctaa
cgggtcactg 180 gagctgtctt cgcagctgcc cgcaggaccc tcaggacttc
tgctttcggc agtgaatccc 240 tgggatgtgc tgctgtgcgt gtcggggact
gtgatcgcag gcgaaaatgc gctggtggtg 300 gcgctcatcg catccactcc
cgcgctgcgc acgcccatgt ttgtgctcgt gggtagtctg 360 gccactgctg
acctgctggc gggctgtggc ctcatcctac acttcgtgtt ccagtacgtg 420
gtgccctcgg agactgtgag cctgctcatg gtgggcttcc tggtggcgtc cttcgccgcc
480 tcagtcagca gcctgctcgc tatcacagtg gaccgttacc tgtcccttta
caacgcgctc 540 acctactact cgcgccggac cctgttgggc gtgcacctct
tgctagcagc cacctggatc 600 c 601 9 510 DNA Rattus rattus 9
aagcttaaaa tgaacgaaga cccgaaggtc aatttaagcg ggctgcctcg ggactgtata
60 gaagctggta ctccggagaa catctcagcc gctgtcccct cccagggctc
tgttgtggag 120 tcagaacccg agctcgttgt caacccctgg gacattgtct
tgtgcagctc aggaaccctc 180 atctgctgtg aaaatgccgt cgtggtcctt
atcatcttcc acagccccag cctgcgagca 240 cccatgttcc tgctgatagg
cagcctggct cttgcagacc tgctggctgg tctgggactc 300 atcatcaatt
ttgtttttgc ctacctgctt cagtcagaag ccaccaagct ggtcacaatt 360
ggactcattg tcgcctcttt ctctgcctct gtctgcagtt tgctggctat cactgtggac
420 cgctacctct cgctgtatta cgccctgacg taccactccg agaggaccgt
cacctttacc 480 tatgtcatgc tagtgatgct ctggggatcc 510 10 24 DNA
Artificial Sequence Oligonucleotide 10 cttaagcttg tggcatttgg tact
24 11 28 DNA Artificial Sequence Oligonucleotide 11 tctggatcct
tggccaggca gtggaagt 28 12 30 DNA Artificial Sequence PCR Primer 12
gagaattcac tcctgagctc aagatgaact 30 13 30 DNA Artificial Sequence
PCR Primer 13 cgggatcccc gtaactgagc cacttcagat 30 14 1050 DNA Homo
sapiens 14 atgaactcca ccttggatgg taatcagagc agccaccctt tttgcctctt
ggcatttggc 60 tatttggaaa ctgtcaattt ttgccttttg gaagtattga
ttattgtctt tctaactgta 120 ttgattattt ctggcaacat cattgtgatt
tttgtatttc actgtgcacc tttgttgaac 180 catcacacta caagttattt
tatccagact atggcatatg ctgacctttt tgttggggtg 240 agctgcgtgg
tcccttcttt atcactcctc catcaccccc ttccagtaga ggagtccttg 300
acttgccaga tatttggttt tgtagtatca gttctgaaga gcgtctccat ggcttctctg
360 gcctgtatca gcattgatag atacattgcc attactaaac ctttaaccta
taatactctg 420 gttacaccct ggagactacg cctgtgtatt ttcctgattt
ggctatactc gaccctggtc 480 ttcctgcctt cctttttcca ctggggcaaa
cctggatatc atggagatgt gtttcagtgg 540 tgtgcggagt cctggcacac
cgactcctac ttcaccctgt tcatcgtgat gatgttatat 600 gccccagcag
cccttattgt ctgcttcacc tatttcaaca tcttccgcat ctgccaacag 660
cacacaaagg atatcagcga aaggcaagcc cgcttcagca gccagagtgg ggagactggg
720 gaagtgcagg cctgtcctga taagcgctat gccatggtcc tgtttcgaat
cactagtgta 780 ttttacatcc tctggttgcc atatatcatc tacttcttgt
tggaaagctc cactggccac 840 agcaaccgct tcgcatcctt cttgaccacc
tggcttgcta ttagtaacag tttctgcaac 900 tgtgtaattt atagtctctc
caacagtgta ttccaaagag gactaaagcg cctctcaggg 960 gctatgtgta
cttcttgtgc aagtcagact acagccaacg acccttacac agttagaagc 1020
aaaggccctc ttaatggatg tcatatctga 1050 15 349 PRT Homo sapiens 15
Met Asn Ser Thr Leu Asp Gly Asn Gln Ser Ser His Pro Phe Cys Leu 1 5
10 15 Leu Ala Phe Gly Tyr Leu Glu Thr Val Asn Phe Cys Leu Leu Glu
Val 20 25 30 Leu Ile Ile Val Phe Leu Thr Val Leu Ile Ile Ser Gly
Asn Ile Ile 35 40 45 Val Ile Phe Val Phe His Cys Ala Pro Leu Leu
Asn His His Thr Thr 50 55 60 Ser Tyr Phe Ile Gln Thr Met Ala Tyr
Ala Asp Leu Phe Val Gly Val 65 70 75 80 Ser Cys Val Val Pro Ser Leu
Ser Leu Leu His His Pro Leu Pro Val 85 90 95 Glu Glu Ser Leu Thr
Cys Gln Ile Phe Gly Phe Val Val Ser Val Leu 100 105 110 Lys Ser Val
Ser Met Ala Ser Leu Ala Cys Ile Ser Ile Asp Arg Tyr 115 120 125 Ile
Ala Ile Thr Lys Pro Leu Thr Tyr Asn Thr Leu Val Thr Pro Trp 130 135
140 Arg Leu Arg Leu Cys Ile Phe Leu Ile Trp Leu Tyr Ser Thr Leu Val
145 150 155 160 Phe Leu Pro Ser Phe Phe His Trp Gly Lys Pro Gly Tyr
His Gly Asp 165 170 175 Val Phe Gln Trp Cys Ala Glu Ser Trp His Thr
Asp Ser Tyr Phe Thr 180 185 190 Leu Phe Ile Val Met Met Leu Tyr Ala
Pro Ala Ala Leu Ile Val Cys 195 200 205 Phe Thr Tyr Phe Asn Ile Phe
Arg Ile Cys Gln Gln His Thr Lys Asp 210 215 220 Ile Ser Glu Arg Gln
Ala Arg Phe Ser Ser Gln Ser Gly Glu Thr Gly 225 230 235 240 Glu Val
Gln Ala Cys Pro Asp Lys Arg Tyr Ala Met Val Leu Phe Arg 245 250 255
Ile Thr Ser Val Phe Tyr Ile Leu Trp Leu Pro Tyr Ile Ile Tyr Phe 260
265 270 Leu Leu Glu Ser Ser Thr Gly His Ser Asn Arg Phe Ala Ser Phe
Leu 275 280 285 Thr Thr Trp Leu Ala Ile Ser Asn Ser Phe Cys Asn Cys
Val Ile Tyr 290 295 300 Ser Leu Ser Asn Ser Val Phe Gln Arg Gly Leu
Lys Arg Leu Ser Gly 305 310 315 320 Ala Met Cys Thr Ser Cys Ala Ser
Gln Thr Thr Ala Asn Asp Pro Tyr 325 330 335 Thr Val Arg Ser Lys Gly
Pro Leu Asn Gly Cys His Ile 340 345 16 30 DNA Artificial Sequence
PCR Primer 16 aggaagcttt aaatttccaa gccatgaatg 30 17 31 DNA
Artificial Sequence PCR Primer 17 accgaattca gattacattt gatttactat
g 31 18 1086 DNA Homo sapiens 18 atgaatgaat ccaggtggac tgaatggagg
atcctgaaca tgagcagtgg cattgtgaat 60 gtgtccgagc gtcactcctg
cccacttgga tttggccact acagtgtggt ggatgtctgc 120 atcttcgaga
cagtggttat tgtgttgctg acatttctga tcattgctgg gaatctaaca 180
gttatctttg tctttcattg tgctccactg ttacatcatt atactaccag ctatttcatt
240 cagacgatgg catatgctga tcttttcgtt ggagttagct gcttggttcc
tactctgtca 300 cttctccact actccacagg tgtccacgag tcattgactt
gccaggtttt tggatatatc 360 atctcagttc taaaaagtgt ttctatggca
tgtcttgctt gcatcagtgt ggatcgttat 420 cttgcaataa ccaagcctct
ttcctacaat caactggtca ccccttgtcg cttgagaatt 480 tgcattattt
tgatctggat ctactcctgc ctaattttct tgccttcctt ttttggctgg 540
gggaaacctg gttaccatgg tgacattttt gaatggtgtg ccacgtcttg gctcaccagt
600 gcctatttta ctggctttat tgtttgttta ctttatgctc ctgctgcctt
tgttgtctgc 660 ttcacttact tccacatttt caaaatttgc cgtcagcaca
ccaaagagat aaatgaccga 720 agagcccgat tccctagtca tgaggtagat
tcttccagag agactggaca cagccctgac 780 cgtcgctacg ccatggtttt
gtttaggata accagtgtat tttatatgct gtggctcccc 840 tatataattt
actttcttct agaaagctcc cgggtcttgg acaatccaac tctgtccttc 900
ttaacaacct ggcttgcaat aagtaatagt ttttgtaact gtgtaatata cagcctctcc
960 aacagcgttt tccggctagg cctccgaaga ctgtctgaga caatgtgcac
atcctgtatg 1020 tgtgtgaagg atcaggaagc acaagaaccc aaacctagga
aacgggctaa ttcttgctcc 1080 atttga 1086 19 361 PRT Homo sapiens 19
Met Asn Glu Ser Arg Trp Thr Glu Trp Arg Ile Leu Asn Met Ser Ser 1 5
10 15 Gly Ile Val Asn Val Ser Glu Arg His Ser Cys Pro Leu Gly Phe
Gly 20 25 30 His Tyr Ser Val Val Asp Val Cys Ile Phe Glu Thr Val
Val Ile Val 35 40 45 Leu Leu Thr Phe Leu Ile Ile Ala Gly Asn Leu
Thr Val Ile Phe Val 50 55 60 Phe His Cys Ala Pro Leu Leu His His
Tyr Thr Thr Ser Tyr Phe Ile 65 70 75 80 Gln Thr Met Ala Tyr Ala Asp
Leu Phe Val Gly Val Ser Cys Leu Val 85 90 95 Pro Thr Leu Ser Leu
Leu His Tyr Ser Thr Gly Val His Glu Ser Leu 100 105 110 Thr Cys Gln
Val Phe Gly Tyr Ile Ile Ser Val Leu Lys Ser Val Ser 115 120 125 Met
Ala Cys Leu Ala Cys Ile Ser Val Asp Arg Tyr Leu Ala Ile Thr 130 135
140 Lys Pro Leu Ser Tyr Asn Gln Leu Val Thr Pro Cys Arg Leu Arg Ile
145 150 155 160 Cys Ile Ile Leu Ile Trp Ile Tyr Ser Cys Leu Ile Phe
Leu Pro Ser 165 170 175 Phe Phe Gly Trp Gly Lys Pro Gly Tyr His Gly
Asp Ile Phe Glu Trp 180 185 190 Cys Ala Thr Ser Trp Leu Thr Ser Ala
Tyr Phe Thr Gly Phe Ile Val 195 200 205 Cys Leu Leu Tyr Ala Pro Ala
Ala Phe Val Val Cys Phe Thr Tyr Phe 210 215 220 His Ile Phe Lys Ile
Cys Arg Gln His Thr Lys Glu Ile Asn Asp Arg 225 230 235 240 Arg Ala
Arg Phe Pro Ser His Glu Val Asp Ser Ser Arg Glu Thr Gly 245 250 255
His Ser Pro Asp Arg Arg Tyr Ala Met Val Leu Phe Arg Ile Thr Ser 260
265 270 Val Phe Tyr Met Leu Trp Leu Pro Tyr Ile Ile Tyr Phe Leu Leu
Glu 275 280 285 Ser Ser Arg Val Leu Asp Asn Pro Thr Leu Ser Phe Leu
Thr Thr Trp 290 295 300 Leu Ala Ile Ser Asn Ser Phe Cys Asn Cys Val
Ile Tyr Ser Leu Ser 305 310 315 320 Asn Ser Val Phe Arg Leu Gly Leu
Arg Arg Leu Ser Glu Thr Met Cys 325 330 335 Thr Ser Cys Met Cys Val
Lys Asp Gln Glu Ala Gln Glu Pro Lys Pro 340 345 350 Arg Lys Arg Ala
Asn Ser Cys Ser Ile 355 360 20 32 DNA Artificial Sequence PCR
Primer 20 agcgaattct gcccacccca cgccgaggtg ct 32 21 30 DNA
Artificial Sequence PCR Primer 21 tgcggatccg ccagctcttg agcctgcaca
30 22 1382 DNA Homo sapiens 22 ggccttatct ttccagtcgt ccagcatgct
ctgcccaccc cacgccgagg tgcactgacc 60 atgagcctca actcctccct
cagctgcagg aaggagctga gtaatctcac tgaggaggag 120 ggtggcgaag
ggggcgtcat catcacccag ttcatcgcca tcattgtcat caccattttt 180
gtctgcctgg gaaacctggt catcgtggtc accttgtaca agaagtccta cctcctcacc
240 ctcagcaaca agttcgtctt cagcctgact ctgtccaact tcctgctgtc
cgtgttggtg 300 ctgccttttg tggtgacgag ctccatccgc agggaatgga
tctttggtgt agtgtggtgc 360 aacttctctg ccctcctcta cctgctgatc
agctctgcca gcatgctaac cctcggggtc 420 attgccatcg accgctacta
tgctgtcctg taccccatgg tgtaccccat gaagatcaca 480 gggaaccggg
ctgtgatggc acttgtctac atctggcttc actcgctcat cggctgcctg 540
ccacccctgt ttggttggtc atccgtggag tttgacgagt tcaaatggat gtgtgtggct
600 gcttggcacc gggagcctgg ctacacggcc ttctggcaga tctggtgtgc
cctcttcccc 660 tttctggtca tgctggtgtg ctatggcttc atcttccgcg
tggccagggt caaggcacgc 720 aaggtgcact gtggcacagt cgtcatcgtg
gaggaggatg ctcagaggac cgggaggaag 780 aactccagca cctccacctc
ctcttcaggc agcaggagga atgcctttca gggtgtggtc 840 tactcggcca
accagtgcaa agccctcatc accatcctgg tggtcctcgg tgccttcatg 900
gtcacctggg gcccctacat ggttgtcatc gcctctgagg ccctctgggg gaaaagctcc
960 gtctccccga gcctggagac ttgggccaca tggctgtcct ttgccagcgc
tgtctgccac 1020 cccctgatct atggactctg gaacaagaca gttcgcaaag
aactactggg catgtgcttt 1080 ggggaccggt attatcggga accatttgtg
caacgacaga ggacttccag gctcttcagc 1140 atttccaaca ggatcacaga
cctgggcctg tccccacacc tcactgcgct catggcaggt 1200 ggacagcccc
tggggcacag cagcagcacg ggggacactg gcttcagctg ctcccaggac 1260
tcaggtaacc tgcgtgcttt ataagcctct cacctgtcgc gttttccctg tgttgcgttt
1320 cccccgtgtc gcgtttcccc tgtgcaggct caagagctgg cggaggggca
tttcccacgg 1380 tg 1382 23 407 PRT Homo sapiens 23 Met Ser Leu Asn
Ser Ser Leu Ser Cys Arg Lys Glu Leu Ser Asn Leu 1 5 10 15 Thr Glu
Glu Glu Gly Gly Glu Gly Gly Val Ile Ile Thr Gln Phe Ile 20 25 30
Ala Ile Ile Val Ile Thr Ile Phe Val Cys Leu Gly Asn Leu Val Ile 35
40 45 Val Val Thr Leu Tyr Lys Lys Ser Tyr Leu Leu Thr Leu Ser Asn
Lys 50 55 60 Phe Val Phe Ser Leu Thr Leu Ser Asn Phe Leu Leu Ser
Val Leu Val 65 70 75 80 Leu Pro Phe Val Val Thr Ser Ser Ile Arg Arg
Glu Trp Ile Phe Gly 85 90 95 Val Val Trp Cys Asn Phe Ser Ala Leu
Leu Tyr Leu Leu Ile Ser Ser 100 105 110 Ala Ser Met Leu Thr Leu Gly
Val Ile Ala Ile Asp Arg Tyr Tyr Ala 115 120 125 Val Leu Tyr Pro Met
Val Tyr Pro Met Lys Ile Thr Gly Asn Arg Ala 130 135 140 Val Met Ala
Leu Val Tyr Ile Trp Leu His Ser Leu Ile Gly Cys Leu 145 150 155 160
Pro Pro Leu Phe Gly Trp Ser Ser Val Glu Phe Asp Glu Phe Lys Trp 165
170 175 Met Cys Val Ala Ala Trp His Arg Glu Pro Gly Tyr Thr Ala Phe
Trp 180 185 190 Gln Ile Trp Cys Ala Leu Phe Pro Phe Leu Val Met Leu
Val Cys Tyr 195 200 205 Gly Phe Ile Phe Arg Val Ala Arg Val Lys Ala
Arg Lys Val His Cys 210 215 220 Gly Thr Val Val Ile Val Glu Glu Asp
Ala Gln Arg Thr Gly Arg Lys 225 230 235 240 Asn Ser Ser Thr Ser Thr
Ser Ser Ser Gly Ser Arg Arg Asn Ala Phe 245 250 255 Gln Gly Val Val
Tyr Ser Ala Asn Gln Cys Lys Ala Leu Ile Thr Ile 260 265 270 Leu Val
Val Leu Gly Ala Phe Met Val Thr Trp Gly Pro Tyr Met Val 275 280 285
Val Ile Ala Ser Glu Ala Leu Trp Gly Lys Ser Ser Val Ser Pro Ser 290
295 300 Leu Glu Thr Trp Ala Thr Trp Leu Ser Phe Ala Ser Ala Val Cys
His 305 310 315 320 Pro Leu Ile Tyr Gly Leu Trp Asn Lys Thr Val Arg
Lys Glu Leu Leu 325 330 335 Gly Met Cys Phe Gly Asp Arg Tyr Tyr Arg
Glu Pro Phe Val Gln Arg 340 345 350 Gln Arg Thr Ser Arg Leu Phe Ser
Ile Ser Asn Arg Ile Thr Asp Leu 355 360 365 Gly Leu Ser Pro His Leu
Thr Ala Leu Met Ala Gly Gly Gln Pro Leu 370 375 380 Gly His Ser Ser
Ser Thr Gly Asp Thr Gly Phe Ser Cys Ser Gln Asp 385 390 395 400 Ser
Gly Asn Leu Arg Ala Leu 405 24 30 DNA Artificial Sequence PCR
Primer 24 ggaagcttca ggcccaaaga tggggaacat 30 25 30 DNA Artificial
Sequence PCR Primer 25 gtggatccac ccgcggagga cccaggctag 30 26 1697
DNA Homo sapiens 26 actcccaaag tgctgggctt acaggtgtaa gccatcatgt
ccagccgttc agatattcta 60 gttgaattgg agttggtggg ctagtacacc
ttctaaatta aatgagtaaa ggatttagaa 120 tggtgcctga cacacagtag
gtgctacatt catgttagct actattataa acctttcctg 180 cctctgactt
tcagggtctt gcccaccacc agcgatgccc agcccttggt agagcttgaa 240
ccaccttcta taaacaggat ggcggtggag agacaggccc agtccctgag cccatgagga
300 gtgtggcccc ttcaggccca aagatgggga acatcactgc agacaactcc
tcgatgagct 360 gtaccatcga ccataccatc caccagacgc tggccccggt
ggtctatgtt accgtgctgg 420 tggtgggctt cccggccaac tgcctgtccc
tctacttcgg ctacctgcag atcaaggccc 480 ggaacgagct gggcgtgtac
ctgtgcaacc tgacggtggc cgacctcttc tacatctgct 540 cgctgccctt
ctggctgcag tacgtgctgc agcacgacaa ctggtctcac ggcgacctgt 600
cctgccaggt gtgcggcatc ctcctgtacg agaacatcta catcagcgtg ggcttcctct
660 gctgcatctc cgtggaccgc tacctggctg tggcccatcc cttccgcttc
caccagttcc 720 ggaccctgaa ggcggccgtc ggcgtcagcg tggtcatctg
ggccaaggag ctgctgacca 780 gcatctactt cctgatgcac gaggaggtca
tcgaggacga gaaccagcac cgcgtgtgct 840 ttgagcacta ccccatccag
gcatggcagc gcgccatcaa ctactaccgc ttcctggtgg 900 gcttcctctt
ccccatctgc ctgctgctgg cgtcctacca gggcatcctg
cgcgccgtgc 960 gccggagcca cggcacccag aagagccgca aggaccagat
ccagcggctg gtgctcagca 1020 ccgtggtcat cttcctggcc tgcttcctgc
cctaccacgt gttgctgctg gtgcgcagcg 1080 tctgggaggc cagctgcgac
ttcgccaagg gcgttttcaa cgcctaccac ttctccctcc 1140 tgctcaccag
cttcaactgc gtcgccgacc ccgtgctcta ctgcttcgtc agcgagacca 1200
cccaccggga cctggcccgc ctccgcgggg cctgcctggc cttcctcacc tgctccagga
1260 ccggccgggc cagggaggcc tacccgctgg gtgcccccga ggcctccggg
aaaagcgggg 1320 cccagggtga ggagcccgag ctgttgacca agctccaccc
ggccttccag acccctaact 1380 cgccagggtc gggcgggttc cccacgggca
ggttggccta gcctgggtcc tccgcgggtg 1440 gctccacgtg aggcctgagc
cttcagccca cgggcctcag ggcctgccgc ctcctgcttc 1500 cctcgctgcg
gaggcaggga agcccctgta actccggaag cctgctctcg cttgctgagc 1560
ccgctgggac cgccgagggt gggaataagc cccggttggc tcgtgggaat aagccgtgtc
1620 ctctgccgcg gctgcgatgt ggccacgctg gggctgctgg tcgggggaaa
acagtgaact 1680 gcgtcccctg gcctgct 1697 27 365 PRT Homo sapiens 27
Met Gly Asn Ile Thr Ala Asp Asn Ser Ser Met Ser Cys Thr Ile Asp 1 5
10 15 His Thr Ile His Gln Thr Leu Ala Pro Val Val Tyr Val Thr Val
Leu 20 25 30 Val Val Gly Phe Pro Ala Asn Cys Leu Ser Leu Tyr Phe
Gly Tyr Leu 35 40 45 Gln Ile Lys Ala Arg Asn Glu Leu Gly Val Tyr
Leu Cys Asn Leu Thr 50 55 60 Val Ala Asp Leu Phe Tyr Ile Cys Ser
Leu Pro Phe Trp Leu Gln Tyr 65 70 75 80 Val Leu Gln His Asp Asn Trp
Ser His Gly Asp Leu Ser Cys Gln Val 85 90 95 Cys Gly Ile Leu Leu
Tyr Glu Asn Ile Tyr Ile Ser Val Gly Phe Leu 100 105 110 Cys Cys Ile
Ser Val Asp Arg Tyr Leu Ala Val Ala His Pro Phe Arg 115 120 125 Phe
His Gln Phe Arg Thr Leu Lys Ala Ala Val Gly Val Ser Val Val 130 135
140 Ile Trp Ala Lys Glu Leu Leu Thr Ser Ile Tyr Phe Leu Met His Glu
145 150 155 160 Glu Val Ile Glu Asp Glu Asn Gln His Arg Val Cys Phe
Glu His Tyr 165 170 175 Pro Ile Gln Ala Trp Gln Arg Ala Ile Asn Tyr
Tyr Arg Phe Leu Val 180 185 190 Gly Phe Leu Phe Pro Ile Cys Leu Leu
Leu Ala Ser Tyr Gln Gly Ile 195 200 205 Leu Arg Ala Val Arg Arg Ser
His Gly Thr Gln Lys Ser Arg Lys Asp 210 215 220 Gln Ile Gln Arg Leu
Val Leu Ser Thr Val Val Ile Phe Leu Ala Cys 225 230 235 240 Phe Leu
Pro Tyr His Val Leu Leu Leu Val Arg Ser Val Trp Glu Ala 245 250 255
Ser Cys Asp Phe Ala Lys Gly Val Phe Asn Ala Tyr His Phe Ser Leu 260
265 270 Leu Leu Thr Ser Phe Asn Cys Val Ala Asp Pro Val Leu Tyr Cys
Phe 275 280 285 Val Ser Glu Thr Thr His Arg Asp Leu Ala Arg Leu Arg
Gly Ala Cys 290 295 300 Leu Ala Phe Leu Thr Cys Ser Arg Thr Gly Arg
Ala Arg Glu Ala Tyr 305 310 315 320 Pro Leu Gly Ala Pro Glu Ala Ser
Gly Lys Ser Gly Ala Gln Gly Glu 325 330 335 Glu Pro Glu Leu Leu Thr
Lys Leu His Pro Ala Phe Gln Thr Pro Asn 340 345 350 Ser Pro Gly Ser
Gly Gly Phe Pro Thr Gly Arg Leu Ala 355 360 365 28 20 DNA
Artificial Sequence PCR Primer 28 ctggtcctgc actttgctgc 20 29 23
DNA Artificial Sequence PCR Primer 29 agcatcacat aggtccgtgt cac 23
30 24 DNA Artificial Sequence PCR Primer 30 accagaaagg gtgtgggtac
actg 24 31 20 DNA Artificial Sequence PCR Primer 31 ggaacgaaag
ggcactttgg 20 32 20 DNA Artificial Sequence PCR Primer 32
gctgcctcgg gattatttag 20 33 23 DNA Artificial Sequence PCR Primer
33 gcctattagc aggaacatgg gtg 23 34 18 DNA Artificial Sequence
Antisense Sequence 34 gctagcgttc atcgccgc 18 35 18 DNA Artificial
Sequence Missense Sequence 35 ctggactgta tcgccccg 18 36 32 DNA
Artificial Sequence Sense Sequence 36 gatctctaga atgatgtggg
gtgcaggcag cc 32 37 35 DNA Artificial Sequence Antisense Sequence
37 ctagggtacc cggacatcac tgggggagcg ggatc 35 38 31 DNA Artificial
Sequence Sense Sequence 38 gatctctaga atgcagggtg caaatccggc c 31 39
35 DNA Artificial Sequence Antisense Sequence 39 ctagggtacc
cggacctcgc tgggagacct ggaac 35 40 22 DNA Artificial Sequence PCR
Primer 40 atgtggaacg cgacgcccag cg 22 41 22 DNA Artificial Sequence
PCR Primer 41 tcatgtatta atactagatt ct 22 42 38 DNA Artificial
Sequence PCR Primer 42 taccatgtgg aacgcgacgc ccagcgaaga gccggggt 38
43 39 DNA Artificial Sequence PCR Primer 43 cggaattcat gtattaatac
tagattctgt ccaggcccg 39 44 1101 DNA Homo sapiens 44 atgtggaacg
cgacgcccag cgaagagccg gggttcaacc tcacactggc cgacctggac 60
tgggatgctt cccccggcaa cgactcgctg ggcgacgagc tgctgcagct cttccccgcg
120 ccgctgctgg cgggcgtcac agccacctgc gtggcactct tcgtggtggg
tatcgctggc 180 aacctgctca ccatgctggt ggtgtcgcgc ttccgcgagc
tgcgcaccac caccaacctc 240 tacctgtcca gcatggcctt ctccgatctg
ctcatcttcc tctgcatgcc cctggacctc 300 gttcgcctct ggcagtaccg
gccctggaac ttcggcgacc tcctctgcaa actcttccaa 360 ttcgtcagtg
agagctgcac ctacgccacg gtgctcacca tcacagcgct gagcgtcgag 420
cgctacttcg ccatctgctt cccactccgg gccaaggtgg tggtcaccaa ggggcgggtg
480 aagctggtca tcttcgtcat ctgggccgtg gccttctgca gcgccgggcc
catcttcgtg 540 ctagtcgggg tggagcacga gaacggcacc gacccttggg
acaccaacga gtgccgcccc 600 accgagtttg cggtgcgctc tggactgctc
acggtcatgg tgtgggtgtc cagcatcttc 660 ttcttccttc ctgtcttctg
tctcacggtc ctctacagtc tcatcggcag gaagctgtgg 720 cggaggaggc
gcggcgatgc tgtcgtgggt gcctcgctca gggaccagaa ccacaagcaa 780
accgtgaaaa tgctggctgt agtggtgttt gccttcatcc tctgctggct ccccttccac
840 gtagggcgat atttattttc caaatccttt gagcctggct ccttggagat
tgctcagatc 900 agccagtact gcaacctcgt gtcctttgtc ctcttctacc
tcagtgctgc catcaacccc 960 attctgtaca acatcatgtc caagaagtac
cgggtggcag tgttcagact tctgggattc 1020 gaacccttct cccagagaaa
gctctccact ctgaaagatg aaagttctcg ggcctggaca 1080 gaatctagta
ttaatacatg a 1101 45 366 PRT Homo sapiens 45 Met Trp Asn Ala Thr
Pro Ser Glu Glu Pro Gly Phe Asn Leu Thr Leu 1 5 10 15 Ala Asp Leu
Asp Trp Asp Ala Ser Pro Gly Asn Asp Ser Leu Gly Asp 20 25 30 Glu
Leu Leu Gln Leu Phe Pro Ala Pro Leu Leu Ala Gly Val Thr Ala 35 40
45 Thr Cys Val Ala Leu Phe Val Val Gly Ile Ala Gly Asn Leu Leu Thr
50 55 60 Met Leu Val Val Ser Arg Phe Arg Glu Leu Arg Thr Thr Thr
Asn Leu 65 70 75 80 Tyr Leu Ser Ser Met Ala Phe Ser Asp Leu Leu Ile
Phe Leu Cys Met 85 90 95 Pro Leu Asp Leu Val Arg Leu Trp Gln Tyr
Arg Pro Trp Asn Phe Gly 100 105 110 Asp Leu Leu Cys Lys Leu Phe Gln
Phe Val Ser Glu Ser Cys Thr Tyr 115 120 125 Ala Thr Val Leu Thr Ile
Thr Ala Leu Ser Val Glu Arg Tyr Phe Ala 130 135 140 Ile Cys Phe Pro
Leu Arg Ala Lys Val Val Val Thr Lys Gly Arg Val 145 150 155 160 Lys
Leu Val Ile Phe Val Ile Trp Ala Val Ala Phe Cys Ser Ala Gly 165 170
175 Pro Ile Phe Val Leu Val Gly Val Glu His Glu Asn Gly Thr Asp Pro
180 185 190 Trp Asp Thr Asn Glu Cys Arg Pro Thr Glu Phe Ala Val Arg
Ser Gly 195 200 205 Leu Leu Thr Val Met Val Trp Val Ser Ser Ile Phe
Phe Phe Leu Pro 210 215 220 Val Phe Cys Leu Thr Val Leu Tyr Ser Leu
Ile Gly Arg Lys Leu Trp 225 230 235 240 Arg Arg Arg Arg Gly Asp Ala
Val Val Gly Ala Ser Leu Arg Asp Gln 245 250 255 Asn His Lys Gln Thr
Val Lys Met Leu Ala Val Val Val Phe Ala Phe 260 265 270 Ile Leu Cys
Trp Leu Pro Phe His Val Gly Arg Tyr Leu Phe Ser Lys 275 280 285 Ser
Phe Glu Pro Gly Ser Leu Glu Ile Ala Gln Ile Ser Gln Tyr Cys 290 295
300 Asn Leu Val Ser Phe Val Leu Phe Tyr Leu Ser Ala Ala Ile Asn Pro
305 310 315 320 Ile Leu Tyr Asn Ile Met Ser Lys Lys Tyr Arg Val Ala
Val Phe Arg 325 330 335 Leu Leu Gly Phe Glu Pro Phe Ser Gln Arg Lys
Leu Ser Thr Leu Lys 340 345 350 Asp Glu Ser Ser Arg Ala Trp Thr Glu
Ser Ser Ile Asn Thr 355 360 365 46 330 PRT Homo sapiens 46 Met Met
Trp Gly Ala Gly Ser Pro Leu Ala Trp Leu Ser Ala Gly Ser 1 5 10 15
Gly Asn Val Asn Val Ser Ser Val Gly Pro Ala Glu Gly Pro Thr Gly 20
25 30 Pro Ala Ala Pro Leu Pro Ser Pro Lys Ala Trp Asp Val Val Leu
Cys 35 40 45 Ile Ser Gly Thr Leu Val Ser Cys Glu Asn Ala Leu Val
Val Ala Ile 50 55 60 Ile Val Gly Thr Pro Ala Phe Arg Ala Pro Met
Phe Leu Leu Val Gly 65 70 75 80 Ser Leu Ala Val Ala Asp Leu Leu Ala
Gly Leu Gly Leu Val Leu His 85 90 95 Phe Ala Ala Val Phe Cys Ile
Gly Ser Ala Glu Met Ser Leu Val Leu 100 105 110 Val Gly Val Leu Ala
Met Ala Phe Thr Ala Ser Ile Gly Ser Leu Leu 115 120 125 Ala Ile Thr
Val Asp Arg Tyr Leu Ser Leu Tyr Asn Ala Leu Thr Tyr 130 135 140 Tyr
Ser Glu Thr Thr Val Thr Arg Thr Tyr Val Met Leu Ala Leu Val 145 150
155 160 Trp Gly Gly Ala Leu Gly Leu Gly Leu Leu Pro Val Leu Ala Trp
Asn 165 170 175 Cys Leu Asp Gly Leu Thr Thr Cys Gly Val Val Tyr Pro
Leu Ser Lys 180 185 190 Asn His Leu Val Val Leu Ala Ile Ala Phe Phe
Met Val Phe Gly Ile 195 200 205 Met Leu Gln Leu Tyr Ala Gln Ile Cys
Arg Ile Val Cys Arg His Ala 210 215 220 Gln Gln Ile Ala Leu Gln Arg
His Leu Leu Pro Ala Ser His Tyr Val 225 230 235 240 Ala Thr Arg Lys
Gly Ile Ala Thr Leu Ala Val Val Leu Gly Ala Phe 245 250 255 Ala Ala
Cys Trp Leu Pro Phe Thr Val Tyr Cys Leu Leu Gly Asp Ala 260 265 270
His Ser Pro Pro Leu Tyr Thr Tyr Leu Thr Leu Leu Pro Ala Thr Tyr 275
280 285 Asn Ser Met Ile Asn Pro Ile Ile Tyr Ala Phe Arg Asn Gln Asp
Val 290 295 300 Gln Lys Val Leu Trp Ala Val Cys Cys Cys Cys Ser Ser
Ser Lys Leu 305 310 315 320 Pro Phe Arg Ser Arg Ser Pro Ser Asp Val
325 330 47 362 PRT Homo sapiens 47 Met Asn Ala Ser Ala Ala Ser Leu
Asn Asp Ser Gln Val Val Val Val 1 5 10 15 Ala Ala Glu Gly Ala Ala
Ala Ala Ala Thr Ala Ala Gly Gly Pro Asp 20 25 30 Thr Gly Glu Trp
Gly Pro Pro Ala Ala Ala Ala Leu Gly Ala Gly Gly 35 40 45 Gly Ala
Asn Gly Ser Leu Glu Leu Ser Ser Gln Leu Ser Ala Gly Pro 50 55 60
Pro Gly Leu Leu Leu Pro Ala Val Asn Pro Trp Asp Val Leu Leu Cys 65
70 75 80 Ser Val Gly Thr Val Ile Ala Gly Glu Asn Ala Leu Val Val
Ala Leu 85 90 95 Ile Ala Ser Thr Pro Ala Leu Arg Thr Pro Met Phe
Val Leu Val Gly 100 105 110 Ser Leu Ala Thr Ala Asp Leu Leu Ala Gly
Cys Gly Leu Ile Leu His 115 120 125 Phe Val Phe Gln Tyr Leu Val Pro
Ser Glu Thr Val Ser Leu Leu Thr 130 135 140 Val Gly Phe Leu Val Ala
Ser Phe Ala Ala Ser Val Ser Ser Leu Leu 145 150 155 160 Ala Ile Thr
Val Asp Arg Tyr Leu Ser Leu Tyr Asn Ala Leu Thr Tyr 165 170 175 Tyr
Ser Arg Arg Thr Leu Leu Gly Val His Leu Leu Leu Ala Ala Thr 180 185
190 Trp Thr Val Ser Leu Gly Leu Gly Leu Leu Pro Val Leu Gly Trp Asn
195 200 205 Cys Leu Ala Glu Arg Ala Ala Cys Ser Val Val Arg Pro Leu
Ala Arg 210 215 220 Ser His Val Ala Leu Leu Ser Ala Ala Phe Phe Met
Val Phe Gly Ile 225 230 235 240 Met Leu His Leu Tyr Val Arg Ile Cys
Gln Val Val Trp Arg His Ala 245 250 255 His Gln Ile Ala Leu Gln Gln
His Cys Leu Ala Pro Pro His Leu Ala 260 265 270 Ala Thr Arg Lys Gly
Val Gly Thr Leu Ala Val Val Leu Gly Thr Phe 275 280 285 Gly Ala Ser
Trp Leu Pro Phe Ala Ile Tyr Cys Val Val Gly Ser His 290 295 300 Glu
Asp Pro Ala Val Tyr Thr Tyr Ala Thr Leu Leu Pro Ala Thr Tyr 305 310
315 320 Asn Ser Met Ile Asn Pro Ile Ile Tyr Ala Phe Arg Asn Gln Glu
Ile 325 330 335 Gln Arg Ala Leu Trp Leu Leu Leu Cys Gly Cys Phe Gln
Ser Lys Val 340 345 350 Pro Phe Arg Ser Arg Ser Pro Ser Glu Val 355
360 48 334 PRT Homo sapiens 48 Met Asn Glu Asp Leu Lys Val Asn Leu
Ser Gly Leu Pro Arg Asp Tyr 1 5 10 15 Leu Asp Ala Ala Ala Ala Glu
Asn Ile Ser Ala Ala Val Ser Ser Arg 20 25 30 Val Pro Ala Val Glu
Pro Glu Pro Glu Leu Val Val Asn Pro Trp Asp 35 40 45 Ile Val Leu
Cys Thr Ser Gly Thr Leu Ile Ser Cys Glu Asn Ala Ile 50 55 60 Val
Val Leu Ile Ile Phe His Asn Pro Ser Leu Arg Ala Pro Met Phe 65 70
75 80 Leu Leu Ile Gly Ser Leu Ala Leu Ala Asp Leu Leu Ala Gly Ile
Gly 85 90 95 Leu Ile Thr Asn Phe Val Phe Ala Tyr Leu Leu Gln Ser
Glu Ala Thr 100 105 110 Lys Leu Val Thr Ile Gly Leu Ile Val Ala Ser
Phe Ser Ala Ser Val 115 120 125 Cys Ser Leu Leu Ala Ile Thr Val Asp
Arg Tyr Leu Ser Leu Tyr Tyr 130 135 140 Ala Leu Thr Tyr His Ser Glu
Arg Thr Val Thr Phe Thr Tyr Val Met 145 150 155 160 Leu Val Met Leu
Trp Gly Thr Ser Gly Leu Leu Pro Val Met Gly Trp 165 170 175 Asn Cys
Leu Arg Asp Glu Ser Thr Cys Ser Val Val Arg Pro Leu Thr 180 185 190
Lys Asn Asn Ala Ala Ile Leu Ser Val Ser Phe Leu Phe Met Phe Ala 195
200 205 Ile Cys Leu Leu Met Leu Gln Leu Tyr Ile Gln Ile Cys Lys Ile
Val 210 215 220 Met Arg His Ala His Gln Ile Ala Leu Gln His His Phe
Leu Ala Thr 225 230 235 240 Ser His Tyr Val Thr Thr Arg Lys Gly Val
Ser Thr Leu Ala Ile Ile 245 250 255 Leu Gly Thr Phe Ala Ala Cys Trp
Met Pro Phe Thr Leu Tyr Ser Leu 260 265 270 Ile Ala Asp Tyr Thr Tyr
Pro Ser Ile Tyr Thr Tyr Ala Thr Leu Leu 275 280 285 Pro Ala Thr Tyr
Asn Ser Ile Ile Asn Pro Val Ile Tyr Ala Phe Arg 290 295 300 Asn Gln
Glu Ile Gln Lys Ala Leu Cys Leu Ile Cys Cys Gly Cys Ile 305 310 315
320 Pro Ser Ser Leu Ala Gln Arg Ala Arg Ser Pro Ser Asp Val 325 330
49 234 PRT Homo sapiens 49 Met Asn Leu Asn Ser Cys Leu Phe Gly Glu
Thr Val Leu Leu Leu Ile 1 5 10 15 Ile Gly Asn Val Ile Phe Val Phe
His Cys Ala Pro Leu Leu His Thr 20 25 30 Thr Ser Tyr Phe Ile Gln
Thr Met Ala Tyr Ala Asp Leu Phe Val Gly 35 40 45 Val Ser Cys Val
Pro Leu Ser Leu Leu His Val Glu Ser Leu Thr Cys 50 55 60 Gln Phe
Gly Ser Val Leu Lys Ser Val Ser Met Ala Leu Ala Cys Ile 65 70 75 80
Ser Asp Arg Tyr Ala Ile Thr Lys Pro Leu Tyr Asn Leu Val Thr Pro
85 90 95 Arg Leu Arg Cys Ile Leu Ile Trp Tyr Ser Leu Phe Leu Pro
Ser Phe 100 105 110 Phe Trp Gly Lys Pro Gly Tyr His Gly Asp Phe Trp
Cys Ala Ser Trp 115 120 125 Thr Tyr Phe Thr Phe Ile Val Leu Tyr Ala
Pro Ala Ala Val Cys Phe 130 135 140 Thr Tyr Phe Ile Phe Ile Cys Gln
His Thr Lys Ile Arg Ala Arg Phe 145 150 155 160 Ser Glu Pro Asp Arg
Tyr Ala Met Val Leu Phe Arg Ile Thr Ser Val 165 170 175 Phe Tyr Leu
Trp Leu Pro Tyr Ile Ile Tyr Phe Leu Leu Glu Ser Ser 180 185 190 Asn
Ser Phe Leu Thr Thr Trp Leu Ala Ile Ser Asn Ser Phe Cys Asn 195 200
205 Cys Val Ile Tyr Ser Leu Ser Asn Ser Val Phe Gly Leu Arg Leu Ser
210 215 220 Met Cys Thr Ser Cys Ala Pro Asn Cys Ile 225 230 50 361
PRT Homo sapiens 50 Met Asn Glu Ser Arg Trp Thr Glu Trp Arg Ile Leu
Asn Met Ser Ser 1 5 10 15 Gly Ile Val Asn Val Ser Glu Arg His Ser
Cys Pro Leu Gly Phe Gly 20 25 30 His Tyr Ser Val Val Asp Val Cys
Ile Phe Glu Thr Val Val Ile Val 35 40 45 Leu Leu Thr Phe Leu Ile
Ile Ala Gly Asn Leu Thr Val Ile Phe Val 50 55 60 Phe His Cys Ala
Pro Leu Leu His His Tyr Thr Thr Ser Tyr Phe Ile 65 70 75 80 Gln Thr
Met Ala Tyr Ala Asp Leu Phe Val Gly Val Ser Cys Leu Val 85 90 95
Pro Thr Leu Ser Leu Leu His Tyr Ser Thr Gly Val His Glu Ser Leu 100
105 110 Thr Cys Gln Val Phe Gly Tyr Ile Ile Ser Val Leu Lys Ser Val
Ser 115 120 125 Met Ala Cys Leu Ala Cys Ile Ser Val Asp Arg Tyr Leu
Ala Ile Thr 130 135 140 Lys Pro Leu Ser Tyr Asn Gln Leu Val Thr Pro
Cys Arg Leu Arg Ile 145 150 155 160 Cys Ile Ile Leu Ile Trp Ile Tyr
Ser Cys Leu Ile Phe Leu Pro Ser 165 170 175 Phe Phe Gly Trp Gly Lys
Pro Gly Tyr His Gly Asp Ile Phe Glu Trp 180 185 190 Cys Ala Thr Ser
Trp Leu Thr Ser Ala Tyr Phe Thr Gly Phe Ile Val 195 200 205 Cys Leu
Leu Tyr Ala Pro Ala Ala Phe Val Val Cys Phe Thr Tyr Phe 210 215 220
His Ile Phe Lys Ile Cys Arg Gln His Thr Lys Glu Ile Asn Asp Arg 225
230 235 240 Arg Ala Arg Phe Pro Ser His Glu Val Asp Ser Ser Arg Glu
Thr Gly 245 250 255 His Ser Pro Asp Arg Arg Tyr Ala Met Val Leu Phe
Arg Ile Thr Ser 260 265 270 Val Phe Tyr Met Leu Trp Leu Pro Tyr Ile
Ile Tyr Phe Leu Leu Glu 275 280 285 Ser Ser Arg Val Leu Asp Asn Pro
Thr Leu Ser Phe Leu Thr Thr Trp 290 295 300 Leu Ala Ile Ser Asn Ser
Phe Cys Asn Cys Val Ile Tyr Ser Leu Ser 305 310 315 320 Asn Ser Val
Phe Arg Leu Gly Leu Arg Arg Leu Ser Glu Thr Met Cys 325 330 335 Thr
Ser Cys Met Cys Val Lys Asp Gln Glu Ala Gln Glu Pro Lys Pro 340 345
350 Arg Lys Arg Ala Asn Ser Cys Ser Ile 355 360 51 349 PRT Homo
sapiens 51 Met Asn Ser Thr Leu Asp Gly Asn Gln Ser Ser His Pro Phe
Cys Leu 1 5 10 15 Leu Ala Phe Gly Tyr Leu Glu Thr Val Asn Phe Cys
Leu Leu Glu Val 20 25 30 Leu Ile Ile Val Phe Leu Thr Val Leu Ile
Ile Ser Gly Asn Ile Ile 35 40 45 Val Ile Phe Val Phe His Cys Ala
Pro Leu Leu Asn His His Thr Thr 50 55 60 Ser Tyr Phe Ile Gln Thr
Met Ala Tyr Ala Asp Leu Phe Val Gly Val 65 70 75 80 Ser Cys Val Val
Pro Ser Leu Ser Leu Leu His His Pro Leu Pro Val 85 90 95 Glu Glu
Ser Leu Thr Cys Gln Ile Phe Gly Phe Val Val Ser Val Leu 100 105 110
Lys Ser Val Ser Met Ala Ser Leu Ala Cys Ile Ser Ile Asp Arg Tyr 115
120 125 Ile Ala Ile Thr Lys Pro Leu Thr Tyr Asn Thr Leu Val Thr Pro
Trp 130 135 140 Arg Leu Arg Leu Cys Ile Phe Leu Ile Trp Leu Tyr Ser
Thr Leu Val 145 150 155 160 Phe Leu Pro Ser Phe Phe His Trp Gly Lys
Pro Gly Tyr His Gly Asp 165 170 175 Val Phe Gln Trp Cys Ala Glu Ser
Trp His Thr Asp Ser Tyr Phe Thr 180 185 190 Leu Phe Ile Val Met Met
Leu Tyr Ala Pro Ala Ala Leu Ile Val Cys 195 200 205 Phe Thr Tyr Phe
Asn Ile Phe Arg Ile Cys Gln Gln His Thr Lys Asp 210 215 220 Ile Ser
Glu Arg Gln Ala Arg Phe Ser Ser Gln Ser Gly Glu Thr Gly 225 230 235
240 Glu Val Gln Ala Cys Pro Asp Lys Lys Tyr Ala Met Val Leu Phe Arg
245 250 255 Ile Thr Ser Val Phe Tyr Ile Leu Trp Leu Pro Tyr Ile Ile
Tyr Phe 260 265 270 Leu Leu Glu Ser Ser Thr Gly His Ser Asn Arg Phe
Ala Ser Phe Leu 275 280 285 Thr Thr Trp Leu Ala Ile Ser Asn Ser Phe
Cys Asn Cys Val Ile Tyr 290 295 300 Ser Leu Ser Asn Ser Val Phe Gln
Arg Gly Leu Lys Arg Leu Ser Gly 305 310 315 320 Ala Met Cys Thr Ser
Cys Ala Ser Gln Thr Thr Ala Asn Asp Pro Tyr 325 330 335 Thr Val Arg
Ser Lys Gly Pro Leu Asn Gly Cys His Ile 340 345 52 4069 DNA Homo
sapiens 52 aagcttgata tcgaattcct gcagcccggg ggatccacta gttctagagc
ggccgccacc 60 gcggtggagc tccagctttt gttcccttta gtgagggtta
attgcgcgct agaggatctt 120 tgtgaaggaa ccttacttct gtggtgtgac
ataattggac aaactaccta cagagattta 180 aagctctaag gtaaatataa
aatttttaag tgtataatgt gttaaactac tgattctaat 240 tgtttgtgta
ttttagattc caacctatgg aactgatgaa tgggagcagt ggtggaatgc 300
ctttaatgag gaaaacctgt tttgctcaga agaaatgcca tctagtgatg atgaggctac
360 tgctgactct caacattcta ctcctccaaa aaagaagaga aaggtagaag
accccaagga 420 ctttccttca gaattgctaa gttttttgag tcatgctgtg
tttagtaata gaactcttgc 480 ttgctttgct atttacacca caaaggaaaa
agctgcactg ctatacaaga aaattatgga 540 aaaatattct gtaaccttta
taagtaggca taacagttat aatcataaca tactgttttt 600 tcttactcca
cacaggcata gagtgtctgc tattaataac tatgctcaaa aattgtgtac 660
ctttagcttt ttaatttgta aaggggttaa taaggaatat ttgatgtata gtgccttgac
720 tagagatcat aatcagccat accacatttg tagaggtttt acttgcttta
aaaaacctcc 780 cacacctccc cctgaacctg aaacataaaa tgaatgcaat
tgttgttgtt aacttgttta 840 ttgcagctta taatggttac aaataaagca
atagcatcac aaatttcaca aataaagcat 900 ttttttcact gcattctagt
tgtggtttgt ccaaactcat caatgtatct tatcatgtct 960 agatcttccg
aaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca 1020
gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
1080 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
atagtcccgc 1140 ccctaactcc gcccatcccg cccctaactc cgcccagttc
cgcccattct ccgccccatg 1200 gctgactaat tttttttatt tatgcagagg
ccgaggccgc ctcggcctct gagctattcc 1260 agaagtagtg aggaggcttt
tttggaggcc taggcttttg caaaaagctc cctcgagagc 1320 ttggcgtaat
catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 1380
cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
1440 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
gtcgtgccag 1500 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
tgcgtattgg gcgctcttcc 1560 gcttcctcgc tcactgactc gctgcgctcg
gtcgttcggc tgcggcgagc ggtatcagct 1620 cactcaaagg cggtaatacg
gttatccaca gaatcagggg ataacgcagg aaagaacatg 1680 tgagcaaaag
gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 1740
cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
1800 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
cgtgcgctct 1860 cctgttccga ccctgccgct taccggatac ctgtccgcct
ttctcccttc gggaagcgtg 1920 gcgctttctc aatgctcacg ctgtaggtat
ctcagttcgg tgtaggtcgt tcgctccaag 1980 ctgggctgtg tgcacgaacc
ccccgttcag cccgaccgct gcgccttatc cggtaactat 2040 cgtcttgagt
ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 2100
aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
2160 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
agttaccttc 2220 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
ccgctggtag cggtggtttt 2280 tttgtttgca agcagcagat tacgcgcaga
aaaaaaggat ctcaagaaga tcctttgatc 2340 ttttctacgg ggtctgacgc
tcagtggaac gaaaactcac gttaagggat tttggtcatg 2400 agattatcaa
aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 2460
atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
2520 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
cgtcgtgtag 2580 ataactacga tacgggaggg cttaccatct ggccccagtg
ctgcaatgat accgcgagac 2640 ccacgctcac cggctccaga tttatcagca
ataaaccagc cagccggaag ggccgagcgc 2700 agaagtggtc ctgcaacttt
atccgcctcc atccagtcta ttaattgttg ccgggaagct 2760 agagtaagta
gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 2820
gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
2880 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
tcctccgatc 2940 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
ttatggcagc actgcataat 3000 tctcttactg tcatgccatc cgtaagatgc
ttttctgtga ctggtgagta ctcaaccaag 3060 tcattctgag aatagtgtat
gcggcgaccg agttgctctt gcccggcgtc aacacgggat 3120 aataccgcgc
cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 3180
cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
3240 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
aaaaacagga 3300 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
aatgttgaat actcatactc 3360 ttcctttttc aatattattg aagcatttat
cagggttatt gtctcatgcg cgttgacatt 3420 gattattgac tagttattaa
tagtaatcaa ttacggggtc attagttcat agcccatata 3480 tggagttccg
cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 3540
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
3600 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta
catcaagtgt 3660 atcatatgcc aagtacgccc cctattgacg tcaatgacgg
taaatggccc gcctggcatt 3720 atgcccagta catgacctta tgggactttc
ctacttggca gtacatctac gtattagtca 3780 tcgctattac catggtgatg
cggttttggc agtacatcaa tgggcgtgga tagcggtttg 3840 actcacgggg
atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 3900
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
3960 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
agagaaccca 4020 ctgcttaact ggcttatcga aattaatacg actcactata
gggagaccc 4069 53 4069 DNA Homo sapiens 53 gggtctccct atagtgagtc
gtattaattt cgataagcca gttaagcagt gggttctcta 60 gttagccaga
gagctctgct tatatagacc tcccaccgta cacgcctacc gcccatttgc 120
gtcaatgggg cggagttgtt acgacatttt ggaaagtccc gttgattttg gtgccaaaac
180 aaactcccat tgacgtcaat ggggtggaga cttggaaatc cccgtgagtc
aaaccgctat 240 ccacgcccat tgatgtactg ccaaaaccgc atcaccatgg
taatagcgat gactaatacg 300 tagatgtact gccaagtagg aaagtcccat
aaggtcatgt actgggcata atgccaggcg 360 ggccatttac cgtcattgac
gtcaataggg ggcgtacttg gcatatgata cacttgatgt 420 actgccaagt
gggcagttta ccgtaaatag tccacccatt gacgtcaatg gaaagtccct 480
attggcgtta ctatgggaac atacgtcatt attgacgtca atgggcgggg gtcgttgggc
540 ggtcagccag gcgggccatt taccgtaagt tatgtaacgc ggaactccat
atatgggcta 600 tgaactaatg accccgtaat tgattactat taataactag
tcaataatca atgtcaacgc 660 gcatgagaca ataaccctga taaatgcttc
aataatattg aaaaaggaag agtatgagta 720 ttcaacattt ccgtgtcgcc
cttattccct tttttgcggc attttgcctt cctgtttttg 780 ctcacccaga
aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg 840
gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
900 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
tcccgtgttg 960 acgccgggca agagcaactc ggtcgccgca tacactattc
tcagaatgac ttggttgagt 1020 actcaccagt cacagaaaag catcttacgg
atggcatgac agtaagagaa ttatgcagtg 1080 ctgccataac catgagtgat
aacactgcgg ccaacttact tctgacaacg atcggaggac 1140 cgaaggagct
aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 1200
gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
1260 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
gcttcccggc 1320 aacaattaat agactggatg gaggcggata aagttgcagg
accacttctg cgctcggccc 1380 ttccggctgg ctggtttatt gctgataaat
ctggagccgg tgagcgtggg tctcgcggta 1440 tcattgcagc actggggcca
gatggtaagc cctcccgtat cgtagttatc tacacgacgg 1500 ggagtcaggc
aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 1560
ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
1620 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
atgaccaaaa 1680 tcccttaacg tgagttttcg ttccactgag cgtcagaccc
cgtagaaaag atcaaaggat 1740 cttcttgaga tccttttttt ctgcgcgtaa
tctgctgctt gcaaacaaaa aaaccaccgc 1800 taccagcggt ggtttgtttg
ccggatcaag agctaccaac tctttttccg aaggtaactg 1860 gcttcagcag
agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc 1920
acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
1980 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
tagttaccgg 2040 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
acagcccagc ttggagcgaa 2100 cgacctacac cgaactgaga tacctacagc
gtgagcattg agaaagcgcc acgcttcccg 2160 aagggagaaa ggcggacagg
tatccggtaa gcggcagggt cggaacagga gagcgcacga 2220 gggagcttcc
agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 2280
gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
2340 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
atgttctttc 2400 ctgcgttatc ccctgattct gtggataacc gtattaccgc
ctttgagtga gctgataccg 2460 ctcgccgcag ccgaacgacc gagcgcagcg
agtcagtgag cgaggaagcg gaagagcgcc 2520 caatacgcaa accgcctctc
cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca 2580 ggtttcccga
ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc 2640
attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
2700 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag
ctctcgaggg 2760 agctttttgc aaaagcctag gcctccaaaa aagcctcctc
actacttctg gaatagctca 2820 gaggccgagg cggcctcggc ctctgcataa
ataaaaaaaa ttagtcagcc atggggcgga 2880 gaatgggcgg aactgggcgg
agttaggggc gggatgggcg gagttagggg cgggactatg 2940 gttgctgact
aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac 3000
tttccacacc tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg
3060 gagcctgggg actttccaca ccctaactga cacacatttc ggaagatcta
gacatgataa 3120 gatacattga tgagtttgga caaaccacaa ctagaatgca
gtgaaaaaaa tgctttattt 3180 gtgaaatttg tgatgctatt gctttatttg
taaccattat aagctgcaat aaacaagtta 3240 acaacaacaa ttgcattcat
tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt 3300 aaagcaagta
aaacctctac aaatgtggta tggctgatta tgatctctag tcaaggcact 3360
atacatcaaa tattccttat taaccccttt acaaattaaa aagctaaagg tacacaattt
3420 ttgagcatag ttattaatag cagacactct atgcctgtgt ggagtaagaa
aaaacagtat 3480 gttatgatta taactgttat gcctacttat aaaggttaca
gaatattttt ccataatttt 3540 cttgtatagc agtgcagctt tttcctttgt
ggtgtaaata gcaaagcaag caagagttct 3600 attactaaac acagcatgac
tcaaaaaact tagcaattct gaaggaaagt ccttggggtc 3660 ttctaccttt
ctcttctttt ttggaggagt agaatgttga gagtcagcag tagcctcatc 3720
atcactagat ggcatttctt ctgagcaaaa caggttttcc tcattaaagg cattccacca
3780 ctgctcccat tcatcagttc cataggttgg aatctaaaat acacaaacaa
ttagaatcag 3840 tagtttaaca cattatacac ttaaaaattt tatatttacc
ttagagcttt aaatctctgt 3900 aggtagtttg tccaattatg tcacaccaca
gaagtaaggt tccttcacaa agatcctcta 3960 gcgcgcaatt aaccctcact
aaagggaaca aaagctggag ctccaccgcg gtggcggccg 4020 ctctagaact
agtggatccc ccgggctgca ggaattcgat atcaagctt 4069 54 1273 PRT Homo
sapiens 54 Lys Leu Asp Ile Glu Phe Leu Gln Pro Gly Gly Ser Thr Ser
Ser Arg 1 5 10 15 Ala Ala Ala Thr Ala Val Glu Leu Gln Leu Leu Phe
Pro Leu Val Arg 20 25 30 Val Asn Cys Ala Leu Glu Asp Leu Cys Glu
Gly Thr Leu Leu Leu Trp 35 40 45 Cys Asp Ile Ile Gly Gln Thr Thr
Tyr Arg Asp Leu Lys Leu Gly Lys 50 55 60 Tyr Lys Ile Phe Lys Cys
Ile Met Cys Thr Thr Asp Ser Asn Cys Leu 65 70 75 80 Cys Ile Leu Asp
Ser Asn Leu Trp Asn Met Gly Ala Val Val Glu Cys 85 90 95 Leu Gly
Lys Pro Val Leu Leu Arg Arg Asn Ala Ile Gly Tyr Cys Leu 100 105 110
Ser Thr Phe Tyr Ser Ser Lys Lys Glu Glu Lys Gly Arg Arg Pro Gln 115
120 125 Gly Leu Ser Phe Arg Ile Ala Lys Phe Phe Glu Ser Cys Cys Val
Asn 130 135 140 Ser Cys Leu Leu Cys Tyr Leu His His Lys Gly Lys Ser
Cys Thr Ala 145 150 155 160 Ile Gln Glu Asn Tyr Gly Lys Ile Phe Cys
Asn Leu Tyr Lys Ala Gln 165 170 175 Leu His Thr Val Phe Ser Tyr Ser
Thr Gln Ala Ser Val Cys Tyr Leu 180 185 190 Cys Ser Lys Ile Val Tyr
Leu Leu Phe Asn Leu Arg Gly Gly Ile Phe 195 200 205 Asp Val Cys Leu
Asp Arg Ser Ser Ala Ile Pro His Leu Arg Phe Tyr 210 215 220 Leu Leu
Lys Thr Ser His Thr Ser Pro Thr Asn Ile Lys Met Gln Leu 225 230 235
240 Leu Leu Leu Thr Cys Leu Leu Gln Leu Ile Met Val Thr Asn Lys Ala
245
250 255 Ile Ala Ser Gln Ile Ser Gln Ile Lys His Phe Phe His Cys Ile
Leu 260 265 270 Val Val Val Cys Pro Asn Ser Ser Met Tyr Leu Ile Met
Ser Arg Ser 275 280 285 Ser Glu Met Cys Val Ser Gly Val Glu Ser Pro
Gln Ala Pro Gln Gln 290 295 300 Ala Glu Val Cys Lys Ala Cys Ile Ser
Ile Ser Gln Gln Pro Gly Val 305 310 315 320 Glu Ser Pro Gln Ala Pro
Gln Gln Ala Glu Val Cys Lys Ala Cys Ile 325 330 335 Ser Ile Ser Gln
Gln Pro Ser Arg Pro Leu Arg Pro Ser Arg Pro Leu 340 345 350 Arg Pro
Val Pro Pro Ile Leu Arg Pro Met Ala Asp Phe Phe Leu Phe 355 360 365
Met Gln Arg Pro Arg Pro Pro Arg Pro Leu Ser Tyr Ser Arg Ser Ser 370
375 380 Glu Glu Ala Phe Leu Glu Ala Ala Phe Ala Lys Ser Ser Leu Glu
Ser 385 390 395 400 Leu Ala Ser Trp Ser Leu Phe Pro Val Asn Cys Tyr
Pro Leu Thr Ile 405 410 415 Pro His Asn Ile Arg Ala Gly Ser Ile Lys
Cys Lys Ala Trp Gly Ala 420 425 430 Val Ser Leu Thr Leu Ile Ala Leu
Arg Ser Leu Pro Ala Phe Gln Ser 435 440 445 Gly Asn Leu Ser Cys Gln
Leu His Ile Gly Gln Arg Ala Gly Arg Gly 450 455 460 Gly Leu Arg Ile
Gly Arg Ser Ser Ala Ser Ser Leu Thr Asp Ser Leu 465 470 475 480 Arg
Ser Val Val Arg Leu Arg Arg Ala Val Ser Ala His Ser Lys Ala 485 490
495 Val Ile Arg Leu Ser Thr Glu Ser Gly Asp Asn Ala Gly Lys Asn Met
500 505 510 Ala Lys Gly Gln Gln Lys Ala Arg Asn Arg Lys Lys Ala Ala
Leu Leu 515 520 525 Ala Phe Phe His Arg Leu Arg Pro Pro Asp Glu His
His Lys Asn Arg 530 535 540 Arg Ser Ser Gln Arg Trp Arg Asn Pro Thr
Gly Leu Arg Tyr Gln Ala 545 550 555 560 Phe Pro Pro Gly Ser Ser Leu
Val Arg Ser Pro Val Pro Thr Leu Pro 565 570 575 Leu Thr Gly Tyr Leu
Ser Ala Phe Leu Pro Ser Gly Ser Val Ala Leu 580 585 590 Ser Gln Cys
Ser Arg Cys Arg Tyr Leu Ser Ser Val Val Val Arg Ser 595 600 605 Lys
Leu Gly Cys Val His Glu Pro Pro Val Gln Pro Asp Arg Cys Ala 610 615
620 Leu Ser Gly Asn Tyr Arg Leu Glu Ser Asn Pro Val Arg His Asp Leu
625 630 635 640 Ser Pro Leu Ala Ala Ala Thr Gly Asn Arg Ile Ser Arg
Ala Arg Tyr 645 650 655 Val Gly Gly Ala Thr Glu Phe Leu Lys Trp Trp
Pro Asn Tyr Gly Tyr 660 665 670 Thr Arg Arg Thr Val Phe Gly Ile Cys
Ala Leu Leu Lys Pro Val Thr 675 680 685 Phe Gly Lys Arg Val Gly Ser
Ser Ser Gly Lys Gln Thr Thr Ala Gly 690 695 700 Ser Gly Gly Phe Phe
Val Cys Lys Gln Gln Ile Thr Arg Arg Lys Lys 705 710 715 720 Gly Ser
Gln Glu Asp Pro Leu Ile Phe Ser Thr Gly Ser Asp Ala Gln 725 730 735
Trp Asn Glu Asn Ser Arg Gly Ile Leu Val Met Arg Leu Ser Lys Arg 740
745 750 Ile Phe Thr Ile Leu Leu Asn Lys Ser Phe Lys Ser Ile Ser Ile
Tyr 755 760 765 Glu Thr Trp Ser Asp Ser Tyr Gln Cys Leu Ile Ser Glu
Ala Pro Ile 770 775 780 Ser Ala Ile Cys Leu Phe Arg Ser Ser Ile Val
Ala Leu Pro Val Val 785 790 795 800 Ile Thr Thr Ile Arg Glu Gly Leu
Pro Ser Gly Pro Ser Ala Ala Met 805 810 815 Ile Pro Arg Asp Pro Arg
Ser Pro Ala Pro Asp Leu Ser Ala Ile Asn 820 825 830 Gln Pro Ala Gly
Arg Ala Glu Arg Arg Ser Gly Pro Ala Thr Leu Ser 835 840 845 Ala Ser
Ile Gln Ser Ile Asn Cys Cys Arg Glu Ala Arg Val Ser Ser 850 855 860
Ser Pro Val Asn Ser Leu Arg Asn Val Val Ala Ile Ala Thr Gly Ile 865
870 875 880 Val Val Ser Arg Ser Ser Phe Gly Met Ala Ser Phe Ser Ser
Gly Ser 885 890 895 Gln Arg Ser Arg Arg Val Thr Ser Pro Met Leu Cys
Lys Lys Ala Val 900 905 910 Ser Ser Phe Gly Pro Pro Ile Val Val Arg
Ser Lys Leu Ala Ala Val 915 920 925 Leu Ser Leu Met Val Met Ala Ala
Leu His Asn Ser Leu Thr Val Met 930 935 940 Pro Ser Val Arg Cys Phe
Ser Val Thr Gly Glu Tyr Ser Thr Lys Ser 945 950 955 960 Phe Glu Cys
Met Arg Arg Pro Ser Cys Ser Cys Pro Ala Ser Thr Arg 965 970 975 Asp
Asn Thr Ala Pro His Ser Arg Thr Leu Lys Val Leu Ile Ile Gly 980 985
990 Lys Arg Ser Ser Gly Arg Lys Leu Ser Arg Ile Leu Pro Leu Leu Arg
995 1000 1005 Ser Ser Ser Met Pro Thr Arg Ala Pro Asn Ser Ser Ala
Ser Phe 1010 1015 1020 Thr Phe Thr Ser Val Ser Gly Ala Lys Thr Gly
Arg Gln Asn Ala 1025 1030 1035 Ala Lys Lys Gly Ile Arg Ala Thr Arg
Lys Cys Ile Leu Ile Leu 1040 1045 1050 Phe Leu Phe Gln Tyr Tyr Ser
Ile Tyr Gln Gly Tyr Cys Leu Met 1055 1060 1065 Arg Val Asp Ile Asp
Tyr Leu Val Ile Asn Ser Asn Gln Leu Arg 1070 1075 1080 Gly His Phe
Ile Ala His Ile Trp Ser Ser Ala Leu His Asn Leu 1085 1090 1095 Arg
Met Ala Arg Leu Ala Asp Arg Pro Thr Thr Pro Ala His Arg 1100 1105
1110 Gln Arg Met Phe Pro Arg Gln Gly Leu Ser Ile Asp Val Asn Gly
1115 1120 1125 Trp Thr Ile Tyr Gly Lys Leu Pro Thr Trp Gln Tyr Ile
Lys Cys 1130 1135 1140 Ile Ile Cys Gln Val Arg Pro Leu Leu Thr Ser
Met Thr Val Asn 1145 1150 1155 Gly Pro Pro Gly Ile Met Pro Ser Thr
Pro Tyr Gly Thr Phe Leu 1160 1165 1170 Leu Gly Ser Thr Ser Thr Tyr
Ser Ser Leu Leu Pro Trp Cys Gly 1175 1180 1185 Phe Gly Ser Thr Ser
Met Gly Val Asp Ser Gly Leu Thr His Gly 1190 1195 1200 Asp Phe Gln
Val Ser Thr Pro Leu Thr Ser Met Gly Val Cys Phe 1205 1210 1215 Gly
Thr Lys Ile Asn Gly Thr Phe Gln Asn Val Val Thr Thr Pro 1220 1225
1230 Pro His Arg Lys Trp Ala Val Gly Val Tyr Gly Gly Arg Ser Ile
1235 1240 1245 Ala Glu Leu Ser Gly Leu Glu Asn Pro Leu Leu Asn Trp
Leu Ile 1250 1255 1260 Glu Ile Asn Thr Thr His Tyr Arg Glu Thr 1265
1270 55 1310 PRT Homo sapiens 55 Ser Leu Ile Ser Asn Ser Cys Ser
Pro Gly Asp Pro Leu Val Leu Glu 1 5 10 15 Arg Pro Pro Pro Arg Trp
Ser Ser Ser Phe Cys Ser Leu Gly Leu Ile 20 25 30 Ala Arg Arg Ile
Phe Val Lys Glu Pro Tyr Phe Cys Gly Val Thr Leu 35 40 45 Asp Lys
Leu Pro Thr Glu Ile Ser Ser Lys Val Asn Ile Lys Phe Leu 50 55 60
Ser Val Cys Val Lys Leu Leu Ile Leu Ile Val Cys Val Phe Ile Pro 65
70 75 80 Thr Tyr Gly Thr Asp Glu Trp Glu Gln Trp Trp Asn Ala Phe
Asn Glu 85 90 95 Glu Asn Leu Phe Cys Ser Glu Glu Met Pro Ser Ser
Asp Asp Glu Ala 100 105 110 Thr Ala Asp Ser Gln His Ser Thr Pro Pro
Lys Lys Lys Arg Lys Val 115 120 125 Glu Asp Pro Lys Asp Phe Pro Ser
Glu Leu Leu Ser Phe Leu Ser His 130 135 140 Ala Val Phe Ser Asn Arg
Thr Leu Ala Cys Phe Ala Ile Tyr Thr Thr 145 150 155 160 Lys Glu Lys
Ala Ala Leu Leu Tyr Lys Lys Ile Met Glu Lys Tyr Ser 165 170 175 Val
Thr Phe Ile Ser Arg His Asn Ser Tyr Asn His Asn Ile Leu Phe 180 185
190 Phe Leu Thr Pro His Arg His Arg Val Ser Ala Ile Asn Asn Tyr Ala
195 200 205 Gln Lys Leu Cys Thr Phe Ser Phe Leu Ile Cys Lys Gly Val
Asn Lys 210 215 220 Glu Tyr Leu Met Tyr Ser Ala Leu Thr Arg Asp His
Asn Gln Pro Tyr 225 230 235 240 His Ile Cys Arg Gly Phe Thr Cys Phe
Lys Lys Pro Pro Thr Pro Pro 245 250 255 Pro Glu Pro Glu Thr Asn Glu
Cys Asn Cys Cys Cys Leu Val Tyr Cys 260 265 270 Ser Leu Trp Leu Gln
Ile Lys Gln His His Lys Phe His Lys Ser Ile 275 280 285 Phe Phe Thr
Ala Phe Leu Trp Phe Val Gln Thr His Gln Cys Ile Leu 290 295 300 Ser
Cys Leu Asp Leu Pro Lys Cys Val Ser Val Arg Val Trp Lys Val 305 310
315 320 Pro Arg Leu Pro Ser Arg Gln Lys Tyr Ala Lys His Ala Ser Gln
Leu 325 330 335 Val Ser Asn Gln Val Trp Lys Val Pro Arg Leu Pro Ser
Arg Gln Lys 340 345 350 Tyr Ala Lys His Ala Ser Gln Leu Val Ser Asn
His Ser Pro Ala Pro 355 360 365 Asn Ser Ala His Pro Ala Pro Asn Ser
Ala Gln Phe Arg Pro Phe Ser 370 375 380 Ala Pro Trp Leu Thr Asn Phe
Phe Tyr Leu Cys Arg Gly Arg Gly Arg 385 390 395 400 Leu Gly Leu Ala
Ile Pro Glu Val Val Arg Arg Leu Phe Trp Arg Pro 405 410 415 Arg Leu
Leu Gln Lys Ala Pro Ser Arg Ala Trp Arg Asn His Gly His 420 425 430
Ser Cys Phe Leu Cys Glu Ile Val Ile Arg Ser Gln Phe His Thr Thr 435
440 445 Tyr Glu Pro Glu Ala Ser Val Lys Pro Gly Val Pro Asn Glu Ala
Asn 450 455 460 Ser His Leu Arg Cys Ala His Cys Pro Leu Ser Ser Arg
Glu Thr Cys 465 470 475 480 Arg Ala Ser Cys Ile Asn Glu Ser Ala Asn
Ala Arg Gly Glu Ala Val 485 490 495 Cys Val Leu Gly Ala Leu Pro Leu
Pro Arg Ser Leu Thr Arg Cys Ala 500 505 510 Arg Ser Phe Gly Cys Gly
Glu Arg Tyr Gln Leu Thr Gln Arg Arg Tyr 515 520 525 Gly Tyr Pro Gln
Asn Gln Gly Ile Thr Gln Glu Arg Thr Cys Glu Gln 530 535 540 Lys Ala
Ser Lys Arg Pro Gly Thr Val Lys Arg Pro Arg Cys Trp Arg 545 550 555
560 Phe Ser Ile Gly Ser Ala Pro Leu Thr Ser Ile Thr Lys Ile Asp Ala
565 570 575 Gln Val Arg Gly Gly Glu Thr Arg Gln Asp Tyr Lys Asp Thr
Arg Arg 580 585 590 Phe Pro Leu Glu Ala Pro Ser Cys Ala Leu Leu Phe
Arg Pro Cys Arg 595 600 605 Leu Pro Asp Thr Cys Pro Pro Phe Ser Leu
Arg Glu Ala Trp Arg Phe 610 615 620 Leu Asn Ala His Ala Val Gly Ile
Ser Val Arg Cys Arg Ser Phe Ala 625 630 635 640 Pro Ser Trp Ala Val
Cys Thr Asn Pro Pro Phe Ser Pro Thr Ala Ala 645 650 655 Pro Tyr Pro
Val Thr Ile Val Leu Ser Pro Thr Arg Asp Thr Thr Tyr 660 665 670 Arg
His Trp Gln Gln Pro Leu Val Thr Gly Leu Ala Glu Arg Gly Met 675 680
685 Ala Val Leu Gln Ser Ser Ser Gly Gly Leu Thr Thr Ala Thr Leu Glu
690 695 700 Gly Gln Tyr Leu Val Ser Ala Leu Cys Ser Gln Leu Pro Ser
Glu Lys 705 710 715 720 Glu Leu Val Ala Leu Asp Pro Ala Asn Lys Pro
Pro Leu Val Ala Val 725 730 735 Val Phe Leu Phe Ala Ser Ser Arg Leu
Arg Ala Glu Lys Lys Asp Leu 740 745 750 Lys Lys Ile Leu Ser Phe Leu
Arg Gly Leu Thr Leu Ser Gly Thr Lys 755 760 765 Thr His Val Lys Gly
Phe Trp Ser Asp Tyr Gln Lys Gly Ser Ser Pro 770 775 780 Arg Ser Phe
Ile Lys Asn Glu Val Leu Asn Gln Ser Lys Val Tyr Met 785 790 795 800
Ser Lys Leu Gly Leu Thr Val Thr Asn Ala Ser Val Arg His Leu Ser 805
810 815 Gln Arg Ser Val Tyr Phe Val His Pro Leu Pro Asp Ser Pro Ser
Cys 820 825 830 Arg Leu Arg Tyr Gly Arg Ala Tyr His Leu Ala Pro Val
Leu Gln Tyr 835 840 845 Arg Glu Thr His Ala His Arg Leu Gln Ile Tyr
Gln Gln Thr Ser Gln 850 855 860 Pro Glu Gly Pro Ser Ala Glu Val Val
Leu Gln Leu Tyr Pro Pro Pro 865 870 875 880 Ser Ser Leu Leu Ile Val
Ala Gly Lys Leu Glu Val Val Arg Gln Leu 885 890 895 Ile Val Cys Ala
Thr Leu Leu Pro Leu Leu Gln Ala Ser Trp Cys His 900 905 910 Ala Arg
Arg Leu Val Trp Leu His Ser Ala Pro Val Pro Asn Asp Gln 915 920 925
Gly Glu Leu His Asp Pro Pro Cys Cys Ala Lys Lys Arg Leu Ala Pro 930
935 940 Ser Val Leu Arg Ser Leu Ser Glu Val Ser Trp Pro Gln Cys Tyr
His 945 950 955 960 Ser Trp Leu Trp Gln His Cys Ile Ile Leu Leu Leu
Ser Cys His Pro 965 970 975 Asp Ala Phe Leu Leu Val Ser Thr Gln Pro
Ser His Ser Glu Asn Ser 980 985 990 Val Cys Gly Asp Arg Val Ala Leu
Ala Arg Arg Gln His Gly Ile Ile 995 1000 1005 Pro Arg His Ile Ala
Glu Leu Lys Cys Ser Ser Leu Glu Asn Val 1010 1015 1020 Leu Arg Gly
Glu Asn Ser Gln Gly Ser Tyr Arg Cys Asp Pro Val 1025 1030 1035 Arg
Cys Asn Pro Leu Val His Pro Thr Asp Leu Gln His Leu Leu 1040 1045
1050 Leu Ser Pro Ala Phe Leu Gly Glu Gln Lys Gln Glu Gly Lys Met
1055 1060 1065 Pro Gln Lys Arg Glu Gly Arg His Gly Asn Val Glu Tyr
Ser Tyr 1070 1075 1080 Ser Ser Phe Phe Asn Ile Ile Glu Ala Phe Ile
Arg Val Ile Val 1085 1090 1095 Ser Cys Ala Leu Thr Leu Ile Ile Asp
Leu Leu Ile Val Ile Asn 1100 1105 1110 Tyr Gly Val Ile Ser Ser Pro
Ile Tyr Gly Val Pro Arg Tyr Ile 1115 1120 1125 Thr Tyr Gly Lys Trp
Pro Ala Trp Leu Thr Ala Gln Arg Pro Pro 1130 1135 1140 Pro Ile Asp
Val Asn Asn Asp Val Cys Ser His Ser Asn Ala Asn 1145 1150 1155 Arg
Asp Phe Pro Leu Thr Ser Met Gly Gly Leu Phe Thr Val Asn 1160 1165
1170 Cys Pro Leu Gly Ser Thr Ser Ser Val Ser Tyr Ala Lys Tyr Ala
1175 1180 1185 Pro Tyr Arg Gln Arg Met Ala Arg Leu Ala Leu Cys Pro
Val His 1190 1195 1200 Asp Leu Met Gly Leu Ser Tyr Leu Ala Val His
Leu Arg Ile Ser 1205 1210 1215 His Arg Tyr Tyr His Gly Asp Ala Val
Leu Ala Val His Gln Trp 1220 1225 1230 Ala Trp Ile Ala Val Leu Thr
Gly Ile Ser Lys Ser Pro Pro His 1235 1240 1245 Arg Gln Trp Glu Phe
Val Leu Ala Pro Lys Ser Thr Gly Leu Ser 1250 1255 1260 Lys Met Ser
Gln Leu Arg Pro Ile Asp Ala Asn Gly Arg Ala Cys 1265 1270 1275 Thr
Val Gly Gly Leu Tyr Lys Gln Ser Ser Leu Ala Asn Arg Thr 1280 1285
1290 His Cys Leu Thr Gly Leu Ser Lys Leu Ile Arg Leu Thr Ile Gly
1295 1300 1305 Arg Pro 1310 56 1286 PRT Homo sapiens 56 Gln Ala Tyr
Arg Ile Pro Ala Ala Arg Gly Ile His Phe Ser Gly Arg 1 5 10 15 His
Arg Gly Gly Ala Pro Ala Phe Val Pro Phe Ser Glu Gly Leu Arg 20 25
30 Ala Arg Gly Ser Leu Arg Asn Leu Thr Ser Val Val His Asn Trp Thr
35 40 45 Asn Tyr Leu Gln Arg Phe Lys Ala Leu Arg Ile Asn Phe Val
Tyr Asn 50 55 60 Val Leu Asn Tyr Phe Leu Phe Val Tyr Phe Arg Phe
Gln Pro Met Glu 65 70 75 80 Leu Met Asn Gly Ser Ser Gly Gly Met Pro
Leu Met Arg Lys Thr Cys 85 90 95 Phe Ala Gln Lys Lys Cys His
Leu Val Met Met Arg Leu Leu Leu Thr 100 105 110 Leu Asn Ile Leu Leu
Leu Gln Lys Arg Arg Glu Arg Lys Thr Pro Arg 115 120 125 Thr Phe Leu
Gln Asn Cys Val Phe Val Met Leu Cys Leu Val Ile Glu 130 135 140 Leu
Leu Leu Ala Leu Leu Phe Thr Pro Gln Arg Lys Lys Leu His Cys 145 150
155 160 Tyr Thr Arg Lys Leu Trp Lys Asn Ile Leu Pro Leu Val Gly Ile
Thr 165 170 175 Val Ile Ile Ile Thr Tyr Cys Phe Phe Leu Leu His Thr
Gly Ile Glu 180 185 190 Cys Leu Leu Leu Ile Thr Met Leu Lys Asn Cys
Val Pro Leu Ala Phe 195 200 205 Phe Val Lys Gly Leu Ile Arg Asn Ile
Cys Ile Val Pro Leu Glu Ile 210 215 220 Ile Ile Ser His Thr Thr Phe
Val Glu Val Leu Leu Ala Leu Lys Asn 225 230 235 240 Leu Pro His Leu
Pro Leu Asn Leu Lys His Lys Met Asn Ala Ile Val 245 250 255 Val Val
Asn Leu Phe Ile Ala Ala Tyr Asn Gly Tyr Lys Ser Asn Ser 260 265 270
Ile Thr Asn Phe Thr Asn Lys Ala Phe Phe Ser Leu His Ser Ser Cys 275
280 285 Gly Leu Ser Lys Leu Ile Asn Val Ser Tyr His Val Ile Phe Arg
Asn 290 295 300 Val Cys Gln Leu Gly Cys Gly Lys Ser Pro Gly Ser Pro
Ala Gly Arg 305 310 315 320 Ser Met Gln Ser Met His Leu Asn Ser Ala
Thr Arg Cys Gly Lys Ser 325 330 335 Pro Gly Ser Pro Ala Gly Arg Ser
Met Gln Ser Met His Leu Asn Ser 340 345 350 Ala Thr Ile Val Pro Pro
Leu Thr Pro Pro Ile Pro Pro Leu Thr Pro 355 360 365 Pro Ser Ser Ala
His Ser Pro Pro His Gly Leu Ile Phe Phe Ile Tyr 370 375 380 Ala Glu
Ala Glu Ala Ala Ser Ala Ser Glu Leu Phe Gln Lys Gly Gly 385 390 395
400 Phe Phe Gly Gly Leu Gly Phe Cys Lys Lys Leu Pro Arg Glu Leu Gly
405 410 415 Val Ile Met Val Ile Ala Val Ser Cys Val Lys Leu Leu Ser
Ala His 420 425 430 Asn Ser Thr Gln His Thr Ser Arg Lys His Lys Val
Ser Leu Gly Cys 435 440 445 Leu Met Ser Glu Leu Thr His Ile Asn Cys
Val Ala Leu Thr Ala Arg 450 455 460 Phe Pro Val Gly Lys Pro Val Val
Pro Ala Ala Leu Met Asn Arg Pro 465 470 475 480 Thr Arg Gly Glu Arg
Arg Phe Ala Tyr Trp Ala Leu Phe Arg Phe Leu 485 490 495 Ala His Leu
Ala Ala Leu Gly Arg Ser Ala Ala Ala Ser Gly Ile Ser 500 505 510 Ser
Leu Lys Gly Gly Asn Thr Val Ile His Arg Ile Arg Gly Arg Arg 515 520
525 Lys Glu His Val Ser Lys Arg Pro Ala Lys Gly Gln Glu Pro Lys Gly
530 535 540 Arg Val Ala Gly Val Phe Pro Ala Pro Pro Pro Arg Ala Ser
Gln Lys 545 550 555 560 Ser Thr Leu Lys Ser Glu Val Ala Lys Pro Asp
Arg Thr Ile Lys Ile 565 570 575 Pro Gly Val Ser Pro Trp Lys Leu Pro
Arg Ala Leu Ser Cys Ser Asp 580 585 590 Pro Ala Ala Tyr Arg Ile Pro
Val Arg Leu Ser Pro Phe Gly Lys Arg 595 600 605 Gly Ala Phe Ser Met
Leu Thr Leu Val Ser Gln Phe Gly Val Gly Arg 610 615 620 Ser Leu Gln
Ala Gly Leu Cys Ala Arg Thr Pro Arg Ser Ala Arg Pro 625 630 635 640
Leu Arg Leu Ile Arg Leu Ser Ser Val Gln Pro Gly Lys Thr Arg Leu 645
650 655 Ile Ala Thr Gly Ser Ser His Trp Gln Asp Gln Ser Glu Val Cys
Arg 660 665 670 Arg Cys Tyr Arg Val Leu Glu Val Val Ala Leu Arg Leu
His Lys Asp 675 680 685 Ser Ile Trp Tyr Leu Arg Ser Ala Glu Ala Ser
Tyr Leu Arg Lys Lys 690 695 700 Ser Trp Leu Leu Ile Arg Gln Thr Asn
His Arg Trp Arg Trp Phe Phe 705 710 715 720 Cys Leu Gln Ala Ala Asp
Tyr Ala Gln Lys Lys Arg Ile Ser Arg Arg 725 730 735 Ser Phe Asp Leu
Phe Tyr Gly Val Arg Ser Val Glu Arg Lys Leu Thr 740 745 750 Leu Arg
Asp Phe Gly His Glu Ile Ile Lys Lys Asp Leu His Leu Asp 755 760 765
Pro Phe Lys Leu Lys Met Lys Phe Ile Asn Leu Lys Tyr Ile Val Asn 770
775 780 Leu Val Gln Leu Pro Met Leu Asn Gln Gly Thr Tyr Leu Ser Asp
Leu 785 790 795 800 Ser Ile Ser Phe Ile His Ser Cys Leu Thr Pro Arg
Arg Val Asp Asn 805 810 815 Tyr Asp Thr Gly Gly Leu Thr Ile Trp Pro
Gln Cys Cys Asn Asp Thr 820 825 830 Ala Arg Pro Thr Leu Thr Gly Ser
Arg Phe Ile Ser Asn Lys Pro Ala 835 840 845 Ser Arg Lys Gly Arg Ala
Gln Lys Trp Ser Cys Asn Phe Ile Arg Leu 850 855 860 His Pro Val Tyr
Leu Leu Pro Gly Ser Ser Lys Phe Ala Ser Phe Ala 865 870 875 880 Gln
Arg Cys Cys His Cys Tyr Arg His Arg Gly Val Thr Leu Val Val 885 890
895 Trp Tyr Gly Phe Ile Gln Leu Arg Phe Pro Thr Ile Lys Ala Ser Tyr
900 905 910 Met Ile Pro His Val Val Gln Lys Ser Gly Leu Leu Arg Ser
Ser Asp 915 920 925 Arg Cys Gln Lys Val Gly Arg Ser Val Ile Thr His
Gly Tyr Gly Ser 930 935 940 Thr Ala Phe Ser Tyr Cys His Ala Ile Arg
Lys Met Leu Phe Cys Asp 945 950 955 960 Trp Val Leu Asn Gln Val Ile
Leu Arg Ile Val Tyr Ala Ala Thr Glu 965 970 975 Leu Leu Leu Pro Gly
Val Asn Thr Gly Tyr Arg Ala Thr Gln Asn Phe 980 985 990 Lys Ser Ala
His His Trp Lys Thr Phe Phe Gly Ala Lys Thr Leu Lys 995 1000 1005
Asp Leu Thr Ala Val Glu Ile Gln Phe Asp Val Thr His Ser Cys 1010
1015 1020 Thr Gln Leu Ile Phe Ser Ile Phe Tyr Phe His Gln Arg Phe
Trp 1025 1030 1035 Val Ser Lys Asn Arg Lys Ala Lys Cys Arg Lys Lys
Gly Asn Lys 1040 1045 1050 Gly Asp Thr Glu Met Leu Asn Thr His Thr
Leu Pro Phe Ser Ile 1055 1060 1065 Leu Leu Lys His Leu Ser Gly Leu
Leu Ser His Ala Arg His Leu 1070 1075 1080 Leu Thr Ser Tyr Ser Ile
Thr Gly Ser Leu Val His Ser Pro Tyr 1085 1090 1095 Met Glu Phe Arg
Val Thr Leu Thr Val Asn Gly Pro Pro Gly Pro 1100 1105 1110 Pro Asn
Asp Pro Arg Pro Leu Thr Ser Ile Met Thr Tyr Val Pro 1115 1120 1125
Ile Val Thr Pro Ile Gly Thr Phe His Arg Gln Trp Val Asp Tyr 1130
1135 1140 Leu Arg Thr Ala His Leu Ala Val His Gln Val Tyr His Met
Pro 1145 1150 1155 Ser Thr Pro Pro Ile Asp Val Asn Asp Gly Lys Trp
Pro Ala Trp 1160 1165 1170 His Tyr Ala Gln Tyr Met Thr Leu Trp Asp
Phe Pro Thr Trp Gln 1175 1180 1185 Tyr Ile Tyr Val Leu Val Ile Ala
Ile Thr Met Val Met Arg Phe 1190 1195 1200 Trp Gln Tyr Ile Asn Gly
Arg Gly Arg Phe Asp Ser Arg Gly Phe 1205 1210 1215 Pro Ser Leu His
Pro Ile Asp Val Asn Gly Ser Leu Phe Trp His 1220 1225 1230 Gln Asn
Gln Arg Asp Phe Pro Lys Cys Arg Asn Asn Ser Ala Pro 1235 1240 1245
Leu Thr Gln Met Gly Gly Arg Arg Val Arg Trp Glu Val Tyr Ile 1250
1255 1260 Ser Arg Ala Leu Trp Leu Thr Arg Glu Pro Thr Ala Leu Ala
Tyr 1265 1270 1275 Arg Asn Tyr Asp Ser Leu Gly Asp 1280 1285 57
1293 PRT Homo sapiens 57 Trp Val Ser Leu Val Val Leu Ile Ser Ile
Ser Gln Leu Ser Ser Gly 1 5 10 15 Phe Ser Ser Pro Glu Ser Ser Ala
Tyr Ile Asp Leu Pro Pro Tyr Thr 20 25 30 Pro Thr Ala His Leu Arg
Gln Trp Gly Gly Val Val Thr Thr Phe Trp 35 40 45 Lys Val Pro Leu
Ile Leu Val Pro Lys Gln Thr Pro Ile Asp Val Asn 50 55 60 Gly Val
Glu Thr Trp Lys Ser Pro Val Lys Pro Leu Ser Thr Pro Ile 65 70 75 80
Asp Val Leu Pro Lys Pro His His His Gly Asn Ser Asp Asp Tyr Val 85
90 95 Asp Val Leu Pro Ser Arg Lys Val Pro Gly His Val Leu Gly Ile
Met 100 105 110 Pro Gly Gly Pro Phe Thr Val Ile Asp Val Asn Arg Gly
Arg Thr Trp 115 120 125 His Met Ile His Leu Met Tyr Cys Gln Val Gly
Ser Leu Pro Ile Val 130 135 140 His Pro Leu Thr Ser Met Glu Ser Pro
Tyr Trp Arg Tyr Tyr Gly Asn 145 150 155 160 Ile Arg His Tyr Arg Gln
Trp Ala Gly Val Val Gly Arg Ser Ala Arg 165 170 175 Arg Ala Ile Tyr
Arg Lys Leu Cys Asn Ala Glu Leu His Ile Trp Ala 180 185 190 Met Asn
Pro Arg Asn Leu Leu Leu Ile Thr Ser Gln Ser Met Ser Thr 195 200 205
Arg Met Arg Gln Pro Met Leu Gln Tyr Lys Arg Lys Ser Met Ser Ile 210
215 220 Gln His Phe Arg Val Ala Leu Ile Pro Phe Phe Ala Ala Phe Cys
Leu 225 230 235 240 Pro Val Phe Ala His Pro Glu Thr Leu Val Lys Val
Lys Asp Ala Glu 245 250 255 Asp Gln Leu Gly Ala Arg Val Gly Tyr Ile
Glu Leu Asp Leu Asn Ser 260 265 270 Gly Lys Ile Leu Glu Ser Phe Arg
Pro Glu Glu Arg Phe Pro Met Met 275 280 285 Ser Thr Phe Lys Val Leu
Leu Cys Gly Ala Val Leu Ser Arg Val Asp 290 295 300 Ala Gly Gln Glu
Gln Leu Gly Arg Arg Ile His Tyr Ser Gln Asn Asp 305 310 315 320 Leu
Val Glu Tyr Ser Pro Val Thr Glu Lys His Leu Thr Asp Gly Met 325 330
335 Thr Val Arg Glu Leu Cys Ser Ala Ala Ile Thr Met Ser Asp Asn Thr
340 345 350 Ala Ala Asn Leu Leu Leu Thr Thr Ile Gly Gly Pro Lys Glu
Leu Thr 355 360 365 Ala Phe Leu His Asn Met Gly Asp His Val Thr Arg
Leu Asp Arg Trp 370 375 380 Glu Pro Glu Leu Asn Glu Ala Ile Pro Asn
Asp Glu Arg Asp Thr Thr 385 390 395 400 Met Pro Val Ala Met Ala Thr
Thr Leu Arg Lys Leu Leu Thr Gly Glu 405 410 415 Leu Leu Thr Leu Ala
Ser Arg Gln Gln Leu Ile Asp Trp Met Glu Ala 420 425 430 Asp Lys Val
Ala Gly Pro Leu Leu Arg Ser Ala Leu Pro Ala Gly Trp 435 440 445 Phe
Ile Ala Asp Lys Ser Gly Ala Gly Glu Arg Gly Ser Arg Gly Ile 450 455
460 Ile Ala Ala Leu Gly Pro Asp Gly Lys Pro Ser Arg Ile Val Val Ile
465 470 475 480 Tyr Thr Thr Gly Ser Gln Ala Thr Met Asp Glu Arg Asn
Arg Gln Ile 485 490 495 Ala Glu Ile Gly Ala Ser Leu Ile Lys His Trp
Leu Ser Asp Gln Val 500 505 510 Tyr Ser Tyr Ile Leu Ile Asp Leu Lys
Leu His Phe Phe Lys Arg Ile 515 520 525 Val Lys Ile Leu Phe Asp Asn
Leu Met Thr Lys Ile Pro Arg Glu Phe 530 535 540 Ser Phe His Ala Ser
Asp Pro Val Glu Lys Ile Lys Gly Ser Ser Asp 545 550 555 560 Pro Phe
Phe Leu Arg Val Ile Cys Cys Leu Gln Thr Lys Lys Pro Pro 565 570 575
Leu Pro Ala Val Val Cys Leu Pro Asp Gln Glu Leu Pro Thr Leu Phe 580
585 590 Pro Lys Val Thr Gly Phe Ser Arg Ala Gln Ile Pro Asn Thr Val
Leu 595 600 605 Leu Val Pro Leu Gly His His Phe Lys Asn Ser Val Ala
Pro Pro Thr 610 615 620 Tyr Leu Ala Leu Leu Ile Leu Leu Pro Val Ala
Ala Ala Ser Gly Asp 625 630 635 640 Lys Ser Cys Leu Thr Gly Leu Asp
Ser Arg Arg Leu Pro Asp Lys Ala 645 650 655 Gln Arg Ser Gly Thr Gly
Gly Ser Cys Thr Gln Pro Ser Leu Glu Arg 660 665 670 Thr Thr Tyr Thr
Glu Leu Arg Tyr Leu Gln Arg Glu His Glu Ser Ala 675 680 685 Thr Leu
Pro Glu Gly Arg Lys Ala Asp Arg Tyr Pro Val Ser Gly Arg 690 695 700
Val Gly Thr Gly Glu Arg Thr Arg Glu Leu Pro Gly Gly Asn Ala Trp 705
710 715 720 Tyr Leu Tyr Ser Pro Val Gly Phe Arg His Leu Leu Glu Arg
Arg Phe 725 730 735 Leu Cys Ser Ser Gly Gly Arg Ser Leu Trp Lys Asn
Ala Ser Asn Ala 740 745 750 Ala Phe Leu Arg Phe Leu Ala Phe Cys Trp
Pro Phe Ala His Met Phe 755 760 765 Phe Pro Ala Leu Ser Pro Asp Ser
Val Asp Asn Arg Ile Thr Ala Phe 770 775 780 Glu Ala Asp Thr Ala Arg
Arg Ser Arg Thr Thr Glu Arg Ser Glu Ser 785 790 795 800 Val Ser Glu
Glu Ala Glu Glu Arg Pro Ile Arg Lys Pro Pro Leu Pro 805 810 815 Ala
Arg Trp Pro Ile His Cys Ser Trp His Asp Arg Phe Pro Asp Trp 820 825
830 Lys Ala Gly Ser Glu Arg Asn Ala Ile Asn Val Ser Leu Thr His Ala
835 840 845 Pro Gln Ala Leu His Phe Met Leu Pro Ala Arg Met Leu Cys
Gly Ile 850 855 860 Val Ser Gly Gln Phe His Thr Gly Asn Ser Tyr Asp
His Asp Tyr Ala 865 870 875 880 Lys Leu Ser Arg Glu Leu Phe Ala Lys
Ala Ala Ser Lys Lys Ala Ser 885 890 895 Ser Leu Leu Leu Glu Leu Arg
Gly Arg Gly Gly Leu Gly Leu Cys Ile 900 905 910 Asn Lys Lys Asn Ser
Ala Met Gly Arg Arg Met Gly Gly Thr Gly Arg 915 920 925 Ser Gly Arg
Asp Gly Arg Ser Gly Arg Asp Tyr Gly Cys Leu Ile Glu 930 935 940 Met
His Ala Leu His Thr Ser Ala Cys Trp Gly Ala Trp Gly Leu Ser 945 950
955 960 Thr Pro Gly Cys Leu Ile Glu Met His Ala Leu His Thr Ser Ala
Cys 965 970 975 Trp Gly Ala Trp Gly Leu Ser Thr Pro Leu Thr His Ile
Ser Glu Asp 980 985 990 Leu Asp Met Ile Arg Tyr Ile Asp Glu Phe Gly
Gln Thr Thr Thr Arg 995 1000 1005 Met Gln Lys Lys Cys Phe Ile Cys
Glu Ile Cys Asp Ala Ile Ala 1010 1015 1020 Leu Phe Val Thr Ile Ile
Ser Cys Asn Lys Gln Val Asn Asn Asn 1025 1030 1035 Asn Cys Ile His
Phe Met Phe Gln Val Gln Gly Glu Val Trp Glu 1040 1045 1050 Val Phe
Ser Lys Asn Leu Tyr Lys Cys Gly Met Ala Asp Tyr Asp 1055 1060 1065
Leu Ser Arg His Tyr Thr Ser Asn Ile Pro Tyr Pro Leu Tyr Lys 1070
1075 1080 Leu Lys Ser Arg Tyr Thr Ile Phe Glu His Ser Tyr Gln Thr
Leu 1085 1090 1095 Tyr Ala Cys Val Glu Glu Lys Thr Val Cys Tyr Asp
Tyr Asn Cys 1100 1105 1110 Tyr Ala Tyr Leu Arg Leu Gln Asn Ile Phe
Pro Phe Ser Cys Ile 1115 1120 1125 Ala Val Gln Leu Phe Pro Leu Trp
Cys Lys Gln Ser Lys Gln Glu 1130 1135 1140 Phe Tyr Tyr Thr Gln His
Asp Ser Lys Asn Leu Ala Ile Leu Lys 1145 1150 1155 Glu Ser Pro Trp
Gly Leu Leu Pro Phe Ser Ser Phe Leu Glu Glu 1160 1165 1170 Asn Val
Glu Ser Gln Gln Pro His His His Met Ala Phe Leu Leu 1175 1180 1185
Ser Lys Thr Gly Phe Pro His Arg His Ser Thr Thr Ala Pro Ile 1190
1195 1200 His Gln Phe His Arg Leu Glu Ser Lys Ile His Lys Gln Leu
Glu 1205 1210 1215 Ser Val Val His Ile Ile His Leu Lys Ile Leu Tyr
Leu Pro Ser 1220 1225 1230 Phe Lys Ser Leu Val Val Cys Pro Ile Met
Ser His His Arg Ser 1235 1240
1245 Lys Val Pro Ser Gln Arg Ser Ser Ser Ala Gln Leu Thr Leu Thr
1250 1255 1260 Lys Gly Asn Lys Ser Trp Ser Ser Thr Ala Val Ala Ala
Ala Leu 1265 1270 1275 Glu Leu Val Asp Pro Pro Gly Cys Arg Asn Ser
Ile Ser Ser Leu 1280 1285 1290 58 1279 PRT Homo sapiens 58 Gly Leu
Pro Ile Val Ser Arg Ile Asn Phe Asp Lys Pro Val Lys Gln 1 5 10 15
Trp Val Leu Leu Ala Arg Glu Leu Cys Leu Tyr Arg Pro Pro Thr Val 20
25 30 His Ala Tyr Arg Pro Phe Ala Ser Met Gly Arg Ser Cys Tyr Asp
Ile 35 40 45 Leu Glu Ser Pro Val Asp Phe Gly Ala Lys Thr Asn Ser
His Arg Gln 50 55 60 Trp Gly Gly Asp Leu Glu Ile Pro Val Ser Gln
Thr Ala Ile His Ala 65 70 75 80 His Cys Thr Ala Lys Thr Ala Ser Pro
Trp Arg Leu Ile Arg Arg Cys 85 90 95 Thr Ala Lys Glu Ser Pro Ile
Arg Ser Cys Thr Gly His Asn Ala Arg 100 105 110 Arg Ala Ile Tyr Arg
His Arg Gln Gly Ala Tyr Leu Ala Tyr Asp Thr 115 120 125 Leu Asp Val
Leu Pro Ser Gly Gln Phe Thr Val Asn Ser Pro Pro Ile 130 135 140 Asp
Val Asn Gly Lys Ser Leu Leu Ala Leu Leu Trp Glu His Thr Ser 145 150
155 160 Leu Leu Thr Ser Met Gly Gly Gly Arg Trp Ala Val Ser Gln Ala
Gly 165 170 175 His Leu Pro Val Met Arg Gly Thr Pro Tyr Met Gly Tyr
Glu Leu Met 180 185 190 Thr Pro Leu Ile Thr Ile Asn Asn Ser Ile Ile
Asn Val Asn Ala His 195 200 205 Glu Thr Ile Thr Leu Ile Asn Ala Ser
Ile Ile Leu Lys Lys Glu Glu 210 215 220 Tyr Glu Tyr Ser Thr Phe Pro
Cys Arg Pro Tyr Ser Leu Phe Cys Gly 225 230 235 240 Ile Leu Pro Ser
Cys Phe Cys Ser Pro Arg Asn Ala Gly Glu Ser Lys 245 250 255 Arg Cys
Arg Ser Val Gly Cys Thr Ser Gly Leu His Arg Thr Gly Ser 260 265 270
Gln Gln Arg Asp Pro Glu Phe Ser Pro Arg Arg Thr Phe Ser Asn Asp 275
280 285 Glu His Phe Ser Ser Ala Met Trp Arg Gly Ile Ile Pro Cys Arg
Arg 290 295 300 Ala Arg Ala Thr Arg Ser Pro His Thr Leu Phe Ser Glu
Leu Gly Val 305 310 315 320 Leu Thr Ser His Arg Lys Ala Ser Tyr Gly
Trp His Asp Ser Lys Arg 325 330 335 Ile Met Gln Cys Cys His Asn His
Glu His Cys Gly Gln Leu Thr Ser 340 345 350 Asp Asn Asp Arg Arg Thr
Glu Gly Ala Asn Arg Phe Phe Ala Gln His 355 360 365 Gly Gly Ser Cys
Asn Ser Pro Ser Leu Gly Thr Gly Ala Glu Ser His 370 375 380 Thr Lys
Arg Arg Ala His His Asp Ala Cys Ser Asn Gly Asn Asn Val 385 390 395
400 Ala Gln Thr Ile Asn Trp Arg Thr Thr Tyr Ser Ser Phe Pro Ala Thr
405 410 415 Ile Asn Arg Leu Asp Gly Gly Gly Ser Cys Arg Thr Thr Ser
Ala Leu 420 425 430 Gly Pro Ser Gly Trp Leu Val Tyr Cys Ile Trp Ser
Arg Ala Trp Val 435 440 445 Ser Arg Tyr His Cys Ser Thr Gly Ala Arg
Trp Ala Leu Pro Tyr Arg 450 455 460 Ser Tyr Leu His Asp Gly Glu Ser
Gly Asn Tyr Gly Thr Lys Thr Asp 465 470 475 480 Arg Asp Arg Cys Leu
Thr Asp Ala Leu Val Thr Val Arg Pro Ser Leu 485 490 495 Leu Ile Tyr
Thr Leu Asp Phe Lys Thr Ser Phe Leu Ile Lys Asp Leu 500 505 510 Gly
Glu Asp Pro Phe Ser His Asp Gln Asn Pro Leu Thr Val Phe Val 515 520
525 Pro Leu Ser Val Arg Pro Arg Arg Lys Asp Gln Arg Ile Phe Leu Arg
530 535 540 Ser Phe Phe Ser Ala Arg Asn Leu Leu Leu Ala Asn Lys Lys
Thr Thr 545 550 555 560 Ala Thr Ser Gly Gly Leu Phe Ala Gly Ser Arg
Ala Thr Asn Ser Phe 565 570 575 Ser Glu Gly Asn Trp Leu Gln Gln Ser
Ala Asp Thr Lys Tyr Cys Pro 580 585 590 Ser Ser Val Ala Val Val Arg
Pro Pro Leu Gln Glu Leu Cys Ser Thr 595 600 605 Ala Tyr Ile Pro Arg
Ser Ala Asn Pro Val Thr Ser Gly Cys Cys Gln 610 615 620 Trp Arg Val
Val Ser Tyr Arg Val Gly Leu Lys Thr Ile Val Thr Gly 625 630 635 640
Gly Ala Ala Val Gly Leu Asn Gly Gly Phe Val His Thr Ala Gln Leu 645
650 655 Gly Ala Asn Asp Leu His Arg Thr Glu Ile Pro Thr Ala Ala Leu
Arg 660 665 670 Lys Arg His Ala Ser Arg Arg Glu Lys Gly Gly Gln Val
Ser Gly Lys 675 680 685 Arg Gln Gly Arg Asn Arg Arg Ala His Glu Gly
Ala Ser Arg Gly Lys 690 695 700 Arg Leu Val Ser Leu Ser Cys Arg Val
Ser Pro Pro Leu Thr Ala Ser 705 710 715 720 Ile Phe Val Met Leu Val
Arg Gly Ala Glu Pro Met Glu Lys Arg Gln 725 730 735 Gln Arg Gly Leu
Phe Thr Val Pro Gly Leu Leu Leu Ala Phe Cys Ser 740 745 750 His Val
Leu Ser Cys Val Ile Pro Phe Cys Gly Pro Tyr Tyr Arg Leu 755 760 765
Val Ser Tyr Arg Ser Pro Gln Pro Asn Asp Arg Ala Gln Arg Val Ser 770
775 780 Glu Arg Gly Ser Gly Arg Ala Pro Asn Thr Gln Thr Ala Ser Pro
Arg 785 790 795 800 Ala Leu Ala Asp Ser Leu Met Gln Leu Ala Arg Gln
Val Ser Arg Leu 805 810 815 Glu Ser Gly Gln Ala Gln Arg Asn Cys Glu
Leu Ala His Ser Leu Gly 820 825 830 Thr Pro Gly Phe Thr Leu Tyr Ala
Ser Gly Ser Tyr Val Val Trp Asn 835 840 845 Cys Glu Arg Ile Thr Ile
Ser His Arg Lys Gln Leu Pro Leu Arg Gln 850 855 860 Ala Leu Glu Gly
Ala Phe Cys Lys Ser Leu Gly Leu Gln Lys Ser Leu 865 870 875 880 Leu
Thr Thr Ser Gly Ile Ala Gln Arg Pro Arg Arg Pro Arg Pro Leu 885 890
895 His Lys Lys Lys Leu Val Ser His Gly Ala Glu Asn Gly Arg Asn Trp
900 905 910 Ala Glu Leu Gly Ala Gly Trp Ala Glu Leu Gly Ala Gly Leu
Trp Leu 915 920 925 Leu Thr Asn Asp Ala Cys Phe Ala Tyr Phe Cys Leu
Leu Gly Ser Leu 930 935 940 Gly Thr Phe His Thr Trp Leu Leu Thr Asn
Asp Ala Cys Phe Ala Tyr 945 950 955 960 Phe Cys Leu Leu Gly Ser Leu
Gly Thr Phe His Thr Leu Thr Asp Thr 965 970 975 His Phe Gly Arg Ser
Arg His Asp Lys Ile His Val Trp Thr Asn His 980 985 990 Asn Asn Ala
Val Lys Lys Met Leu Tyr Leu Asn Leu Cys Tyr Cys Phe 995 1000 1005
Ile Cys Asn His Tyr Lys Leu Gln Thr Ser Gln Gln Gln Leu His 1010
1015 1020 Ser Phe Tyr Val Ser Gly Ser Gly Gly Gly Val Gly Gly Phe
Leu 1025 1030 1035 Lys Gln Val Lys Pro Leu Gln Met Trp Tyr Gly Leu
Ser Leu Val 1040 1045 1050 Lys Ala Leu Tyr Ile Lys Tyr Ser Leu Leu
Thr Pro Leu Gln Ile 1055 1060 1065 Lys Lys Leu Lys Val His Asn Phe
Ala Leu Leu Ile Ala Asp Thr 1070 1075 1080 Leu Cys Leu Cys Gly Val
Arg Lys Asn Ser Met Leu Leu Leu Leu 1085 1090 1095 Cys Leu Leu Ile
Lys Val Thr Glu Tyr Phe Ser Ile Ile Phe Leu 1100 1105 1110 Tyr Ser
Ser Ala Ala Phe Ser Phe Val Val Ile Ala Lys Gln Ala 1115 1120 1125
Arg Val Leu Leu Leu Asn Thr Ala Leu Lys Lys Leu Ser Asn Ser 1130
1135 1140 Glu Gly Lys Ser Leu Gly Ser Ser Thr Phe Leu Phe Phe Phe
Gly 1145 1150 1155 Gly Val Glu Cys Glu Ser Ala Val Ala Ser Ser Ser
Leu Asp Gly 1160 1165 1170 Ile Ser Ser Glu Gln Asn Arg Phe Ser Ser
Leu Lys Ala Phe His 1175 1180 1185 His Cys Ser His Ser Ser Val Pro
Val Gly Ile Asn Thr Gln Thr 1190 1195 1200 Ile Arg Ile Ser Ser Leu
Thr His Tyr Thr Leu Lys Asn Phe Ile 1205 1210 1215 Phe Thr Leu Glu
Leu Ile Ser Val Gly Ser Leu Ser Asn Tyr Val 1220 1225 1230 Thr Pro
Gln Lys Gly Ser Phe Thr Lys Ile Leu Arg Ala Ile Asn 1235 1240 1245
Pro His Arg Glu Gln Lys Leu Glu Leu His Arg Gly Gly Gly Arg 1250
1255 1260 Ser Arg Thr Ser Gly Ser Pro Gly Leu Gln Glu Phe Asp Ile
Lys 1265 1270 1275 Leu 59 1284 PRT Homo sapiens 59 Gly Ser Pro Tyr
Ser Glu Ser Tyr Phe Arg Ala Ser Ala Val Gly Ser 1 5 10 15 Leu Val
Ser Gln Arg Ala Leu Leu Ile Thr Ser His Arg Thr Arg Leu 20 25 30
Pro Pro Ile Cys Val Asn Gly Ala Glu Leu Leu Arg His Phe Gly Lys 35
40 45 Ser Arg Phe Trp Cys Gln Asn Lys Leu Pro Leu Thr Ser Met Gly
Trp 50 55 60 Arg Leu Gly Asn Pro Arg Glu Ser Asn Arg Tyr Pro Arg
Pro Leu Met 65 70 75 80 Tyr Cys Gln Asn Arg Ile Thr Met Val Ile Ala
Met Thr Asn Thr Met 85 90 95 Tyr Cys Gln Val Gly Lys Ser His Lys
Val Met Tyr Trp Ala Cys Gln 100 105 110 Ala Gly His Leu Pro Ser Leu
Thr Ser Ile Gly Gly Val Leu Gly Ile 115 120 125 Tyr Thr Cys Thr Ala
Lys Trp Ala Val Tyr Arg Lys Ser Thr His Arg 130 135 140 Gln Trp Lys
Val Pro Ile Gly Val Thr Met Gly Thr Tyr Val Ile Ile 145 150 155 160
Asp Val Asn Gly Arg Gly Ser Leu Gly Gly Gln Pro Gly Gly Pro Phe 165
170 175 Thr Val Ser Tyr Val Thr Arg Asn Ser Ile Tyr Gly Leu Thr Asn
Asp 180 185 190 Pro Val Ile Asp Tyr Tyr Leu Val Asn Asn Gln Cys Gln
Arg Ala Asp 195 200 205 Asn Asn Pro Asp Lys Cys Phe Asn Asn Ile Glu
Lys Gly Arg Val Val 210 215 220 Phe Asn Ile Ser Val Ser Pro Leu Phe
Pro Phe Leu Arg His Phe Ala 225 230 235 240 Phe Leu Phe Leu Leu Thr
Gln Lys Arg Trp Lys Lys Met Leu Lys Ile 245 250 255 Ser Trp Val His
Glu Trp Val Thr Ser Asn Trp Ile Ser Thr Ala Val 260 265 270 Arg Ser
Leu Arg Val Phe Ala Pro Lys Asn Val Phe Gln Ala Leu Leu 275 280 285
Lys Phe Cys Tyr Val Ala Arg Tyr Tyr Pro Val Leu Thr Pro Gly Lys 290
295 300 Ser Asn Ser Val Ala Ala Tyr Thr Ile Leu Arg Met Thr Trp Leu
Ser 305 310 315 320 Thr His Gln Ser Gln Lys Ser Ile Leu Arg Met Ala
Gln Glu Asn Tyr 325 330 335 Ala Val Leu Pro Pro Val Ile Thr Leu Arg
Pro Thr Tyr Phe Gln Arg 340 345 350 Ser Glu Asp Arg Arg Ser Pro Leu
Phe Cys Thr Thr Trp Gly Ile Met 355 360 365 Leu Ala Leu Ile Val Gly
Asn Arg Ser Met Lys Pro Tyr Gln Thr Thr 370 375 380 Ser Val Thr Pro
Arg Cys Leu Gln Trp Gln Gln Arg Cys Ala Asn Tyr 385 390 395 400 Leu
Ala Asn Tyr Leu Leu Leu Pro Gly Asn Asn Thr Gly Trp Arg Arg 405 410
415 Ile Lys Leu Gln Asp His Phe Cys Ala Arg Pro Phe Arg Leu Ala Gly
420 425 430 Leu Leu Leu Ile Asn Leu Glu Pro Val Ser Val Gly Leu Ala
Val Ser 435 440 445 Leu Gln His Trp Gly Gln Met Val Ser Pro Pro Val
Ser Leu Ser Thr 450 455 460 Arg Arg Gly Val Arg Gln Leu Trp Met Asn
Glu Ile Asp Arg Ser Leu 465 470 475 480 Arg Val Pro His Leu Ser Ile
Gly Asn Cys Gln Thr Lys Phe Thr His 485 490 495 Ile Tyr Phe Arg Leu
Ile Asn Phe Ile Phe Asn Leu Lys Gly Ser Arg 500 505 510 Arg Ser Phe
Leu Ile Ile Ser Pro Lys Ser Leu Asn Val Ser Phe Arg 515 520 525 Ser
Thr Glu Arg Gln Thr Pro Lys Arg Ser Lys Asp Leu Leu Glu Ile 530 535
540 Leu Phe Phe Cys Ala Ser Ala Ala Cys Lys Gln Lys Asn His Arg Tyr
545 550 555 560 Gln Arg Trp Phe Val Cys Arg Ile Lys Ser Tyr Gln Leu
Phe Phe Arg 565 570 575 Arg Leu Ala Ser Ala Glu Arg Arg Tyr Gln Ile
Leu Ser Phe Cys Ser 580 585 590 Arg Ser Ala Thr Thr Ser Arg Thr Leu
His Arg Leu His Thr Ser Leu 595 600 605 Cys Ser Cys Tyr Gln Trp Leu
Leu Pro Val Ala Ile Ser Arg Val Leu 610 615 620 Pro Gly Trp Thr Gln
Asp Asp Ser Tyr Arg Ile Arg Arg Ser Gly Arg 625 630 635 640 Ala Glu
Arg Gly Val Arg Ala His Ser Pro Ala Trp Ser Glu Arg Pro 645 650 655
Thr Pro Asn Asp Thr Tyr Ser Val Ser Ile Glu Lys Ala Pro Arg Phe 660
665 670 Pro Lys Gly Glu Arg Arg Thr Gly Ile Arg Ala Ala Gly Ser Glu
Gln 675 680 685 Glu Ser Ala Arg Gly Ser Phe Gln Gly Glu Thr Pro Gly
Ile Phe Ile 690 695 700 Val Leu Ser Gly Phe Ala Thr Ser Asp Leu Ser
Val Asp Phe Cys Asp 705 710 715 720 Ala Arg Gln Gly Gly Gly Ala Tyr
Gly Lys Thr Pro Ala Thr Arg Pro 725 730 735 Phe Tyr Gly Ser Trp Pro
Phe Ala Gly Leu Leu Leu Thr Cys Ser Phe 740 745 750 Leu Arg Tyr Pro
Leu Ile Leu Trp Ile Thr Val Leu Pro Pro Leu Ser 755 760 765 Glu Leu
Ile Pro Leu Ala Ala Ala Glu Arg Pro Ser Ala Ala Ser Gln 770 775 780
Ala Arg Lys Arg Lys Ser Ala Gln Tyr Ala Asn Arg Leu Ser Pro Arg 785
790 795 800 Val Gly Arg Phe Ile Asn Ala Ala Gly Thr Thr Gly Phe Pro
Thr Gly 805 810 815 Lys Arg Ala Val Ser Ala Thr Gln Leu Met Val Ser
Ser Leu Ile Arg 820 825 830 His Pro Arg Leu Tyr Thr Leu Cys Phe Arg
Leu Val Cys Cys Val Glu 835 840 845 Leu Ala Asp Asn Asn Phe Thr Gln
Glu Thr Ala Met Thr Met Ile Thr 850 855 860 Pro Ser Ser Arg Gly Ser
Phe Leu Gln Lys Pro Arg Pro Pro Lys Lys 865 870 875 880 Pro Pro His
Tyr Phe Trp Asn Ser Ser Glu Ala Glu Ala Ala Ser Ala 885 890 895 Ser
Ala Ile Lys Lys Ile Ser Gln Pro Trp Gly Gly Glu Trp Ala Glu 900 905
910 Leu Gly Gly Val Arg Gly Gly Met Gly Gly Val Arg Gly Gly Thr Met
915 920 925 Val Ala Asp Leu Arg Cys Met Leu Cys Ile Leu Leu Pro Ala
Gly Glu 930 935 940 Pro Gly Asp Phe Pro His Leu Val Ala Asp Leu Arg
Cys Met Leu Cys 945 950 955 960 Ile Leu Leu Pro Ala Gly Glu Pro Gly
Asp Phe Pro His Pro Asn His 965 970 975 Thr Phe Arg Lys Ile Thr Asp
Thr Leu Met Ser Leu Asp Lys Pro Gln 980 985 990 Leu Glu Cys Ser Glu
Lys Asn Ala Leu Phe Val Lys Phe Val Met Leu 995 1000 1005 Leu Leu
Tyr Leu Pro Leu Ala Ala Ile Asn Lys Leu Thr Thr Thr 1010 1015 1020
Ile Ala Phe Ile Leu Cys Phe Arg Phe Arg Gly Arg Cys Gly Arg 1025
1030 1035 Phe Phe Lys Ala Ser Lys Thr Ser Thr Asn Val Val Trp Leu
Ile 1040 1045 1050 Met Ile Ser Ser Gln Gly Thr Ile His Gln Ile Phe
Leu Ile Asn 1055 1060 1065 Pro Phe Thr Asn Lys Ala Lys Gly Thr Gln
Phe Leu Ser Ile Val 1070 1075 1080 Ile Asn Ser Arg His Ser Met Pro
Val Trp Ser Lys Lys Lys Gln 1085 1090 1095 Tyr Val Met Ile Ile Thr
Val Met Pro Thr Tyr Lys Gly Tyr Arg
1100 1105 1110 Ile Phe Phe His Asn Phe Leu Val Gln Cys Ser Phe Phe
Leu Cys 1115 1120 1125 Gly Val Asn Ser Lys Ala Ser Lys Ser Ser Ile
Thr Lys His Ser 1130 1135 1140 Met Thr Gln Lys Thr Gln Phe Arg Lys
Val Leu Gly Val Phe Tyr 1145 1150 1155 Leu Ser Leu Leu Phe Trp Arg
Ser Arg Met Leu Arg Val Ser Ser 1160 1165 1170 Ser Leu Ile Ile Thr
Arg Trp His Phe Phe Ala Lys Gln Val Phe 1175 1180 1185 Leu Ile Lys
Gly Ile Pro Pro Leu Leu Pro Phe Ile Ser Ser Ile 1190 1195 1200 Gly
Trp Asn Leu Lys Tyr Thr Asn Asn Asn Gln Phe Asn Thr Leu 1205 1210
1215 Tyr Thr Lys Phe Tyr Ile Tyr Leu Arg Ala Leu Asn Leu Cys Arg
1220 1225 1230 Phe Val Gln Leu Cys His Thr Thr Glu Val Arg Phe Leu
His Lys 1235 1240 1245 Asp Pro Leu Ala Arg Asn Pro Ser Leu Lys Gly
Thr Lys Ala Gly 1250 1255 1260 Ala Pro Pro Arg Trp Arg Pro Leu Asn
Trp Ile Pro Arg Ala Ala 1265 1270 1275 Gly Ile Arg Tyr Gln Ala 1280
60 354 PRT Homo sapiens 60 Met Gly Asn His Thr Trp Glu Gly Cys His
Val Asp Ser Arg Val Asp 1 5 10 15 His Leu Phe Pro Pro Ser Leu Tyr
Ile Phe Val Ile Gly Val Gly Leu 20 25 30 Pro Thr Asn Cys Leu Ala
Leu Trp Ala Ala Tyr Arg Gln Val Gln Gln 35 40 45 Arg Asn Glu Leu
Gly Val Tyr Leu Met Asn Leu Ser Ile Ala Asp Leu 50 55 60 Leu Tyr
Ile Cys Thr Leu Pro Leu Trp Val Asp Tyr Phe Leu His His 65 70 75 80
Asp Asn Trp Ile His Gly Pro Gly Ser Cys Lys Leu Phe Gly Phe Ile 85
90 95 Phe Tyr Thr Asn Ile Tyr Ile Ser Ile Ala Phe Leu Cys Cys Ile
Ser 100 105 110 Val Asp Arg Tyr Leu Ala Val Ala His Pro Leu Arg Phe
Ala Arg Leu 115 120 125 Arg Arg Val Lys Thr Ala Val Ala Val Ser Ser
Val Val Trp Ala Thr 130 135 140 Glu Leu Gly Ala Asn Ser Ala Pro Leu
Phe His Asp Glu Leu Phe Arg 145 150 155 160 Asp Arg Tyr Asn His Thr
Phe Cys Phe Glu Lys Phe Pro Met Glu Gly 165 170 175 Trp Val Ala Trp
Met Asn Leu Tyr Arg Val Phe Val Gly Phe Leu Phe 180 185 190 Pro Trp
Ala Leu Met Leu Leu Ser Tyr Arg Gly Ile Leu Arg Ala Val 195 200 205
Arg Gly Ser Val Ser Thr Glu Arg Gln Glu Lys Ala Lys Ile Lys Arg 210
215 220 Leu Ala Leu Ser Leu Ile Ala Ile Val Leu Val Cys Phe Ala Pro
Tyr 225 230 235 240 His Val Leu Leu Leu Ser Arg Ser Ala Ile Tyr Leu
Gly Arg Pro Trp 245 250 255 Asp Cys Gly Phe Glu Glu Arg Val Phe Ser
Ala Tyr His Ser Ser Leu 260 265 270 Ala Phe Thr Ser Leu Asn Cys Val
Ala Asp Pro Ile Leu Tyr Cys Leu 275 280 285 Val Asn Glu Gly Ala Arg
Ser Asp Val Ala Lys Ala Leu His Asn Leu 290 295 300 Leu Arg Phe Leu
Ala Ser Asp Lys Pro Gln Glu Met Ala Asn Ala Ser 305 310 315 320 Leu
Thr Leu Glu Thr Pro Leu Thr Ser Lys Arg Asn Ser Thr Ala Lys 325 330
335 Ala Met Thr Gly Ser Trp Ala Ala Thr Pro Pro Pro Arg Gly Thr Arg
340 345 350 Cys Ser
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