U.S. patent application number 10/290198 was filed with the patent office on 2003-06-12 for sirp proteins and uses thereof.
This patent application is currently assigned to Max-Planck-Gesellschaft Zur Foderung Der Wissenschaften E.V.. Invention is credited to Chen, Zhengjun, Kharitonenkov, Alexei, Ullrich, Axel.
Application Number | 20030109002 10/290198 |
Document ID | / |
Family ID | 26706645 |
Filed Date | 2003-06-12 |
United States Patent
Application |
20030109002 |
Kind Code |
A1 |
Ullrich, Axel ; et
al. |
June 12, 2003 |
SIRP proteins and uses thereof
Abstract
The present invention features isolated, purified, or enriched
nucleic acid encoding a SIRP polypeptide and isolated, purified, or
enriched SIRP polypeptide and uses thereof.
Inventors: |
Ullrich, Axel; (Munchen,
DE) ; Kharitonenkov, Alexei; (Martinsried, DE)
; Chen, Zhengjun; (Graefelfing, DE) |
Correspondence
Address: |
FOLEY AND LARDNER
SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
Max-Planck-Gesellschaft Zur
Foderung Der Wissenschaften E.V.
|
Family ID: |
26706645 |
Appl. No.: |
10/290198 |
Filed: |
November 8, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10290198 |
Nov 8, 2002 |
|
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08999689 |
Nov 14, 1997 |
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60030964 |
Nov 15, 1996 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 530/350; 536/23.2 |
Current CPC
Class: |
Y02A 50/30 20180101;
Y02A 50/473 20180101; C12N 9/1205 20130101; A61K 38/00 20130101;
C07K 16/18 20130101; C12N 9/16 20130101; C07K 14/4703 20130101;
C07K 16/40 20130101 |
Class at
Publication: |
435/69.1 ;
435/320.1; 435/325; 530/350; 536/23.2 |
International
Class: |
C12P 021/02; C12N
005/06; C07K 014/47; C07H 021/04 |
Claims
What is claimed is:
1. An isolated, purified, or enriched nucleic acid encoding a SIRP
polypeptide.
2. The nucleic acid of claim 1, wherein said SIRP polypeptide is a
mammalian SIRP polypeptide.
3. The nucleic acid of claim 2, wherein said mammalian SIRP
polypeptide is a human SIRP polypeptide.
4. A nucleic acid probe for the detection of nucleic acid encoding
a SIRP polypeptide in a sample.
5. Recombinant nucleic acid encoding a SIRP polypeptide and a
vector or a promoter effective to initiate transcription in a host
cell.
6. An isolated, purified, recombinant, or enriched SIRP
polypeptide.
7. The SIRP polypeptide of claim 5, wherein said SIRP polypeptide
is a mammalian SIRP polypeptide.
8. The SIRP polypeptide of claim 6, wherein said SIRP polypeptide
is a human SIRP polypeptide.
9. A purified antibody having specific binding affinity to a SIRP
polypeptide.
10. A hybridoma which produces an antibody having specific binding
affinity to a SIRP polypeptide.
11. A method of detecting a compound capable of binding to a SIRP
polypeptide comprising the steps of incubating said compound with
said SIRP polypeptide and detecting the presence of said compound
bound to said SIRP polypeptide.
12. A method of screening potential agents useful for treatment of
a disease or condition characterized by an abnormality in a signal
transduction pathway, wherein said signal transduction pathway
includes an interaction between a SIRP polypeptide and a natural
binding partner, comprising the step of assaying said potential
agents for those able to promote or disrupt said interaction as an
indication of a useful said agent.
13. A method for diagnosis of a disease or condition characterized
by an abnormality in a signal transduction pathway, wherein said
signal transduction pathway includes an interaction between a SIRP
polypeptide and a natural binding partner, comprising the step of
detecting the level of said interaction as an indication of said
disease or condition.
14. A method for treatment of an organism having a disease or
condition characterized by an abnormality in a signal transduction
pathway, wherein said signal transduction pathway includes an
interaction between a SIRP polypeptide and a natural binding
partner comprising the step of promoting or disrupting said
interaction.
15. An isolated nucleic acid molecule comprising a nucleotide
sequence that; (a) encodes a polypeptide having the full length
amino acid sequence set forth in SEQ ID NO.: 5, SEQ ID NO: 6, SEQ
ID NO: 7, or SEQ ID NO: 8; (b) the complement of the nucleotide
sequence of (a) or; (c) hybridizes under highly stringent
conditions to the nucleotide sequence of (a) and encodes a
naturally occurring SIRP protein.
16. A nucleic acid molecule comprising a nucleotide sequence that
encodes (a) a SIRP protein having the full length amino acid
sequence of sequence set forth in SEQ ID NO.: 5, SEQ ID NO: 6, SEQ
ID NO: 7, or SEQ ID NO: 8 except that it lacks one of the following
segments of amino acid residues: extracellular domain,
transmembrane domain, cytoplasmic domain, tyrosine bearing SH2
binding region in the cytoplasmic domain; or (b) the complement of
the nucleotide sequence of (a).
17. An isolated nucleic acid molecule comprising a nucleotide
sequence that encodes a polypeptide having the full length amino
acid sequence set forth in SEQ ID NO: 5; SEQ ID NO: 6, SEQ ID NO:
7, or SEQ ID NO: 8 except that it lacks at least one, but not more
than two, of the domains selected from the group consisting of the
extracellular domain, the transmembrane domain, and the SHP-2
binding domain.
18. A recombinant vector containing the nucleotide sequence of any
one of claims 14-17.
19. A genetically engineered host cell containing the nucleotide
sequence of any one of claims 14-18.
Description
FIELD OF THE INVENTION
[0001] This invention relates to receptor tyrosine kinase
associated proteins and their uses.
BACKGROUND OF THE INVENTION
[0002] Signal transduction is a fundamental mechanism whereby
external stimuli are relayed to the interior of cells. A key aspect
of signal transduction involves the reversible phosphorylation of
tyrosine residues on proteins. The phosphorylation state of
tyrosine residues on a protein is modified through the reciprocal
actions of tyrosine kinases (TKs) and tyrosine phosphatases
(TPs).
[0003] For example, a variety of polypeptide growth factors and
hormones mediate their cellular effects by interacting with cell
surface receptors and soluble or cytoplasmic polypeptide containing
molecules having tyrosine kinase enzymatic activity (for review,
see Williams, et al. Cell 61:203-212 (1990); Carpenter, et al. J.
Biol. Chem. 265:7709-7712 (1990)). The interaction of these ligands
with their receptors induces a series of events which include
receptor dimerization and stimulation of protein tyrosine kinase
activity. Tyrosine autophosphorylation on multiple sites creates
specific binding sites for target proteins, which bind to the
activated receptor with their SH2 domains (for review, see
Schlessinger and Ullrich, Neuron 9:383-391, (1992)).
[0004] SH2 (src homology 2) domains are conserved sequences of
about 100 amino acids found in cytoplasmic non-receptor tyrosine
kinases such as pp60src, PLC-.gamma., GAP and v-crk (Mayer, et al.,
Nature 332:272-275 (1988); Pawson, Oncogene 3:491-495 (1988)).
While having distinct catalytic domains, all these molecules share
conserved SH2 and SH3 (src homology 3) domains and the ability to
associate with receptors with tyrosine kinase activity (Anderson,
et al. Science 250:979-982 (1990)).
[0005] Tyrosine kinase activation and receptor autophosphorylation
are prerequisites for the association between growth factor
receptors and SH2 domain-containing proteins (Margolis, et al.,
Mol. Cell. Biol. 10:435-441- (1990); Kumjian et al., Proc. Natl.
Acad. Sci. USA 86:6232-8239 (1989); Kazlauskas, et al., Science
247:1578-1581 (1990)). In particular, the carboxy-terminal
(C-terminal) fragment of the epidermal growth factor receptor
(EGFR), which contains all the known autophosphorylation sites,
binds specifically to the SH2 domains of GAP and PLC-.gamma. (see
below). Hence, a major site of association exists between the SH2
domain of these substrate proteins and the tyrosine phosphorylated
C-terminal tail of the EGFR.
[0006] Target proteins which bind to activated receptors have been
identified by analysis of proteins that co-immunoprecipitate with
growth factor receptors, or that bind to receptors attached
to-immobilized matrices (Morrison, et al., Cell 58:649-657 (1989);
Kazlauskas, et al., EMBO J. 9:3279-3286 (1990)).
[0007] Ohnishi et al. J. Biol. Chem. 271:25569-25574 (1996), not
admitted to be prior art, described that a brain specific
immunoglobulin-like molecule with tyrosine-based activation motifs,
BIT, is associated with protein-tyrosine phosphatase SH-PTP2,
whereby two SH2 domains of SH-PTP2 simultaneously interact with two
phosphotyrosines of BIT-TAM.
[0008] Phosphotyrosine phosphatases (PTPs) are involved with
negative or positive regulation of growth factor-specific cell
responses such as mitosis, differentiation, migration, survival,
transformation or death. For example, SHP-2 is a phosphotyrosine
phosphatase which contains a SH2 domain. SHP-2 is a positive signal
transducer for a number of receptor tyrosine kinases (RTKs) and
cytokine receptors.
SUMMARY OF THE INVENTION
[0009] Within the scope of this invention, applicant has identified
a novel mammalian protein family of at least fifteen members
designated SIgnal Regulatory Proteins (SIRPs). In particular,
Applicant has cloned and sequenced the coding sequences of 4
members of SIRPs, SIRP1 and SIRP4 from human, and SIRP.alpha.1 and
SIRP.beta.1 from mouse. In this regard, the present invention
relates to SIRP polypeptides, nucleic acids encoding such
polypeptides, cells, tissues and animals containing such
polypeptides or nucleic acids, antibodies to such polypeptides or
nucleic acids, assays utilizing such polypeptides or nucleic acids,
and methods relating to all of the foregoing.
[0010] SIRP family proteins play a general role in the regulation
of signals that define diverse physiological and pathological
processes. Thus, the present invention provides several agents and
methods useful for diagnosing, treating, and preventing various
diseases or conditions associated with abnormalities in these
pathways as well as assay systems useful for screening for
therapeutically effective agents.
[0011] In particular, SIRP polypeptides are involved in various
signal transduction pathways such as the negative regulation of
signals generated by receptor tyrosine kinases, including, but not
limited to, receptors for EGF, insulin and platelet derived growth
factor (PDGF). For example, acting like a tumor suppressor, SIRP4
exerts negative regulatory effects on growth factor and hormone
induced cellular responses such as DNA synthesis. Oncogenesis may
be associated with mutant SIRPs or not enough SIRPs. Restoring
SIRPs to their normal levels such as by gene therapy could restore
the cells to a normal growth pattern. Insulin receptor activity is
also regulated by SIRPS. Overexpression of SIRPs may be involved in
type II diabetes where sufficient insulin is present but insulin
signaling is deficient. A compound that inhibits the negative
regulation of insulin signaling by SIRPs, such as by interfering
with the interaction between SIRP and SHP-2 may lead to enhanced
insulin signaling.
[0012] All SIRP proteins have a receptor-like, or Immunoglubulin
(Ig) like extracellular domain and a transmembrane domain. There
are two subtypes of SIRPs distinguished by the presence or absence
of a cytoplasmic SHP-2 binding domain. For example, SIRP4 has a
cytoplasmic domain while SIRP1 doesn't. The cytoplasmic domain of
SIRP4 contains two SHP-2 binding regions each having two tyrosine
residues.
[0013] The growth inhibitory effect of SIRP4 depends on
phosphorylation of tyrosines and is related to reduced MAP kinase
activation. SIRP4 becomes a substrate of activated receptor
tyrosine kinases (RTKs) upon EGF, insulin or PDGF stimulation. In
its tyrosine phosphorylated form, SIRP4 binds a phosphotyrosine
phosphatase, SHP-2, via SH2 interactions. Once SIRP4 binds SHP-2,
it activates the catalytic activity of SHP-2 and becomes a
substrate of SHP-2. This direct activation of SHP-2 could induce
activation of Src or other Src family kinases. The above described
interaction allows SIRP4 to participate in major signal
transduction pathways involving SHP-2.
[0014] SHP-2 has two SH2 domains and is required for signaling
downstream of a variety of RTKs. SHP-2 has been reported to bind
directly to RTKs such as PDGF receptor, EGF receptor, and cKit in
response to stimulation by their ligands. Insulin receptor
substrate 1 (IRS-1) also associates with SHP-2 in response to
insulin.
[0015] SIRP4 also binds SHP-1 and Grb2, both of which contain a
SH-2 domain. Grb2 is an adapter molecule and one of its functions
is to link growth factor receptors to downstream effector proteins.
Grb2 is known to bind tyrosine-phosphorylated SHP-2 in response to
PDGF stimulation.
[0016] The full length nucleic acid sequences encoding hSIRP1,
hSIRP4, mSIRP.alpha.1 and mSIRP.beta.1 proteins are set forth
respectively in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ
ID NO: 4. The coding regions are nt 41-1237 of SEQ ID NO: 1, nt
13-1524 of SEQ ID NO: 2, nt 59-1597 of SEQ ID NO: 3, and nt 86-1261
of SEQ ID NO: 4.
[0017] The full length amino acid sequences of hSIRP1, hSIRP4,
mSIRP.alpha.1 and mSIRP.beta.1 are set forth respectively in SEQ ID
NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8. The first two
Ig-like domains of hSIRP1 is from aa 54-227; the third Ig-like
domain is from aa 250-330; the extracelluar domain next to the
membrane is from aa 336-366; and the transmembrane domain is from
aa 367-398. The first two Ig-like domains of hSIRP4 is from aa
1-227; the third Ig-like domain is from aa 250-336; the
extracelluar domain next to the membrane, the transmembrane domain,
and the cytoplasmic domain immediate next to the membrane are from
aa 347-407; and the rest of the cytoplasmic domain is from aa
408-503.
[0018] Thus, in a first aspect the invention features an isolated,
purified, enriched or recombinant nucleic acid encoding a SIRP
polypeptide. Preferably such nucleic acid encodes a mammalian SIRP
polypeptide, more preferably it encodes a human SIRP
polypeptide.
[0019] By "isolated" in reference to nucleic acid is meant a
polymer of 2 (preferably 21, more preferably 39, most preferably
75) or more nucleotides conjugated to each other, including DNA or
RNA that is isolated from a-natural source or that is synthesized.
The isolated nucleic acid of the present invention is unique in the
sense that it is not found in a pure or separated state in nature.
Use of the term "isolated" indicates that a naturally occurring
sequence has been removed from its normal cellular environment.
Thus, the sequence may be in a cell-free solution or placed in a
different cellular environment. The term does not imply that the
sequence is the only nucleotide chain present, but does indicate
that it is the predominate sequence present (at least 10-20% more
than any other nucleotide sequence) and is essentially free (about
90-95% pure at least) of non-nucleotide material naturally
associated with it. Therefore, the term does not encompass an
isolated chromosome encoding one or more SIRP polypeptides.
[0020] By the use of the term "enriched" in reference to nucleic
acid is meant that the specific DNA or RNA sequence constitutes a
significantly higher fraction (2-5 fold) of the total DNA or RNA
present in the cells or solution of interest than in normal or
diseased cells or in the cells from which the sequence was taken.
This could be caused by a person by preferential reduction in the
amount of other DNA or RNA present, or by a preferential increase
in the amount of the specific DNA or RNA sequence, or by a
combination of the two. However, it should be noted that enriched
does not imply that there are no other DNA or RNA sequences
present, just that the relative amount of the sequence of interest
has been significantly increased in a useful manner and preferably
separate from a sequence library. The term significant here is used
to indicate that the level of increase is useful to the person
making such an increase, and generally means an increase relative
to other nucleic acids of about at least 2 fold, more preferably at
least 5 to 10 fold or even more. The term also does not imply that
there is no DNA or RNA from other sources. The other source DNA
may, for example, comprise DNA from a yeast or bacterial genome, or
a cloning vector such as pUC19. This term distinguishes from
naturally occurring events, such as viral infection, or tumor type
growths, in which the level of one mRNA may be naturally increased
relative to other species of mRNA. That is, the term is meant to
cover only those situations in which a person has intervened to
elevate the proportion of the desired nucleic acid.
[0021] It is also advantageous for some purposes that a nucleotide
sequence be in purified form. The term "purified" in reference to
nucleic acid does not require absolute purity (such as a
homogeneous preparation); instead, it represents an indication that
the sequence is relatively purer than in the natural environment
(compared to the natural level this level should be at least 2-5
fold greater, e.g., in terms of mg/ml). Individual clones isolated
from a cDNA library may be purified to electrophoretic homogeneity.
The claimed DNA molecules obtained from these clones could be
obtained directly from total DNA or from total RNA. The cDNA clones
are not naturally occurring, but rather are preferably obtained via
manipulation of a partially purified naturally occurring substance
(messenger RNA). The construction of a cDNA library from mRNA
involves the creation of a synthetic substance (cDNA) and pure
individual cDNA clones can be isolated from the synthetic library
by clonal selection of the cells carrying the cDNA library. Thus,
the process which includes the construction of a cDNA library from
mRNA and isolation of distinct cDNA clones yields an approximately
10.sup.6-fold purification of the native message. Thus,
purification of at least one order of magnitude, preferably two or
three orders, and more preferably four or five orders of magnitude
is expressly contemplated.
[0022] By "SIRP polypeptide" is meant 9 or more contiguous amino
acids set forth in the full length amino acid sequence of SEQ ID
NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. The SIRP
polypeptides can be encoded by full-length nucleic acid sequences
or any portion of a full-length nucleic acid sequence, so long as a
functional activity of the polypeptide is retained. Preferred
functional activities include the ability to bind to a receptor
tyrosine kinase or a SH-2 domain bearing protein such as SHP-2,
SHP-1 or Grb-2. A non full-length SIRP polypeptide may be used to
elicit an antibody against the polypeptide and the full-length
polypeptide using techniques known to those skilled in the art. The
present invention also encompasses deletion mutants lacking one or
more isolated SIRP domains (e.g., Ig-like domain, transmembrane
domain, SH2 binding domain, and tyrosine residues), and
complementary sequences capable of hybridizing to full length SIRP
protein under stringent hybridization conditions.
[0023] In preferred embodiments, isolated nucleic acid comprises,
consists essentially of, or consists of a nucleic acid sequence set
forth in the full length nucleic acid sequence SEQ ID NO: 1, SEQ ID
NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 or at least 27, 30, 45, 60 or
90 contiguous nucleotides thereof and the SIRP polypeptide
comprises, consists essentially of, or consists of at least 9, 10,
15, 20, 30, 50, 100, 200, or 300 contiguous amino acids of a SIRP
polypeptide.
[0024] By "comprising" it is meant including, but not limited to,
whatever follows the word "comprising". Thus, use of the term
"comprising" indicates that the listed elements are required or
mandatory, but that other elements are optional and may or may not
be present. By "consisting of" is meant including, and limited to,
whatever follows the phrase "consisting of". Thus, the phrase
"consisting of" indicates that the listed elements are required or
mandatory, and that no other elements may be present. By
"consisting essentially of" is meant including any elements listed
after the phrase, and limited to other elements that do not
interfere with or contribute to the activity or action specified in
the disclosure for the listed elements. Thus, the phrase
"consisting essentially of" indicates that the listed elements are
required or mandatory, but that other elements are optional and may
or may not be present depending upon whether or not they affect the
activity or action of the listed elements.
[0025] Compositions and probes of the present invention may contain
human nucleic acids encoding a SIRP polypeptide but are
substantially free of nucleic acid not encoding SIRP polypeptide.
The human nucleic acid encoding a SIRP polypeptide is at least 18
contiguous bases of the nucleotide sequence set forth in SEQ ID NO:
1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 and will selectively
hybridize to human genomic DNA encoding a SIRP polypeptide, or is
complementary to such a sequence. The nucleic acid may be isolated
from a natural source by cDNA cloning or subtractive hybridization;
the natural source may be blood, semen, and tissue of various
organisms including eukaryotes, mammals, birds, fish, plants,
gorillas, rhesus monkeys, chimpanzees and humans; and the nucleic
acid may be synthesized by the triester method or by using an
automated DNA synthesizer. In yet other preferred embodiments the
nucleic acid is a conserved or unique region, for example those
useful for the design of hybridization probes to facilitate
identification and cloning of additional polypeptides, the design
of PCR probes to facilitate cloning of additional polypeptides, and
obtaining antibodies to polypeptide regions.
[0026] By "conserved nucleic acid regions", are meant regions
present on two or more nucleic acids encoding a SIRP polypeptide,
to which a particular nucleic acid sequence can hybridize to under
lower stringency conditions. Examples of lower stringency
conditions suitable for screening for nucleic acid encoding SIRP
polypeptides are provided in Abe, et al. J. Biol. Chem., 19:13361
(1992) (hereby incorporated by reference herein in its entirety,
including any drawings). Preferably, conserved regions differ by no
more than 7 out of 20 nucleotides.
[0027] By "unique nucleic acid region" is meant a sequence present
in a full length nucleic acid coding for a SIRP polypeptide that is
not present in a sequence coding for any other naturally occurring
polypeptide. Such regions preferably comprise 12 or 20 contiguous
nucleotides present in the full length nucleic acid encoding a SIRP
polypeptide.
[0028] The invention also features a nucleic acid probe for the
detection of a nucleic acid encoding a SIRP polypeptide in a
sample. The nucleic acid probe contains nucleic acid that will
hybridize to at least one sequence set forth in SEQ ID NO: 1, SEQ
ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.
[0029] In preferred embodiments the nucleic acid probe hybridizes
to nucleic acid encoding at least 12, 27, 30, 35, 40, 50, 100, 200,
or 300 contiguous amino acids of the full-length sequence set forth
in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.
Various low or high stringency hybridization conditions may be used
depending upon the specificity and selectivity desired.
[0030] By "high stringency hybridization conditions" is meant those
hybridizing conditions that (1) employ low ionic strength and high
temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium
citrate/0.1% SDS at 50.degree. C.; (2) employ during hybridization
a denaturing agent such as formamide, for example, 50% (vol/vol)
formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%
polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with
750 mM NaCl, 75 mM sodium citrate at 42.degree. C.; or (3) employ
50% formamide, 5.times.SSC (0.75 M NaCl, 0.075 M Sodium
pyrophosphate, 5.times.Denhardt's solution, sonicated salmon sperm
DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfate at 42.degree. C.,
with washes at 42.degree. C. in 0.2.times.SSC and 0.1% SDS. Under
stringent hybridization conditions only highly complementary
nucleic acid sequences hybridize. Preferably, such conditions
prevent hybridization of nucleic acids having 1 or 2 mismatches out
of 20 contiguous nucleotides.
[0031] Methods for using the probes include detecting the presence
or amount of SIRP RNA in a sample by contacting the sample with a
nucleic acid probe under conditions such that hybridization occurs
and detecting the presence or amount of the probe bound to SIRP
RNA. The nucleic acid duplex formed between the probe and a nucleic
acid sequence coding for a SIRP polypeptide may be used in the
identification of the sequence of the nucleic acid detected (for
example see, Nelson et al., in Nonisotopic DNA Probe Techniques, p.
275 Academic Press, San Diego (Kricka, ed., 1992) hereby
incorporated by reference herein in its entirety, including any
drawings). Kits for performing such methods may be constructed to
include a container means having disposed therein a nucleic acid
probe.
[0032] The invention also features recombinant nucleic acid,
preferably in a cell or an organism. The recombinant nucleic acid
may contain a sequence (coding sequence or noncoding sequence) or a
segment of sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID
NO: 3, or SEQ ID NO: 4 and a vector or a promoter effective to
initiate transcription in a host cell. The recombinant nucleic acid
can alternatively contain a transcriptional initiation region
functional in a cell, a sequence complimentary to an RNA sequence
encoding a SIRP polypeptide and a transcriptional termination
region functional in a cell.
[0033] In another aspect the invention features an isolated,
enriched or purified SIRP polypeptide.
[0034] By "isolated" in reference to a polypeptide is meant a
polymer of 2 (preferably 7, more preferably 13, most preferably 25)
or more amino acids conjugated to each other, including
polypeptides that are isolated from a natural source or that are
synthesized. The isolated polypeptides of the present invention are
unique in the sense that they are not found in a pure or separated
state in nature. Use of the term "isolated" indicates that a
naturally occurring sequence has been removed from its normal
cellular environment. Thus, the sequence may be in a cell-free
solution or placed in a different cellular environment. The term
does not imply that the sequence is the only amino acid chain
present, but that it is the predominate sequence present (at least
10-20% more than any other sequence) and is essentially free (about
90-95% pure at least) of non-amino acid material naturally
associated with it.
[0035] By the use of the term "enriched" in reference to a
polypeptide is meant that the specific amino acid sequence
constitutes a significantly higher fraction (2-5 fold) of the total
of amino acids present in the cells or solution of interest than in
normal or diseased cells or in the cells from which the sequence
was taken. This could be caused by a person by preferential
reduction in the amount of other amino acids present, or by a
preferential increase in the amount of the specific amino acid
sequence of interest, or by a combination of the two. However, it
should be noted that enriched does not imply that there are no
other amino acid sequences present, just that the relative amount
of the sequence of interest has been significantly increased. The
term significant here is used to indicate that the level of
increase is useful to the person making such an increase, and
generally means an increase relative to other amino acids of about
at least 2 fold, more preferably at least 5 to 10 fold or even
more. The term also does not imply that there is no amino acid from
other sources. The other source amino acid may, for example,
comprise amino acid encoded by a yeast or bacterial genome, or a
cloning vector such as pUC19. The term is meant to cover only those
situations in which man has intervened to elevate the proportion of
the desired amino acid.
[0036] It is also advantageous for some purposes that an amino acid
sequence be in purified form. The term "purified" in reference to a
polypeptide does not require absolute purity (such as a homogeneous
preparation); instead, it represents an indication that the
sequence is relatively purer than in the natural environment
(compared to the natural level this level should be at least 2-5
fold greater, e.g., in terms of mg/ml). Purification of at least
one order of magnitude, preferably two or three orders, and more
preferably four or five orders of magnitude is expressly
contemplated. The substance is preferably free of contamination at
a functionally significant level, for example 90%, 95%, or 99%
pure.
[0037] In preferred embodiments SIRP polypeptides contain at least
9, 10, 15, 20, or 30 contiguous amino acids of the full-length
sequence set forth in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or
SEQ ID NO: 8.
[0038] In yet another aspect the invention features a purified
antibody (e.g., a monoclonal or polyclonal antibody) having
specific binding affinity to a SIRP polypeptide. The antibody
contains a sequence of amino acids that is able to specifically
bind to a SIRP polypeptide.
[0039] By "specific binding affinity" is meant that the antibody
will bind to a hSIRP polypeptide at a certain detectable amount but
will not bind other polypeptides to the same extent, under
identical conditions. The present invention also encompasses
antibodies that can distinguish hSIRP1 from hSIRP2 or hSIRP3 or can
otherwise distinguish between the various SIRPs.
[0040] Antibodies having specific binding affinity to a SIRP
polypeptide may be used in methods for detecting the presence
and/or amount of a SIRP polypeptide is a sample by contacting the
sample with the antibody under conditions such that an
immunocomplex forms and detecting the presence and/or amount of the
antibody conjugated to the SIRP polypeptide. Diagnostic kits for
performing such methods may be constructed to include a first
container means containing the antibody and a second container
means having a conjugate of a binding partner of the antibody and a
label.
[0041] In another aspect the invention features a hybridoma which
produces an antibody having specific binding affinity to a SIRP
polypeptide.
[0042] By "hybridoma" is meant an immortalized cell line which is
capable of secreting an antibody, for example a SIRP antibody.
[0043] In preferred embodiments the SIRP antibody comprises a
sequence of amino acids that is able to specifically bind a SIRP
polypeptide.
[0044] Another aspect of the invention features a method of
detecting the presence or amount of a compound capable of binding
to a SIRP polypeptide. The method involves incubating the compound
with a SIRP polypeptide and detecting the presence or amount of the
compound bound to the SIRP polypeptide.
[0045] In preferred embodiments, the compound inhibits an activity
of SIRP. The present invention also features compounds capable of
binding and inhibiting SIRP polypeptide that are identified by
methods described above.
[0046] In another aspect the invention features a method of
screening potential agents useful for treatment of a disease or
condition characterized by an abnormality in a signal transduction
pathway that contains an interaction between a SIRP polypeptide and
a natural binding partner (NBP). The method involves assaying
potential agents for those able to promote or disrupt the
interaction as an indication of a useful agent.
[0047] By "NBP" is meant a natural binding partner of a SIRP
polypeptide that naturally associates with a SIRP polypeptide. The
structure (primary, secondary, or tertiary) of the particular
natural binding partner will influence the particular type of
interaction between the SIRP polypeptide and the natural binding
partner. For example, if the natural binding partner comprises a
sequence of amino acids complementary to the SIRP polypeptide,
covalent bonding may be a possible interaction. Similarly, other
structural characteristics may allow for other corresponding
interactions. The interaction is not limited to particular residues
and specifically may involve phosphotyrosine, phosphoserine, or
phosphothreonine residues. A broad range of sequences may be
capable of interacting with SIRP polypeptides. One example of a
natural binding partner may be SHP-2, which is described above.
Other examples include, but are not limited to, SHP-1 and Grb2.
Using techniques well known in the art, one may identify several
natural binding partners for SIRP polypeptides such as by utilizing
a two-hybrid screen.
[0048] By "screening" is meant investigating an organism for the
presence or absence of a property. The process may include
measuring or detecting various properties, including the level of
signal transduction and the level of interaction between a SIRP
polypeptide and a NBP.
[0049] By "disease or condition" is meant a state in an organism,
e.g., a human, which is recognized as abnormal by members of the
medical community. The disease or condition may be characterized by
an abnormality in one or more signal transduction pathways in a
cell wherein one of the components of the signal transduction
pathway is either a SIRP polypeptide or a NBP. Specific diseases or
disorders which might be treated or prevented, based upon the
affected cells include cancers and diabetes.
[0050] In preferred embodiments, the methods described herein
involve identifying a patient in need of treatment. Those skilled
in the art will recognize that various techniques may be used to
identify such patients.
[0051] By "abnormality" is meant an a level which is statistically
different from the level observed in organisms not suffering from
such A disease or condition and may be characterized as either an
excess amount, intensity or duration of signal or a deficient
amount, intensity or duration of signal. The abnormality in signal
transduction may be realized as an abnormality in cell function,
viability or differentiation state. The present invention is based
in part on the determination that such abnormality in a pathway can
be alleviated by action at the SHP-2-SIRP interaction site in the
pathway. An abnormal interaction level may also either be greater
or less than the normal level and may impair the normal performance
or function of the organism. Thus, it is also possible to screen
for agents that will be useful for treating a disease or condition,
characterized by an abnormality in the signal transduction pathway,
by testing compounds for their ability to affect the interaction
between a SIRP polypeptide and SHP-2, since the complex formed by
such interaction is part of the signal transduction pathway.
However, the disease or condition may be characterized by an
abnormality in the signal transduction pathway even if the level of
interaction between the SIRP polypeptide and NBP is normal.
[0052] By "interact" is meant any physical association between
polypeptides, whether covalent or non-covalent. This linkage can
include many chemical mechanisms, for instance covalent binding,
affinity binding, intercalation, coordinate binding and
complexation. Examples of non-covalent bonds include electrostatic
bonds, hydrogen bonds, and Van der Waals bonds. Furthermore, the
interactions between polypeptides may either be direct or indirect.
Thus, the association between two given polypeptides may be
achieved with an intermediary agent, or several such agents, that
connects the two proteins of interest (e.g., a SIRP polypeptide and
SHP-2). Another example of an indirect interaction is the
independent production, stimulation, or inhibition of both a SIRP
polypeptide and SHP-2 by a regulatory agent. Depending upon the
type of interaction present, various methods may be used to measure
the level of interaction. For example, the strengths of covalent
bonds are often measured in terms of the energy required to break a
certain number of bonds (i.e., kcal/mol) Non-covalent interactions
are often described as above, and also in terms of the distance
between the interacting molecules. Indirect interactions may be
described in a number of ways, including the number of intermediary
agents involved, or the degree of control exercised over the SIRP
polypeptide relative to the control exercised over SHP-2 or another
NBP.
[0053] By "disrupt" is meant that the interaction between the SIRP
polypeptide and SHP-2 or a NBP is reduced either by preventing
expression of the SIRP polypeptide, or by preventing expression of
SHP-2 or NBP, or by specifically preventing interaction of the
naturally synthesized proteins or by interfering with the
interaction of the proteins.
[0054] By "promote" is meant that the interaction between a SIRP
polypeptide and SHP-2 or NBP is increased either by increasing
expression of a SIRP polypeptide, or by increasing expression of
SHP-2 or a NBP, or by decreasing the dephosphorylating activity of
the corresponding regulatory PTP (or other phosphatase acting on
other phosphorylated signaling components) by promoting interaction
of the SIRP polypeptide and SHP-2 or NBP or by prolonging the
duration of the interaction. Covalent binding can be promoted
either by direct condensation of existing side chains or by the
incorporation of external bridging molecules. Many bivalent or
polyvalent linking agents are useful in coupling polypeptides, such
as an antibody, to other molecules. For example, representative
coupling agents can include organic compounds such as thioesters,
carbodiimides, succinimide esters, diisocyanates, glutaraldehydes,
diazobenzenes and hexamethylene diamines. This listing is not
intended to be exhaustive of the various classes of coupling agents
known in the art but, rather, is exemplary of the more common
coupling agents. (See Killen and Lindstrom 1984, J. Immunol.
133:1335-2549; Jansen, F. K., et al., 1982, Immunological Rev.
62:185-216; and Vitetta et al., supra).
[0055] By "signal transduction pathway" is meant the sequence of
events that involves the transmission of a message from an
extracellular protein to the cytoplasm through a cell membrane. The
signal ultimately will cause the cell to perform a particular
function, for example, to uncontrollably proliferate and therefore
cause cancer. Various mechanisms for the signal transduction
pathway (Fry et al., Protein Science., 2:1785-1797, 1993) provide
possible methods for measuring the amount or intensity of a given
signal. Depending upon the particular disease associated with the
abnormality in a signal transduction pathway, various symptoms may
be detected. Those skilled in the art recognize those symptoms that
are associated with the various other diseases described herein.
Furthermore, since some adapter molecules recruit secondary signal
transducer proteins towards the membrane, one measure of signal
transduction is the concentration and localization of various
proteins and complexes. In addition, conformational changes that
are involved in the transmission of a signal may be observed using
circular dichroism and fluorescence studies.
[0056] In another aspect the invention features a method of
diagnosis of an organism for a disease or condition characterized
by an abnormality in a signal transduction pathway that contains an
interaction between a SIRP polypeptide and SHP-2 or a NBP. The
method involves detecting the level of interaction as an indication
of said disease or condition.
[0057] By "organism" is meant any living creature. The term
includes mammals, and specifically humans. Preferred organisms
include mice, as the ability to treat or diagnose mice is often
predictive of the ability to function in other organisms such as
humans.
[0058] By "diagnosis" is meant any method of identifying a symptom
normally associated with a given disease or condition. Thus, an
initial diagnosis may be conclusively established as correct by the
use of additional confirmatory evidence such as the presence of
other symptoms. Current classification of various diseases and
conditions is constantly changing as more is learned about the
mechanisms causing the diseases or conditions. Thus, the detection
of an important symptom, such as the detection of an abnormal level
of interaction between SIRP polypeptides and SHP-2 or NBPs may form
the basis to define and diagnose a newly named disease or
condition. For example, conventional cancers are classified
according to the presence of a particular set of symptoms. However,
a subset of these symptoms may both be associated with an
abnormality in a particular signaling pathway, such as the
ras.sup.21 pathway and in the future these diseases may be
reclassified as ras.sup.21 pathway diseases regardless of the
particular symptoms observed.
[0059] Yet another aspect of the invention features a method for
treatment of an organism having a disease or condition
characterized by an abnormality in a signal transduction pathway.
The signal transduction pathway contains an interaction between a
SIRP polypeptide and SHP-2 or a NBP and the method involves
promoting or disrupting the interaction, including methods that
target the SIRP:NBP interaction directly, as well as methods that
target other points along the pathway.
[0060] By "dominant negative mutant protein" is meant a mutant
protein that interferes with the normal signal transduction
pathway. The dominant negative mutant protein contains the domain
of interest (e.g., an SIRP polypeptide or SHP-2 or a NBP), but has
a mutation preventing proper signaling, for example by preventing
binding of a second domain from the same protein. One example of a
dominant negative protein is described in Millauer et al., Nature
Feb. 10, 1994. The agent is preferably a peptide which blocks or
promotes interaction of the SIRP polypeptide and SHP-2 or another
NBP. The peptide may be recombinant, purified, or placed in a
pharmaceutically acceptable carrier or diluent.
[0061] An EC.sub.50 or IC.sub.50 of less than or equal to 100 .mu.M
is preferable, and even more preferably less than or equal to 50
.mu.M, and most preferably less that or equal to 20 .mu.M. Such
lower EC.sub.50's or IC.sub.50's are advantageous since they allow
lower concentrations of molecules to be used in vivo or in vitro
for therapy or diagnosis. The discovery of molecules with such low
EC.sub.50's and IC.sub.50's enables the design and synthesis of
additional molecules having similar potency and effectiveness. In
addition, the molecule may have an EC.sub.50 or IC.sub.50 less than
or equal to 100 .mu.M at one or more, but not all cells chosen from
the group consisting of parathyroid cell, bone osteoclast,
juxtaglomerular kidney cell, proximal tubule kidney cell, distal
tubule kidney cell, cell of the thick ascending limb of Henle's
loop and/or collecting duct, central nervous system cell,
keratinocyte in the epidermis, parafollicular cell in the thyroid
(C-cell), intestinal cell, trophoblast in the placenta, platelet,
vascular smooth muscle cell, cardiac atrial cell, gastrin-secreting
cell, glucagon-secreting cell, kidney mesangial cell, mammary cell,
beta cell, fat/adipose cell, immune cell and GI tract cell.
[0062] By "therapeutically effective amount" is meant an amount of
a pharmaceutical composition having a therapeutically relevant
effect. A therapeutically relevant effect relieves to some extent
one or more symptoms of the disease or condition in the patient; or
returns to normal either partially or completely one or more
physiological or biochemical parameters associated with or
causative of the disease or condition. Generally, a therapeutically
effective amount is between about 1 nmole and 1 .mu.mole of the
molecule, depending on its EC.sub.50 or IC.sub.50 and on the age
and size of the patient, and the disease associated with the
patient.
[0063] In another aspect, the invention describes a polypeptide
comprising a recombinant SIRP polypeptide or a unique fragment
thereof. By "unique fragment," is meant an amino acid sequence
present in a full-length SIRP polypeptide that is not present in
any other naturally occurring polypeptide. Preferably, such a
sequence comprises 6 contiguous amino acids present in the full
sequence. More preferably, such a sequence comprises 12 contiguous
amino acids present in the full sequence. Even more preferably,
such a sequence comprises 18 contiguous amino acids present in the
full sequence.
[0064] By "recombinant SIRP polypeptide" is meant to include a
polypeptide produced by recombinant DNA techniques such that it is
distinct from a naturally occurring polypeptide either in its
location (e.g., present in a different cell or tissue than found in
nature), purity or structure. Generally, such a recombinant
polypeptide will be present in a cell in an amount different from
that normally observed in nature.
[0065] In another aspect, the invention describes a recombinant
cell or tissue containing a purified nucleic acid coding for a SIRP
polypeptide. In such cells, the nucleic acid may be under the
control of its genomic regulatory elements, or may be under the
control of exogenous regulatory elements including an exogenous
promoter. By "exogenous" it is meant a promoter that is not
normally coupled in vivo transcriptionally to the coding sequence
for the SIRP polypeptide.
[0066] In another aspect, the invention features a SIRP polypeptide
binding agent able to bind to a SIRP polypeptide. The binding agent
is preferably a purified antibody which recognizes an epitope
present on a SIRP polypeptide. Other binding agents include
molecules which bind to the SIRP polypeptide and analogous
molecules which bind to a SIRP polypeptide.
[0067] By "purified" in reference to an antibody is meant that the
antibody is distinct from naturally occurring antibody, such as in
a purified form. Preferably, the antibody is provided as a
homogeneous preparation by standard techniques. Uses of antibodies
to the cloned polypeptide include those to be used as therapeutics,
or as diagnostic tools.
[0068] In another aspect, the invention provides a nucleic acid
molecule comprising a nucleotide sequence that encodes a
polypeptide having the full length amino acid sequence set forth in
SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 except
that it lacks at least one of the domains selected from the group
consisting of the extracellular Ig like domain, the transmembrane
domain, and the SHP-2 binding domains. Such deletion mutants are
useful in the design of assays for protein inhibitors. The nucleic
acid molecules described above may be, for example, cDNA or genomic
DNA and may be placed in a recombinant vector or expression vector.
In such a vector, the nucleic acid preferably is operatively
associated with the regulatory nucleotide sequence containing
transcriptional and translational regulatory information that
controls expression of the nucleotide sequence in a host cell.
[0069] Thus, the invention also provides a genetically engineered
host cell containing any of the nucleotide sequences described
herein and the nucleic acid preferably is operatively associated
with the regulatory nucleotide sequence containing transcriptional
and translational regulatory information that controls expression
of the nucleotide sequence in a host cell. Such host cells may
obviously be either prokaryotic or eukaryotic.
[0070] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof, and from the claims.
BRIEF DESCRIPTION OF THE FIGURES
[0071] FIG. 1 shows the deduced amino acid sequences of SIRP4 and
SIRP1. Identical amino acids are boxed. The putative signal
sequence and transmembrane region are indicated by thin and thick
overlines, respectively. Three Ig-like domains are indicated by
stippled overlines. Potential tyrosine phosphorylation sites are
shown in bold, the C-terminal proline rich region is shaded. The
location of oligonucleotides flanking the Ex region is indicated by
stars.
[0072] FIG. 2 shows the alignment of extracellular regions
including the first Ig-like domain of 15 SIRP family members. Ex1
shows amino acids encoded by the initial PCR fragment that was used
for screening and GST-fusion protein construction. Ex2-11 are
derived from PCR and cDNA sequences, Ex12-15 from genomic isolates.
Numbering is according to FIG. 1.
[0073] FIG. 3 shows the alignment of amino acid sequences of human
SIRP4, mouse SIRP1, human SIRP.alpha.1 and mouse SIRP.beta.1.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0074] The present invention relates to SIRP polypeptides, nucleic
acids encoding such polypeptides, cells, tissues and animals
containing such nucleic acids, antibodies to such polypeptides,
assays utilizing such polypeptides, and methods relating to all of
the foregoing. Those skilled in the art will recognize that many of
the methods described below in relation to SIRP1, SIRP4, SHP-2,
SHP-1 and Grb2 could also be utilized with respect to the other
members of this group.
[0075] Various other features and aspects of the invention are:
Nucleic Acid Encoding A SIRP Polypeptide; A Nucleic Acid Probe for
the Detection of SIRP; Probe Based Method And Kit For Detecting
SIRP; DNA Constructs Comprising a SIRP Nucleic Acid Molecule and
Cells Containing These Constructs; Purified SIRP Polypeptides; SIRP
Antibody And Hybridoma; An Antibody Based Method And Kit For
Detecting SIRP; Isolation of Compounds Which Interact With SIRP;
Compositions; Disruption of Protein Complexes; Antibodies to
Complexes; Pharmaceutical Formulations and Modes of Administration;
Identification of Agents; Purification and Production of Complexes;
Derivatives of Complexes; and Evaluation of Disorders.
[0076] All of these aspects and features are explained in detail
with respect to another protein involved with signal transduction,
PYK-2, in PCT publication WO 96/18738, which is incorporated herein
by reference in its entirety, including any drawings. Those skilled
in the art will readily appreciate that such description can be
easily adapted to SIRP as well, and is equally applicable to the
present invention.
EXAMPLES
[0077] The examples below are non-limiting and are merely
representative of various aspects and features of the procedures
used to identify the full-length nucleic acid and amino acid
sequences of a series of SIRP proteins. Experiments demonstrating
SIRP expression, interaction and signaling activities are also
provided.
[0078] Material and Methods
[0079] Cell Culture and Transient Expression
[0080] MM5/C1, Rat1-IR, A431 or human fibroblast cells were grown
until confluency, starved for 18 hours in serum-free medium, and
either left untreated or were POV--(1 mM sodium orthovanadate, 3 mM
H.sub.2O.sub.2), insulin--(100 nM), EGF--(1 nM), or PDGF--(100 pM)
stimulated for different time intervals as indicated. SIRP4, SHP-2
(Vogel, et al., Science 259:1611-1614 (1994)) or SHP-2C463A mutant
(Stein-Gerlach, et al. J. Biol. Chem. 270:24635-24637 (1995)) cDNAs
were transiently cotransfected in BHK-IR, BHK-EGFR or
BHK-.beta.PDGFR cells using the calcium precipitation method (Chen,
et al. Mol. Cell. Biol. 7:2745-2752 (1987)). After stimulation,
cells were lysed in buffer containing 50 mM HEPES, pH 7.5, 150 mM
NaCl, 1% Triton X-100, 10% glycerol, 1 mM POV, 1 mM EDTA, 1 mM
PMSF, 1 mg/ml leupeptin, 1 mg/ml aprotinin.
[0081] Immunoprecipitation and Western Blotting
[0082] SHP-2 immunoprecipitations were performed with polyclonal
anti-SHP-2 antibodies (Vogel, et al., Science 259:1611-1614
(1994)). Overexpressed SIRP4 or endogenous SIRP4-like proteins were
immunoprecipitated by polyclonal anti-Ex1 antibodies raised by
immunizing rabbits with a GST-fusion protein containing the Ex1
fragment (FIG. 2). Western blots were labeled with monoclonal
anti-phosphotyrosine antibodies 5E2 (Fendly, et al., Cancer Res.
50:1550-1558 (1990)), and after stripping, reprobed with monoclonal
anti-SHP-2 antibodies (Transduction Laboratories), or polyclonal
anti-SIRP4-CT antibodies, raised against a GST-fusion protein
containing the C-terminal part of SIRP4 (amino acids 336-503). For
immunolabeling goat anti-mouse or -rabbit horseradish peroxidase
conjugates (Bio-Rad) and the ECL detection system (Amersham) were
used.
[0083] To obtain 293 cells stably expressing SIRP4 (293/SIRP4),
cells were transfected with SIRP4 cDNA in pLXSN (Miller, et al.
Biotechniques 7:980-988 (1989)) using the calcium precipitation
method, followed by selection with G418 (1 mg/ml). SIRP4 was
immunoprecipitated from quiescent or POV-stimulated (1 mM)
293/SIRP4 cells with polyclonal anti-Ex1 antibodies. Subsequently,
crude lysates of [.sup.35S]-methionine labeled 293 cells expressing
different SH2 domain containing proteins were added to the affinity
matrix and incubated for 2 h at 4.degree. C. The is immunocomplexes
were washed, separated by SDS-PAGE and analyzed by
autoradiography.
[0084] Enzymatic Deglycosylation
[0085] To perform in vitro deglycosylation SHP-2 immunocomplexes or
the 110 kDa protein preparation were first denatured in the
presence of 1% SDS at 100.degree. C. for 5 min. Deglycosylation was
done in potassium phosphate buffer (40 mM, pH 7.0), containing 20
mM EDTA, 1% b-mercaptoethanol, 1% Triton X-100 and 0.5 Unit of
Endoglycosidase F/N-Glycosidase F (Boehringer Mannheim) at
37.degree. C. for 16 hours.
[0086] Protein Purification
[0087] Approximately 10.sup.10 Rat1-IR cells were used to purify
the 110 kDa protein. Starved Rat1-IR cells were insulin-stimulated
(100 nM) for 10 min, washed briefly with ice-cold hypotonic buffer
containing 20 mM HEPES, pH 7.5, 1 mM POV, 1 mM EDTA, 1 mM PMSF, 1
mg/ml leupeptin, 1 mg/ml aprotinin, scraped into the same buffer
and homogenized. Obtained cell extracts were pelleted at 1000 rpm
for 15 min, and supernatants were spun at 48.000 g for 1 hour.
Membranes were solubilized in lysis buffer as described above. hIR
was depleted from membrane extracts using an affinity column with
monoclonal anti-hIR antibody 83-14 (Redemann et al., Mol. Cell.
Biol. 12:491-498 (1992)), covalently coupled to Protein A-Sepharose
beads (Pharmacia). Depleted extracts were applied onto a
WGA-agarose 6 MB column (Sigma), and glycoproteins were eluted with
0.3 M N-acetyl-glucosamine in HNTG (20 mM HEPES (pH 7.5), 150 mM
NaCl, 0.1% Triton X-100, 10% glycerol, 1 mM POV). After
concentration protein extracts were applied onto an
anti-phosphotyrosine antibody column (Sigma). Bound proteins were
eluted with 20 mM phosphotyrosine in HNTG. The eluate was subjected
to SDS-PAGE, proteins were transferred to a PVDF membrane
(Millipore) and stained with Coomassie blue. The protein of 110 kD
apparent molecular weight was microsequenced. The following five
tryptic peptides were obtained: PIYSFIGGEHFPR, IVEPDTEIK, YGFSPR,
IKEVAHVNLEVR, VAAGDSAT.
[0088] Biological Assays
[0089] To produce retroviruses expressing pLXSN, wild type SIRP4
and mutated SIRP4 constructs, BOSC 23 cells were transiently
transfected by expression plasmids as described (Pear, et al. Proc.
Natl. Acad. Sci. 90:8392-8396 (1993)). To obtain NIH3T3 cells
stably expressing wild type SIRP4, SIRP4-4Y or SIRP4-DCT mutants
subconfluent NIH3T3 cells (10.sup.5 cells per 6 cm dish) were
incubated with supernatants of transfected BOSC 23 cells for 4 h in
the presence of Polybrene (4 mg/ml), followed by selection with
G418 (1 mg/ml).
[0090] To perform focus formation assays cell lines 3T3/pLXSN,
3T3/SIRP4, 3T3/SIRP4-4Y or 3T3/SIRP4-DCT were superinfected for 4
hours with equal volumes of v-fms-virus supernatant
(10.sup.5cells/6 cm dish). Cells were cultivated for 14 days in 4%
FCS with medium change every second day. Cell foci were stained
with Crystal violet (0.1% crystal violet, 30% methanol).
Example 1
Identification and Cloning of Signal Regulatory Proteins
[0091] Western blot of mammalian cells with anti-phosphotyrosine
antibodies and anti-SHP-2 antibodies was used to identify tyrosine
phosphorylated SHP-2 associated proteins.
[0092] Western blots containing anti-SHP-2 immunoprecipitates from
starved or POV-treated mouse MM5/C1 mammary carcinoma, rat
fibroblast Rat1-IR or human epidermal carcinoma A431 cells were
incubated with anti-phosphotyrosine antibodies or anti-SHP-2
antibodies. Samples were deglycosylated with or treated without
Endoglycosidase F/N-Glycosidase F (Endo.F/F). As a control,
insulin-stimulated Rat1-IR cell lysates were immunoprecipitated
with preimmune rabbit serum (aNS).
[0093] Samples from each purification step (i.e., solubilized crude
membrane extract, hIR-depleted extracts, concentrated eluate from
WGA-agarose beads, and eluate from anti-phosphotyrosine antibody
column) were analyzed by 10% SDS-PAGE and visualized by silver
staining and in Western blots using monoclonal anti-phosphotyrosine
antibodies.
[0094] A major tyrosine phosphorylated protein was revealed in
analysis of anti-SHP-2 immunoprecipitates from both pervanadate
(POV) and growth factor stimulated cells. This phosphoprotein
migrated at 120 kDa, 110 kDa and 90 kDa positions in mouse mammary
tumor (MM5/C1) cells, Rat1 cells overexpressing the human insulin
receptor (Rat1-IR), and human epidermoid carcinoma (A431) cells,
respectively.
[0095] Upon in vitro deglycosylation, this glycoprotein was reduced
to 65 kDa apparent molecular weight (MW) in all cases. This
indicated that the same SHP-2 binding protein of 65 kDa was
differentially glycosylated in a species specific manner.
[0096] In some cell lines such as A431, other tyrosine
phosphorylated proteins in the 90-120 kDa range remained unaffected
by the deglycosylation treatment. These proteins may represent Gab1
and/or the human homologue of the Drosophila DOS protein.
[0097] Insulin treated Rat1-IR were used to purify the 110 kDa
SHP-2 binding glycoprotein using standard chromatography
procedures. Approximately 4 mg of the glycoprotein that copurified
with SHP-2 were obtained and subject to microsequence analysis.
This yielded five peptide sequences: PIYSFIGGEHFPR, IVEPDTEIK,
YGFSPR, IKEVAHVNLEVR, VAAGDSAT. Computer aided search in the EST
database led to the identification of a 305 bp rat sequence
(accession Nr.: H31804) and subsequent human cDNA fragment of 2 kb
(EMBL databank, accession Nr.: U6701) containing matching and
homologous sequences, respectively.
[0098] Specific primers flanking the very 5' portion of this
sequence were used to amplify a 360 bp human DNA fragment (encoding
Ex1 in FIG. 2) which was used to screen a human placenta cDNA
library.
[0099] Several positive clones were isolated. One clone of 2.4 kb
encoded a polypeptide of 503 amino acids designated SIRP4 (for
SIgnal Regulating Protein 4) with a calculated mass of 57,000. The
deduced sequence identifies SIRP4 as a transmembrane protein with
three Ig-like domains and a cytoplasmic portion containing four
potential tyrosine phosphorylation sites and one proline-rich
region.
[0100] A second cDNA clone, SIRP1, is also identified. This protein
is highly homologous to SIRP4 within the Ig-like domains (Ig-1:
83%; Ig-2: 88%; Ig-3: 83%), but displays striking sequence
divergence at the amino terminus and upstream of the transmembrane
domain which gives rise to a shorter protein that still contains a
transmembrane-like region but lacks the cytoplasmic C-terminal
portion.
[0101] SIRP4 and SIRP1 are members of a novel protein family. This
protein family has a variety of distinct sequence isoforms as
evidenced by comparison of fifteen cDNA and genomic sequences
within the first Ig-like domain (FIG. 2). Two major classes exist
in SIRP family distinguished by the presence or absence of a
cytoplasmic SHP-2 binding domain.
Example 2
Analyzing the Functions of SIRP4
[0102] SIRP4 Binds to SHP-2 and Serves as a Substrate for SHP-2,
IR, EGFR, and .beta.PDGFR
[0103] The identity of SIRP4 as SHP-2 binding protein and substrate
was confirmed by expression of the SIRP4 cDNA either alone or in
combination with SHP-2 or an enzymatically inactive mutant
SHP-2C463A in BHK cells. BHK cells stably express human EGF-,
insulin- or .beta.PDGF receptors.
[0104] Immunoprecipitations were performed with a polyclonal
antibody raised against a GST-fusion protein containing the
extracellular Ex1 region (FIG. 2).
[0105] Western blots containing anti-SIRP4 immunoprecipitations
from quiescent or ligand-stimulated BHK-IR, BHK-EGFR or
BHK-.beta.PDGF cells were labeled with anti-phosphotyrosine,
anti-SHP-2 and anti-SIRP4 antibodies, respectively.
[0106] Anti-SIRP4 immunoprecipitation revealed a tyrosine
phosphorylated protein of 85-90 kDa upon ligand stimulation which
associated with SHP-2.
[0107] The results suggested SIRP4 to be a direct substrate of
SHP-2 since expression of the SHP-2 mutant SHP-2C463A led to a
significant increase in its phosphotyrosine content (even in
starved cells) while coexpression of wt SHP-2 resulted in
dephosphorylation. The MW of overexpressed SIRP4 matches that of
the endogenous protein detected in SHP-2 immunoprecipitates from
A431 cells.
[0108] Endogenous SIRP4-like proteins were immunoprecipitated from
untreated or EGF-stimulated A431 cells, from quiescent or
PDGF-treated human fibroblasts, or from starved or
insulin-stimulated HBL-100 cells. As a control, ligand-stimulated
cell lysates were immunoprecipitated with preimmune rabbit serum
(aNS). Immunoblots were probed with monoclonal anti-phosphotyrosine
and monoclonal anti-SHP-2 antibodies.
[0109] Polyclonal anti-Ex1 antibodies immunoprecipitate a protein
of 85-90 kDa apparent MW from A431, HBL-100 tumor cells and human
fibroblasts. This protein was tyrosine phosphorylated upon EGF,
insulin or PDGF stimulation, respectively, and coprecipitated with
SHP-2 in a ligand dependent manner.
[0110] These data indicate the existence of SIRP4 in several human
cell lines where SIRP4 serves as a substrate for insulin-, EGF- and
.beta.PDGF receptors, binds SHP-2 in its tyrosine phosphorylated
form and serves as a substrate for the phosphatase activity of
SHP-2. The interaction of SHP-2 with SIRP4 likely involves one or
both SH2 domains of SHP-2 as suggested by the requirement of
phosphotyrosine residues and the abrogation of detectable
association by mutation of critical residues in SHP-2 SH2
domains.
[0111] In vitro binding assays were performed to determine whether
SIRP4 is able to interact with other SH2 domain-containing
proteins. SIRP4-associated [.sup.35S]-Methionine labeled proteins
were resolved on SDS-PAGE and detected by autoradiography. The
result shows that SIRP4 associates with both SHP-1 and Grb2 but not
p85, Shc, Grb7, PLC-g, c-src, Nck, Vav, GAP, or ISGF-3.
[0112] A catalytically inactive SHP-1 mutant has recently been
shown to bind an as yet unidentified tyrosine phosphorylated
protein of 90-95-kDa in human 293 cells. This tyrosine
phosphorylated protein is likely to be SIRP4 or one of its family
members.
[0113] Effects of SIRP4 on Cell Growth and Transformation
[0114] To investigate the biological function of SIRP4, three
stable transfectants of NIH3T3 cells were constructed to express
wild type SIRP4 or SIRP4 mutants carrying either point mutations of
the putative SHP-2 tyrosine binding sites (SIRP4-4Y) or a deletion
of most of the cytoplasmic region (SIRP4-DCT).
[0115] Ligand-stimulated [.sup.3H]-thymidine incorporation of
NIH3T3 cells expressing empty vector (3T3/pLXSN), wild type SIRP4
(3T3/SIRP4), SIRP4-4Y (3T3/SIRP4-4Y) or SIRP4-DCT (3T3/SIRP4-DCT,
amino acids 402-503 are deleted) mutants. Cells were grown to
confluence in 24-well dishes (Nunc), starved for 24 h in DMEM/0.5%
FCS, stimulated with different concentrations of insulin or EGF for
18 h, then incubated with 0.5 mCi [.sup.3H]-thymidine per well for
4 h. Incorporation into DNA was determined as described (Redemann,
et al. Mol. Cell. Biol. 12:491-498 (1992)).
[0116] Upon stimulation of cells with insulin, EGF and PDGF,
control cells showed growth factor-induced DNA synthesis as
measured by [.sup.3H]-thymidine incorporation. Overexpression of
SIRP4 led to a decrease of [.sup.3H]-thymidine incorporation. In
contrast, both SIRP4 mutants had nearly no effect on DNA synthesis.
The observed inhibitory effect on DNA synthesis must be connected
to SIRP4 tyrosine phosphorylation and/or its association with SHP-2
since wt SIRP4 became tyrosine phosphorylated and bound to SHP-2
upon ligand stimulation, and SIRP4 mutants did not.
[0117] SIRP4 effected growth inhibition upon insulin or EGF
stimulation is correlated with reduced MAP kinase activation in
3T3/SIRP4 cells. 3T3/pLXSN, 3T3/SIRP4 or 3T3/SIRP4-4Y cells were
starved for 18 hours in DMEM/0.5% FCS and stimulated with insulin
or EGF for the time indicated. MAP kinase was detected in Western
blots by using polyclonal erk1 and erk2 antibodies (Santa Cruz). In
contrast, expression of SIRP4 mutants defective in SHP-2 binding
had no effect on MAP kinase activation. Similar observations were
made upon stimulation of the cells with PDGF.
[0118] These data strongly indicate that SIRP4 represents a novel
regulatory-element in the pathway that leads to MAP kinase
activation.
[0119] We next determined the consequence of SIRP4 overexpression
on oncogene mediated transformation of NIH3T3 cells. To examine the
ability of SIRP4 to influence the formation of cell foci,
subconfluent 3T3/pLXSN, 3T3/SIRP4, 3T3/SIRP4-4Y or 3T3/SIRP4-DCT
cells were infected with v-fms virus supernatants. As measured by
focus formation, transformation by a v-fms retrovirus was
significantly suppressed in cells overexpressing wt SIRP4 but not
in cells expressing mutant SIRP4.
[0120] Previous reports have described certain SHP-2 binding
proteins of 110-130 kDa apparent MW in mouse, rat or hamster cells.
Tyrosine hyperphosphorylation of these proteins was observed when
an enzymatically inactive SHP-2 mutant was overexpressed. In
addition, disruption of SHP-2 function induced a variety of
negative effects on growth factor-induced cellular signals. Our
experiments strongly indicate that these proteins belong to the
SIRP family and that the biological effects previously observed are
due to the function of these SIRP proteins.
[0121] Without being bound by any theory, applicant proposes that
tyrosine docking sites on SIRP proteins for either SHP-2 and/or
other SH2 proteins such as SHP-1 or Grb2 play a significant role
since the inhibitory effect of SIRP4 on NIH3T3 cell proliferation
and transformation depends on phosphorylation of tyrosines.
[0122] One or both of the SHP phosphatases may tightly regulate the
SIRP4 phosphorylation state.
[0123] SIRP4 may also act in its phosphorylated state as a
"trapping" protein that sequesters SHP-2 from activated RTKs. The
sequestion makes SHP-2 unavailable for other positive regulatory
functions such as an adapter which recruits the Grb2-SOS complex to
activated receptors. Such a function is supported by the
observation that SHP-2 has higher affinity to the tyrosine
phosphorylated form of SIRP4 than to autophosphorylated insulin and
EGF receptors (Yamauchi, et al., J. Biol. Chem. 270:17716-17722,
Yamauchi, et al. J. Biol. Chem. 270:14871-14874 (1995)).
[0124] A third possibility is based on the membrane-spanning
structural features of the SIRP4 variant. The high degree of
sequence diversity within the Ig-domains is reminiscent of
immunoglobulin variable regions and suggests a role of
extracellular determinants in the SIRP related signal transduction.
Structurally defined interaction of SIRP with specific receptors,
soluble ligands, extracellular matrix components or other factors
may result in specific regulatory consequences for intracellular
signaling events.
[0125] All publications referenced are incorporated by reference
herein, including the nucleotide sequences, amino acid sequences,
drawings and tables in each publication. All the compounds
disclosed and referred to in the publications mentioned above are
incorporated by reference herein, including those compounds
disclosed and referred to in articles cited by the publications
mentioned above.
[0126] Other embodiments of this invention are disclosed in the
following claims. As will be obvious to those skilled in the art,
may variations and modifications may be made without departing from
the spirit and scope of the invention.
Sequence CWU 1
1
26 1 3804 DNA Homo sapiens 1 cacagacgtt tggacagagc aggctcctaa
ggtctccaga atgcccgtgc cagcctcctg 60 gccccacctt cctagtcctt
tcctgctgat gacgctactg ctggggagac tcacaggagt 120 ggcaggtgag
gacgagctac aggtgattca gcctgaaaag tccgtatcag ttgcagctgg 180
agagtcggcc actctgcgct gtgctatgac gtccctgatc cctgtggggc ccatcatgtg
240 gtttagagga gctggagcag gccgggaatt aatctacaat cagaaagaag
gccacttccc 300 acgggtaaca actgtttcag aactcacaaa gagaaacaac
ctgaactttt ccatcagcat 360 cagtaacatc accccagcag acgccggcac
ctactactgt gtgaagttcc ggaaagggag 420 ccctgacgac gtggagttta
agtctggagc aggcactgag ctgtctgtgc gcgccaaacc 480 ctctgccccc
gtggtatcgg gccctgcggt gagggccaca cctgagcaca cagtgagctt 540
cacctgcgag tcccatggct tctctcccag agacatcacc ctgaaatggt tcaaaaatgg
600 gaatgagctc tcagacttcc agaccaacgt ggaccccgca ggagacagtg
tgtcctacag 660 catccacagc acagccaggg tggtgctgac ccgtggggac
gttcactctc aagtcatctg 720 cgagatggcc cacatcacct tgcaggggga
ccctcttcgt gggactgcca acttgtctga 780 ggccatccga gttccaccca
ccttggaggt tactcaacag cccatgaggg cagagaacca 840 ggcaaacgtc
acctgccagg tgagcaattt ctacccccgg ggactacagc tgacctggtt 900
ggagaatgga aatgtgtccc ggacagaaac agcttcgacc ctcatagaga acaaggatgg
960 cacctacaac tggatgagct ggctcctggt gaacacctgt gcccacaggg
acgatgtggt 1020 gctcacctgt caggtggagc atgatgggca gcaagcagtc
agcaaaagct atgccctgga 1080 gatctcagca caccagaagg agcacggctc
agatatcacc catgaaccag cgctggctcc 1140 tactgctcca ctcctcgtag
ctctcctcct gggccccaag ctgctactgg tggttggtgt 1200 ctctgccatc
tacatctgct ggaaacagaa ggcctgactg accctcagtc tctgctgcct 1260
cctcctttct tgagaagctc agcctgagag aaggagctgg cgagaacctt ccccacactc
1320 agctccaaac gcctcctctc ccaggtcatc tgcctgccca cacgctcctg
ttccaccttc 1380 acaagaccat gatgccccaa agcagtgtct ctattcacgg
tcctgagcag gggccatggg 1440 attgggctct gggcactgac tcatggcacc
tccctagaag gtgagaaaca ctccaaatct 1500 aaacacacca ggacttctcc
catccgtcgc cttgggactg gccataaacc acagactctc 1560 tccaggctct
caagagttat cctgtcttct ggattcctgc ctaccccaac tcccccagcc 1620
ttgttgaggt tctctactgc ctcctgaata cacatgaacc cctataccaa ttttaagaaa
1680 aaaatgattc tctttcctct ttgtccaagc atcctatccc tcaaacccaa
aaagaaagaa 1740 gctctccctt ctctctctgt gatggagaca gtatttcttc
tagtatcctg cagccttccc 1800 agtcctgctg cttgtggtag aaattgctgc
cacagcccaa cattgaggag ccctcgatga 1860 ctgcccttta caactcatat
tcagttctgc ctccaaaatg catgtgtcca cttacatgag 1920 atggtaaatg
tttaacaatg gactttctga aagggaaaaa ccaaaagctg ttttgcagtg 1980
cttgccaatt tctctagtgt aataactccc aacctgacca atttcagcac tgccaacagt
2040 taaacaacca gattcgaaga ttcctgaaat ttaacaattg gttttcaggg
cccagtccaa 2100 gcctgctgct ggaaacctca gagttaaatc cctattctcc
acacctctca cctccaccac 2160 ccctccctgt cccagccagc atcatctctt
tggggaccac tcctctggct ttcatttttc 2220 agccacagtg attctttgga
aaagtcaaat catatcactt ctctgcttct tccccaacac 2280 agctgcatgg
tcccgctctc cctccttcaa gtctctgctc aatgtcactt cattaaaggc 2340
ggccttctat aaactacctt gtataaaata ttatttattt tctctatccc ggcattctaa
2400 tttctcttat cctaattaat ttttctttag cccttatttt gatgagtatt
atgccgaata 2460 caggcagccc tcacttttca tggccagtgc aagattgcaa
aaagactgtg caacctgaaa 2520 cccaggaaag cagtctccat agtcaatcag
aaaaacaatg atcattctgt gacctttacc 2580 attttttgtc aaaatattag
aaactctcac actctcagtt acaaatgtag aggacaatga 2640 aaatataatg
aaataaatat ttatttgtgc actacaattc aaagcattag aaacattgaa 2700
gtcaatggcg tttcttgtaa atgtatccag atgaggttgg aagagtgctt gacctttttg
2760 tatatttcta atatggagtg atatagtttg gctctgtgtc tccatccaaa
tctcatctta 2820 aattgtaatc tgcatgtgtt gtgggaatgg gacctaggta
ggaggtgact gaatacatgg 2880 gggcggactt cccccttgct gttcttgtga
tagtgagttc tcataagatc tcagtgagtt 2940 ctcatgagat ctggtttttt
gaaagtgtgt ggcaagtccc ccttcgctct ctctctctct 3000 ctccctcctg
ccaccatgtg aagaaggtgc ctgcttcctt ttctccttcc accatggttg 3060
taagtttcct gaggcctccc agtcatgctt cctgttaagc ctgtggaact gtgagtccaa
3120 ttaaacctct tttattcata aaatatccag tttctggtag ttctttatag
cagtgtgaga 3180 atgggctaat acacggagca agcatcgttc tttcattttt
atttatttta ttttttgaga 3240 tggagtttca ccttattccc aggctggagt
gcaatgtcgt gatcttggct cactgcaacc 3300 cccgcctcca gggttcaagt
gattctcctg cctcagcctc ctgagtagct gggattacag 3360 gcatgtacca
ccacacccag ctaattttgt atttttagta gagatggggt ttctccatgt 3420
tgatcagact agtcttgaac tcccgacctc aggtgatcca cctgtcttgg cctcccaaag
3480 tgctgggatt acaggcatga gccaccatgc ctagccagca agcatcattt
ctattatacc 3540 ttggtgtttg cctctttcta agtttggact agcttccaac
atcttatccc ttgaattttc 3600 aatattgtgg aatcactcca gaagatcctt
tcatgtgaag ttttttgctg gcatttcaac 3660 ctttgggaca tcttcagccc
ttttattacc actcctctcc catttgtggc agtttgcgtt 3720 tactacctcc
ctctggctgc ctatctgaag ttcctgcatc agggtctaca ttgccacagt 3780
caactatttg tacttctaga attc 3804 2 2433 DNA Homo sapiens 2
cagccgcggc ccatggagcc cgccggcccg gcccccggcc gcctcgggcc gctgctctgc
60 ctgctgctcg ccgcgtcctg cgcctggtca ggagtggcgg gtgaggagga
gctgcaggtg 120 attcagcctg acaagtccgt atcagttgca gctggagagt
cggccattct gcactgcact 180 gtgacctccc tgatccctgt ggggcccatc
cagtggttca gaggagctgg accagcccgg 240 gaattaatct acaatcaaaa
agaaggccac ttcccccggg taacaactgt ttcagagtcc 300 acaaagagag
aaaacatgga cttttccatc agcatcagta acatcacccc agcagatgcc 360
ggcacctact actgtgtgaa gttccggaaa gggagccctg acacggagtt taagtctgga
420 gcaggcactg agctgtctgt gcgtgccaaa ccctctgccc ccgtggtatc
gggccctgcg 480 gcgagggcca cacctcagca cacagtgagc ttcacctgcg
agtcccacgg cttctcaccc 540 agagacatca ccctgaaatg gttcaaaaat
gggaatgagc tctcagactt ccagaccaac 600 gtggaccccg taggagagag
cgtgtcctac agcatccaca gcacagccaa ggtggtgctg 660 acccgcgagg
acgttcactc tcaagtcatc tgcgaggtgg cccacgtcac cttgcagggg 720
gaccctcttc gtgggactgc caacttgtct gagaccatcc gagttccacc caccttggag
780 gttactcaac agcccgtgag ggcagagaac caggtgaatg tcacctgcca
ggtgaggaag 840 ttctaccccc agagactaca gctgacctgg ttggagaatg
gaaacgtgtc ccggacagaa 900 acggcctcaa ccgttacaga gaacaaggat
ggtacctaca actggatgag ctggctcctg 960 gtgaatgtat ctgcccacag
ggatgatgtg aagctcacct gccaggtgga gcatgacggg 1020 cagccagcgg
tcagcaaaag ccatgacctg aaggtctcag cccacccgaa ggagcagggc 1080
tcaaataccg ccgctgagaa cactggatct aatgaacgga acatctatat tgtggtgggt
1140 gtggtgtgca ccttgctggt ggccctactg atggcggccc tctacctcgt
ccgaatcaga 1200 cagaagaaag cccagggctc cacttcttct acaaggttgc
atgagcccga gaagaatgcc 1260 agagaaataa cacaggacac aaatgatatc
acatatgcag acctgaacct gcccaagggg 1320 aagaagcctg ctccccaggc
tgcggagccc aacaaccaca cggagtatgc cagcattcag 1380 accagcccgc
agcccgcgtc ggaggacacc ctcacctatg ctgacctgga catggtccac 1440
ctcaaccgga cccccaagca gccggccccc aagcctgagc cgtccttctc agagtacgcc
1500 agcgtccagg tcccgaggaa gtgaatggga ccgtggtttg ctctagcacc
catctctacg 1560 cgctttcttg tcccacaggg agccgccgtg atgagcacag
ccaacccagt tcccggaggg 1620 ctggggcggt gcaggctctg ggacccaggg
gccagggtgg ctcttctctc cccacccctc 1680 cttggctctc cagcacttcc
tgggcagcca cggccccctc ccccaacatt gccacacacc 1740 tggaggctga
cgttgccaaa ccagccaggg aaccaacctg ggaagtggcc agaactgcct 1800
ggggtccaag aactcttgtg cctccgtcca tcaccatgtg ggttttgaag accctcgact
1860 gcctccccga tgctccgaag cctgatcttc cagggtgggg aggagaaaat
cccacctccc 1920 ctgacctcca ccacctccac caccaccacc accaccacca
ccaccactac caccaccacc 1980 caactggggc tagagtgggg aagatttccc
ctttagatca aactgcccct tccatggaaa 2040 agctggaaaa aaactctgga
acccatatcc aggcttggtg aggttgctgc caacagtcct 2100 ggcctccccc
atccctaggc aaagagccat gagtcctgga ggaggagagg acccctccca 2160
aaggactgga agcaaaaccc tctgcttcct tgggtccctc caagactccc tggggcccaa
2220 ctgtgttgct ccacccggac ccatctctcc cttctagacc tgagcttgcc
cctccagcta 2280 gcactaagca acatctcgct gtaagcgcct gtaaattact
gtgaaatgtg aaacgtgcaa 2340 tcttgaaact gaggtgttag aaaacttgat
ctgtggtgtt ttgttttgtt ttttttctta 2400 aaacaacagc aacgtgaaaa
aaaaaaaaaa aaa 2433 3 3645 DNA Mus sp. 3 gcccgcctgc cgagcgcgct
caccgccgct ctccctcctt gctctgcagc cgcggcccat 60 ggagcccgcc
ggcgcccctg gccgcctagg gccgctgctg ctctgcctgc tgctctccgc 120
gtcctgtttc tgtacaggag tcacggggaa agaactgaag gtgactcagc ctgagaaatc
180 agtgtctgtt gctgctgggg attcgaccgt tctgaactgc actttgacct
ccttgttgcc 240 ggtgggaccc attaagtggt acagaggagt aggcaaagcc
ggctgtttga tctacagttt 300 cacaggagaa cactttcctc gagttacaaa
tgtttcagat gctactaaga gaaacaatat 360 ggacttttcc atccgtatca
gtaatgtcac cccagaagat gccggtacct actactgtgt 420 gaagttccag
aaaggaccat cagagcctga cacagaaata caatctggag ggggaacaga 480
ggtctatgta ctcgccaaac cttctccacc ggaggatccc cccaggagac aggggcatac
540 tgaccagaaa gtgaacttca cctgcaagtc tcatggcttc tctccccgga
atatcaccct 600 gaagtggttc aaagatgggc aagaactcca ccccttggag
accaccgtga accctagtgg 660 aaagaatgtc tcctacaaca tctccagcac
agtcagggtg gtactaaact ccatggatgt 720 tcattctaag gtcatctgcg
aggtagccca catcaccttg gatagaagcc ctcttcgtgg 780 gattgctaac
ctgtctaact tcatccgagt ttcacccacc gtgaaggtca cccaacagtc 840
cccgacgtca atgaaccagg tgaacctcac ctgccgggat gagaggttct accccgagga
900 tctccagctg atctggctgg agaatggaaa cgtatcacgg aatgacacgc
ccaagaatct 960 cacaaagaac acggatggga cctataatta cacaagcttg
ttcctggtga actcatctgc 1020 tcatagagag gacgtggtgt tcacgtgcca
ggtgaagcac gaccaacagc cagcgatcac 1080 ccgaaaccat accgtgctgg
gacttgccca ctcgagtgat caagggagca tgcaaacctt 1140 ccctggtaat
aatgctaccc acaactggaa tgtcttcatc ggtgtgggcg tggcgtgtgc 1200
tttgctcgta gtcctgctga tggctgctct ctacctcctc cggatcaaac agaagaaagc
1260 caaggggtca acatcttcca cacggttgca cgagcccgag aagaacgcca
gggaaataac 1320 ccaggtacag tctttgatcc aggacacaaa tgacatcaac
gacatcacat acgcagacct 1380 gaatctccca aagagaagga agcccgcacc
cggctccctt gagttcctta acaaccacac 1440 agaatatgca agcattgaga
caggcaaagt gcctaggcca gaggataccc tcacctatgc 1500 tgacctggac
atggtccacc tcagccgggc acagccagcc cccaagcctg agccatcttt 1560
ctcagagtat gctagtgtcc aggtccagag gaagtgaatg gggctgtggt ctgtactagg
1620 ccccatcccc acaagttttc ttgtcctaca tggagtggcc atgacgagga
catccagcca 1680 gccaatcctg tccccagaag gccaggtggc acgggtccta
ggaccagggg taagggtggc 1740 ctttgtcttc cctccgtggc tcttcaacac
ctcttgggca ccacgtcccc ttcttccgga 1800 ggctgggtct tgcagaacca
gagggcgaac tggagaaatc tgcctggaat ccaagaagtg 1860 ttgtgcctcg
gcccatcact cgtgggctcg gatcctggtc ttggcaaccc caggttgcgt 1920
ccttgatgtt ccagagcttg gtcttctgtg tggagaagag ctcaccatct ctacccaact
1980 tgagctttgg gaccagactc cctttagatc aaaccgcccc atctgtggaa
gaactacacc 2040 agaagtcgac aagttttcag ccaacagtgt ctggcctccc
cacctcccag gctgactagc 2100 ctggggagaa ggaaccctct cctcctagac
cagcagagac tccctgggca tgttcagtgt 2160 ggccccacct cccttccagt
cccagcttgc ttcctccagc tagcactaac tcagcagcat 2220 cgctctgtgg
acgcctgtaa attattgaga aatgtgaact gtgcagtctt aaagctaagg 2280
tgttagaaaa tttgatttat gctgtttagt tgttgttggg tttcttttct ttttaatttc
2340 tttttctttt ttgatttttt ttctttccct taaaacaaca gcagccagca
tcttggctct 2400 ttgtcatgtg ttgaatggtt gggtcttgtg aagtctgagg
tctaacagtt tattgtcctg 2460 gaaggatttt cttacagcag aaacagattt
ttttcaaatt cccagaatcc tgaggaccaa 2520 gaaggatccc tcagctgcta
cttccagcac gcagcgtcac tgggacgaac caggccctgt 2580 tcttacaagg
ccacatggcg ggcctttgcc tccatggcta ctgtggtaag tgcagccttg 2640
tctgacccaa tgctgaccta atgttggcca ttccacattg aggggacaag gtcagtgatg
2700 ccccccttgg ctcacaagca cttcagaggc atgcagagag aagggacact
cgtccagctc 2760 tctgaggtaa tcagtgcaag gaggagtccg ttttttgcca
gcaaacctca gcaggatcac 2820 actggaacag aacctggtca tacctgtgac
aacacagctg tgagccaggg caaaccaccc 2880 actgtcactg gctcgagagt
ctgggagagc tctgacccga caccctttaa actggatgcc 2940 ggggcctggc
tgggcaatgc caagtggtta tggcaaccct gactatctgg tcttaacatg 3000
tagctcagga agtggaggcg ctaatgtccc caatccctgg ggattcctga ttccagctat
3060 tcatgtaagc agagccaacc tgcctatttc tgtagggtgc gactgggatg
ttaggagcac 3120 agcaaggacc cagctctgta gggctggtga cctgatacct
tctcataatg gcatctagaa 3180 gttaggctga gttgcctcac tggcccagca
aaccagaact tgtctttggc cgggccatgt 3240 tcttgggctg tcttctaatt
ccaaagggtt ggttggtaaa gctccacccc cttctcctct 3300 gcctaaagac
ataacatgtg tatacacaca cgggtgtata gatgagttaa aagaatgtcc 3360
tcgctggcat cctaattttg tcttaagttt ttttggaggg agaaaggaac aaggcaaggg
3420 aagatgtgta gctttggctt taaccaggca gcctgggggc tcccaagcct
atggaaccct 3480 ggtacaaaga agagaacaga agcgccctgt gaggagtggg
atttgttttt ctgtagacca 3540 gatgagaagg aaacaggccc tgttttgtac
atagttgcaa cttaaaattt ttggcttgca 3600 aaatattttt gtaataaaga
tttctgggta acaataaaaa aaaaa 3645 4 2020 DNA Mus sp. 4 ccctcactaa
agggaacaaa agctggagct ccaccgcggt ggcggccgct ctagaactag 60
tggatccccc gggctgcagg caaccatgct tctcctagat gcctggaccc acattcctca
120 ctgtgtcctg ctgttgatcc tgcttctggg acttaaagga gcagctatga
gagagctgaa 180 ggtgatccaa cctgttaaat cattttttgt tggtgctgga
gggtcagcca ctctgaactg 240 cacagtgaca tctctcctcc ctgtggggcc
catgaggtgg tacaggggta taggacaaag 300 tcgactcttg atatactcgt
tcacaggaga aggcttcccc agaataacaa atacttcaga 360 tactacaaag
agaaacaaca tggacttttc catccgtatc agtaatgtca ctcctgctga 420
ttcgggtacc tactactgtg tgaagttcca gagaggacca tcagactttt acactgagat
480 tcagtctgga ggtggcactg agttgtcagt acttgctaaa ccatcttcac
ctatggtctc 540 cggtcctgca gccagagctg tccctcagca gacagtgacc
tttacatgca gatcccatgg 600 attctttccg cggaacctca cgctgaagtg
gttcaagaat ggagatgaga tctctcactt 660 ggaaacttct gtggaaccgg
aagaaacaag tgtctcctat agagtttcca gcacagtcca 720 ggtggtgttg
gaacctaggg atgtccgctc tcagatcatc tgtgaagtgg atcatgtcac 780
tttagatcga gcccctctca gagggattgc tcacatctct gagttcattc aagttccacc
840 caccctggag atccgccagc agccaacaat ggtttggaat gtgataaatg
ttacctgcca 900 aatacagaag ttctatcctc caagttttca gttgacctgg
ttagagaatg gaaatatatc 960 ccggagagaa gtacctttta cacttacagt
aaacaaggat ggaacttaca actggatcag 1020 ctgtctcttg gtgaacatat
ctgcccttga ggagaacatg gtagtgacat gccaggttga 1080 gcatgatgga
caagcagaag tcattgaaac ccatactgtg gtggtcactg aacatcagag 1140
agtgaaaggt actgctacca agtctggtga ggtcttcacc ccacccttat gtctaaatgt
1200 aaattgggct ttatttttta tgtataaggt aacattcttg attattgtag
cattatcctg 1260 acaactacaa agtaaaatgt taacgtcata tttcattccc
aacttctcac acgtctcaca 1320 tatctttcca ctaatagatt aaatagttaa
gaatggaagg tatcatcaaa ttccagtatc 1380 ttgccccttc cctgttttac
ctaacatttg tgaacatcct tatgctcatg tgtttccttt 1440 accatatctt
tactgactcc attacatttt agatatttcc taaatatagt gtcctaatgg 1500
agtgaaattt caacgggtca cctgacaacc tgtttgtaca cacacacaca cacacacaca
1560 cacacacaca cacacagcat atgatctgga ctaatgaaat aaaggaaaat
caaatgtcca 1620 ttggagcact gctatcacta aggtataagg aaaacttgct
agcaaagtat ttcttttcaa 1680 cttgttacga tgctagcagt tagtttgcat
tagattggac ccatttatgt gaatatcttt 1740 ttccttctct taaaacaaca
aaaaagatcc tcaactccag tgacttttga aaaactcatg 1800 ttccttggca
tccctccttt gctgtgagtt cattggctgg ataaacactg ggtcgcctaa 1860
ttatctataa atatgccagt taaaaatgtc aaggttagaa agcatcagtc catacagtgc
1920 aaatatagtc cacagtgggt gctcaggtaa atcatgatat tttcatttaa
aatatacatt 1980 caataaaatt aactgtagtt caaaaaaaaa aaaaaaaaaa 2020 5
398 PRT Homo sapiens 5 Met Pro Val Pro Ala Ser Trp Pro His Leu Pro
Ser Pro Phe Leu Leu 1 5 10 15 Met Thr Leu Leu Leu Gly Arg Leu Thr
Gly Val Ala Gly Glu Asp Glu 20 25 30 Leu Gln Val Ile Gln Pro Glu
Lys Ser Val Ser Val Ala Ala Gly Glu 35 40 45 Ser Ala Thr Leu Arg
Cys Ala Met Thr Ser Leu Ile Pro Val Gly Pro 50 55 60 Ile Met Trp
Phe Arg Gly Ala Gly Ala Gly Arg Glu Leu Ile Tyr Asn 65 70 75 80 Gln
Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Glu Leu Thr 85 90
95 Lys Arg Asn Asn Leu Asn Phe Ser Ile Ser Ile Ser Asn Ile Thr Pro
100 105 110 Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly
Ser Pro 115 120 125 Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu
Leu Ser Val Arg 130 135 140 Ala Lys Pro Ser Ala Pro Val Val Ser Gly
Pro Ala Val Arg Ala Thr 145 150 155 160 Pro Glu His Thr Val Ser Phe
Thr Cys Glu Ser His Gly Phe Ser Pro 165 170 175 Arg Asp Ile Thr Leu
Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp 180 185 190 Phe Gln Thr
Asn Val Asp Pro Ala Gly Asp Ser Val Ser Tyr Ser Ile 195 200 205 His
Ser Thr Ala Arg Val Val Leu Thr Arg Gly Asp Val His Ser Gln 210 215
220 Val Ile Cys Glu Met Ala His Ile Thr Leu Gln Gly Asp Pro Leu Arg
225 230 235 240 Gly Thr Ala Asn Leu Ser Glu Ala Ile Arg Val Pro Pro
Thr Leu Glu 245 250 255 Val Thr Gln Gln Pro Met Arg Ala Glu Asn Gln
Ala Asn Val Thr Cys 260 265 270 Gln Val Ser Asn Phe Tyr Pro Arg Gly
Leu Gln Leu Thr Trp Leu Glu 275 280 285 Asn Gly Asn Val Ser Arg Thr
Glu Thr Ala Ser Thr Leu Ile Glu Asn 290 295 300 Lys Asp Gly Thr Tyr
Asn Trp Met Ser Trp Leu Leu Val Asn Thr Cys 305 310 315 320 Ala His
Arg Asp Asp Val Val Leu Thr Cys Gln Val Glu His Asp Gly 325 330 335
Gln Gln Ala Val Ser Lys Ser Tyr Ala Leu Glu Ile Ser Ala His Gln 340
345 350 Lys Glu His Gly Ser Asp Ile Thr His Glu Pro Ala Leu Ala Pro
Thr 355 360 365 Ala Pro Leu Leu Val Ala Leu Leu Leu Gly Pro Lys Leu
Leu Leu Val 370 375 380 Val Gly Val Ser Ala Ile Tyr Ile Cys Trp Lys
Gln Lys Ala 385 390 395 6 503 PRT Homo sapiens 6 Met Glu Pro Ala
Gly Pro Ala Pro Gly Arg Leu Gly Pro Leu Leu Cys 1 5 10 15 Leu Leu
Leu Ala Ala Ser Cys Ala Trp Ser Gly Val Ala Gly Glu Glu 20 25 30
Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala Ala Gly 35
40 45 Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro Val
Gly 50 55 60 Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu
Leu Ile Tyr 65 70 75 80
Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Glu Ser 85
90 95 Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn Ile
Thr 100 105 110 Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg
Lys Gly Ser 115 120 125 Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr
Glu Leu Ser Val Arg 130 135 140 Ala Lys Pro Ser Ala Pro Val Val Ser
Gly Pro Ala Ala Arg Ala Thr 145 150 155 160 Pro Gln His Thr Val Ser
Phe Thr Cys Glu Ser His Gly Phe Ser Pro 165 170 175 Arg Asp Ile Thr
Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp 180 185 190 Phe Gln
Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr Ser Ile 195 200 205
His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His Ser Gln 210
215 220 Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu
Arg 225 230 235 240 Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val Pro
Pro Thr Leu Glu 245 250 255 Val Thr Gln Gln Pro Val Arg Ala Glu Asn
Gln Val Asn Val Thr Cys 260 265 270 Gln Val Arg Lys Phe Tyr Pro Gln
Arg Leu Gln Leu Thr Trp Leu Glu 275 280 285 Asn Gly Asn Val Ser Arg
Thr Glu Thr Ala Ser Thr Val Thr Glu Asn 290 295 300 Lys Asp Gly Thr
Tyr Asn Trp Met Ser Trp Leu Leu Val Asn Val Ser 305 310 315 320 Ala
His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His Asp Gly 325 330
335 Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser Ala His Pro
340 345 350 Lys Glu Gln Gly Ser Asn Thr Ala Ala Glu Asn Thr Gly Ser
Asn Glu 355 360 365 Arg Asn Ile Tyr Ile Val Val Gly Val Val Cys Thr
Leu Leu Val Ala 370 375 380 Leu Leu Met Ala Ala Leu Tyr Leu Val Arg
Ile Arg Gln Lys Lys Ala 385 390 395 400 Gln Gly Ser Thr Ser Ser Thr
Arg Leu His Glu Pro Glu Lys Asn Ala 405 410 415 Arg Glu Ile Thr Gln
Asp Thr Asn Asp Ile Thr Tyr Ala Asp Leu Asn 420 425 430 Leu Pro Lys
Gly Lys Lys Pro Ala Pro Gln Ala Ala Glu Pro Asn Asn 435 440 445 His
Thr Glu Tyr Ala Ser Ile Gln Thr Ser Pro Gln Pro Ala Ser Glu 450 455
460 Asp Thr Leu Thr Tyr Ala Asp Leu Asp Met Val His Leu Asn Arg Thr
465 470 475 480 Pro Lys Gln Pro Ala Pro Lys Pro Glu Pro Ser Phe Ser
Glu Tyr Ala 485 490 495 Ser Val Gln Val Pro Arg Lys 500 7 512 PRT
Mus sp. 7 Met Glu Pro Ala Gly Ala Pro Gly Arg Leu Gly Pro Leu Leu
Leu Cys 1 5 10 15 Leu Leu Leu Ser Ala Ser Cys Phe Cys Thr Gly Val
Thr Gly Lys Glu 20 25 30 Leu Lys Val Thr Gln Pro Glu Lys Ser Val
Ser Val Ala Ala Gly Asp 35 40 45 Ser Thr Val Leu Asn Cys Thr Leu
Thr Ser Leu Leu Pro Val Gly Pro 50 55 60 Ile Lys Trp Tyr Arg Gly
Val Gly Lys Ala Gly Cys Leu Ile Tyr Ser 65 70 75 80 Phe Thr Gly Glu
His Phe Pro Arg Val Thr Asn Val Ser Asp Ala Thr 85 90 95 Lys Arg
Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Asn Val Thr Pro 100 105 110
Glu Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Gln Lys Gly Pro Ser 115
120 125 Glu Pro Asp Thr Glu Ile Gln Ser Gly Gly Gly Thr Glu Val Tyr
Val 130 135 140 Leu Ala Lys Pro Ser Pro Pro Glu Asp Pro Pro Arg Arg
Gln Gly His 145 150 155 160 Thr Asp Gln Lys Val Asn Phe Thr Cys Lys
Ser His Gly Phe Ser Pro 165 170 175 Arg Asn Ile Thr Leu Lys Trp Phe
Lys Asp Gly Gln Glu Leu His Pro 180 185 190 Leu Glu Thr Thr Val Asn
Pro Ser Gly Lys Asn Val Ser Tyr Asn Ile 195 200 205 Ser Ser Thr Val
Arg Val Val Leu Asn Ser Met Asp Val His Ser Lys 210 215 220 Val Ile
Cys Glu Val Ala His Ile Thr Leu Asp Arg Ser Pro Leu Arg 225 230 235
240 Gly Ile Ala Asn Leu Ser Asn Phe Ile Arg Val Ser Pro Thr Val Lys
245 250 255 Val Thr Gln Gln Ser Pro Thr Ser Met Asn Gln Val Asn Leu
Thr Cys 260 265 270 Arg Asp Glu Arg Phe Tyr Pro Glu Asp Leu Gln Leu
Ile Trp Leu Glu 275 280 285 Asn Gly Asn Val Ser Arg Asn Asp Thr Pro
Lys Asn Leu Thr Lys Asn 290 295 300 Thr Asp Gly Thr Tyr Asn Tyr Thr
Ser Leu Phe Leu Val Asn Ser Ser 305 310 315 320 Ala His Arg Glu Asp
Val Val Phe Thr Cys Gln Val Lys His Asp Gln 325 330 335 Gln Pro Ala
Ile Thr Arg Asn His Thr Val Leu Gly Leu Ala His Ser 340 345 350 Ser
Asp Gln Gly Ser Met Gln Thr Phe Pro Gly Asn Asn Ala Thr His 355 360
365 Asn Trp Asn Val Phe Ile Gly Val Gly Val Ala Cys Ala Leu Leu Val
370 375 380 Val Leu Leu Met Ala Ala Leu Tyr Leu Leu Arg Ile Lys Gln
Lys Lys 385 390 395 400 Ala Lys Gly Ser Thr Ser Ser Thr Arg Leu His
Glu Pro Glu Lys Asn 405 410 415 Ala Arg Glu Ile Thr Gln Val Gln Ser
Leu Ile Gln Asp Thr Asn Asp 420 425 430 Ile Asn Asp Ile Thr Tyr Ala
Asp Leu Asn Leu Pro Lys Arg Arg Lys 435 440 445 Pro Ala Pro Gly Ser
Leu Glu Phe Leu Asn Asn His Thr Glu Tyr Ala 450 455 460 Ser Ile Glu
Thr Gly Lys Val Pro Arg Pro Glu Asp Thr Leu Thr Tyr 465 470 475 480
Ala Asp Leu Asp Met Val His Leu Ser Arg Ala Gln Pro Ala Pro Lys 485
490 495 Pro Glu Pro Ser Phe Ser Glu Tyr Ala Ser Val Gln Val Gln Arg
Lys 500 505 510 8 391 PRT Mus sp. 8 Met Leu Leu Leu Asp Ala Trp Thr
His Ile Pro His Cys Val Leu Leu 1 5 10 15 Leu Ile Leu Leu Leu Gly
Leu Lys Gly Ala Ala Met Arg Glu Leu Lys 20 25 30 Val Ile Gln Pro
Val Lys Ser Phe Phe Val Gly Ala Gly Gly Ser Ala 35 40 45 Thr Leu
Asn Cys Thr Val Thr Ser Leu Leu Pro Val Gly Pro Met Arg 50 55 60
Trp Tyr Arg Gly Ile Gly Gln Ser Arg Leu Leu Ile Tyr Ser Phe Thr 65
70 75 80 Gly Glu Gly Phe Pro Arg Ile Thr Asn Thr Ser Asp Thr Thr
Lys Arg 85 90 95 Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Asn Val
Thr Pro Ala Asp 100 105 110 Ser Gly Thr Tyr Tyr Cys Val Lys Phe Gln
Arg Gly Pro Ser Asp Phe 115 120 125 Tyr Thr Glu Ile Gln Ser Gly Gly
Gly Thr Glu Leu Ser Val Leu Ala 130 135 140 Lys Pro Ser Ser Pro Met
Val Ser Gly Pro Ala Ala Arg Ala Val Pro 145 150 155 160 Gln Gln Thr
Val Thr Phe Thr Cys Arg Ser His Gly Phe Phe Pro Arg 165 170 175 Asn
Leu Thr Leu Lys Trp Phe Lys Asn Gly Asp Glu Ile Ser His Leu 180 185
190 Glu Thr Ser Val Glu Pro Glu Glu Thr Ser Val Ser Tyr Arg Val Ser
195 200 205 Ser Thr Val Gln Val Val Leu Glu Pro Arg Asp Val Arg Ser
Gln Ile 210 215 220 Ile Cys Glu Val Asp His Val Thr Leu Asp Arg Ala
Pro Leu Arg Gly 225 230 235 240 Ile Ala His Ile Ser Glu Phe Ile Gln
Val Pro Pro Thr Leu Glu Ile 245 250 255 Arg Gln Gln Pro Thr Met Val
Trp Asn Val Ile Asn Val Thr Cys Gln 260 265 270 Ile Gln Lys Phe Tyr
Pro Pro Ser Phe Gln Leu Thr Trp Leu Glu Asn 275 280 285 Gly Asn Ile
Ser Arg Arg Glu Val Pro Phe Thr Leu Thr Val Asn Lys 290 295 300 Asp
Gly Thr Tyr Asn Trp Ile Ser Cys Leu Leu Val Asn Ile Ser Ala 305 310
315 320 Leu Glu Glu Asn Met Val Val Thr Cys Gln Val Glu His Asp Gly
Gln 325 330 335 Ala Glu Val Ile Glu Thr His Thr Val Val Val Thr Glu
His Gln Arg 340 345 350 Val Lys Gly Thr Ala Thr Lys Ser Gly Glu Val
Phe Thr Pro Pro Leu 355 360 365 Cys Leu Asn Val Asn Trp Ala Leu Phe
Phe Met Tyr Lys Val Thr Phe 370 375 380 Leu Ile Ile Val Ala Leu Ser
385 390 9 13 PRT Rattus sp. 9 Pro Ile Tyr Ser Phe Ile Gly Gly Glu
His Phe Pro Arg 1 5 10 10 9 PRT Rattus sp. 10 Ile Val Glu Pro Asp
Thr Glu Ile Lys 1 5 11 6 PRT Rattus sp. 11 Tyr Gly Phe Ser Pro Arg
1 5 12 12 PRT Rattus sp. 12 Ile Lys Glu Val Ala His Val Asn Leu Glu
Val Arg 1 5 10 13 8 PRT Rattus sp. 13 Val Ala Ala Gly Asp Ser Ala
Thr 1 5 14 107 PRT Unknown Organism Description of Unknown Organism
Mus sp. or Homosapiens 14 Asp Glu Leu Gln Val Ile Gln Pro Glu Lys
Ser Val Ser Val Ala Ala 1 5 10 15 Gly Glu Ser Ala Thr Leu Arg Cys
Ala Met Thr Ser Leu Ile Pro Val 20 25 30 Gly Pro Ile Met Trp Phe
Arg Gly Ala Gly Ala Gly Arg Glu Leu Ile 35 40 45 Tyr Asn Gln Lys
Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Glu 50 55 60 Leu Thr
Lys Arg Asn Asn Leu Asp Phe Ser Ile Ser Ile Ser Asn Ile 65 70 75 80
Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Glu Gly 85
90 95 Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala 100 105 15 107
PRT Unknown Organism Description of Unknown Organism Mus sp. or
Homosapiens 15 Asp Glu Leu Gln Val Ile Gln Pro Glu Lys Ser Val Ser
Val Ala Ala 1 5 10 15 Gly Glu Ser Ala Thr Leu Arg Cys Ala Met Thr
Ser Leu Ile Pro Val 20 25 30 Gly Pro Ile Met Trp Phe Arg Gly Ala
Gly Ala Gly Arg Glu Leu Ile 35 40 45 Tyr Asn Gln Lys Glu Gly His
Phe Pro Arg Val Thr Thr Val Ser Glu 50 55 60 Leu Thr Lys Arg Asn
Asn Leu Asp Phe Ser Ile Ser Ile Ser Asn Ile 65 70 75 80 Thr Pro Ala
Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly 85 90 95 Ser
Pro Asp Asp Val Glu Phe Lys Ser Gly Ala 100 105 16 107 PRT Unknown
Organism Description of Unknown Organism Mus sp. or Homosapiens 16
Glu Glu Leu Gln Val Ile Gln Pro Glu Lys Ser Val Leu Val Ala Ala 1 5
10 15 Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro
Val 20 25 30 Gly Pro Ile Met Trp Phe Arg Gly Ala Gly Ala Gly Arg
Glu Leu Ile 35 40 45 Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val
Thr Thr Val Ser Asp 50 55 60 Leu Thr Lys Arg Asn Asn Met Asp Phe
Ser Ile Arg Ile Gly Asn Ile 65 70 75 80 Thr Pro Ala Asp Ala Gly Thr
Tyr Tyr Cys Val Lys Phe Arg Lys Gly 85 90 95 Ser Pro Asp Asp Val
Glu Phe Lys Ser Gly Ala 100 105 17 107 PRT Unknown Organism
Description of Unknown Organism Mus sp. or Homosapiens 17 Glu Glu
Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala Pro 1 5 10 15
Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro Val 20
25 30 Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu
Ile 35 40 45 Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr
Val Ser Glu 50 55 60 Ser Thr Lys Arg Glu Asn Met Asn Phe Ser Ile
Ser Ile Ser Asn Ile 65 70 75 80 Thr Pro Ala Asp Ala Gly Thr Tyr Tyr
Cys Val Lys Phe Arg Lys Gly 85 90 95 Ser Pro Asp Asp Val Glu Phe
Lys Ser Gly Ala 100 105 18 107 PRT Unknown Organism Description of
Unknown Organism Mus sp. or Homosapiens 18 Asp Glu Leu Gln Val Ile
Gln Pro Asp Lys Ser Val Ser Val Ala Ala 1 5 10 15 Gly Glu Ser Ala
Thr Leu Arg Cys Ala Met Thr Ser Leu Ile Pro Val 20 25 30 Gly Pro
Ile Met Trp Phe Arg Gly Ala Gly Ala Gly Arg Glu Leu Ile 35 40 45
Ser Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Glu 50
55 60 Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn
Ile 65 70 75 80 Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe
Arg Lys Gly 85 90 95 Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala
100 105 19 107 PRT Unknown Organism Description of Unknown Organism
Mus sp. or Homosapiens 19 Asp Glu Leu Gln Val Ile Gln Pro Glu Lys
Ser Val Ser Val Ala Pro 1 5 10 15 Gly Glu Ser Ala Thr Leu Arg Cys
Ala Met Thr Ser Leu Ile Pro Val 20 25 30 Gly Pro Ile Met Trp Phe
Arg Gly Ala Gly Ala Gly Arg Glu Leu Ile 35 40 45 Ser Asn Gln Lys
Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Glu 50 55 60 Leu Thr
Lys Arg Asn Asn Leu Asp Phe Ser Ile Ser Ile Ser Asn Ile 65 70 75 80
Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly 85
90 95 Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala 100 105 20 106
PRT Unknown Organism Description of Unknown Organism Mus sp. or
Homosapiens 20 Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser
Val Ala Pro 1 5 10 15 Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr
Ser Leu Ile Pro Val 20 25 30 Gly Pro Ile Gln Trp Phe Arg Gly Ala
Gly Pro Ala Arg Glu Leu Ile 35 40 45 Tyr Asn Gln Lys Glu Gly His
Phe Pro Arg Val Thr Thr Val Ser Asp 50 55 60 Leu Thr Lys Arg Asn
Asn Leu Asp Phe Ser Ile Ser Ile Ser Asn Ile 65 70 75 80 Thr Pro Ala
Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly 85 90 95 Ser
Pro Asp Val Glu Phe Lys Ser Gly Ala 100 105 21 106 PRT Unknown
Organism Description of Unknown Organism Mus sp. or Homosapiens 21
Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala Pro 1 5
10 15 Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro
Val 20 25 30 Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg
Glu Leu Ile 35 40 45 Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val
Thr Thr Val Ser Glu 50 55 60 Ser Thr Lys Arg Glu Asn Leu Asp Phe
Ser Ile Ser Ile Ser Asn Ile 65 70 75 80 Thr Pro Ala Asp Ala Gly Thr
Tyr Tyr Cys Val Lys Phe Arg Lys Gly 85 90 95 Ser Pro Asp Val Glu
Phe Lys Ser Gly Ala 100 105 22 107 PRT Unknown Organism Description
of Unknown Organism Mus sp. or Homosapiens 22 Asp Glu Leu Gln Val
Ile Gln Ser Glu Lys Ser Val Ser Val Ala Ala 1 5 10 15 Gly Glu Ser
Ala Ala Leu His Cys Ala Met Thr Ser Leu Ile Pro Val 20 25 30 Gly
Pro Ile Met Trp Phe Arg Gly Ala Gly Ala Gly Arg Glu Leu Ile 35 40
45 Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Glu
50 55 60 Leu Thr Lys Arg Asn Asn Leu Asp Phe Ser Ile Ser Ile Ser
Asn Ile 65 70 75 80 Thr Pro Ala Asp Ala Gly Thr
Tyr Tyr Cys Val Lys Phe Arg Lys Gly 85 90 95 Ser Pro Asp Asp Val
Glu Phe Lys Ser Gly Ala 100 105 23 107 PRT Unknown Organism
Description of Unknown Organism Mus sp. or Homosapiens 23 Asp Glu
Leu Gln Val Ile Gln Pro Glu Lys Ser Val Ser Val Ala Ala 1 5 10 15
Gly Glu Ser Ala Thr Leu Arg Cys Ala Met Thr Ser Leu Ile Pro Val 20
25 30 Gly Pro Ile Met Trp Phe Arg Gly Ala Gly Ala Gly Arg Glu Leu
Ile 35 40 45 Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr
Val Ser Glu 50 55 60 Leu Thr Lys Arg Asn Asn Leu Asp Phe Ser Ile
Arg Ile Ser Asn Ile 65 70 75 80 Thr Pro Ala Asp Ala Gly Thr Tyr Tyr
Cys Val Lys Phe Arg Lys Gly 85 90 95 Ser Pro Asp Asp Val Glu Phe
Lys Ser Gly Ala 100 105 24 107 PRT Unknown Organism Description of
Unknown Organism Mus sp. or Homosapiens 24 Asp Glu Leu Gln Val Ile
Gln Pro Glu Ala Phe Val Ser Val Ala Ala 1 5 10 15 Gly Glu Met Ala
Thr Leu Asn Cys Thr Val Thr Ser Leu Leu Pro Val 20 25 30 Gly Pro
Ile Gln Trp Phe Arg Gly Ala Cys Pro Gly Gln Lys Leu Ile 35 40 45
Tyr Ser Pro Lys Arg Cys His Ser Pro Arg Val Thr Thr Ile Ser Asp 50
55 60 Gln Arg Lys Arg Asn Ser Thr Asp Tyr Ser Ile Arg Ile Ser Ser
Ile 65 70 75 80 Thr Leu Glu Asp Ala Gly Thr Tyr Tyr Cys Met Lys Leu
Arg Arg Ala 85 90 95 Ile Pro Ala Asn Val Glu Ile Lys Ser Gly Thr
100 105 25 107 PRT Unknown Organism Description of Unknown Organism
Mus sp. or Homosapiens 25 Glu Glu Leu Gln Met Ile Gln Pro Glu Lys
Leu Leu Leu Val Thr Val 1 5 10 15 Gly Lys Thr Ala Thr Leu His Cys
Thr Val Thr Ser Leu Leu Pro Val 20 25 30 Gly Pro Val Leu Trp Phe
Arg Gly Val Gly Pro Gly Arg Glu Leu Ile 35 40 45 Tyr Asn Gln Lys
Glu Gly His Phe Pro Arg Val Thr Arg Val Ser Asp 50 55 60 Leu Thr
Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Ser Ile 65 70 75 80
Thr Pro Ala Val Val Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly 85
90 95 Ser Pro Glu Asn Val Glu Phe Lys Ser Gly Pro 100 105 26 106
PRT Unknown Organism Description of Unknown Organism Mus sp. or
Homosapiens 26 Glu Glu Leu Gln Val Ile Gln Pro Glu Lys Ser Val Ser
Val Ala Ala 1 5 10 15 Gly Glu Ser Ala Ala Leu Gln Cys Thr Val Thr
Ser Leu Asn Pro Val 20 25 30 Gly Pro Ile Gln Arg Phe Arg Gly Ala
Gly Pro Gly Arg Lys Leu Ile 35 40 45 Tyr His Gln Lys Glu Gly His
Phe Pro Arg Val Thr Thr Val Ser Asp 50 55 60 Leu Thr Lys Arg Thr
Asn Met Asp Phe Ser Ile Cys Ile Ser Asn Ile 65 70 75 80 Thr Pro Ala
Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Gln Lys Gly 85 90 95 Ser
Pro Asp Val Glu Leu Lys Ser Gly Ala 100 105
* * * * *