U.S. patent application number 10/442754 was filed with the patent office on 2003-10-23 for androgen receptor coactivators.
Invention is credited to Chang, Chawnshang.
Application Number | 20030198990 10/442754 |
Document ID | / |
Family ID | 31721354 |
Filed Date | 2003-10-23 |
United States Patent
Application |
20030198990 |
Kind Code |
A1 |
Chang, Chawnshang |
October 23, 2003 |
Androgen receptor coactivators
Abstract
Disclosed are androgen receptor-associated proteins, designated
ARA24, ARA54, ARA55, and Rb, that have been demonstrated to
interact with the androgen receptor to alter levels of androgen
receptor-mediated transcriptional activation. Certain of these
proteins interact with the androgen receptor in an
androgen-dependent manner, whereas certain proteins may induce
transcriptional activation in the presence of other ligands, such
as E2 or HF. Also disclosed is a method of detecting androgenic or
antiandrogenic activity using these proteins in a mammalian
two-hybrid transient transfection assay.
Inventors: |
Chang, Chawnshang;
(Pittsford, NY) |
Correspondence
Address: |
NEEDLE & ROSENBERG, P.C.
SUITE 1000
999 PEACHTREE STREET
ATLANTA
GA
30309-3915
US
|
Family ID: |
31721354 |
Appl. No.: |
10/442754 |
Filed: |
May 20, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10442754 |
May 20, 2003 |
|
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09354221 |
Jul 15, 1999 |
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Current U.S.
Class: |
435/6.14 ;
435/320.1; 435/325; 435/69.1; 435/7.2; 530/350; 536/23.5 |
Current CPC
Class: |
C07K 14/4705 20130101;
G01N 2333/723 20130101; C07K 14/721 20130101 |
Class at
Publication: |
435/6 ; 435/7.2;
435/69.1; 435/320.1; 435/325; 530/350; 536/23.5 |
International
Class: |
C12Q 001/68; G01N
033/53; G01N 033/567; C07H 021/04; C12P 021/02; C12N 005/06; C07K
014/72 |
Claims
We claim:
1. An isolated polynucleotide comprising a sequence selected from
the group consisting of SEQ ID NO:1 and SEQ ID NO:3.
2. A genetic construct comprising a promoter capable of causing
expression of a protein coding region in a cell, the promoter
operably connected to a protein coding region encoding the
expression of a polypeptide from coding regions of ARA54 or
ARA55.
3. The genetic construct of claim 2 wherein the polypeptide encoded
by the protein coding sequence comprises a sequence selected from
the group consisting of SEQ ID NO:2 and SEQ ID NO:4.
4. A eukaryotic host cell comprising the genetic construct of claim
2.
5. A method for testing the androgenic or antiandrogenic effect of
a chemical compound comprising the steps of: (a) transfecting a
host cell with at least one genetic construct capable of producing
in the host cell a polypeptide selected from the group consisting
of ARA54, ARA55, ARA24, and Rb, the host cell also producing human
androgen receptor protein; (b) exposing the cell to the chemical
compound; and (c) measuring the level of transcriptional activity
caused by the androgen receptor.
6. The method of claim 5 wherein the host cell is a prostate
cell.
7. The method of claim 5, wherein the cell is a eukaryotic cell
that lacks native endogenous androgen receptor, the cell having
also an introduced genetic construct producing androgen receptor
protein.
8. The method of claim 5, wherein the genetic construct comprises a
DNA sequence selected from the group consisting of SEQ ID NO:1, SEQ
ID NO:3, SEQ ID NO:5, and SEQ ID NO:7.
9. The method of claim 5, wherein the cell is transfected with a
genetic construct comprising a reporter gene expressible in the
cell, the expression of said reporter gene being susceptible to
detection and quantitation.
10. The method of claim 9, wherein the reporter gene is selected
from the group consisting of a chloramphenicol acetyltransferase
gene and a luciferase gene.
11. A method for testing the androgenic or antiandrogenic effect of
a chemical compound comprising the steps of: (a) transfecting a
host cell with at least one genetic construct capable of producing
in the host cell human androgen receptor protein and a polypeptide
selected from the group consisting of ARA54, ARA55, ARA24, and Rb;
(b) exposing the cell to the chemical compound; and (c) measuring
the interaction between AR and an AR co-activator.
12. A method as claimed in claim 11 wherein the co-activator is
selected from the group consisting of ARA54, ARA55, ARA24 and Rb.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] Not applicable.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not applicable.
BACKGROUND OF THE INVENTION
[0003] Androgens constitute a class of hormones that control the
development and proper function of mammalian male reproductive
systems, including the prostate and epididymis. Androgens also
affect the physiology of many non-reproductive systems, including
muscle, skin, pituitary, lymphocytes, hair growth, and brain.
Androgens exert their effect by altering the level of gene
expression of specific genes in a process that is mediated by
binding of androgen to an androgen receptor. The androgen receptor,
which is a member of the steroid receptor super family, plays an
important role in male sexual differentiation and in prostate cell
proliferation. Binding of androgen by the androgen receptor allows
the androgen receptor to interact with androgen responsive element
(AREs), DNA sequences found on genes whose expression is regulated
by androgen.
[0004] Androgen-mediated regulation of gene expression is a
complicated process that may involve multiple co-activators (Adler
et al., Proc. National Acad. Sci. USA 89:6319-6325, 1992). A
fundamental question in the field of steroid hormone biology is how
specific androgen-activated transcription can be achieved in vivo
when several different receptors recognize the same DNA sequence.
For example, the androgen receptor (AR), the glucocorticoid
receptor (GR), and the progesterone receptor (PR) all recognize the
same sequence but activate different transcription activities. Some
have speculated that accessory factors may selectively interact
with the androgen receptor to determine the specificity of gene
activation by the androgen receptor.
[0005] Prostate cancer is the most common malignant neoplasm in
aging males in the United States. Standard treatment includes the
surgical or chemical castration of the patient in combination with
the administration of anti-androgens such as 17 .beta. estradiol
(E2) or hydroxyflutamide (HF). However, most prostate cancers
treated with androgen ablation and anti-androgens progress from an
androgen-dependant to an androgen-independent state, causing a high
incidence of relapse within 18 months (Crawford, Br. J. Urology 70:
suppl. 1, 1992). The mechanisms by which prostate cancer cells
become resistant to hormonal therapy remain unclear. One hypothesis
that has been advanced is that over the course of treatment, a
mutation in the AR occurs which alters the receptor's sensitivity
to other steroid hormones or anti-androgens, such as E2 and HF,
thereby causing the progression from androgen-dependent to
androgen-independent prostrate cancer. This hypothesis is supported
by transient transfection assays in which it has been shown that
anti-androgens may have an agonistic activity that stimulates
mutant AR (mAR) mediated transcription.
[0006] Recently, A1B1 was identified as estrogen receptor
coactivator that is expressed at higher levels in ovarian cancer
cell lines and breast cancer cells than in noncancerous cells
(Anzick, et al. Science 277:965-968, 1997). This result suggests
that steroid hormone receptor cofactors may play an important role
in the progression of certain diseases, such as hormone responsive
tumors.
[0007] The identification, isolation, and characterization of genes
that encode factors involved in the regulation of gene expression
by androgen receptors will facilitate the development of screening
assays to evaluate the potential efficacy of drugs in the treatment
of prostate cancers.
BRIEF SUMMARY OF THE INVENTION
[0008] The present invention includes an isolated polynucleotide
that encodes a co-activator for human androgen receptor, the
polynucleotide comprising a sequence that encodes a polypeptide
selected from the group consisting of an ARA54 polypeptide, an
ARA55 polypeptide, an ARA24 polypeptide, and an Rb polypeptide.
[0009] Another aspect of the present invention is a genetic
construct comprising a promoter functional in a prokaryotic or
eukaryotic cell operably connected to a polynucleotide that encodes
a polypeptide selected from the group consisting of an ARA54
polypeptide, an ARA55 polypeptide, an ARA24 polypeptide and an Rb
polypeptide.
[0010] The present invention provides a method for screening
candidate pharmaceutical molecules for the ability to promote or
inhibit the interaction of ARs and AREs to modulate androgenic
activity comprising the steps of:
[0011] (a) providing a genetic construct comprising a promoter
functional in a eukaryotic cell operably connected to a
polynucleotide comprising a sequence that encodes a polypeptide
selected from the group consisting of an ARA54 polypeptide, an
ARA55 polypeptide, an ARA24 polypeptide, and a retinoblastoma
polypeptide;
[0012] (b) cotransforming a suitable eukaryotic cell with the
construct of step a, and a construct comprising at least a portion
of an expressible androgen receptor sequence;
[0013] (c) culturing the cells in the presence of a candidate
pharmaceutical molecule; and
[0014] (d) assaying the transcriptional activity induced by the
androgen receptor.
[0015] It is an object of the present invention to a provide a
genetic construct capable of expressing a factor involved in
co-activation of the human androgen receptor.
[0016] It is an object of the present invention to provide a method
for evaluating the ability of candidate pharmaceutical molecules to
modulate the effect of androgen receptor coactivators on gene
expression.
[0017] Other objects, features, and advantages of the present
invention will become apparent upon reading the specification and
claims.
DETAILED DESCRIPTION OF THE INVENTION
[0018] Transactivation of genes by the androgen receptor is a
complicated system that involves many different coactivators. It is
not currently known just how many factors are involved in androgen
receptor-mediated regulation of gene expression. The identification
and/or characterization of four androgen receptor coactivators is
reported herein. Inclusion of one or more of these coactivators in
an assay for androgenic and antiandrogenic activity is expected to
increase the sensitivity of the assay. Information about these
coactivators is valuable in the design of pharmaceutical agents
intended to enhance or interfere with normal coactivator function.
A preliminary assessment of the efficacy of a potential therapeutic
agent can be made by evaluating the effect of the agent on the
ability of the coactivator to enhance transactivation by the
androgen receptor.
[0019] One aspect of the present invention is an isolated
polynucleotide that encodes a co-activator for human androgen
receptor, the polynucleotide comprising a sequence that encodes a
polypeptide selected from the group consisting of an ARA54
polypeptide, an ARA55 polypeptide, an ARA24 polypeptide and an Rb
polypeptide.
[0020] Another aspect of the present invention is a genetic
construct comprising a promoter functional in a prokaryotic or
eukaryotic cell operably connected to a polynucleotide that encodes
a polypeptide selected from the group consisting of an ARA54
polypeptide, an ARA55 polypeptide, an ARA24 polypeptide and an Rb
polypeptide.
[0021] The present invention includes a method for screening
candidate pharmaceutical molecules for the ability to promote or
inhibit the ARs and AREs to result in modulation of androgenic
effect comprising the steps of:
[0022] (a) providing a genetic construct comprising a promoter
functional in a eukaryotic cell operably connected to a
polynucleotide comprising a sequence that encodes a polypeptide
selected from the group consisting of an ARA54 polypeptide, an
ARA55 polypeptide, an ARA24 polypeptide, and a retinoblastoma
polypeptide;
[0023] (b) cotransforming a suitable eukaryotic cell with the
construct of step a, and a construct comprising at least a portion
of an expressible androgen receptor sequence;
[0024] (c) culturing the cells in the presence of a candidate
pharmaceutical molecule; and
[0025] (d) assaying the transcriptional activity induced by the
androgen receptor gene.
[0026] The human androgen receptor is comprised of a ligand binding
domain (LBD), a DNA binding domain (DBD), a hinge domain containing
nuclear localization signals, and a transactivation domain in the
hyper-variable N-terminus. Truncation or deletion of the LBD
results in constitutive transactivation by the N-terminal
domain.
[0027] In certain cases, progression of prostate cancer from
androgen dependent- to androgen independent-stage may be caused by
a mutation in the LBD that alters the ligand specificity of the mAR
(Taplan et al., New Engl. J. Med. 332:1393-1398 (1995); Gaddipati
et al., Cancer Res. 54:2861-2864 (1994)). We examined whether
differential steroid specificity of wild type (wt) AR and mAR
involves the use of different androgen receptor-associated (ARA)
proteins or coactivators by these receptors.
[0028] As described in the examples, a yeast two-hybrid system with
mART887S as bait was used to screen the human prostate cDNA
library. The sequences of two clones encoding a putative
coactivators (designated ARA54 and ARA55) are shown in SEQ ID NO:1
and SEQ ID NO:3, respectively. The putative amino acid sequences of
ARA54 and ARA55 are shown in SEQ ID NO:2 and SEQ ID NO:4,
respectively. Also provided are the DNA and amino acid sequences of
ARA24 (SEQ ID NO:5 and SEQ ID NO:6, respectively) and Rb (SEQ ID
NO:7 and SEQ ID NO:8, respectively). These coactivators were
further characterized as detailed below. It is expected that some
minor variations from SEQ ID NOs:1-8 associated with nucleotide
additions, deletions, and mutations, whether naturally occurring or
introduced in vitro, will not affect coactivation by the expression
product or polypeptide.
[0029] Briefly, ARA54 is a 54 kDa protein that interacts with AR in
an androgen-dependent manner. Coexpression of ARA54 and AR in a
mammalian two-hybrid system demonstrated that reporter gene
activity was enhanced in an androgen-dependent manner. ARA54
functions as a coactivator relatively specific for AR-mediated
transcription. However, ARA54 may also function as a general
coactivator of the transcriptional activity for other steroid
receptors through their cognate ligands and response elements.
ARA54 was found to enhance the transcriptional activity of AR and
PR up to 6 fold and 3-5 fold, respectively. In contrast, ARA54 has
only marginal effects (less than 2 fold) on glucocorticoid receptor
(GR) and estrogen receptor (ER) in DU145 cells.
[0030] Coexpression of ARA54 with known AR coactivators SRC-1 or
ARA70 revealed that each of these coactivators may contribute
individually to achieve maximal AR-mediated transcriptional
activity. Moreover, when ARA54 was expressed simultaneously with
SRC-1 or ARA70, the increase in AR-mediated transactivation was
additive but not synergistic relative to that observed in the
presence of each coactivator alone.
[0031] The C-terminal domain of ARA54 (a.a. 361-471 of SEQ ID NO:1)
serves as a dominant negative inhibitor of AR-mediated gene
expression of target genes. Coexpression of exogenous full-length
ARA54 can reduce this squelching effect in a dose-dependent
manner.
[0032] ARA54 enhanced transactivation of wtAR in the presence of
DHT (10.sup.-10 to 10.sup.-8 M) by about 3-5 fold. However,
transactivation of wtAR was enhanced only marginally with E2
(10.sup.-9-10.sup.-7 M) or HF (10.sup.-7-10.sup.-5 M) as the
ligand. The ability of ARA54 to enhance transactivation by two
mutant receptors (mARt877a and mARe708k) that exhibit differential
sensitivities to E2 and HF (Yeh et al., Proc. Natl. Acad. Sci. USA,
in press (1998)) was also examined. The mutant mARt877a, which is
found in many prostate tumors (23), was activated by E2
(10.sup.-9-10.sup.-7 M) and HF (10.sup.-7-10.sup.-5 M), and ARA54
could further enhance E2- or HF-mediated AR transactivation. In
contrast, the mutant mARe708k, first identified in a yeast genetic
screening (Wang, C., Ph.D. Thesis of University of
Wisconsin--Madison (1997)), exhibited ligand specificity and
response to ARE54 comparable to that of wtAR.
[0033] It is expected that any polypeptide having substantial
homology to ARA54 that still actuates the same biological effect
can function as "an ARA54 polypeptide." With the sequence
information disclosed herein, one skilled in the art can obtain any
ARA54 polypeptide using standard molecular biological techniques.
An ARA54 polypeptide is a polypeptide that is capable of enhancing
transactivation of AR in an androgen-dependent manner, enhancing E2
or HF transactivation by the mutant receptor mARt877a, and reducing
inhibition of AR-mediated gene expression caused by overexpression
of the C-terminal domain of ARA54 (a.a. 361-471 of SEQ ID NO:1).
The sequence information presented in this application can be used
to identify, clone or sequence allelic variations in the ARA54
genes as well as the counterpart genes from other mammalian species
it is also contemplate that truncations of the native coding region
can be made to express smaller polypeptides that will retain the
same biological activity.
[0034] The polynucleotide sequence of ARA55 (SEQ ID NO:3) exhibits
high homology to the C-terminus of mouse hic5 (hydrogen peroxide
inducible clone) (Pugh, B., Curr. Opin. Cell Biol. 8:303-311
(1996)), and like hic5, ARA55 expression is induced by TGFb.
Cotransfection assays of transcriptional activation, which are
described in detail below, revealed that ARA55 is able to bind to
both wtAR and mART887S in a ligand-dependent manner to enhance AR
transcriptional activities. ARA55 enhanced transcriptional
activation by wtAR in the presence of 10.sup.-9M DHT or T, but not
10.sup.-9M E2 or HF. In contrast, ARA5S can enhance transcriptional
activation by mART887S in the presence of DHT, testosterone (T),
E2, or HF. ARA55 did not enhance transcriptional activation of
mARe708k in the presence of E2, but can enhance transcription in
the presence of DHT or T. The C-terminal domain of ARA55 (amino
acids 251-444 of SEQ ID NO:3) is sufficient for binding to ARs, but
does not enhance transcriptional activation by ARs.
[0035] The invention is not limited to the particular ARA55
polypeptide disclosed in SEQ ID NO:4. It is expected that any ARA55
polypeptide could be used in the practice of the present invention.
By "an ARA55 polypeptide" it meant a polypeptide that is capable of
enhancing transactivation of wtAR, the mutant receptor mARt877a, in
the presence of DHT, E2, or HF or intact receptor mARe708k in the
presence of DHT or T. Such polypeptides include allelic variants
and the corresponding genes from other mammalian species as well as
truncations.
[0036] The AR N-terminal domain comprises a polymorphic
poly-glutamine (Q) stretch and a polymorphic poly-glycine (G)
stretch that account for variability in the length of human AR cDNA
observed. The length of the poly-Q region (normally 11-33 residues
in length) is inversely correlated with the risk of prostate
cancer, and directly correlated with the SBMA, or Kennedy's disease
(La Spada et al., Nature (London) 352:77-79 (1991)). The incidence
of higher grade, distant metastatic, and fatal prostate cancer is
higher in men having shorter AR poly-Q stretches.
[0037] As described in the examples, experiments undertaken to
identify potential coactivators that interact with the AR poly-Q
region led to the isolation of a clone encoding a coactivator,
designated ARA24, that interacts with the poly-Q region. The
sequences of the ARA24 clone and its putative translation product
is shown in SEQ ID NO:5 and SEQ ID NO:6.
[0038] The ARA24 clone has an ORF that is identical to the
published ORF for human Ran, an abundant, ras-like small GTPase
(Beddow et al. Proc. Natl. Acad. Sci. USA 92:3328-3332, 1995).
Overexpression of ARA24 in the presence of DHT does enhance
transcriptional activation by AR over that observed in cells
transfected with AR alone. Moreover, expression of antisense ARA24
(ARA24 as) does reduce DHT-induced transcriptional activation.
[0039] An ARA24 polypeptide is one that interacts with the poly-Q
region of an AR. An ARA24 polypeptide is further characterized by
its ability to increase transactivation when overexpressed in
eukaryotic cells having some endogenous ARA24, but expression of an
ARA24 antisense RNA reduces AR receptor transactivation.
[0040] Androgen receptor mutations do not account for all cases of
androgen-independent tumors, because some androgen-independent
tumors retain wild-type AR. A significant percentage of
androgen-insensitive tumors have been correlated with reduced
expression of retinoblastoma protein (Rb) (Bookstein, et al.,
Science 247:712-715, (1990)), expression a truncated Rb protein
(Bookstein, et al. Proc. Natl. Acad. Sci. USA 87:7762-7766 (1990)),
or a missing Rb allele (Brooks, et al. Prostate 26:35-39, (1995)).
The prostate cancer cell line DU145 has an abnormal short mRNA
transcript of Rb exon 21 (Sarkar, et al. Prostate 21:145-152
(1992)) and transfecton of the wild-type Rb gene into DU145 cells
was shown to repress the malignant phenotype (Bookstein, et al.
Proc. Natl. Acad. Sci. USA 87:7762-7766 (1990)).
[0041] Rb functions in the control of cell proliferation and
differentiation (Weinberg, R. A., Cell 81:323-330 (1995);
Kranenburg et al., FEBS Lett. 367:103-106 (1995)). In resting
cells, hypophophorylated Rb prevents inappropriate entry of cells
into the cell division cycle. Phosphorylation of Rb by
cyclin-dependent kinases relieves Rb-mediated growth suppression,
and allows for cell proliferation(Dowdy et al., Cell 73:499-511
(1993); Chen et al., Cell 58:1193-1198 (1989)). Conversely,
dephosphorylation of Rb during G1 progression induces growth arrest
or cell differentiation(Chen et al. (1989); Mihara et al., Science
246:1300-1303 (1989)). In dividing cells, Rb is dephosphorylated
during mitotic exit and G1 entry(Ludlow et al., Mol. Cell. Biol.
13:367-372 (1993)). This dephosphorylation activates Rb for the
ensuing G1 phase of the cell cycle, during which Rb exerts it
growth suppressive effects.
[0042] We investigated the role of Rb in AR transactivation as
detailed in the examples. We found that Rb can induce
transcriptional activity of wtAR or mARs877t in the presence of
DHT, E2, or HF, and mARe708k in the presence of DHT. We also
discovered that Rb and ARA70 transciptional activity act
synergistically to enhance transciptional activity of ARs. The
sequence of the cloned Rb gene and the deduced amino acid sequence
of the ORF are shown in SEQ ID NO:7 and SEQ ID NO:8, respectively.
An Rb polypeptide is a polypeptide that is substantially homologous
to SEQ ID NO:8, that interacts with the N-terminal domain of AR,
and which acts synergistically with ARA70 in enhancing
transactivation by AR.
[0043] In the examples, various eukaryotic cell types, including
yeast, prostate cells having mutant AR and cells lacking AR, were
used to evaluate the ability of the putative androgen coactivators
to enhance transactivation by AR. It is expected that in the method
of the present invention, any eukaryotic cell could be employed in
an assay for AR activity. This feature allows the investigator
flexibility in designing assays.
[0044] As described below, cells were transfected using a calcium
phosphate technique. It is expected that the method of the present
invention could be practiced using any transfection means
including, for example, electroporation or particle
bombardment.
[0045] Changes in the level of transactivation by AR can be
assessed by any means, including measuring changes in the level of
mRNA for a gene under the control of AR, or by quantitating the
amount of a particular protein expressed using an antibody specific
for a protein, the expression of which is under the control of AR.
Most conveniently, transactivation by AR can be assessed by means
of a reporter gene.
[0046] As used herein, a reporter gene is a gene under the control
of an androgen receptor, the gene encoding a protein susceptible to
quantitation by a colormetric or fluorescent assay. In the examples
below, a chloramphenicol acetyltransferase or a luciferase gene
were used as reporter genes. The gene may either be resident in a
chromosome of the host cell, or may be introduced into the host
cell by cotransfection with the coactivator gene.
[0047] The following nonlimiting examples are intended to be purely
illustrative.
EXAMPLES
[0048] Plasmid Construction
[0049] A human prostate library in pACT2 yeast expression vector (a
gift from Dr. S. Elledge) consists of the GAL4 activation domain
(GAL4AD, a.a. 768-881) fused with human prostate cDNA.
[0050] pSG5 wtAR was constructed as described previously (Yeh and
Chang, Proc. Natl. Acad. Sci USA 93:5517-5521, 1996).
[0051] pGAL0-AR (wild-type) was obtained from D. Chen (University
of Massachusetts). pGAL0 contains the GAL4 DNA binding domain
(DBD).
[0052] For construction of pAS2-wtAR or -mAR, the C-terminal
fragments (aa 595-918) from wtAR, mARt877s (Dr. S. P. Balk, Beth
Israel Hospital, Boston, Mass.), or mARe708k (H. Shim, Hyogo
Medical College, Japan) were inserted in pAS2 yeast expression
vector (Clontech). Another AR mutant (mARv888m), derived from
androgen insensitive syndrome patient, was constructed as
previously described (Mowszowicz, et al. Endocrine 1:203-209,
1993).
[0053] pGAL4-VP16 was used to construct a fusion of ARA70.
pGAL4-VP16 contains the GAL4 DBD linked to the acidic activation
domain of VP16.
[0054] pCMX-Gal-N-RB and pCMX-VP!6-AR were constructed by inserting
fragments Rb (aa 370-928) and AR (aa 590-918) into pCMX-gal-N and
pCMX-VP16, respectively. The sequence of construction junction was
verified by sequencing.
[0055] pYX-ARA24/Ran was constructed by placing the ARA24 gene
under the control of the gal-1 promoter of yeast expression plasmid
pYX243 (Ingenus). A cDNA fragment encoding the AR poly-Q stretch
and its flanking regions (AR a.a. 11-208) was ligated to a PAS2
yeast expression plasmid for use as bait in the two hybrid assay.
AR cDNAs of different poly-Q lengths that span the same AR poly-Q
region as our bait plasmid were constructed in pAS2 in the same
way, for yeast two-hybrid liquid culture .beta.-gal assay. These AR
bait plasmids with poly-Q lengths of 1, 25, 49 were all transformed
into yeast Y190 and found to not be autonomously active.
pCMV-antisense ARA24/Ran (ARA24 as) expression plasmid was
constructed by inserting a 334-bp Bgl II fragment of ARA24/Ran,
which spans 5'-untranslated region and the translation start codon
of ARA24/Ran (nucleotides 1-334 of SEQ ID NO:5), into pCMV vector
in the antisense orientation. The MMTV-CAT and MMTV-Luc reporter
genes were used for AR transactivation assay. pSG5-AR and
pSV-.beta.gal are under the regulation of SV40 promoter and
.beta.-globulin gene intron-1 enhancer. p6R-ARQ1, p6R-ARQ25,
p6R-ARQ49 were kindly provided by Dr. Roger L. Meisfield
(Chamberlain, et al. Nucleic Acids Res. 22:3181-3186, 1994)
[0056] pSG5-GAL4DBD-ARA24 was generated by inserting the coding
sequence of Gal4DBD-ARA24 hybrid protein into pSG5 vector.
pVP16-ARN-Q1, pVP16-ARN-Q25, pVP16-ARN-Q25, pVP16-ARN-Q35,
pVP16-ARN-Q49 were generated by inserting each poly-Q AR N-terminal
domain (a.a. 34-555) into pVP16 vector (Clontech) to be expressed
as a VP16AD hybrid protein. GAL0AR plasmid, which contains GAL4DBD
fused to E region of human AR, was a gift from Dr. D. Chen. The
pSG5-CAT reporter plasmid (Clontech) contains five GAL4 binding
sites upstream of the E1b TATA box, linked to the CAT gene.
[0057] pSG5-AR and pSG5-ARA70 were constructed as previously
described (Yeh and Chang, Proc. Natl. Acad. Sci USA 93:5517-5521,
1996). Two mutants of the AR gene (mAR877 derived from prostate
cancer, codon 877 mutation Thr to Ala; and mAR708 derived from
partial androgen insensitive syndrome (PIAS), codon 708 mutation
Glu to Lys), were provided by S. Balk (Beth Israel Hospital,
Boston) and H. Shima (Hyogo Medical College, Japan),
respectively.
[0058] Clones used in the two-hybrid system to evaluate the role of
Rb in AR transactivation were made by ligating an Rb fragment (aa
371-928) to the DBD of GAL4. Similarly, near full-length (aa
36-918) AR (nAR) and AR-LBD (aa 590-918) fragments ligated to
transcriptional activator VP16.
[0059] Screening of Prostate cDNA Library by a Yeast Two-Hybrid
System for ARAs Associated With the Ligand Binding Domain
[0060] To identify ARA coactivators interact with the LBD, a
pACT2-prostate cDNA library was cotransformed into Y190 yeast cells
with a plasmid of pAS2 mAR(mART877S) which contains GAL4DBD (aa
1-147) fused with the C-terminal domain of this mAR. Transformants
were selected for growth on SD plates with 3-aminotriazole (25 mM)
and DHT (100 nM) lacking histidine, leucine and tryptophan (-3SD
plates). Colonies were also filter-assayed for .beta.-galactosidase
activity. Plasmid DNA from positive cDNA clones were found to
interact with mtARt877s but not GAL4TR4 was isolated from yeast,
amplified in E. coli, and the inserts confirmed by DNA
sequencing.
[0061] To identify clones that interact with the poly-Q region of
the N-terminal domain, the AR poly-Q stretch (aa 11-208) was
inserted into the pAS2 yeast expression plasmid and cotransformed
into Y190 yeast cells with a human brain cDNA library fused to the
Gal4 activation domain. Transformants were selected for growth on
SD plates lacking histidine, leucine and tryptophan and
supplemented with 3-aminotriazole (40 mM).
[0062] Amplification and Characterization of ARA Clones
[0063] Full length DNA sequences comprising two coactivators,
designated ARA54 (SEQ ID NO:1) and ARA55 (SEQ ID NO:3), that were
found to interact with mARt877s were isolated by 5'RACE PCR using
Marathon cDNA Amplification Kit (Clontech) according to the
manufacturer's protocol.
[0064] The missing 5' coding region of the ARA54 gene was isolated
from H1299 cells using the gene-specific antisense primer shown in
SEQ ID NO:9 and following PCR reaction conditions: 94.degree. C.
for 1 min, 5 cycles of 94.degree. C. for 5 sec.fwdarw.72.degree. C.
for 3 min, 5 cycles of 94.degree. C. for 5 sec.fwdarw.70.degree. C.
for 3 min, then 25 cycles of 94.degree. C. for 5
sec.fwdarw.68.degree. C. for 3 min. The PCR product was subcloned
into pT7-Blue vector (Novagen) and sequenced.
[0065] ARA55 was amplified by PCR from the HeLa cell line using an
ARA55-specific antisense primer (SEQ ID NO:10) and the PCR reaction
conditions described for isolation of ARA54.
[0066] Using the 5'RACE-PCR method, we were able to isolate a 1721
bp DNA fragment (SEQ ID NO:1) from the H1299 cell line with an open
reading frame that encodes a novel protein 474 amino acids in
length (SEQ ID NO:2). The in-vitro translation product is a
polypeptide with an apparent molecular mass of 54.+-.2 kDA,
consistent with the calculated molecular weight (53.8 kDa). The
middle portion of ARA54 (a.a. 220-265 of SEQ ID NO:2) contains a
cysteine-rich region that may form a zinc finger motif called the
RING finger, defined as CX.sub.2CX.sub.9-27CXHX.sub.2CX.sub.2C-
X.sub.6-17CX.sub.2C (SEQ ID NO: 11), a domain conserved among
several human transcriptional factor or proto-oncogeny proteins,
including BRCA1, RING1, PML and MEL-18 (Miki et al., Science
266:66-71 (1994); Borden et al., EMBO J. 14:1532-1541 (1995);
Lovering et al., Proc. Natl. Acad. Sci. USA 90:2112-2116 (1993);
Blake et al., Oncogene 6: 653-657 (1991); Ishida et al, Gene
129:249-255 (1993)). In addition, ARA54 also contains a second
cysteine-rich motif which has a B box like structure located at 43
amino acids downstream from the RING finger motif. However, ARA54
differs from members of the RING finger-B-box family in that it
lacks a predicted coiled-coil domain immediately C-terminal to the
B box domain, which is highly conserved in the RING finger-B-box
family. Therefore, ARA54 may represent a new subgroup of this
family.
[0067] The full-length human ARA55 has an open reading frame that
encodes a 444 aa polypeptide (SEQ ID NO:4) with a predicted
molecular weight of 55 kD that ARA55 shares 91% homology with mouse
hic5. Human ARA55 has four LIM motifs in the C-terminal region. An
LIM motif is a cysteine-rich zinc-binding motif with consensus
sequence: CX.sub.2CX.sub.16-23HX.sub.2C-
X.sub.2CX.sub.2CX.sub.16-21CX.sub.2 (C, H, D) (SEQ ID NO:12)
(Sadler, et al., J. Cell Biol. 119:1573-1587 (1992)). Although the
function of the LIM motif has not been fully defined, some data
suggest that it may play a role in protein-protein interaction
(Schmeichel & Beckerle, Cell 79:211-219, 1994). Among all
identified SR associated proteins, only ARA55 and thyroid hormone
interacting protein 6 (Trip 6) (Lee, et al. Mol. Endocrinol.
9:243-254 (1995)) have LIM motifs.
[0068] A clone that showed strong interaction with the poly-Q bait
was identified and subsequently subjected to sequence analysis.
This clone contains 1566 bp insert (SEQ ID NO:5) with an open
reading frame encoding a 216:aa polypeptide (SEQ ID NO:6) with a
calculated molecular weight of 24 kDa. GenBank sequence comparison
showed that this clone has the same open reading frame sequence as
Ran/TC4, an abundant ras-like small GTPase involved in
nucleocytoplasmic transport that is found in a wide variety of cell
types (Beddow et al., Proc. Natl. Acad. Sci. U.S.A. 92:3328-3332,
1995). Accordingly, the factor was designated ARA24/Ran. The cDNA
sequence of the ARA24 clone (SEQ ID NO:5)(GenBank accession number
AF052578) is longer than that of the published ORF for human Ran,
in that it includes 24 and 891 bp of 5'- and 3'-untranslated
regions, respectively.
[0069] Northern Blotting
[0070] The total RNA (25 .mu.g) was fractionated on a 1%
formaldehyde-MOPS agarose gel, transferred onto a Hybond-N nylon
membrane (Amersham) and prehybridized. A probe corresponding to the
900 bp C-terminus of ARA55 or an ARA54-specific sequence was
.sup.32P-labeled in vitro using Random Primed DNA Labeling Kit
(Boehringer-Mannheim) according to the manufacture's protocol and
hybridized overnight. After washing, the blot was exposed and
quantified by Molecular Dynamics PhosphorImager. .beta.-actin was
used to monitor the amount of total RNA in each lane.
[0071] Northern blot analysis indicated the presence of a 2 kb
ARA55 transcript in Hela and prostate PC3 cells. The transcript was
not detected in other tested cell lines, including HepG2, H1299,
MCF7, CHO, PC12, P19, and DU145 cells. The ARA54 transcript was
found in H1299 cells, as well as in prostate cancer cell lines PC3
and LNCaP.
[0072] Co-Immunoprecipitation of AR and ARAs
[0073] Lysates from in-vitro translated full-length of AR and ARA54
were incubated with or without 10.sup.-8 M DHT in the modified RIPA
buffer (50 mM Tris-HCL pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.1% NP40, 1
mM PMSF, aprotinin, leupeptin, pepstatin, 0.25% Na-deoxycholate,
0.25% gelatin) and rocked at 4.degree. C. for 2 hr. The mixture was
incubated with rabbit anti-His.tag polyclonal antibodies for
another 2 hr and protein A/G PLUS -Agarose (Santa Cruz) were added
and incubated at 4.degree. C. for additional 2 hr. The conjugated
beads were washed 4 times with RIPA buffer, boiled in SDS sample
buffer and analyzed by 8% SDS/PAGE and visualized by STORM 840
(Molecular Dynamics).
[0074] ARA54 and AR were found in a complex when immunoprecipitated
in the presence of 10.sup.-8 M DHT, but not in the absence of DHT.
This result suggests that ARA54 interacts with AR in an
androgen-dependent manner.
[0075] Interaction between recombinant full length human AR and
ARA24/Ran proteins further examined by co-immunoprecipitation,
followed by SDS-PAGE and western blotting. Results of the
co-immunoprecipitation assay indicate that ARA24/Ran interacts
directly with AR. The phosphorylation state of bound guanine
nucleotide to the small GTPases does not affect this
interaction.
[0076] AR Pull-Down Assay Using GST-Rb
[0077] Full-length Rb fused to glutathione-S-transferase
(ST-Rb.sub.1-928) was expressed and purified from E. coli. strain
Bl21pLys as described recently (Zarkowska & Mittnacht, J. Biol.
Chem. 272:12738-12746, 1997). Approximately 2 .mu.g of His-tag
column purified baculovirus AR was mixed with GST-loaded
glutathione-Sepharose beads in 1 ml of NET-N (20 mM Tris-HCL (pH
8.0, 100 mM NaCl, 1 mM EDTA, 0.5% (v/v) Noniodet P-40) and
incubated with gentle rocking for 3 hr at 4.degree. C.
[0078] Following low-speed centrifugation to pellet the beads, the
clarified supernatant was mixed with GST-Rb-loaded
glutathione-Sepharose beads in the presence or absence of 10 mM DHT
and incubated for an additional 3 hr with gentle rocking at
4.degree. C. The pelleted beads were washed 5 times with NET-N,
mixed with SDS-sample buffer, boiled, and the proteins separated by
electrophoresis on a 7.5% polyacrylamide gel. A Western blot of the
gel was incubated with anti-AR polyclonal antibody NH27 and
developed with alkaline phosphatase-conjugated secondary
antibodies.
[0079] AR was coprecipitated with GST-Rb, but not GST alone,
indicating that AR and Rb are associated in a complex together.
[0080] Transfection Studies
[0081] Human prostate cancer DU145 or PC3 cells, or human lung
carcinoma cells NCI H1299 were grown in Dulbecco's minimal
essential medium (DMEM) containing penicillin (25 U/ml),
streptomycin (25 .mu.g/ml), and 5% fetal calf serum (FCS). One hour
before transfection, the medium was changed to DMEM with 5%
charcoal-stripped FCS. Phenol red-free and serum-free media were
used on the experiments employing E2 or TGF.beta., respectively. A
.beta.-galactosidase expression plasmid, pCMV-.beta.-gal, was used
as an internal control for transfection efficiency.
[0082] Cells were transfected using the calcium phosphate technique
(Yeh, et al. Molec. Endocrinol. 8:77-88, 1994). The medium was
changed 24 hr posttransfection and the cells treated with either
steroid hormones or hydroxyflutamide, and cultured for an
additional 24 hr. Cells were harvested and assayed for CAT activity
after the cell lysates were normalized by using
.beta.-galactosidase as an internal control. Chloramphenicol
acetyltransferase (CAT) activity was visualized by PhosphorImager
(Molecular Dynamics) and quantitated by ImageQuant software
(Molecular Dynamics).
[0083] Mammalian Two-Hybrid Assay
[0084] The mammalian two-hybrid system employed was essentially the
protocol of Clontech (California), with the following
modifications. In order to obtain better expression, the GAL4DBD
(a.a. 1-147) was fused to pSG5 under the control of an SV40
promoter, and named pGAL0. The hinge and LBD of wtAR were then
inserted into pGAL0. Similarly, the VP16 activation domain was
fused to pCMX under the control of a CMV promoter, and designated
pCMX-VP16 (provided by Dr. R. M. Evan).
[0085] The DHT-dependent interaction between AR and ARA54 was
confirmed in prostate DU145 cells using two-hybrid system with CAT
reporter gene assay. Transient transfection of either ARA54 or wtAR
alone showed negligible transcriptional activity. However,
coexpression of AR with ARA54 in the presence of 10.sup.-8 M DHT
significantly induced CAT activity.
[0086] ARA54 functions as a coactivator relatively specific for
AR-mediated transcription. ARA54 induces the transcriptional
activity of AR and PR by up to 6 fold and 3-5 fold, respectively.
In contrast, ARA54 showed only marginal effects (less than 2 fold)
on GR and ER in DU145 cells. These data suggest that ARA54 is less
specific to AR as relative to ARA70, which shows higher specificity
to AR. However, we can not rule out the possibility that ARA54
might be more general to other steroid receptors in other cell
types under different conditions.
[0087] Coexpression of ARA54 with SRC-1 or ARA70 was found to
enhance AR transcriptional activity additively rather than
synergistically. These results indicate that these cofactors may
contribute individually to the proper or maximal AR-mediated
transcriptional activity.
[0088] Since the C-terminal region of ARA54 (a.a. 361-471 of SEQ ID
NO:2) isolated from prostate cDNA library has shown to be
sufficient to interact with AR in yeast two-hybrid assays, we
further investigated whether it could squelch the effect of ARA54
on AR-activated transcription in H1299 cells, which contain
endogenous ARA54. The C-terminal region of ARA54 inhibits
AR-mediated transcription by up to 70%; coexpression of exogenous
full-length ARA54 reverses this squelching effect in a
dose-dependent manner. These results demonstrate that the
C-terminal domain of ARA54 can serve as a dominant negative
inhibitor, and that ARA54 is required for the proper or maximal AR
transactivation in human H1299 cells.
[0089] Examination of the effect of ARA54 on the transcriptional
activities of wtAR and mtARs in the presence of DHT, E2 and HF
revealed differential ligand specificity. Translational activation
of wtAR occurred in the presence of DHT (10.sup.-10 to 10.sup.-8
M); coexpression of ARA54 enhanced transactivation by another 3-5
fold. However, wtAR responded only marginally to E2
(10.sup.-9-10.sup.-7 M) or HF (10.sup.-7-10.sup.-5 M) in the
presence or absence of ARA54. As expected, the positive control,
ARA70, is able to enhance the AR transcriptional activity in the
presence of 10.sup.-9-10.sup.-7 M E2 and 10.sup.-7-10.sup.-5 HF,
that matches well with previous reports (Yeh, PNAS, Miyamoto,
PNAS).
[0090] The AR mutants Art877a, which is found in many prostate
tumors (23), and Are708k, found in a yeast genetic screening (24)
and a patient with partial androgen insensitivity, exhibited
differential specificity for lignands. In the absence of ARA54,
Art877a responded to E2 (10.sup.-9-10.sup.-7 M) and HF
(10.sup.-7-10.sup.-5 M), and ARA54 could further enhance E2- or
HF-mediated AR transactivation. These results suggested that mtARs
might also require cofactors for the proper or maximal DHT-, E2-,
or HF-mediated AR transcriptional activity. The DHT response of
mARe708k was only a slightly less sensitive than that of wtAR or
mARt877s, whereas E2 and HF exhibited no agonistic activity toward
ARe708k. Together, these results imply that the change of residue
708 on AR might be critical for the interaction of the
antiandrogen-ARe708k-ARA5- 4 complex, and that both AR structure
and coactivators may play a role in determining ligand
specificity.
[0091] CAT activity in DU145 cells cotransfected with a plasmid
encoding the hormone binding domain of wtAR fused to the GAL4 DBD
(GAL0AR) and a plasmid encoding full-length ARA55 fused to the
activation domain of VP16 (VP16-ARA55) was significantly induced by
the cotransfection of VP16-ARA55 and GAL0AR in the presence of 10
nM DHT, but not induced by E2 or HF. Combination of GAL0 empty
vector and VP16-ARA55 did not show any CAT activity. Combination of
GAL0AR and VP16 vector showed negligible CAT activity. These
results indicate that ARA55 interacts with AR in an
androgen-dependent manner.
[0092] Transient transfection assays were conducted to investigate
the role of ARA55 in the transactivation activity of AR. DU145
cells were cotransfected with MMTV-CAT reporter, increasing amounts
of ARA55 and wtAR under eukaryotic promoter control. Ligand-free AR
has minimal MMTV-CAT reporter activity in the presence or absence
of ARA55. ARA55 alone also has only minimal reporter activity
Addition of 10 nM DHT resulted in 4.3 fold increase of AR
transcriptional activity and ARA55 further increased this induction
by 5.3 fold (from 4.3 fold to 22.8 fold) in a dose-dependent
manner. The induced activity reached a plateau at the ratio of
AR:ARA55 to 1:4.5. Similar results were obtained using PC3 cells
with DU145 cells, or using a CAT reporter gene under the control of
a 2.8 kb promoter region of a PSA gene. The C-terminus of ARA55
(ARA55.sub.251-444) (a.a. 251-444 of SEQ ID NO:4) did not enhance
CAT activity. Cotransfection of PC3 cells, which contain endogenous
ARA55, with ARA55.sub.251-444, AR and MMTV-CAT reporter in the
presence of 10 nM DHT demonstrated dramatically reduced AR
transcriptional activity relative to cells transfected with AR and
MMTV-CAT alone. These results demonstrate that ARA55 is required
for the proper or maximal AR transcriptional activity in PC3 cells,
and that the C-terminus of ARA55 can serve as a dominant negative
inhibitor.
[0093] The effect of ARA55 on mARt877s and mARe708k in the presence
of DHT and its antagonists, E2, and HF. The mARt877s receptor is
found in LNCaP cells and/or advanced prostate cancers and has a
point mutation at codon 877 (Thr to Ser) (Gaddipati et al., Cancer
Res. 54:2861-2864 (1994); Veldscholte et al., Biochem. Biophys.
Commun. 173:534-540 (1990)). The mARe708k receptor, has a point
mutation at codon 708 (Glu to Lys), was isolated by a yeast genetic
screening and exhibits reduced sensitivity to HF and E2 relative to
wtAR (Wang, C., PhD thesis of University of Wisconsin--Madison
(1997)). The transcriptional activities of wtAR, mARt877s, and
mARe708k are induced by DHT (10.sup.-11 to 10.sup.-8 M). ARA55
enhanced the transactivation of all three receptors by 4-8 fold. In
the presence of E2 or HF, wtAR responded marginally only at higher
concentrations (10.sup.-7 M for E2 and 10.sup.-5 M for HF).
Cotransfection of wtAR with ARA55 at a 1:4.5 ratio, however,
increases AR transcriptional activity at 10.sup.-8-10.sup.-7 M for
E2 or 10.sup.-6 to 10.sup.-5 M for HF. Compared to wtAR, the LNCaP
mAR responded much better to E2 and HF and ARA55 significantly
enhanced its transcriptional activity. ARA55 may be needed for the
proper or maximal DHT-, E2-, or HF-mediated AR transcriptional
activity.
[0094] The effect of ARA55 on transcriptional activation by GR, PR,
and ER was tested in DU145 cells. ARA55 is relatively specific to
AR, although it may also enhance GR and PR to a lesser degree, and
has only a marginal effect on ER. ARA70 shows much higher
specificity to AR than ARA55, relative to the other tested steroid
receptors. Although ARA55 enhances AR-mediated transcription to a
greater degree than GR-, PR-, or ER-mediated transcription, it
appears to be less specific than ARA70.
[0095] Because the amino acid sequence of ARA55 has very high
homology to mouse hic5, and early studies hic5 suggested this mouse
gene expression can be induced by the negative TGF.beta. (Shibanuma
et al., J. Biol. Chem. 269:267G7-26774 (1994)), we were interested
to see whether ARA55 could serve as a bridge between TGF.beta. and
AR steroid hormone system. Northern blot--analysis indicated that
TGF.beta. treatment (5 ng/ml) could induce ARA55 mRNA by 2-fold in
PC3 cells. In the same cells, TGF.TM. treatment increased AR
transcriptional activity by 70%. This induction is weak relative to
the affect achieved upon transfection of PC3 cells with exogenous
ARA5S (70% vs. 4 fold). This may be related to the differences in
the ratios of AR and ARA55. The best ratio of AR:ARA55 for maximal
AR transcriptional activity is 1:4.5. Whether other mechanisms may
also be involve in this TGF.beta.-induced AR transcriptional
activity will be an interesting question to investigate. The
unexpected discovery that TGF.beta. may increase AR transcriptional
activity via induction of ARA55 in prostate may represent the first
evidence to link a negative regulatory protein function in a
positive manner, by inducing the transcriptional activity of AR,
the major promoter for the prostate tumor growth.
[0096] The ability of ARA55 to induce transcriptional activity of
both wtAR and mARt877s in the presence of DHT, E2, and HF suggests
an important role for ARA55 in the progression of prostate cancer
and the development of resistance to hormonal therapy. Evaluation
of molecules that interfere with the function of ARA55 may aid in
the identification of potential chemotherapeutic
pharmaceuticals.
[0097] Human small lung carcinoma H1299 cell line, which has no
endogenous AR protein, were transfected with AR and ARA24/Ran.
Because ARA24/Ran is one of the most abundant and ubiquitously
expressed proteins in various cells, both sense and antisense
ARA24/Ran mammalian expression plasmids were tested. Overexpression
of sense ARA24/Ran did not significantly enhance the AR
transactivation, a result that is not surprising, in view of the
abundance of endogenous ARA24/RAN. However, expression of antisense
ARA24/Ran (ARA24as) markedly decreased DHT-induced CAT activity in
a dose dependent manner. Furthermore, increasing the DHT
concentration from 0.1 nM to 10 nM DHT resulted in strong induction
of AR transactivation and decreased the inhibitory effect of ARA24
as effect, indicating that increased DHT concentration can
antagonize the negative effect of ARA24 as.
[0098] The affinity between ARA24/Ran and AR is inversely related
to the length of AR poly-Q stretch. AR transactivation decreases
with increasing AR poly-Q length. Reciprocal two-hybrid assays with
exchanged fusion partners, Gal4DBD-ARA24/Ran and VP16AD-ARNs (a.a.
34-555 with poly-Q lengths of 1, 25, 35, 49 residues) were
conducted using mammalian CHO cells. These results consistently
show that the affinity between ARA24/Ran and AR poly-Q region is
inversely correlated with AR poly-Q length in both yeast and
mammalian CHO cells.
[0099] The regulation of AR transactivation by ARA24/Ran correlates
with their affinity. These results suggest that ARA24/Ran could
achieve differential transactivation of AR, with ARs having
different poly-Q length could existing in a single cell or cell
system. ARA24 as was again used in the ARE-Luc transfection assays
to address the role of AR poly-Q length in the regulation of AR by
ARA24/Ran. ARs of poly-Q lengths 1, 25, and 49 residues, and
increasing amounts (1, 2, and 4 .mu.g) of ARA24 as expression
vectors were co-transfected with equal amounts of reporter plasmid
(pMMTV-Luc) in CHO cells. Although the basal reporter activity is
slightly affected by increasing amounts of antisense ARA24/Ran,
ARA24 as showed a more significant decrease of AR transactivation.
As AR poly-Q length increased, the ARA24 as effect on AR
transactivation decreased. These results suggest that the affinity
of ARA24/Ran for AR and the effect of decreasing ARA24/Ran on AR
transactivation faded over the expansion of AR poly-Q length.
[0100] Coexpression of Rb and AR expression plasmids in DU145 cells
using the mammalian two-hybrid system resulted in a 3 fold increase
in CAT activity by cotransfection of near full length AR (nAR,
amino acids 36-918) and Rb. Cells cotransfected with nAR and PR-LBD
or Rb and ARA70 did not show increased CAT activity. Surprisingly,
addition of 10 nM DHT made very little difference in the
interaction between Rb and nAR. The inability of Rb to interact
with AR-LBD suggest that interaction site of AR is located in
N-terminal domain (aa 36 to 590). Together, our data suggest the
interaction between Rb and AR is unique in the following ways:
first, the interaction is androgen-independent and binding is
specific but relatively weak as compared to other AR associated
protein, such as ARA70 (3 fold vs. 12 fold induced CAT activity in
mammalian two-hybrid assay, data not shown). Second, unlike most
identified steroid receptor associated proteins that bind to
C-terminal domain of steroid receptor, Rb binds to N-terminal
domain of AR. Third, no interaction occurred between Rb and ARA70,
two AR associated proteins in DU145 cells.
[0101] DU145 cells containing mutated Rb (Singh et al., Nature 374:
562-565 (1995)) were cultured with charcoal-stripped FCS in the
presence or absence of 1 nM DHT. No AR transcriptional activity was
observed in DU145 cells transiently transfected with wild type AR
and Rb at the ratio of 1:3 in the absence of DHT. When However, AR
transcriptional activity could be induced 5-fold when wild type AR
was expressed in the presence of 1 nM DHT. Cotransfection of Rb
with AR can further enhance the AR transcriptional activity from
5-fold to 21-fold in the presence of 1 nM DHT. As a control,
cotransfection of ARA70, the first identified AR coactivator, can
further enhance in DU145 cells transcriptional activity from 5-fold
to 36-fold. In DU145 cells transfected with Rb, ARA70, and AR, the
induction of AR transcriptional activity was synergistically
increased from 5-fold to 64-fold. Upon transfection of wild type AR
without Rb or ARA70, only marginal induction (less than 2-fold) was
detected in the presence of 10 nM E2 or 1 .mu.M HF. In contrast,
cotransfection of the wild type AR with Rb or ARA70 can enhance the
AR transcriptional activity to 12-fold (E2) or 3-4 fold (HF), and
cotransfection of Rb and ARA.sub.70 with AR can further enhance the
AR transcriptional activity to 36-fold (E2 or 12-fold (HF). We then
extended these findings to two different AR mutants: mARt877s from
a prostate cancer patient and mARe708k from a
partial-androgen-insensitive patient. Similar inductions were
obtained when wild type AR was replaced by mARt877s. In contrast,
while similar induction was also detected in the presence of 1 nM
DHT when we replace wild type AR with mARe708k, there was almost no
induction by cotransfection of meAR708k with Rb and/or ARA70 in the
presence of 10 nM E2 or 1 .mu.M HF. These results indicated that Rb
and ARA70 can synergistically induce the transcriptional activity
of wild type AR and mAR877 in the presence of 1 nM DHT, 10 nM E2 or
1 .mu.M HF. However, Rb and ARA70 synergistically induce the
transcriptional activity of mAR708 only in the presence of 1 nM
DHT, but not 10 nM E2 or 1 .mu.M HF. The fact that Rb and ARA70 can
induce transcriptional activity of both wild type AR and mutated AR
that occur in many prostate tumors may also argue strongly the
importance of Rb and ARA70 in normal prostate as well as prostate
tumor. Also, the differential induction of DHT vs. E2/HF may
suggest the position of 708 in AR may play vital role for the
recognition of androgen vs anti-androgens to AR.
[0102] We also examined the effect of Rb and ARA70 on the
transcriptional activity of other steroid receptors through their
cognate DNA response elements [MMTV-CAT for AR, glucocorticoid
receptor (GR), and progesterone receptor (PR); ERE-CAT for estrogen
receptor (ER)]. Although Rb and ARA70 can synergistically induce AR
transcriptional activity up to 64-fold, Rb and ARA70 can only have
marginal induction on the transcriptional activity of GR, PR, and
ER in DU145 cells. These results suggest that Rb and ARA70 are more
specific coactivators for AR in prostate DU145 cells. However, it
cannot be ruled out that possibly the assay conditions in prostate
DU145 cells are particularly favorable for Rb and ARA70 to function
as coactivators for AR only, and Rb and ARA70 may function as
stronger coactivators for ER, PR, and GR in other cells or
conditions. Failure of Rb to induce transactivation by mutant
AR888, which is unable to bind androgen, suggests that while
interaction between Rb and AR is androgen-independent, the AR-Rb
(and AR-ARA70) complexes require a ligand for the transactivation
activity.
[0103] The activity of Rb in cell cycle control is related
essentially to its ability to bind to several proteins, thus
modulating their activity. To date, many cellular proteins have
been reported which bind to Rb (Weinberg, R.A., Cell 81:323-330
(1995)). These include a number of transcription factors, a
putative regulator of ras, a nuclear structural protein, a protein
phosphatase, and several protein kinases. Whether all of these
proteins actually complex, and are regulated by Rb, in cells
remains to be seen.
[0104] Much attention has been given to the functional interaction
between Rb and transcription factors. To date, several of these
factors have been shown to form complexes with Rb in cells. Such
complex formation and subsequent function studies have revealed
that the modulating activity of Rb can take the form of repression
of transcription as with E2F (Weintraub et al., Nature 375:812-815
(1995)), or activation as with NF-IL6 (Chen et al., Proc. Natl.
Acad. Sci. USA 93:465-469 (1996)) and the hBrm/BRG1 complex (Singh
et al., (1995)). Here, we show that Rb can bind to AR and induce
the AR transcriptional activity. To our knowledge, this is the
first demonstration of a negative growth regulatory protein
functioning in a positive manner, by initiating transcription via a
signal transduction mechanism involving binding to a nuclear
receptor. When place in the context of regulating the cell cycle
and differentiation, these data suggest a previously undescribed
function for Rb which underscores the importance of this protein in
regulating transcription by direct binding to transcription-factor,
but this protein can also regulate transcription by stimulating at
least one type of signal transduction mechanism.
[0105] A relationship between Rb expression and response to
endocrine therapy of human breast tumor has been suggested
(Anderson et al., J. Pathology 180:65-70 (1996)). Other studies
indicate that Rb gene alterations can occur in all grades and
stages of prostate cancer, in localized as well as metastatic
disease (Brooks et al., Prostate 26:35-39 (1995)). How Rb function
may be linked to androgen-dependent status in prostate tumor
progression remains unclear. One possible explanation is that Rb
alteration may be a necessary event in prostate carcinogenesis for
a subset of prostatic neoplasms, which may be also true for the AR
expression in prostate tumors.
[0106] All publications cited in this application are incorporated
by reference.
[0107] The present invention is not limited to the exemplified
embodiment, but is intended to encompass all such modifications and
variations as come within the scope of the following claims.
Sequence CWU 1
1
12 1 1721 DNA Homo sapien CDS (40)..(1464) misc_feature
(1120)..(1452) Coding sequence and polypeptide region for the
C-terminal domain. 1 ggtctctggt ctcccctctc tgagcactct gaggtcctt atg
tcg tca gaa gat 54 Met Ser Ser Glu Asp 1 5 cga gaa gct cag gag gat
gaa ttg ctg gcc ctg gca agt att tac gat 102 Arg Glu Ala Gln Glu Asp
Glu Leu Leu Ala Leu Ala Ser Ile Tyr Asp 10 15 20 gga gat gaa ttt
aga aaa gca gag tct gtc caa ggt gga gaa acc agg 150 Gly Asp Glu Phe
Arg Lys Ala Glu Ser Val Gln Gly Gly Glu Thr Arg 25 30 35 atc tat
ttg gat ttg cca cag aat ttc aag ata ttt gtg agc ggc aat 198 Ile Tyr
Leu Asp Leu Pro Gln Asn Phe Lys Ile Phe Val Ser Gly Asn 40 45 50
tca aat gag tgt ctc cag aat agt ggc ttt gaa tac acc att tgc ttt 246
Ser Asn Glu Cys Leu Gln Asn Ser Gly Phe Glu Tyr Thr Ile Cys Phe 55
60 65 ctg cct cca ctt gtg ctg aac ttt gaa ctg cca cca gat tat cca
tcc 294 Leu Pro Pro Leu Val Leu Asn Phe Glu Leu Pro Pro Asp Tyr Pro
Ser 70 75 80 85 tct tcc cca cct tca ttc aca ctt agt ggc aaa tgg ctg
tca cca act 342 Ser Ser Pro Pro Ser Phe Thr Leu Ser Gly Lys Trp Leu
Ser Pro Thr 90 95 100 cag cta tct gct cta tgc aag cac tta gac aac
cta tgg gaa gaa cac 390 Gln Leu Ser Ala Leu Cys Lys His Leu Asp Asn
Leu Trp Glu Glu His 105 110 115 cgt ggc agc gtg gtc ctg ttt gcc tgg
atg caa ttt ctt aag gaa gag 438 Arg Gly Ser Val Val Leu Phe Ala Trp
Met Gln Phe Leu Lys Glu Glu 120 125 130 acc cta gca tac ttg aat att
gtc tct cct ttt gag ctc aag att ggt 486 Thr Leu Ala Tyr Leu Asn Ile
Val Ser Pro Phe Glu Leu Lys Ile Gly 135 140 145 tct cag aaa aaa gtg
cag aga agg aca gct caa gct tct ccc aac aca 534 Ser Gln Lys Lys Val
Gln Arg Arg Thr Ala Gln Ala Ser Pro Asn Thr 150 155 160 165 gag cta
gat ttt gga gga gct gct gga tct gat gta gac caa gag gaa 582 Glu Leu
Asp Phe Gly Gly Ala Ala Gly Ser Asp Val Asp Gln Glu Glu 170 175 180
att gtg gat gag aga gca gtg cag gat gtg gaa tca ctg tca aat ctg 630
Ile Val Asp Glu Arg Ala Val Gln Asp Val Glu Ser Leu Ser Asn Leu 185
190 195 atc cag gaa atc ttg gac ttt gat caa gct cag cag ata aaa tgc
ttt 678 Ile Gln Glu Ile Leu Asp Phe Asp Gln Ala Gln Gln Ile Lys Cys
Phe 200 205 210 aat agt aaa ttg ttc ctg tgc agt atc tgt ttc tgt gag
aag ctg ggt 726 Asn Ser Lys Leu Phe Leu Cys Ser Ile Cys Phe Cys Glu
Lys Leu Gly 215 220 225 agt gaa tgc atg tac ttc ttg gag tgc agg cat
gtg tac tgc aaa gcc 774 Ser Glu Cys Met Tyr Phe Leu Glu Cys Arg His
Val Tyr Cys Lys Ala 230 235 240 245 tgt ctg aag gac tac ttt gaa atc
cag atc aga gat ggc cag gtt caa 822 Cys Leu Lys Asp Tyr Phe Glu Ile
Gln Ile Arg Asp Gly Gln Val Gln 250 255 260 tgc ctc aac tgc cca gaa
cca aag tgc cct tcg gtg gcc act cct ggt 870 Cys Leu Asn Cys Pro Glu
Pro Lys Cys Pro Ser Val Ala Thr Pro Gly 265 270 275 cag gtc aaa gag
tta gtg gaa gca gag tta ttt gcc cgt tat gac cgc 918 Gln Val Lys Glu
Leu Val Glu Ala Glu Leu Phe Ala Arg Tyr Asp Arg 280 285 290 ctt ctc
ctc cag tcc tcc ttg gac ctg atg gca gat gtg gtg tac tgc 966 Leu Leu
Leu Gln Ser Ser Leu Asp Leu Met Ala Asp Val Val Tyr Cys 295 300 305
ccc cgg ccg tgc tgc cag ctg cct gtg atg cag gaa cct ggc tgc acc
1014 Pro Arg Pro Cys Cys Gln Leu Pro Val Met Gln Glu Pro Gly Cys
Thr 310 315 320 325 atg ggt atc tgc tcc agc tgc aat ttt gcc ttc tgt
act ttg tgc agg 1062 Met Gly Ile Cys Ser Ser Cys Asn Phe Ala Phe
Cys Thr Leu Cys Arg 330 335 340 ttg acc tac cat ggg gtc tcc cca tgt
aag gtg act gca gag aaa tta 1110 Leu Thr Tyr His Gly Val Ser Pro
Cys Lys Val Thr Ala Glu Lys Leu 345 350 355 atg gac tta cga aat gaa
tac ctg caa gcg gat gag gct aat aaa aga 1158 Met Asp Leu Arg Asn
Glu Tyr Leu Gln Ala Asp Glu Ala Asn Lys Arg 360 365 370 ctt ttg gat
caa agg tat ggt aag aga gtg att cag aag gca ctg gaa 1206 Leu Leu
Asp Gln Arg Tyr Gly Lys Arg Val Ile Gln Lys Ala Leu Glu 375 380 385
gag atg gaa agt aag gag tgg cta gag aag aac tca aag agc tgc cca
1254 Glu Met Glu Ser Lys Glu Trp Leu Glu Lys Asn Ser Lys Ser Cys
Pro 390 395 400 405 tgt tgt gga act ccc ata gag aaa tta gac gga tgt
aac aag atg aca 1302 Cys Cys Gly Thr Pro Ile Glu Lys Leu Asp Gly
Cys Asn Lys Met Thr 410 415 420 tgt act ggc tgt atg caa tat ttc tgt
tgg att tgc atg ggt tct ctc 1350 Cys Thr Gly Cys Met Gln Tyr Phe
Cys Trp Ile Cys Met Gly Ser Leu 425 430 435 tct aga gca aac cct tac
aaa cat ttc aat gac cct ggt tca cca tgt 1398 Ser Arg Ala Asn Pro
Tyr Lys His Phe Asn Asp Pro Gly Ser Pro Cys 440 445 450 ttt aac cgg
ctg ttt tat gct gtg gat gtt gac gac gat att tgg gaa 1446 Phe Asn
Arg Leu Phe Tyr Ala Val Asp Val Asp Asp Asp Ile Trp Glu 455 460 465
gat gag gta gaa gac tag ttaactactg ctcaagatat ttaactactg 1494 Asp
Glu Val Glu Asp 470 475 ctcaagatat ggaagtggat tgtttttccc taatcttccg
tcaagtacac aaagtaactt 1554 tgcgggatat ttagggtact attcattcac
tcttcctgcg tagaagatat ggaagaacga 1614 ggtttatatt ttcatgtggt
actactgaag aaggtgcatt gatacatttt taaatgtaag 1674 ttgagaaaaa
tttataagcc aaaggttcag aaaattaaac tacagaa 1721 2 474 PRT Homo sapien
2 Met Ser Ser Glu Asp Arg Glu Ala Gln Glu Asp Glu Leu Leu Ala Leu 1
5 10 15 Ala Ser Ile Tyr Asp Gly Asp Glu Phe Arg Lys Ala Glu Ser Val
Gln 20 25 30 Gly Gly Glu Thr Arg Ile Tyr Leu Asp Leu Pro Gln Asn
Phe Lys Ile 35 40 45 Phe Val Ser Gly Asn Ser Asn Glu Cys Leu Gln
Asn Ser Gly Phe Glu 50 55 60 Tyr Thr Ile Cys Phe Leu Pro Pro Leu
Val Leu Asn Phe Glu Leu Pro 65 70 75 80 Pro Asp Tyr Pro Ser Ser Ser
Pro Pro Ser Phe Thr Leu Ser Gly Lys 85 90 95 Trp Leu Ser Pro Thr
Gln Leu Ser Ala Leu Cys Lys His Leu Asp Asn 100 105 110 Leu Trp Glu
Glu His Arg Gly Ser Val Val Leu Phe Ala Trp Met Gln 115 120 125 Phe
Leu Lys Glu Glu Thr Leu Ala Tyr Leu Asn Ile Val Ser Pro Phe 130 135
140 Glu Leu Lys Ile Gly Ser Gln Lys Lys Val Gln Arg Arg Thr Ala Gln
145 150 155 160 Ala Ser Pro Asn Thr Glu Leu Asp Phe Gly Gly Ala Ala
Gly Ser Asp 165 170 175 Val Asp Gln Glu Glu Ile Val Asp Glu Arg Ala
Val Gln Asp Val Glu 180 185 190 Ser Leu Ser Asn Leu Ile Gln Glu Ile
Leu Asp Phe Asp Gln Ala Gln 195 200 205 Gln Ile Lys Cys Phe Asn Ser
Lys Leu Phe Leu Cys Ser Ile Cys Phe 210 215 220 Cys Glu Lys Leu Gly
Ser Glu Cys Met Tyr Phe Leu Glu Cys Arg His 225 230 235 240 Val Tyr
Cys Lys Ala Cys Leu Lys Asp Tyr Phe Glu Ile Gln Ile Arg 245 250 255
Asp Gly Gln Val Gln Cys Leu Asn Cys Pro Glu Pro Lys Cys Pro Ser 260
265 270 Val Ala Thr Pro Gly Gln Val Lys Glu Leu Val Glu Ala Glu Leu
Phe 275 280 285 Ala Arg Tyr Asp Arg Leu Leu Leu Gln Ser Ser Leu Asp
Leu Met Ala 290 295 300 Asp Val Val Tyr Cys Pro Arg Pro Cys Cys Gln
Leu Pro Val Met Gln 305 310 315 320 Glu Pro Gly Cys Thr Met Gly Ile
Cys Ser Ser Cys Asn Phe Ala Phe 325 330 335 Cys Thr Leu Cys Arg Leu
Thr Tyr His Gly Val Ser Pro Cys Lys Val 340 345 350 Thr Ala Glu Lys
Leu Met Asp Leu Arg Asn Glu Tyr Leu Gln Ala Asp 355 360 365 Glu Ala
Asn Lys Arg Leu Leu Asp Gln Arg Tyr Gly Lys Arg Val Ile 370 375 380
Gln Lys Ala Leu Glu Glu Met Glu Ser Lys Glu Trp Leu Glu Lys Asn 385
390 395 400 Ser Lys Ser Cys Pro Cys Cys Gly Thr Pro Ile Glu Lys Leu
Asp Gly 405 410 415 Cys Asn Lys Met Thr Cys Thr Gly Cys Met Gln Tyr
Phe Cys Trp Ile 420 425 430 Cys Met Gly Ser Leu Ser Arg Ala Asn Pro
Tyr Lys His Phe Asn Asp 435 440 445 Pro Gly Ser Pro Cys Phe Asn Arg
Leu Phe Tyr Ala Val Asp Val Asp 450 455 460 Asp Asp Ile Trp Glu Asp
Glu Val Glu Asp 465 470 3 1335 DNA Homo sapien CDS (1)..(1335)
misc_feature (750)..(1332) Coding sequence and polypeptide region
for the C-terminal binding domain 3 atg cca agg tca ggg gct ccc aaa
gag cgc cct gcg gag cct ctc acc 48 Met Pro Arg Ser Gly Ala Pro Lys
Glu Arg Pro Ala Glu Pro Leu Thr 1 5 10 15 cct ccc cca tcc tat ggc
cac cag cca aca ggg cag tct ggg gag tct 96 Pro Pro Pro Ser Tyr Gly
His Gln Pro Thr Gly Gln Ser Gly Glu Ser 20 25 30 tca gga gcc tcg
ggg gac aag gac cac ctg tac agc acg gta tgc aag 144 Ser Gly Ala Ser
Gly Asp Lys Asp His Leu Tyr Ser Thr Val Cys Lys 35 40 45 cct cgg
tcc cca aag cct gca gcc ccg gcc gcc cct cca ttc tcc tct 192 Pro Arg
Ser Pro Lys Pro Ala Ala Pro Ala Ala Pro Pro Phe Ser Ser 50 55 60
tcc agc ggt gtc ttg ggt acc ggg ctc tgt gag cta gat cgg ttg ctt 240
Ser Ser Gly Val Leu Gly Thr Gly Leu Cys Glu Leu Asp Arg Leu Leu 65
70 75 80 cag gaa ctt aat gcc act cag ttc aac atc aca gat gaa atc
atg tct 288 Gln Glu Leu Asn Ala Thr Gln Phe Asn Ile Thr Asp Glu Ile
Met Ser 85 90 95 cag ttc cca tct agc aag gtg gct tca gga gag cag
aag gag gac cag 336 Gln Phe Pro Ser Ser Lys Val Ala Ser Gly Glu Gln
Lys Glu Asp Gln 100 105 110 tct gaa gat aag aaa aga ccc agc ctc cct
tcc agc ccg tct cct ggc 384 Ser Glu Asp Lys Lys Arg Pro Ser Leu Pro
Ser Ser Pro Ser Pro Gly 115 120 125 ctc cca aag gct tct gcc acc tca
gcc act ctg gag ctg gat aga ctg 432 Leu Pro Lys Ala Ser Ala Thr Ser
Ala Thr Leu Glu Leu Asp Arg Leu 130 135 140 atg gcc tca ctc cct gac
ttc cgc gtt caa aac cat ctt cca gcc tct 480 Met Ala Ser Leu Pro Asp
Phe Arg Val Gln Asn His Leu Pro Ala Ser 145 150 155 160 ggg cca act
cag cca ccg gtg gtg agc tcc aca aat gag ggc tcc cca 528 Gly Pro Thr
Gln Pro Pro Val Val Ser Ser Thr Asn Glu Gly Ser Pro 165 170 175 tcc
cca cca gag ccg act gca aag ggc agc cta gac acc atg ctg ggg 576 Ser
Pro Pro Glu Pro Thr Ala Lys Gly Ser Leu Asp Thr Met Leu Gly 180 185
190 ctg ctg cag tcc gac ctc agc cgc cgg ggt gtt ccc acc cag gcc aaa
624 Leu Leu Gln Ser Asp Leu Ser Arg Arg Gly Val Pro Thr Gln Ala Lys
195 200 205 ggc ctc tgt ggc tcc tgc aat aaa cct att gct ggg caa gtg
gtg acg 672 Gly Leu Cys Gly Ser Cys Asn Lys Pro Ile Ala Gly Gln Val
Val Thr 210 215 220 gct ctg ggc cgc gcc tgg cac ccc gag cac ttc gtt
tgc gga ggc tgt 720 Ala Leu Gly Arg Ala Trp His Pro Glu His Phe Val
Cys Gly Gly Cys 225 230 235 240 tcc acc gcc ctg gga ggc agc agc ttc
ttc gag aag gat gga gcc ccc 768 Ser Thr Ala Leu Gly Gly Ser Ser Phe
Phe Glu Lys Asp Gly Ala Pro 245 250 255 ttc tgc ccc gag tgc tac ttt
gag cgc ttc tcg cca aga tgt ggc ttc 816 Phe Cys Pro Glu Cys Tyr Phe
Glu Arg Phe Ser Pro Arg Cys Gly Phe 260 265 270 tgc aac cag ccc atc
cga cac aag atg gtg acc gcc ttg ggc act cac 864 Cys Asn Gln Pro Ile
Arg His Lys Met Val Thr Ala Leu Gly Thr His 275 280 285 tgg cac cca
gag cat ttc tgc tgc gtc agt tgc ggg gag ccc ttc gga 912 Trp His Pro
Glu His Phe Cys Cys Val Ser Cys Gly Glu Pro Phe Gly 290 295 300 gat
gag ggt ttc cac gag cgc gag ggc cgc ccc tac tgc cgc cgg gac 960 Asp
Glu Gly Phe His Glu Arg Glu Gly Arg Pro Tyr Cys Arg Arg Asp 305 310
315 320 ttc ctg cag ctg ttc gcc ccg cgc tgc cag ggc tgc cag ggc ccc
atc 1008 Phe Leu Gln Leu Phe Ala Pro Arg Cys Gln Gly Cys Gln Gly
Pro Ile 325 330 335 ctg gat aac tac atc tcg gcg ctc agc ctg ctc tgg
cac ccg gac tgt 1056 Leu Asp Asn Tyr Ile Ser Ala Leu Ser Leu Leu
Trp His Pro Asp Cys 340 345 350 ttc gtc tgc agg gaa tgc ttc gcg ccc
ttc tcg gga ggc agc ttt ttc 1104 Phe Val Cys Arg Glu Cys Phe Ala
Pro Phe Ser Gly Gly Ser Phe Phe 355 360 365 gag cac gag ggc cgc ccg
ttg tgc gag aac cac ttc cac gca cga cgc 1152 Glu His Glu Gly Arg
Pro Leu Cys Glu Asn His Phe His Ala Arg Arg 370 375 380 ggc tcg ctg
tgc ccc acg tgt ggc ctc cct gtg acc ggc cgc tgc gtg 1200 Gly Ser
Leu Cys Pro Thr Cys Gly Leu Pro Val Thr Gly Arg Cys Val 385 390 395
400 tcg gcc ctg ggt cgc cgc ttc cac ccg gac cac ttc gca tgc acc ttc
1248 Ser Ala Leu Gly Arg Arg Phe His Pro Asp His Phe Ala Cys Thr
Phe 405 410 415 tgc ctg cgc ccg ctc acc aag ggg tcc ttc cag gag cgc
gcc ggc aag 1296 Cys Leu Arg Pro Leu Thr Lys Gly Ser Phe Gln Glu
Arg Ala Gly Lys 420 425 430 ccc tac tgc cag ccc tgc ttc ctg aag ctc
ttc ggc tga 1335 Pro Tyr Cys Gln Pro Cys Phe Leu Lys Leu Phe Gly
435 440 445 4 444 PRT Homo sapien 4 Met Pro Arg Ser Gly Ala Pro Lys
Glu Arg Pro Ala Glu Pro Leu Thr 1 5 10 15 Pro Pro Pro Ser Tyr Gly
His Gln Pro Thr Gly Gln Ser Gly Glu Ser 20 25 30 Ser Gly Ala Ser
Gly Asp Lys Asp His Leu Tyr Ser Thr Val Cys Lys 35 40 45 Pro Arg
Ser Pro Lys Pro Ala Ala Pro Ala Ala Pro Pro Phe Ser Ser 50 55 60
Ser Ser Gly Val Leu Gly Thr Gly Leu Cys Glu Leu Asp Arg Leu Leu 65
70 75 80 Gln Glu Leu Asn Ala Thr Gln Phe Asn Ile Thr Asp Glu Ile
Met Ser 85 90 95 Gln Phe Pro Ser Ser Lys Val Ala Ser Gly Glu Gln
Lys Glu Asp Gln 100 105 110 Ser Glu Asp Lys Lys Arg Pro Ser Leu Pro
Ser Ser Pro Ser Pro Gly 115 120 125 Leu Pro Lys Ala Ser Ala Thr Ser
Ala Thr Leu Glu Leu Asp Arg Leu 130 135 140 Met Ala Ser Leu Pro Asp
Phe Arg Val Gln Asn His Leu Pro Ala Ser 145 150 155 160 Gly Pro Thr
Gln Pro Pro Val Val Ser Ser Thr Asn Glu Gly Ser Pro 165 170 175 Ser
Pro Pro Glu Pro Thr Ala Lys Gly Ser Leu Asp Thr Met Leu Gly 180 185
190 Leu Leu Gln Ser Asp Leu Ser Arg Arg Gly Val Pro Thr Gln Ala Lys
195 200 205 Gly Leu Cys Gly Ser Cys Asn Lys Pro Ile Ala Gly Gln Val
Val Thr 210 215 220 Ala Leu Gly Arg Ala Trp His Pro Glu His Phe Val
Cys Gly Gly Cys 225 230 235 240 Ser Thr Ala Leu Gly Gly Ser Ser Phe
Phe Glu Lys Asp Gly Ala Pro 245 250 255 Phe Cys Pro Glu Cys Tyr Phe
Glu Arg Phe Ser Pro Arg Cys Gly Phe 260 265 270 Cys Asn Gln Pro Ile
Arg His Lys Met Val Thr Ala Leu Gly Thr His 275 280 285 Trp His Pro
Glu His Phe Cys Cys Val Ser Cys Gly Glu Pro Phe Gly 290 295 300 Asp
Glu Gly Phe His Glu Arg Glu Gly Arg Pro Tyr Cys Arg Arg Asp 305 310
315 320 Phe Leu Gln Leu Phe Ala Pro Arg Cys Gln Gly Cys Gln Gly Pro
Ile 325 330 335 Leu Asp Asn Tyr Ile Ser Ala Leu Ser Leu Leu Trp His
Pro Asp Cys 340 345 350 Phe Val Cys Arg Glu Cys Phe Ala Pro Phe Ser
Gly Gly Ser Phe Phe 355 360 365 Glu His Glu Gly Arg Pro Leu Cys Glu
Asn His Phe His Ala Arg Arg 370 375 380 Gly Ser
Leu Cys Pro Thr Cys Gly Leu Pro Val Thr Gly Arg Cys Val 385 390 395
400 Ser Ala Leu Gly Arg Arg Phe His Pro Asp His Phe Ala Cys Thr Phe
405 410 415 Cys Leu Arg Pro Leu Thr Lys Gly Ser Phe Gln Glu Arg Ala
Gly Lys 420 425 430 Pro Tyr Cys Gln Pro Cys Phe Leu Lys Leu Phe Gly
435 440 5 1566 DNA Homo sapien CDS (25)..(675) 3'UTR (676)..(1566)
5'UTR (1)..(24) 5 ggcgcttctg gaaggaacgc cgcg atg gct gcg cag gga
gag ccc cag gtc 51 Met Ala Ala Gln Gly Glu Pro Gln Val 1 5 cag ttc
aaa ctt gta ttg gtt ggt gat ggt ggt act gga aaa acg acc 99 Gln Phe
Lys Leu Val Leu Val Gly Asp Gly Gly Thr Gly Lys Thr Thr 10 15 20 25
ttc gtg aaa cgt cat ttg act ggt gaa ttt gag aag aag tat gta gcc 147
Phe Val Lys Arg His Leu Thr Gly Glu Phe Glu Lys Lys Tyr Val Ala 30
35 40 acc ttg ggt gtt gag gtt cat ccc cta gtg ttc cac acc aac aga
gga 195 Thr Leu Gly Val Glu Val His Pro Leu Val Phe His Thr Asn Arg
Gly 45 50 55 cct att aag ttc aat gta tgg gac aca gcc ggc cag gag
aaa ttc ggt 243 Pro Ile Lys Phe Asn Val Trp Asp Thr Ala Gly Gln Glu
Lys Phe Gly 60 65 70 gga ctg aga gat ggc tat tat atc caa gcc cag
tgt gcc atc ata atg 291 Gly Leu Arg Asp Gly Tyr Tyr Ile Gln Ala Gln
Cys Ala Ile Ile Met 75 80 85 ttt gat gta aca tcg aga gtt act tac
aag aat gtg cct aac tgg cat 339 Phe Asp Val Thr Ser Arg Val Thr Tyr
Lys Asn Val Pro Asn Trp His 90 95 100 105 aga gat ctg gta cga gtg
tgt gaa aac atc ccc att gtg ttg tgt ggc 387 Arg Asp Leu Val Arg Val
Cys Glu Asn Ile Pro Ile Val Leu Cys Gly 110 115 120 aac aaa gtg gat
att aag gac agg aaa gtg aag gcg aaa tcc att gtc 435 Asn Lys Val Asp
Ile Lys Asp Arg Lys Val Lys Ala Lys Ser Ile Val 125 130 135 ttc cac
cga aag aag aat ctt cag tac tac gac att tct gcc aaa agt 483 Phe His
Arg Lys Lys Asn Leu Gln Tyr Tyr Asp Ile Ser Ala Lys Ser 140 145 150
aac tac aac ttt gaa aag ccc ttc ctc tgg ctt gct agg aag ctc att 531
Asn Tyr Asn Phe Glu Lys Pro Phe Leu Trp Leu Ala Arg Lys Leu Ile 155
160 165 gga gac cct aac ttg gaa ttt gtt gcc atg cct gct ctc gcc cca
cca 579 Gly Asp Pro Asn Leu Glu Phe Val Ala Met Pro Ala Leu Ala Pro
Pro 170 175 180 185 gaa gtt gtc atg gac cca gct ttg gca gca cag tat
gag cac gac tta 627 Glu Val Val Met Asp Pro Ala Leu Ala Ala Gln Tyr
Glu His Asp Leu 190 195 200 gag gtt gct cag aca act gct ctc ccg gat
gag gat gat gac ctg tga 675 Glu Val Ala Gln Thr Thr Ala Leu Pro Asp
Glu Asp Asp Asp Leu 205 210 215 gaatgaagct ggagcccagc gtcagaagtc
tagttttata ggcagctgtc ctgtgatgtc 735 agcggtgcag cgtgtgtgcc
acctcattat tatctagcta agcggaacat gtgctttatc 795 tgtgggatgc
tgaaggagat gagtgggctt cggagtgaat gtggcagttt aaaaaataac 855
ttcattgttt ggacctgcat atttagctgt ttggacgcag ttgattcctt gagtttcata
915 tataagactg ctgcagtcac atcacaatat tcagtggtga aatcttgttt
gttactgtca 975 ttcccattcc ttttctttag aatcagaata aagttgtatt
tcaaatatct aagcaagtga 1035 actcatccct tgtttataaa tagcatttgg
aaaccactaa agtagggaag ttttatgcca 1095 tgttaatatt tgaattgcct
tgcttttatc acttaatttg aaatctattg ggttaatttc 1155 tccctatgtt
tatttttgta catttgagcc atgtcacaca aactgatgat gacaggtcag 1215
cagtattcta tttggttaga agggttacat ggtgtaaata ttagtgcagt taagctaaag
1275 cagtgtttgc tccaccttca tattggctag gtagggtcac ctagggaagc
acttgctcaa 1335 aatctgtgac ctgtcagaat aaaaatgtgg tttgtacata
tcaaatagat attttaaggg 1395 taatattttc ttttatggca aaagtaatca
tgttttaatg tagaacctca aacaggatgg 1455 aacatcagtg gatggcagga
ggttgggaat tcttgctgtt aaaaataatt acaaattttg 1515 cactttttgt
ttgaatgtta gatgcttagt gtgaagttga tacgcaagcc g 1566 6 216 PRT Homo
sapien 6 Met Ala Ala Gln Gly Glu Pro Gln Val Gln Phe Lys Leu Val
Leu Val 1 5 10 15 Gly Asp Gly Gly Thr Gly Lys Thr Thr Phe Val Lys
Arg His Leu Thr 20 25 30 Gly Glu Phe Glu Lys Lys Tyr Val Ala Thr
Leu Gly Val Glu Val His 35 40 45 Pro Leu Val Phe His Thr Asn Arg
Gly Pro Ile Lys Phe Asn Val Trp 50 55 60 Asp Thr Ala Gly Gln Glu
Lys Phe Gly Gly Leu Arg Asp Gly Tyr Tyr 65 70 75 80 Ile Gln Ala Gln
Cys Ala Ile Ile Met Phe Asp Val Thr Ser Arg Val 85 90 95 Thr Tyr
Lys Asn Val Pro Asn Trp His Arg Asp Leu Val Arg Val Cys 100 105 110
Glu Asn Ile Pro Ile Val Leu Cys Gly Asn Lys Val Asp Ile Lys Asp 115
120 125 Arg Lys Val Lys Ala Lys Ser Ile Val Phe His Arg Lys Lys Asn
Leu 130 135 140 Gln Tyr Tyr Asp Ile Ser Ala Lys Ser Asn Tyr Asn Phe
Glu Lys Pro 145 150 155 160 Phe Leu Trp Leu Ala Arg Lys Leu Ile Gly
Asp Pro Asn Leu Glu Phe 165 170 175 Val Ala Met Pro Ala Leu Ala Pro
Pro Glu Val Val Met Asp Pro Ala 180 185 190 Leu Ala Ala Gln Tyr Glu
His Asp Leu Glu Val Ala Gln Thr Thr Ala 195 200 205 Leu Pro Asp Glu
Asp Asp Asp Leu 210 215 7 4839 DNA Homo sapien CDS (138)..(2924) 7
tccggttttt ctcaggggac gttgaaatta tttttgtaac gggagtcggg agaggacggg
60 gcgtgccccg cgtgcgcgcg cgtcgtcctc cccggcgctc ctccacagct
cgctggctcc 120 cgccgcggaa aggcgtc atg ccg ccc aaa acc ccc cga aaa
acg gcc gcc 170 Met Pro Pro Lys Thr Pro Arg Lys Thr Ala Ala 1 5 10
acc gcc gcc gct gcc gcc gcg gaa ccc ccg gca ccg ccg ccg ccg ccc 218
Thr Ala Ala Ala Ala Ala Ala Glu Pro Pro Ala Pro Pro Pro Pro Pro 15
20 25 cct cct gag gag gac cca gag cag gac agc ggc ccg gag gac ctg
cct 266 Pro Pro Glu Glu Asp Pro Glu Gln Asp Ser Gly Pro Glu Asp Leu
Pro 30 35 40 ctc gtc agg ctt gag ttt gaa gaa aca gaa gaa cct gat
ttt act gca 314 Leu Val Arg Leu Glu Phe Glu Glu Thr Glu Glu Pro Asp
Phe Thr Ala 45 50 55 tta tgt cag aaa tta aag ata cca gat cat gtc
aga gag aga gct tgg 362 Leu Cys Gln Lys Leu Lys Ile Pro Asp His Val
Arg Glu Arg Ala Trp 60 65 70 75 tta act tgg gag aaa gtt tca tct gtg
gat gga gta ttg gga ggt tat 410 Leu Thr Trp Glu Lys Val Ser Ser Val
Asp Gly Val Leu Gly Gly Tyr 80 85 90 att caa aag aaa aag gaa ctg
tgg gga atc tgt atc ttt att gca gca 458 Ile Gln Lys Lys Lys Glu Leu
Trp Gly Ile Cys Ile Phe Ile Ala Ala 95 100 105 gtt gac cta gat gag
atg tcg ttc act ttt act gag cta cag aaa aac 506 Val Asp Leu Asp Glu
Met Ser Phe Thr Phe Thr Glu Leu Gln Lys Asn 110 115 120 ata gaa atc
agt gtc cat aaa ttc ttt aac tta cta aaa gaa att gat 554 Ile Glu Ile
Ser Val His Lys Phe Phe Asn Leu Leu Lys Glu Ile Asp 125 130 135 acc
agt acc aaa gtt gat aat gct atg tca aga ctg ttg aag aag tat 602 Thr
Ser Thr Lys Val Asp Asn Ala Met Ser Arg Leu Leu Lys Lys Tyr 140 145
150 155 gat gta ttg ttt gca ctc ttc agc aaa ttg gaa agg aca tgt gaa
ctt 650 Asp Val Leu Phe Ala Leu Phe Ser Lys Leu Glu Arg Thr Cys Glu
Leu 160 165 170 ata tat ttg aca caa ccc agc agt tcg ata tct act gaa
ata aat tct 698 Ile Tyr Leu Thr Gln Pro Ser Ser Ser Ile Ser Thr Glu
Ile Asn Ser 175 180 185 gca ttg gtg cta aaa gtt tct tgg atc aca ttt
tta tta gct aaa ggg 746 Ala Leu Val Leu Lys Val Ser Trp Ile Thr Phe
Leu Leu Ala Lys Gly 190 195 200 gaa gta tta caa atg gaa gat gat ctg
gtg att tca ttt cag tta atg 794 Glu Val Leu Gln Met Glu Asp Asp Leu
Val Ile Ser Phe Gln Leu Met 205 210 215 cta tgt gtc ctt gac tat ttt
att aaa ctc tca cct ccc atg ttg ctc 842 Leu Cys Val Leu Asp Tyr Phe
Ile Lys Leu Ser Pro Pro Met Leu Leu 220 225 230 235 aaa gaa cca tat
aaa aca gct gtt ata ccc att aat ggt tca cct cga 890 Lys Glu Pro Tyr
Lys Thr Ala Val Ile Pro Ile Asn Gly Ser Pro Arg 240 245 250 aca ccc
agg cga ggt cag aac agg agt gca cgg ata gca aaa caa cta 938 Thr Pro
Arg Arg Gly Gln Asn Arg Ser Ala Arg Ile Ala Lys Gln Leu 255 260 265
gaa aat gat aca aga att att gaa gtt ctc tgt aaa gaa cat gaa tgt 986
Glu Asn Asp Thr Arg Ile Ile Glu Val Leu Cys Lys Glu His Glu Cys 270
275 280 aat ata gat gag gtg aaa aat gtt tat ttc aaa aat ttt ata cct
ttt 1034 Asn Ile Asp Glu Val Lys Asn Val Tyr Phe Lys Asn Phe Ile
Pro Phe 285 290 295 atg aat tct ctt gga ctt gta aca tct aat gga ctt
cca gag gtt gaa 1082 Met Asn Ser Leu Gly Leu Val Thr Ser Asn Gly
Leu Pro Glu Val Glu 300 305 310 315 aat ctt tct aaa cga tac gaa gaa
att tat ctt aaa aat aaa gat cta 1130 Asn Leu Ser Lys Arg Tyr Glu
Glu Ile Tyr Leu Lys Asn Lys Asp Leu 320 325 330 gat gca aga tta ttt
ttg gat cat gat aaa act ctt cag act gat tct 1178 Asp Ala Arg Leu
Phe Leu Asp His Asp Lys Thr Leu Gln Thr Asp Ser 335 340 345 ata gac
agt ttt gaa aca cag aga aca cca cga aaa agt aac ctt gat 1226 Ile
Asp Ser Phe Glu Thr Gln Arg Thr Pro Arg Lys Ser Asn Leu Asp 350 355
360 gaa gag gtg aat gta att cct cca cac act cca gtt agg act gtt atg
1274 Glu Glu Val Asn Val Ile Pro Pro His Thr Pro Val Arg Thr Val
Met 365 370 375 aac act atc caa caa tta atg atg att tta aat tca gca
agt gat caa 1322 Asn Thr Ile Gln Gln Leu Met Met Ile Leu Asn Ser
Ala Ser Asp Gln 380 385 390 395 cct tca gaa aat ctg att tcc tat ttt
aac aac tgc aca gtg aat cca 1370 Pro Ser Glu Asn Leu Ile Ser Tyr
Phe Asn Asn Cys Thr Val Asn Pro 400 405 410 aaa gaa agt ata ctg aaa
aga gtg aag gat ata gga tac atc ttt aaa 1418 Lys Glu Ser Ile Leu
Lys Arg Val Lys Asp Ile Gly Tyr Ile Phe Lys 415 420 425 gag aaa ttt
gct aaa gct gtg gga cag ggt tgt gtc gaa att gga tca 1466 Glu Lys
Phe Ala Lys Ala Val Gly Gln Gly Cys Val Glu Ile Gly Ser 430 435 440
cag cga tac aaa ctt gga gtt cgc ttg tat tac cga gta atg gaa tcc
1514 Gln Arg Tyr Lys Leu Gly Val Arg Leu Tyr Tyr Arg Val Met Glu
Ser 445 450 455 atg ctt aaa tca gaa gaa gaa cga tta tcc att caa aat
ttt agc aaa 1562 Met Leu Lys Ser Glu Glu Glu Arg Leu Ser Ile Gln
Asn Phe Ser Lys 460 465 470 475 ctt ctg aat gac aac att ttt cat atg
tct tta ttg gcg tgc gct ctt 1610 Leu Leu Asn Asp Asn Ile Phe His
Met Ser Leu Leu Ala Cys Ala Leu 480 485 490 gag gtt gta atg gcc aca
tat agc aga agt aca tct cag aat ctt gat 1658 Glu Val Val Met Ala
Thr Tyr Ser Arg Ser Thr Ser Gln Asn Leu Asp 495 500 505 tct gga aca
gat ttg tct ttc cca tgg att ctg aat gtg ctt aat tta 1706 Ser Gly
Thr Asp Leu Ser Phe Pro Trp Ile Leu Asn Val Leu Asn Leu 510 515 520
aaa gcc ttt gat ttt tac aaa gtg atc gaa agt ttt atc aaa gca gaa
1754 Lys Ala Phe Asp Phe Tyr Lys Val Ile Glu Ser Phe Ile Lys Ala
Glu 525 530 535 ggc aac ttg aca aga gaa atg ata aaa cat tta gaa cga
tgt gaa cat 1802 Gly Asn Leu Thr Arg Glu Met Ile Lys His Leu Glu
Arg Cys Glu His 540 545 550 555 cga atc atg gaa tcc ctt gca tgg ctc
tca gat tca cct tta ttt gat 1850 Arg Ile Met Glu Ser Leu Ala Trp
Leu Ser Asp Ser Pro Leu Phe Asp 560 565 570 ctt att aaa caa tca aag
gac cga gaa gga cca act gat cac ctt gaa 1898 Leu Ile Lys Gln Ser
Lys Asp Arg Glu Gly Pro Thr Asp His Leu Glu 575 580 585 tct gct tgt
cct ctt aat ctt cct ctc cag aat aat cac act gca gca 1946 Ser Ala
Cys Pro Leu Asn Leu Pro Leu Gln Asn Asn His Thr Ala Ala 590 595 600
gat atg tat ctt tct cct gta aga tct cca aag aaa aaa ggt tca act
1994 Asp Met Tyr Leu Ser Pro Val Arg Ser Pro Lys Lys Lys Gly Ser
Thr 605 610 615 acg cgt gta aat tct act gca aat gca gag aca caa gca
acc tca gcc 2042 Thr Arg Val Asn Ser Thr Ala Asn Ala Glu Thr Gln
Ala Thr Ser Ala 620 625 630 635 ttc cag acc cag aag cca ttg aaa tct
acc tct ctt tca ctg ttt tat 2090 Phe Gln Thr Gln Lys Pro Leu Lys
Ser Thr Ser Leu Ser Leu Phe Tyr 640 645 650 aaa aaa gtg tat cgg cta
gcc tat ctc cgg cta aat aca ctt tgt gaa 2138 Lys Lys Val Tyr Arg
Leu Ala Tyr Leu Arg Leu Asn Thr Leu Cys Glu 655 660 665 cgc ctt ctg
tct gag cac cca gaa tta gaa cat atc atc tgg acc ctt 2186 Arg Leu
Leu Ser Glu His Pro Glu Leu Glu His Ile Ile Trp Thr Leu 670 675 680
ttc cag cac acc ctg cag aat gag tat gaa ctc atg aga gac agg cat
2234 Phe Gln His Thr Leu Gln Asn Glu Tyr Glu Leu Met Arg Asp Arg
His 685 690 695 ttg gac caa att atg atg tgt tcc atg tat ggc ata tgc
aaa gtg aag 2282 Leu Asp Gln Ile Met Met Cys Ser Met Tyr Gly Ile
Cys Lys Val Lys 700 705 710 715 aat ata gac ctt aaa ttc aaa atc att
gta aca gca tac aag gat ctt 2330 Asn Ile Asp Leu Lys Phe Lys Ile
Ile Val Thr Ala Tyr Lys Asp Leu 720 725 730 cct cat gct gtt cag gag
aca ttc aaa cgt gtt ttg atc aaa gaa gag 2378 Pro His Ala Val Gln
Glu Thr Phe Lys Arg Val Leu Ile Lys Glu Glu 735 740 745 gag tat gat
tct att ata gta ttc tat aac tcg gtc ttc atg cag aga 2426 Glu Tyr
Asp Ser Ile Ile Val Phe Tyr Asn Ser Val Phe Met Gln Arg 750 755 760
ctg aaa aca aat att ttg cag tat gct tcc acc agg ccc cct acc ttg
2474 Leu Lys Thr Asn Ile Leu Gln Tyr Ala Ser Thr Arg Pro Pro Thr
Leu 765 770 775 tca cca ata cct cac att cct cga agc cct tac aag ttt
cct agt tca 2522 Ser Pro Ile Pro His Ile Pro Arg Ser Pro Tyr Lys
Phe Pro Ser Ser 780 785 790 795 ccc tta cgg att cct gga ggg aac atc
tat att tca ccc ctg aag agt 2570 Pro Leu Arg Ile Pro Gly Gly Asn
Ile Tyr Ile Ser Pro Leu Lys Ser 800 805 810 cca tat aaa att tca gaa
ggt ctg cca aca cca aca aaa atg act cca 2618 Pro Tyr Lys Ile Ser
Glu Gly Leu Pro Thr Pro Thr Lys Met Thr Pro 815 820 825 aga tca aga
atc tta gta tca att ggt gaa tca ttc ggg act tct gag 2666 Arg Ser
Arg Ile Leu Val Ser Ile Gly Glu Ser Phe Gly Thr Ser Glu 830 835 840
aag ttc cag aaa ata aat cag atg gta tgt aac agc gac cgt gtg ctc
2714 Lys Phe Gln Lys Ile Asn Gln Met Val Cys Asn Ser Asp Arg Val
Leu 845 850 855 aaa aga agt gct gaa gga agc aac cct cct aaa cca ctg
aaa aaa cta 2762 Lys Arg Ser Ala Glu Gly Ser Asn Pro Pro Lys Pro
Leu Lys Lys Leu 860 865 870 875 cgc ttt gat att gaa gga tca gat gaa
gca gat gga agt aaa cat ctc 2810 Arg Phe Asp Ile Glu Gly Ser Asp
Glu Ala Asp Gly Ser Lys His Leu 880 885 890 cca gga gag tcc aaa ttt
cag cag aaa ctg gca gaa atg act tct act 2858 Pro Gly Glu Ser Lys
Phe Gln Gln Lys Leu Ala Glu Met Thr Ser Thr 895 900 905 cga aca cga
atg caa aag cag aaa atg aat gat agc atg gat acc tca 2906 Arg Thr
Arg Met Gln Lys Gln Lys Met Asn Asp Ser Met Asp Thr Ser 910 915 920
aac aag gaa gag aaa tga ggatctcagg accttggtgg acactgtgta 2954 Asn
Lys Glu Glu Lys 925 cacctctgga ttcattgtct ctcacagatg tgactgtata
actttcccag gttctgttta 3014 tggccacatt taatatcttc agctcttttt
gtggatataa aatgtgcaga tgcaattgtt 3074 tgggtgattc ctaagccact
tgaaatgtta gtcattgtta tttatacaag attgaaaatc 3134 ttgtgtaaat
cctgccattt aaaaagttgt agcagattgt ttcctcttcc aaagtaaaat 3194
tgctgtgctt tatggatagt aagaatggcc ctagagtggg agtcctgata acccaggcct
3254 gtctgactac tttgccttct tttgtagcat ataggtgatg tttgctcttg
tttttattaa 3314 tttatatgta tattttttta atttaacatg aacaccctta
gaaaatgtgt cctatctatc 3374 ttccaaatgc aatttgattg actgcccatt
caccaaaatt atcctgaact cttctgcaaa 3434 aatggatatt attagaaatt
agaaaaaaat tactaatttt acacattaga ttttatttta 3494 ctattggaat
ctgatatact gtgtgcttgt tttataaaat tttgctttta attaaataaa 3554
agctggaagc aaagtataac catatgatac tatcatacta ctgaaacaga tttcatacct
3614 cagaatgtaa aagaacttac tgattatttt cttcatccaa cttatgtttt
taaatgagga
3674 ttattgatag tactcttggt ttttatacca ttcagatcac tgaatttata
aagtacccat 3734 ctagtacttg aaaaagtaaa gtgttctgcc agatcttagg
tatagaggac cctaacacag 3794 tatatcccaa gtgcactttc taatgtttct
gggtcctgaa gaattaagat acaaattaat 3854 tttactccat aaacagactg
ttaattatag gagccttaat ttttttttca tagagatttg 3914 tctaattgca
tctcaaaatt attctgccct ccttaatttg ggaaggtttg tgttttctct 3974
ggaatggtac atgtcttcca tgtatctttt gaactggcaa ttgtctattt atcttttatt
4034 tttttaagtc agtatggtct aacactggca tgttcaaagc cacattattt
ctagtccaaa 4094 attacaagta atcaagggtc attatgggtt aggcattaat
gtttctatct gattttgtgc 4154 aaaagcttca aattaaaaca gctgcattag
aaaaagaggc gcttctcccc tcccctacac 4214 ctaaaggtgt atttaaacta
tcttgtgtga ttaacttatt tagagatgct gtaacttaaa 4274 ataggggata
tttaaggtag cttcagctag cttttaggaa aatcactttg tctaactcag 4334
aattattttt aaaaagaaat ctggtcttgt tagaaaacaa aattttattt tgtgctcatt
4394 taagtttcaa acttactatt ttgacagtta ttttgataac aatgacacta
gaaaacttga 4454 ctccatttca tcattgtttc tgcatgaata tcatacaaat
cagttagttt ttaggtcaag 4514 ggcttactat ttctgggtct tttgctacta
agttcacatt agaattagtg ccagaatttt 4574 aggaacttca gagatcgtgt
attgagattt cttaaataat gcttcagata ttattgcttt 4634 attgcttttt
tgtattggtt aaaactgtac atttaaaatt gctatgttac tattttctac 4694
aattaatagt ttgtctattt taaaataaat tagttgttaa gagtcttaat ggtctgatgt
4754 tgtgttcttt gtattaagta cactaatgtt ctcttttctg tctaggagaa
gatagataga 4814 agataactct cctagtatct catcc 4839 8 928 PRT Homo
sapien 8 Met Pro Pro Lys Thr Pro Arg Lys Thr Ala Ala Thr Ala Ala
Ala Ala 1 5 10 15 Ala Ala Glu Pro Pro Ala Pro Pro Pro Pro Pro Pro
Pro Glu Glu Asp 20 25 30 Pro Glu Gln Asp Ser Gly Pro Glu Asp Leu
Pro Leu Val Arg Leu Glu 35 40 45 Phe Glu Glu Thr Glu Glu Pro Asp
Phe Thr Ala Leu Cys Gln Lys Leu 50 55 60 Lys Ile Pro Asp His Val
Arg Glu Arg Ala Trp Leu Thr Trp Glu Lys 65 70 75 80 Val Ser Ser Val
Asp Gly Val Leu Gly Gly Tyr Ile Gln Lys Lys Lys 85 90 95 Glu Leu
Trp Gly Ile Cys Ile Phe Ile Ala Ala Val Asp Leu Asp Glu 100 105 110
Met Ser Phe Thr Phe Thr Glu Leu Gln Lys Asn Ile Glu Ile Ser Val 115
120 125 His Lys Phe Phe Asn Leu Leu Lys Glu Ile Asp Thr Ser Thr Lys
Val 130 135 140 Asp Asn Ala Met Ser Arg Leu Leu Lys Lys Tyr Asp Val
Leu Phe Ala 145 150 155 160 Leu Phe Ser Lys Leu Glu Arg Thr Cys Glu
Leu Ile Tyr Leu Thr Gln 165 170 175 Pro Ser Ser Ser Ile Ser Thr Glu
Ile Asn Ser Ala Leu Val Leu Lys 180 185 190 Val Ser Trp Ile Thr Phe
Leu Leu Ala Lys Gly Glu Val Leu Gln Met 195 200 205 Glu Asp Asp Leu
Val Ile Ser Phe Gln Leu Met Leu Cys Val Leu Asp 210 215 220 Tyr Phe
Ile Lys Leu Ser Pro Pro Met Leu Leu Lys Glu Pro Tyr Lys 225 230 235
240 Thr Ala Val Ile Pro Ile Asn Gly Ser Pro Arg Thr Pro Arg Arg Gly
245 250 255 Gln Asn Arg Ser Ala Arg Ile Ala Lys Gln Leu Glu Asn Asp
Thr Arg 260 265 270 Ile Ile Glu Val Leu Cys Lys Glu His Glu Cys Asn
Ile Asp Glu Val 275 280 285 Lys Asn Val Tyr Phe Lys Asn Phe Ile Pro
Phe Met Asn Ser Leu Gly 290 295 300 Leu Val Thr Ser Asn Gly Leu Pro
Glu Val Glu Asn Leu Ser Lys Arg 305 310 315 320 Tyr Glu Glu Ile Tyr
Leu Lys Asn Lys Asp Leu Asp Ala Arg Leu Phe 325 330 335 Leu Asp His
Asp Lys Thr Leu Gln Thr Asp Ser Ile Asp Ser Phe Glu 340 345 350 Thr
Gln Arg Thr Pro Arg Lys Ser Asn Leu Asp Glu Glu Val Asn Val 355 360
365 Ile Pro Pro His Thr Pro Val Arg Thr Val Met Asn Thr Ile Gln Gln
370 375 380 Leu Met Met Ile Leu Asn Ser Ala Ser Asp Gln Pro Ser Glu
Asn Leu 385 390 395 400 Ile Ser Tyr Phe Asn Asn Cys Thr Val Asn Pro
Lys Glu Ser Ile Leu 405 410 415 Lys Arg Val Lys Asp Ile Gly Tyr Ile
Phe Lys Glu Lys Phe Ala Lys 420 425 430 Ala Val Gly Gln Gly Cys Val
Glu Ile Gly Ser Gln Arg Tyr Lys Leu 435 440 445 Gly Val Arg Leu Tyr
Tyr Arg Val Met Glu Ser Met Leu Lys Ser Glu 450 455 460 Glu Glu Arg
Leu Ser Ile Gln Asn Phe Ser Lys Leu Leu Asn Asp Asn 465 470 475 480
Ile Phe His Met Ser Leu Leu Ala Cys Ala Leu Glu Val Val Met Ala 485
490 495 Thr Tyr Ser Arg Ser Thr Ser Gln Asn Leu Asp Ser Gly Thr Asp
Leu 500 505 510 Ser Phe Pro Trp Ile Leu Asn Val Leu Asn Leu Lys Ala
Phe Asp Phe 515 520 525 Tyr Lys Val Ile Glu Ser Phe Ile Lys Ala Glu
Gly Asn Leu Thr Arg 530 535 540 Glu Met Ile Lys His Leu Glu Arg Cys
Glu His Arg Ile Met Glu Ser 545 550 555 560 Leu Ala Trp Leu Ser Asp
Ser Pro Leu Phe Asp Leu Ile Lys Gln Ser 565 570 575 Lys Asp Arg Glu
Gly Pro Thr Asp His Leu Glu Ser Ala Cys Pro Leu 580 585 590 Asn Leu
Pro Leu Gln Asn Asn His Thr Ala Ala Asp Met Tyr Leu Ser 595 600 605
Pro Val Arg Ser Pro Lys Lys Lys Gly Ser Thr Thr Arg Val Asn Ser 610
615 620 Thr Ala Asn Ala Glu Thr Gln Ala Thr Ser Ala Phe Gln Thr Gln
Lys 625 630 635 640 Pro Leu Lys Ser Thr Ser Leu Ser Leu Phe Tyr Lys
Lys Val Tyr Arg 645 650 655 Leu Ala Tyr Leu Arg Leu Asn Thr Leu Cys
Glu Arg Leu Leu Ser Glu 660 665 670 His Pro Glu Leu Glu His Ile Ile
Trp Thr Leu Phe Gln His Thr Leu 675 680 685 Gln Asn Glu Tyr Glu Leu
Met Arg Asp Arg His Leu Asp Gln Ile Met 690 695 700 Met Cys Ser Met
Tyr Gly Ile Cys Lys Val Lys Asn Ile Asp Leu Lys 705 710 715 720 Phe
Lys Ile Ile Val Thr Ala Tyr Lys Asp Leu Pro His Ala Val Gln 725 730
735 Glu Thr Phe Lys Arg Val Leu Ile Lys Glu Glu Glu Tyr Asp Ser Ile
740 745 750 Ile Val Phe Tyr Asn Ser Val Phe Met Gln Arg Leu Lys Thr
Asn Ile 755 760 765 Leu Gln Tyr Ala Ser Thr Arg Pro Pro Thr Leu Ser
Pro Ile Pro His 770 775 780 Ile Pro Arg Ser Pro Tyr Lys Phe Pro Ser
Ser Pro Leu Arg Ile Pro 785 790 795 800 Gly Gly Asn Ile Tyr Ile Ser
Pro Leu Lys Ser Pro Tyr Lys Ile Ser 805 810 815 Glu Gly Leu Pro Thr
Pro Thr Lys Met Thr Pro Arg Ser Arg Ile Leu 820 825 830 Val Ser Ile
Gly Glu Ser Phe Gly Thr Ser Glu Lys Phe Gln Lys Ile 835 840 845 Asn
Gln Met Val Cys Asn Ser Asp Arg Val Leu Lys Arg Ser Ala Glu 850 855
860 Gly Ser Asn Pro Pro Lys Pro Leu Lys Lys Leu Arg Phe Asp Ile Glu
865 870 875 880 Gly Ser Asp Glu Ala Asp Gly Ser Lys His Leu Pro Gly
Glu Ser Lys 885 890 895 Phe Gln Gln Lys Leu Ala Glu Met Thr Ser Thr
Arg Thr Arg Met Gln 900 905 910 Lys Gln Lys Met Asn Asp Ser Met Asp
Thr Ser Asn Lys Glu Glu Lys 915 920 925 9 30 DNA Artificial
Sequence Description of Artificial Sequence Oligonucleotide 9
ttctgtagtt taattttctg aacctttggc 30 10 27 DNA Artificial Sequence
Description of Artificial Sequence Oligonucleotide 10 tcagccgaag
agcttcagga agcaggg 27 11 32 PRT Homo sapien VARIANT (2)..(3)
VARIANT (6)..(13) VARIANT (15) VARIANT (17)..(18) VARIANT
(20)..(21) VARIANT (23)..(28) VARIANT (30)..(31) 11 Cys Xaa Xaa Cys
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa His 1 5 10 15 Xaa Xaa
Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys 20 25 30 12
50 PRT Homo sapien VARIANT (2)..(3) VARIANT (5)..(20) VARIANT
(22)..(23) VARIANT (25)..(26) VARIANT (28)..(29) VARIANT (31)..(46)
VARIANT (48)..(49) 12 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa His Xaa Xaa Cys Xaa
Xaa Cys Xaa Xaa Cys Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 35 40 45 Xaa Cys 50
* * * * *