AU2004203835B2 - Androgen receptor coactivators - Google Patents

Description

I

S&FRef: 538800D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT Name and Address of Applicant Actual Inventor(s): Address for Service: Invention Title: University of Rochester, of Office of Technology Transfer, 518 Hylan Building, Rochester, New York, 14627-0140, United States of America Chawnshang Chang Spruson Ferguson St Martins Tower Level 31 Market Street Sydney NSW 2000 (CCN 3710000177) Androgen receptor coactivators The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845c ANDROGEN RECEPTOR COACTIVATORS BACKGROUND OF THE INVENTION 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.

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 the glucocorticoid receptor and the progesterone receptor (PR) all -1recognize 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.

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 3 estradiol (E2) or hydroxyflutamide (HF) However, most prostate cancers treated with androgen ablation and anti-androgens progress from an androgendependant to an androgen-independent state, causing a high incidence of relapse within 18 months (C-aWf'rd'd;_'r. J.

Urolocy 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.

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.

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 -2- NOV.2007 17:37 SPRUSON FERGUSON 92615486 O. 4360 P. 7 3 0 o efficacy of drugs in the treatment of prostate cancers.

O Brief Summary of the invention Z In a first embodiment of the invention there is provided an isolated polynucleotide comprising the sequence set forth in SEQ ID NO:3, In a second embodiment of the invention there is provided an isolated polynucleotide comprising a promoter capable of causing expression of a protein 00 coding region in a cell, the promoter operably connected to a protein coding region of M an ARA55 polypeptide set forth in SEQ ID NO:4.

In a third embodiment of the invention there is provided a eukaryotic host cell comprising the isolated polynucleotide of the second embodiment.

1N In a fourth embodiment of the invention there is provided an isolated polynucleotide encoding the ARA55 polypeptide set forth in SEQ ID NO: 4.

In a fifth embodiment of the invention there is provided a genetic construct is comprising a nucleic acid sequence encoding the ARA55 polypeptide of SEQ ID NO: 4 operably linked to a heterologous promoter.

In a sixth embodiment of the invention there is provided a method for testing the androgenic or antiandrogenic effect of a chemical compound comprising the steps of: 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 the host cell also producing human androgen receptor protein; exposing the cell to the chemical compound; and measuring the level of transcriptional activity caused by the androgen receptor.

In a seventh embodiment of the invention there is provided a method for testing the androgenic or antiandrogenic effect of a chemical compound comprising the steps of: transfecting a host cell with at least one genetic construct capable of producing in the host cell human androgen receptor protein and a polypeptide of exposing the cell to the chemical compound; and measuring the interaction between AR and an AR co-activator.

In an eighth embodiment of the invention there is provided an isolated s3 polynucleotide encoding an ARA55 polypeptide comprising amino acids 251-444 of 1023061-1 COMS ID No: ARCS-168793 Received by IP Australia: Time 17:44 Date 2007-11-15 NOV.2007 17:37 SPRUSON FERGUSON 92615486 10. 4360 P. 8- 3a o SEQ ID NO: 4 and a LIM motif, wherein the ARA55 polypeptide enhances the transcription activity of an Androgen Receptor.

o In a ninth embodiment of the invention there is provided an isolated z polynucleotide encoding an ARA55 polypeptide comprising a sequence corresponding s to amino acids 251-444 of SEQ ID NO: 4 and a protein motif called a LIM motif, wherein the ARA55 polypeptide is capable of enhancing the transcription activity of a Smutant Androgen Receptor, causing an enhancement of the transcription activity of en O the mutant Androgen Receptor.

en O In a tenth embodiment of the invention there is provided an isolated N io polynucleotide encoding an ARA55 polypeptide comprising amino acids 251-444 of SSEQ IM NO: 4, wherein the ARA55 polypeptide inhibits the transcription activity of an Androgen Receptor.

In an eleventh embodiment of the invention there is provided a method of screening molecules for the ability to modulate Androgen Receptor or Progesterone is Receptor activity comprising the steps of: providing a genetic construct to a eukaryotic organism comprising a promoter functional in a eukaryotic cell operably connected to a polynucleotide comprising a sequence that encodes an ARA55 polypeptide; cotransformnning a suitable eukaryotic cell with the construct of step a, and a construct comprising at least a portion of aa expressible androgen receptor sequence; culturing the cells in the presence of a candidate molecule; and assaying the transcriptional activity induced by the androgen receptor gene; wherein an increase or decrease in Androgen Receptor activity relative to ARA55 and AR or Progesterone Receptor activity relative to ARA55 and PR without the molecule is modulation.

In a twelfth embodiment of the invention there is provided a method of screening for molecules that modulate Androgen Receptor or Progesterone Receptor activity comprising the steps of: incubating a compound with a cell, wherein the cell comprises and assaying for Androgen Receptor or Progesterone Receptor activity; wherein an increase or decrease in Androgen Receptor activity relative to ARA55 and AR or Progesterone Receptor activity relative to ARA55 and PR without the 3s compound is modulation.

1023061-1 COMS ID No: ARCS-168793 Received by IP Australia: Time 17:44 Date 2007-11-15 NOV. 2007 17:38 SPRUSON FERGUSON 92615486 NO.4360 P. 9 3b 0 o In a thirteenth embodiment of the invention there is provided a method of screening for molecules that modulate mutant Androgen Receptor activity comprising O the steps of: incubating a compound with a cell, wherein the cell comprises ARA55, and assaying for mutant Androgen Receptor activity; wherein an increase Sor decrease in mutant Androgen Receptor activity relative to ARA55 and AR without 00 the compound is modulation.

o There is also described herein an isolated polynucleotide that encodes a coactivator for human androgen receptor, the polynucleotide comprising a sequence that encodes a polypeptide selected from the group consisting of an ARA54 polypeptide, C, an ARA55 polypeptide, an ARA24 polypeptide, and an Rb polypeptide.

Also described herein 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.

There is also described herein 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: 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; cotransforming a suitable eukaryotic cell with the construct of step and a construct comprising at least a portion of an expressible androgen receptor sequence; culturing the cells in the presence of a candidate pharmaceutical molecule; and assaying the transcriptional activity induced by the androgen receptor.

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.

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.

1023061-1.

COMS ID No: ARCS-168793 Received by IP Australia: Time 17:44 Date 2007-11-15 NOV. 2007 17:38 SPRUSON FERGUSON 92615486 NO.4360 P. 3c 0 SOther objects, features, and advantages of the present invention will become apparent upon reading the specification and claims.

The present invention aims to achieve at least one of the stated objects.

Detailed Description of the Invention Transactivation of genes by the androgen receptor is a complicated system that involves many different coactivators, It is not currently known just how many 00 factors c 0 0 1023061-1 COMS ID No: ARCS-168793 Received by IP Australia: Time 17:44 Date 2007-11-15 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.

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.

Another aspect of the present invention is a genetic construct comprising a promoter functional in a prokaryotic or eukaryptic 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.

The present invention includes a method for screening [R:\LIBFF]06185spcc.doc:GCC 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: 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; 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; .cu.

1 turing the cells in the presence of a candidate pharmaceutical molecule; and assaying the transcriptional activity induced by the androgen receptor gene.

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.

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.

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 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:l-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.

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--demanostrated that reporter gene activity was enhanced in an androgendependent 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.

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.

The C-terminal domain of ARA54 361-471 of SEQ ID NO:1) serves as a dominant negative inhibitor of ARmediated gene expression of target genes. Coexpression of exogenous full-length ARA54 can reduce this squelching effect in a dose-dependent manner.

ARA54 enhanced transactivation of wtAR in the presence of DHT (10-10 to 10 8

M)

by about 3-5 fold. However, transactivation ofwtAR was enhanced only marginally with E2 (109-10 7 M) or HF (107-10 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. (1998), "From estrogen to androgen receptor: a new pathway for sex hormones in prostate". Proc Natl Acad Sci USA 95(10):5527-32) was also examined. The mutant mARt877a, which is found in many prostate tumors, was activated by E2 (109-10- 7 M) and HF (10" 7 10" 5 and ARA54 could further enhance E2- or HF-mediated AR transactivation. In contrast, the mutant mARe708k, first identified in a yeast genetic screening (Wang, Ph.D. Thesis of University of Wisconsin-Madison (1997)), exhibited ligand specificity and response to ARE54 comparable to that ofwtAR.

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 overexpressing of the Cterminal domain of ARA54 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 contemplated that truncations of the native coding region can be made to express small polypeptides that will retain the same biological activity.

The polynucleotide sequence of ARA55 (SEQ ID NO:3) exhibits high homology to the C-terminus of mouse [R:\LIBZZ]06185.doc: KOB (hydrogen peroxide inducible clone) (Pugh, Curr. Opin.

Cell Biol. 8:303-311 (1996)), and like hic5, 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- 9 M DHT or T, but not 10-9M E2 or HF. In contrast, ARA55 can enhance transcriptional activation by mART887S in the presence of DHT, testosterone 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.

The invention is not limited to the particular 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.

The AR N-terminal domain comprises a polymorphic polyglutamine 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.

-8- 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.

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 (ARA24as) does reduce DHTinduced transcriptional activation.

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.

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 wildtype Rb gene into DU145 cells was shown to repress the malignant phenotype (Bookstein, et al. Proc. Natl. Acad.

Sci. USA 87:7762-7766 (1990)).

Rb functions in the control of cell proliferation and differentiation(Weinberg, 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.

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.

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.

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.

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.

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.

The following nonlimiting examples are intended to be purely illustrative.

Examples Plasmid construction A human prostate library in pACT2 yeast expression vector pACT2 (Hua SB, et al.

(1998) "Construction of a modular yeast two-hybrid cDNA library from human EST clones for the human genome protein linkage map", Gene. 215(1):143-52) consists of the GAL4 activation domain (GAL4AD, a.a. 768-881) fused with human prostate cDNA.

wtAR was constructed as described previously (Yeh and Chang, Proc. Natl.

Acad. Sci. USA 93:5517-5521, 1996).

pGALO-AR (wild-type) PGALO-AR (Hofbauer LC, et al. (2002) "Regulation of osteoprotegerin production by androgens and anti-androgens in human osteoblastic lineage cells". Eur J Endocrinol. 147(2):269-73). pGALO contains the GAL4 DNA binding domain (DBD).

For construction of pAS2-wtAR or -mAR, the C-terminal fragments (aa 595-918) from wtAR, mARt877s (Dr. S.P. Balk, [R:\LIBZZ]06185.doc:KOB Beth Israel Hospital, Boston, MA), or mARe708k 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).

pGAL4-VP16 was used to construct a fusion of pGAL4-VP16 contains the GAL4 DBD linked to the acidic activation domain of VP16.

pCMX-Gal-N-RB and pCMX-VP16-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.

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 n-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 (ARA24as) expression plasmid was constructed by inserting a 334-bp Bgl II fragment of ARA24/Ran, which spans 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-pgal are under the regulation of SV40 promoter and 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) pSG5-GAL4DBD-ARA24 was generated by inserting the coding sequence of Gal4DBD-ARA24 hybrid protein into -12vector. pVP16-ARN-Ql, pVP16-ARN-Q25, pVP16-ARN-Q25, pVP16pVP16-ARN-Q49 were generated by inserting each poly-Q AR N-terminal domain 34-555) into pVPl6 vector (Clontech) to be expressed as a VP16AD hybrid protein.

GALOAR plasmid, which contains GAL4DBD fused to E region of human AR, was a gift from Dr. D. Chen. The reporter plasmid (Clontech) contains five GAL4 binding sites upstream of the Elb TATA box, linked to the CAT gene.

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.

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.

Screening of prostate cDNA library by a yeast two-hybrid system for ARAs associated with the ligand binding domain To identify ARA coactivators interact with the LBD, a pACT2-prostate cDNA library was cotransformed into Y190 yeast cells with a plasmid of pAS2mAR(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 (25mM) and DHT (100nM) lacking histidine, leucine and tryptophan (-3SD plates).

Colonies were also filter-assayed for P-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.

-13- 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 3aminotriazole (40 mM).

Amplification and characterization of ARA clones Full length DNA sequences comprising two coactivators, designated ARA54 (SEQ ID NO:l)and ARA55 (SEQ ID NO:3), that were found to interact with mARt877s were isolated by PCR using Marathon cDNA Amplification Kit(Clontech) according to the manufacturer's protocol.

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 0 C for 1 min, 5 cycles of 94 0 C for 5 sec-72 0

C

for 3 min, 5 cycles of 94 0 C for 5 sec-70 0 C for 3 min, then 25 cycles of 94 0 C for 5 sec-68 0 C for 3 min. The PCR product was subcloned into pT7-Blue vector (Novagen) and sequenced.

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.

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 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 2 CXg, 2 7

CXHX

2

CXCX

6 1 7

CX

2 C (SEQ ID NO: 11) -14a 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 coiledcoil 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.

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 2 CX1, 23

HX

2

CX

2

CX

2 CXi6.2,CX 2 (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.

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 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. 92:3328-333.2, 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 and 3 '-untranslated regions, respectively.

Northern Blotting The total RNA 2 5kg) 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 32 P-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. P-actin was used to monitor the amount of total RNA in each lane.

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.

Co-immunoprecipitation of AR and ARAs Lysates from in-vitro translated full-length of AR and ARA54 were incubated with or without 10- 8 M DHT in the modified RIPA buffer (50mM Tris-HCL pH 7.4, 150mM NaC1, EDTA, 0.1% NP40, 1mM PMSF, aprotinin, leupeptin, pepstatin, 0.25% Na-deoxycholate, 0.25% gelatin) and rocked at 4 0 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 0 C for additional 2 hr. The conjugated beads were washed 4 times with RIPA buffer, boiled in SDS sample buffer and analyzed -16by 8% SDS/PAGE and visualized by STORM 840 (Molecular Dynamics).

ARA54 and AR were found in a complex when immunoprecipitated in the presence of 10- 8 M DHT, but not in the absence of DHT. This result suggests that ARA54 interacts with AR in an androgen-dependent manner.

Interaction between recombinant full length human AR and ARA24/Ran proteins further examined by coimmunoprecipitation, 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.

AR pull-down assay using GST-Rb Full-length Rb fused to glutathione-S-transferase (ST-Rbl.

92 was expressed and purified from E. coli. strain Bl21pLys as described recently (Zarkowska Mittnacht, J.

Biol. Chem, 272:12738-12746, 1997). Approximately 2 ig of His-tag column purified baculovirus AR was mixed with GSTloaded glutathione-Sepharose beads in 1 ml of NET-N (20 mM Tris-HCL(pH 8.0, 100 mM NaC1, 1 mM EDTA, Noniodet and incubated with gentle rocking for 3 hr at 4 0

C.

Following low-speed centrifugation to pellet the beads, the clarified supernatant was mixed with GST-Rbloaded glutathione-Sepharose beads in the presence or absence of 10 mM DHT and incubated for an additional 3 hr with gentle rocking at 4 0 C. The pelleted beads were washed times with NET-N, mixed with SDS-sample buffer, boiled, and the proteins separated by electrophoresis on a polyacrylamide gel. A Western blot of the gel was incubated with anti-AR polyclonal antibody NH27 and developed with alkaline phosphatase-conjugated secondary antibodies.

AR was coprecipitated with GST-Rb, but not GST alone, indicating that AR and Rb are associated in a complex together.

-17- Transfection Studies 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 (25U/ml), streptomycin (25,g/ml), and 5% fetal calf serum (FCS). One hour before transfection, the medium was changed to DMEM with 5% charcoal-stripped FCS. Phenol redfree and serum-free media were used on the experiments employing E2 or TGF(, respectively. A -galactosidase expression plasmid, pCMV-3-gal, was used as an internal control for transfection efficiency.

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 P-galactosidase as an internal control.

Chloramphenicol acetyltransferase (CAT) activity was visualized by PhosphorImager (Molecular Dynamics) and quantitated by ImageQuant software (Molecular Dynamics).

Mammalian Two-Hybrid Assay 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 1-147) was fused to under the control of an SV40 promoter, and named pGALO.

The hinge and LBD of wtAR were then inserted into pGALO.

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).

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 -18- ARA54 in the presence of 10- M DHT significantly induced CAT activity.

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 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.

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.

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.

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- 10 to 10-8 coexpression of ARA54 enhanced transactivation by another 3-5 fold. However, wtAR responded only marginally to E2 (10-9-10- 7 M) or HF -19- (10-7-10- 5 M) in the presence or absence of ARA54. As expected, the positive control, is able to enhance the AR transcriptional activity in the presence of 10-9-10 7

M

E2 and 10-7-10 5 HF, that matches well with previous reports (Yeh et al. (1998), "From estrogen to androgen receptor: a new pathway for sex hormones in prostate". Proc Natl Acad Sci USA 95(10):5527-32, and Miyamoto et al. (1998) "Promotion of agonist activity of antiandrogens by the androgen receptor co-activator, ARA70, in human prostate cancer DU145 cells" Proc Natl Acad Sci USA 95(13):7379-84).

The AR mutants Art877a, which is found in many prostate tumors, and Are708k, found in a yeast genetic screening and a patient with partial androgen insensitivity, exhibited differential specificity for ligands. In the absence of ARA54, Art877a responded to E2 (10-9-10- 7 M) and HF (10-7-10 5 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 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 or AR might be critical for the interaction of the antiandrogen-ARe708k-ARA54 complex, and that both AR structure and coactivators may play a role in determining ligand specificity.

CAT activity in DU145 cells cotransfected with a plasmid encoding the hormone binding domain of wtAR fused to the GAL4 DBD (GALOAR) 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 GALOAR in the presence of 10 nM DHT, but not induced by E2 or HF. Combination of GALO empty vector and VP16-ARA55 did not show any CAT activity. Combination of GALOAR and VP16 vector showed negligible CAT activity. These results indicate that interacts with AR in an androgen-dependent manner.

Transient transfection assays were conducted to investigate the role of ARA55 in the transactivation activity to AR. DU145 cells were cotransfected with MMTV-CAT reporter, increasing amounts of ARA55 and wtAR under [R:\LIBZZ]06185.doc:KOB eukaryotic promoter control. Ligand-free AR has minimal MMTV-CAT reporter activity in the presence or absence of 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 2 44 4 251-444 of SEQ ID NO:4) did not enhance CAT activity.

Cotransfection of PC3 cells, which contain endogenous with ARA552.

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 is required for the proper or maximal AR transcriptional activity in PC3 cells, and that the Cterminus of ARA55 can serve as a dominant negative inhibitor.

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, PhD thesis of University of Wisconsin -Madison (1997)). The transcriptional activities of wtAR, mARt877s, and mARe708k are induced by DHT (10- 11 to 10-8 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- 7

M

for E2 and 10- 5 M for HF). Cotransfection of wtAR with -21at a 1:4.5 ratio, however, increases AR transcriptional activity at 10--10- 7 M for E2 or 10-6 to 10

G

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.

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 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 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 TGFi(Shibanuma et al., J. Biol. Chem. 269:26767-26774 (1994)), we were interested to see whether ARA55 could serve as a bridge between TGFP and AR steroid hormone system. Northern blot analysis indicated that TGFB treatment (5 ng/ml) could induce ARA55 mRNA by 2-fold in PC3 cells. In the same cells, TGF5 treatment increased AR transcriptional activity by 70%. This induction is weak relative to the affect achieved upon transfection of PC3 cells with exogenous ARA55 (70% vs. 4 fold). This may be related to the differences in the ratios of AR and The best ratio of AR:ARA55 for maximal AR transcriptional activity is 1:4.5. Whether other mechanisms may also be involve in this TGFB-induced AR transcriptional activity will be an interesting question to investigate. The unexpected discovery that TGFP 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 -22inducing the transcriptional activity of AR, the major promoter for the prostate tumor growth.

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.

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 ARA24as effect, indicating that increased DHT concentration can antagonize the negative effect of ARA24as.

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 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.

The regulation of AR transactivation by ARA24/Ran -23correlates 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. ARA24as 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 2, and 4 gg) of ARA24as 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, ARA24as showed a more significant decrease of AR transactivation. As AR poly-Q length increased, the ARA24as 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.

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 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 Nterminal 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 androgenindependent 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 twohybrid assay, data not shown). Second, unlike most ,identified steroid receptor associated proteins that bind to C-terminal domain of steroid receptor, Rb binds to Nterminal domain of AR. Third, no interaction occurred -24between Rb and ARA70, two AR associated proteins in DU145 cells.

DU145 cells containing mutated Rb (Singh et al., Nature 374: 562-565 (1995)) were cultured with charcoalstripped 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 the first identified AR coactivator, can further enhance in DU145 cells transcriptional activity from 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 AM 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 and cotransfection of Rb and ARA 70 with AR can further enhance the AR transcriptional activity to 36fold (E2 or 12-fold We then extended these findings to two different AR mutants: mARt877s from a prostate cancer patient and mARe708k from a partial-androgeninsensitive 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 AM 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 iM 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 gM 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.

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 and progesterone receptor ERE-CAT for estrogen receptor Although Rb and 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 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 androgenindependent, the AR-Rb (and AR-ARA70) complexes require a ligand for the transactivation activity.

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, 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 -26several protein kinases. Whether all of these proteins actually complex, and are regulated by Rb, in cells remains to be seen.

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.

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 androgendependent 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.

-27- All publications cited in this application are incorporated by reference.

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.

-28-

Claims (19)

1. An isolated polynucleotide comprising the sequence set forth in SEQ O ID NO:3.

2. An isolated polynucleotide comprising a promoter capable of causing expression of a protein coding region in a cell, the promoter operably connected to a protein coding region of an ARA55 polypeptide set forth in SEQ ID NO:4.

3. The isolated polynucleotide of claim 2 wherein the protein coding 00 region comprises a sequence set forth in SEQ ID NO: 3. o

4. A eukaryotic host cell comprising the isolated polynucleotide of claim 2 or 3. 0

5. A eukaryotic host cell according to claim 4, substantially as N hereinbefore described with reference to any one of the Examples.

6. An isolated polynucleotide encoding the ARA55 polypeptide set forth in SEQ ID NO: 4.

7. A genetic construct comprising a nucleic acid sequence encoding the polypeptide of SEQ ID NO: 4 operably linked to a heterologous promoter,

8. A genetic construct according to claim 7, substantially as hereinbefore described with reference to any one of the Examples.

9. A method for testing the androgenic or antiandrogenic effect of a chemical compound comprising the steps of: transfecting a host cell with at least one genetic construct capable of producing in the host cell a polypeptide of ARA55, the host cell also producing human androgen receptor protein; exposing the cell to the chemical compound; and measuring the level of transcriptional activity caused by the androgen receptor. The method of claim 9, wherein the host cell is a prostate cell.

11. The method of claim 9 or 10, 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.

12. The method of any one of claims 9 to 11, wherein the genetic construct comprises a nucleic acid sequence of SEQ ID NO:3.

13. The method of any one of claims 9 to 12, 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, COMS ID No: ARCS-168793 Received by IP Australia: Time 17:44 Date 2007-11-15 NOV. 2007 17:39 SPRUSON FERGUSON 92615486 O. 4360 P. 12 0 O

14. The method of claim 13, wherein the reporter gene is selected from the group consisting of a chloramphenicol, acetyltransferase gene, and a luciferase O gene. A method for testing the androgenic or antiandrogenic effect of a s chemical compound comprising the steps of: 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 00 from the group consisting of o exposing the cell to the chemical compound; and measuring the interaction between AR and an AR co-activator. S16. The method of claim 15, wherein the co-activator is N1

17. An isolated polynucleotide encoding an ARA55 polypeptide comprising amino acids 251-444 of SEQ ID NO; 4 and a LIM motif, wherein the polypeptide enhances the transcription activity of an Androgen Receptor. is 18. The isolated polynucleotide of claim 17, wherein the polypeptide enhances the transcription activity of an Androgen Receptor at least 4 fold.

19. The isolated polynucleotide of claim 17 or 18, wherein the polypeptide enhances the transcription activity of an Androgen Receptor at least 8 zo fold. The isolated polynucleotide of any one of claims 17 to 19, wherein the enhancement comprises an interaction between 17 estradiol (E2) and the mutant Androgen Receptor.

21. The isolated polynucleotide of any one of claims 17 to 19, wherein the enhancement comprises an interaction between hydroxyflutamide (HF) and the mutant Androgen Receptor.

22. The isolated polynucleotide of any one of claims 17 to 19, wherein the enhancement comprises an interaction between dihydrotestosterone (DHT) and the mutant Androgen Receptor,

23. An isolated polynucleotide encoding an ARA55 polypeptide comprising a sequence corresponding to amino acids 251-444 of SEQ ID NO: 4 and a protein motif called a LIM motif, wherein the ARA55 polypeptide is capable of enhancing the transcription activity of a mutant Androgen Receptor, causing an enhancement of the transcription activity of the mutant Androgen Receptor.

1022061-1 COMS ID No: ARCS-168793 Received by IP Australia: Time 17:44 Date 2007-11-15 OV. 2007 17:39 SPRUSON FERGUSON 92615486 NO. 4360-P. 31 0 24. The isolated polynucleotide of claim 23, wherein the enhancement c, comprises an interaction between 17 0 estradiol (E2) and the mutant Androgen o0 Receptor, The isolated polynucleotide of claim 23, wherein the enhancement comprises an interaction between hydroxyflutamide (HF) and the mutant Androgen Receptor. S26. The isolated polynucleotide of claim 23, wherein the enhancement 0n 00 comprises an interaction between dihydrotestosterone (DHT) and the mutant M Androgen Receptor. 1 27. The isolated polynucleotide of claim 23, wherein the enhancement g comprises an interaction between testosterone and the mutant Androgen Receptor. c, 28. An isolated polynucleotide encoding an ARA55 polypeptide comprising amino acids 251-444 of SEQ ID NO: 4, wherein the ARA55 polypeptide inhibits the transcription activity of an Androgen Receptor. I 29. A method of screening molecules for the ability to modulate Androgen Receptor or Progesterone Receptor activity comprising the steps of: providing a genetic construct to a eukaryotic organism comprising a promoter functional in a eukaryotic cell operably connected to a polynucleotide comprising a sequence that encodes an ARA55 polypeptide; cotransforming a suitable eukaryotic cell with the construct of step and a construct comprising at least a portion of an expressible androgen receptor sequence; culturing the cells in the presence of a candidate molecule; and assaying the transcriptional activity induced by the androgen receptor gene; wherein an increase or decrease in Androgen Receptor activity relative to and AR or Progesterone Receptor activity relative to ARA55 and PR without the molecule is modulation. A method of screening for molecules that modulate Androgen Receptor or Progesterone Receptor activity comprising the steps of: incubating a compound with a cell, wherein the cell comprises and assaying for Androgen Receptor or Progesterone Receptor activity; wherein an increase or decrease in Androgen Receptor activity relative to ARA55 and AR or Progesterone Receptor activity relative to ARA55 and PR without the compound is modulation. 1023 6l. I COMS ID No: ARCS-168793 Received by IP Australia: Time 17:44 Date 2007-11-15 15-NOV.2007 17:39 15.~OV 267 1:39SRUSQN F7RUO 92615486 O.46 P.1 NO. 4360 P. 14 32 o31. The method of claim 30, wherein the cell further comprises DBT. 32. The method of claim 30 or 31, wherein the assay is a two-hybrid o system with a reporter gene. Z33. The method of claim 32, wherein the reporter gene is a CAT gene. 34. The method of any one of claims 30 to 33 further comprising identifying compounds that enhance Androgen Receptor or Progesterone Receptor en activity. 00 35. The method of any one of claims 30 to 34, further comprising o identifying compounds that inhibit Androgen Receptor or Progesterone Receptor z0 activity. 36. The method of claim 34 or 35 further comprising synthesizing the Ci identified compound. 37. A method of screening for molecules that modulate mutant Androgen Receptor activity comprising the steps of: incubating a compound with a cell, wherein the cell comprises ARASS, and assaying for mutant Androgen Receptor activity; wherein an increase or decrease in mutant Androgen Receptor activity relative to ARA55 and AR without the compound is modulation. 38. The method of claim 37, wherein the mutant Androgen Receptor is mArt8l7s. 39. The method of claim 37, wherein the mutant Andirogen Receptor is mAre708k, The method of any one of claims 37 to 39, wherein the cell further comprises F-2. 41. The method of any one of claims 37 to 40, wherein the cell fixrther comprises HP. Dated 15 November, 2007 University of Rochester Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON 1021061.1 COMS ID No: ARCS-168793 Received by IP Australia: Time 17:44 Date 2007-11-15 SEQUENCE LISTING <110> Chang, Chawnshang <120> Androgen Receptor Coactivators <130> 920920.90011 <140> <141> <150> US 60/100,243 <151> 1998-09-14 <160> 12 <170> PatentIn Ver. <210> 1 <211> 1721 <212> DNA <213> Homo sapien <220> <221> CDS <222> (40) (1464) <220> <221> misc feature <222> (1120) (1452) <223> Coding sequence and polypeptide region for the C-terminal domain. <220> <221> miscfeature <222> (697) (834) <223> Coding sequence and polypeptide region which may form a cystein-rich RING finger motif. <220> <221> miscfeature <222> (964) .(1089) <223> Coding sequence and polypeptide region for a cystein-rich B box like structure. <400> J. gqtctctggt ctcccctctc tgagcactct gaggtcctt atg tcg tca gaa gat Met Ser Ser Glu Asp cga gaa gct Arg Glu Ala gga gat gaa Gly Asp Glu atc tat ttg Ile Tyr Leu cag gag Gln Glu gat gaa ttg ctg Asp Giu Leu Leu ctg gca agt att Leu Ala Ser Ile tac gat Tyr Asp aga aaa gca gag Arg Lys Ala Giu gtc caa ggt gga Val Gin Gly Gly gaa acc agg Giu Thr Arg agc ggc aat Ser Gly Asn gat ttg cca cag aat ttc aag ata. ttt Asp Leu Pro Gin Asn Phe Lys Ile Phe tca aat Ser Asn gag tgt ctc cag Glu Cys Leu Gin agt ggc ttt gaa. Ser Gly Phe.Giu acc att tgc ttt Thr Ile Cys Phe cct cca ctt gtg Pro Pro Leu Val Ctg Leu aac ttt gaa ctg Asn Phe Giu Leu cca. gat tat cca Pro Asp Tyr Pro tct tcc cca. oct Ser Ser Pro Pro ttc aca ctt agt Phe Thr Leu Ser ggc Gly aaa, tgg ctg tca. Lys Trp Leu Ser oca. act Pro Thr 100 cag cta tct Gin Leu Ser cgt ggc agc Arg Giy Ser 120 cta tgc aag cac Leu Cys Lys His gac aac cta tgg Asp Asn Leu Trp, gaa. gaa. cac Giu Glu His 115 gtg gtC ctg ttt Val Val Leu Phe tgg atg caa. ttt ctt aag gaa gag Trp Met Gin Phe Leu Lys Giu Glu 130 acc cta gca tao ttg aat att Thr Leu Ala Tyr Leu Asn Ile 135 140 gtc tot cot ttt Vai Ser Pro Phe gag Giu 145 ctc aag att ggt Leu Lys Ile Gly cag aaa. aaa gtg Gin Lys Lys Val cag Gin 155 aga agg aca gct Arg Arg Thr Ala caa. Gin 160 got tct ccc aac Ala Ser Pro Asn gag cta gat ttt Giu Leu Asp Phe gga. got got gga Gly Ala Ala Gly tot Ser 175 gat gta gac caa Asp Val Asp Gin gag gaa Giu Giu IS0 att gtg gat gag aga gca gtg cag gat gtg gaa tca ctg tca aat ctg Ile Val Asp Giu Arg Ala Val Gin Asp Val Giu Ser Leu Ser Asn Leu 185 195 atc Ile aa t Asn agt Ser 230 tgt Cys tgc Cys cag Gin ctt Leu ccc Pro 310 atg Met ttg Leu atg Met cag Gin agt Ser 215 gaa Giu ctg Leu ctc Leu gtc Val ctc Leu 295 cgg Arg ggt Gly acc Thr gac A~sp gaa Giu 200 aaa Lys tgc Cys aag Lys aac Asn aaa Lys 280 ctc Leu ccg Pro atc Ile tac T~yr tta Leu 360 atc Ile t tg Leu atg Met gac Asp tgc Cys 265 gag Giu cag Gin tgC Cys tgc Cys cat His 345 cga Arg t tg Leu ttc Phe tac Tyr tac 250 cca. Pro tta Leu tcC Ser tgC Cys tcc Ser 330 ggg Giy aat Asn gac Asp ctg Leu ttc Phe 235 ttt Phe gaa Giu gtg Val tcC Ser cag Gin 315 agc Ser gtc V7al gaa Glu ttt Phe tgc Cys 220 t tg Leu gaa Giu cca Pro gaa Giu t tg Leu 300 ctg Leu tgc Cys tcc Ser tac Tyr ggt Gly 380 gat caa gct Asp Gin Ala 205 agt atc tgt Ser Ile Cys gag tgc agg Giu Cys Arg atc cag atc lie Gin Ile 255 aag tgc cct Lys Cys Pro 270 gca gag tta Ala Glu Leu 285 gac ctg atg Asp Leu Met cct gtg atg Pro Val Met aat ttt gcc Asn Phe Ala 335 cca tgt aag Pro Cys Lys 350 ctg caa gcg Leu Gin Ala 365 aag aga gtg Lys Arg Vai cag Gin ttC Phe cat His 240 aga Arg tcg Ser ttt Phe gca Ala cag Gin 320 ttc Phe gtg Vai gat Asp att Ile cag Gin tgt Cys 225 gtg Val gat Asp gtg, Vai gcc Ala gat Asp 305 gaa Giu tgt Cys act Thr gag Giu cag Gin 385 a ta Ile 210 gag Giu tac Tyr ggc Gly gcc Ala cgt Arg 290 gtg Val cct Pro act Thr gca Ala gct Ala 370 aag Lys aa a Lys aag Lys tgc Cys cag Gin act Thr 275 tat Tyr gtg Val 9gc Gly t tg Leu gag Giu 355 aat Asn gca Ala ttt Phe ggt Gly gcc Al a 245 caa Gin ggt Gly cgc Arg tgc Cys acc Thr 325 agg Arg tta Leu aga Arg gaa Glu 678 726 774 822 870 918 966 1014 1062 1110 1158 1206 ctt ttg gat caa agg tat Leu Leu Asp Gin Arg TIyr 375 gag atg gaa agt aag gag tgg cta gag aag aac tca aag agc tgc cca 1254 Glu Met Giu Ser Lys Glu Trp Leu Giu 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 at; aca 1302 Cys Cys Gly-Thr Pro Ile Giu 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 Gin Tyr Phe Cys Trp Ile Cys Met Gly Ser Leu 425 430 435 tct aga gca aac oct tac aaa cat ttc aat gao 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 got gtg gat gtt gac gac gat att tgg gaa 1446 Phe Asn Arg Leu Phe TPyr Ala Val Asp Val Asp Asp Asp Ile Trp Giu 455 460 465 gat gag gta gaa gac tag ttaactactg ctcaagatat ttaactactg 1494 Asp Glu Val Giu Asp 470 475 otoaagatat, ggaagtggat tgtttttoooc taatottocg toaagtaoac aaagtaactt 1554 tgogggatat ttagggtaot attoattoac tottcctgcg tagaagatat ggaagaacga 1614 ggtttatatt ttcatgtggt aotaotgaag aaggtgoatt gataoatttt taaatgtaag 1674 ttgagaaaaa tttataagoo aaaggttcag aaaattaaac taoagaa 1721 <210> 2 <211> 474 <212> PRT <213> Homo sapien <400> 2 Met Ser Ser Giu Asp Arg Glu Ala Gin Giu Asp Giu Leu Leu Ala Leu 1 5 10 Ala Ser Ile Tyr Asp Gly Asp Glu Phe Ar; Lys Ala Giu Ser Val Gin 25 Gly Gly Giu Thr Arg Ile Tyr Leu Asp Leu Pro Gin Asn Phe Lys Ile 40 Phe Val Ser Gly Asn Ser Asn Glu Cys Leu Gin Asn Ser Gly Phe Glu 55 Tyr Pro Trp Leu Phe Giu 145 Ala Val Ser Gin Cys 225 Val Asp Val Ala Asp 305 Glu Thr Asp Leu Trp Leu 130 Leu Ser Asp Leu Ile 210 Giu Tyr Gly Ala Arg 290 Val Pro Sle Tyr Ser Glu 115 Lys Lrs Pro Ser 195 Lys Lys Cys Gin Thr 27S Val Gly Cys Pro Pro 100 Giu Giu Ile Asn Glu 180 Asn Cys Leu Lys Val 260 Pro Asp Tlyr Cys Phe Ser Thr His Giu Gly Thr 165 Giu Leu Phe Gly Ala 245 Gin Gly Arg Cys Thr 325 Leu 70 S er Gin Arg Thr Ser 150 Glu Ile Ile Asn Ser 230 Cys Cys Gin Leu Pro 310 Met Pro Ser Leu Giy Leu 135 Gin Leu Val Gin Ser 215 Giu Leu Leu Val Leu 295 Arg Gly Pro Pro Ser Ser 120 Al a Lys Asp Asp Glu 200 Lys Cys Lys Asn Lys 280 Leu Pro Ile Leu Pro Ala 105 Val1 Tyr Lys Phe Giu 185 Ile Leu Met Asp Cys 265 Glu Gin Cys Cys Val Ser 90 Leu Val1 Leu Val Gly 170 Arg Leu Phe Tyr Tyr 250 Pro Leu Ser Cys Ser 330 Leu 75 Phe Cys Leu Asn Gin Gly Ala Asp Leu Phe 235 Phe Giu Vai Ser Gin 31S Ser Asn Thr Ly s Phe Ile 140 Arg Ala Val Phe Cys 220 Leu Giu Pro Glu Leu 300 Leu Cys Plhe Leu His Ala 125 Val Arg Ala Gin Asp 205 Ser Glu Ile Lys Al a 285 Asp Pro Asn Gl~u Ser Leu 110 Trp Ser Thr Gly Asp 190 Gin Ile Cys Gin Cys 270 Glu Leu Val Phe Leu Gly Asp Met Pro Ala Ser 175 Val Ala Cys Arg Ile 255 Pro Leu Met Met Ala 335 Pro Lys Asn Gin Phe Gin 160 Asp Giu Gin Phe His 240 Arg Ser Phe Ala Gin 320 Phe Cys Thr Leu Cys Arg LeU Thr Tyr His Gly Val Ser Pro Cys Lys Val 340 345 350 Thr Ala Giu Lys Leu Met Asp Leu Arg Asn Giu Tyr Leu Gin Ala Asp 355 360 365 Giu Ala Asn Lys Arg Leu Leu Asp Gin Arg Tyr Giy Lys Arg Val Ile 370 375 380 Gin Lys Ala Leu Giu Giu Met Giu Ser Lys Giu Trp Leu Giu Lys Asn 385 390 395 400 Ser Lys Ser Cys Pro Cys Cys Gly Thr Pro Ile Giu Lys Leu Asp Gly 405 410 415 Cys Asn Lys Met Thr Cys Thr Gly Cys Met Gin 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 Giu Val Giu Asp 465 470 <210> 3 <211> 1335 <212> DNA <213> Homo sapien <220> <221> CDS <222> (1)..(1335) <220> <221> misc feature <222> (750)..(1332) <223> Coding sequence and polypeptide region for the C-terminal binding domain <220> <221> misc feature <222> (631)..(783) <223> Coding sequence and polypeptide region for a cystein rich LIM motif <220> <221> misc feature <222> (808)..(996) <223> Coding sequence and Polypeptide region for a cystein rich LIM motif <220> <221> misc feature <222> (985)..(1137) <223> Coding sequence and polypeptide region for a cystein rich LIM motif <220> <221> misc feature <222> (1162)..(1314) <223> Coding sequence and polypeptide, region for a cystein rich LIM motif <400> 3 atg cca agg tca ggg gct ccc aaa gag cgc. cct gcg gag cct. ctc acc Met Pro Arg Ser Gly Ala Pro Lys Glu Arg Pro Ala Glu Pro Leu Thr 1 C cct ccc cca Pro Pro Pro tca gga gcc Ser Gly Ala tat ggc cac cag Tyr Gly His Gin cca Pro aca ggg cag tct Thr Gly Gin Ser ggg gag tct Gly Giu Ser gta tgc aag Val Cys Lys tcg ggg gac: aag Ser Gly Asp Lys cac ctg tac agc His Leu Tyr Ser acg Thr cct cgg Pro Arg tcc cca aag cct Ser Pro Lys Pro gcc ccg gcc gcc Ala Pro Ala Ala Cca ttc tcc tct Pro Phe Ser Ser tcc Ser agc ggt gtc ttg Ser Gly Val Leu acc ggg ctc tgt Thr Gly Leu Cys cta gat cgg ttg Leu Asp Arg Leu cag gaa ctt aat Gin Glu Leu Asn cag ttc cca tct Gin Phe Pro Ser 100 act cag ttc aac Thr Gin Phe Asn atc Ile aca gat gaa atc Thr Asp Giu Ile atg tct Met Ser agc aag gtg gct Ser Lys Val Ala gga gag cag aag gag gac cag Gly Glu Gin Lys Giu Asp Gin 110 tct gaa gat aag aaa aga ccc Ser Glu Asp Lys Lys Arg Pro 115 agc ctc cct tcc agc Ser Leu Pro Ser Ser 120 ccg tct cct ggc *Pro Ser Pro Gly 125 ctc cca aag got Leu Pro Lys Ala 130 tot gcc acc tca goc act ctg Ser Ala Thr 135 gag ctg gat aga ctg Glu Leu Asp Arg Leu Ser Ala Thr Leu goc tca ctc cct Ala Ser Leu Pro ttc ogo gtt oaa Phe Arg Val Gin oat Ott oca goo His Leu Pro Ala ggg oca act oag Gly Pro Thr Gin cog gtg gtg ago Pro Val Val Ser aca aat gag ggo Thr Asn Glu Gly too ooa Ser Pro 175 too oca oca gag cog act gca aag Ser Pro Pro Glu Pro Thr Ala Lys ggc Gly 185 cgg Arg ago ota gao acc Ser Leu Asp Thr ggt gtt coo aco Gly Val Pro Thr 205 atg ctg ggg Met Leu Gly 190 cag goo aaa Gin Ala Lys otg otg oag Leu Leu Gin 1195 gao oto ago Asp Leu Ser ggc oto Gly Leu 210 tgt ggo too tgo Cys Gly Ser Cys aat Asn 215 aaa oct att gct. Lys Pro Ile Ala ggg Gly 220 oaa gtg gtg acg Gin Val Val Thr got Al a 225 ctg ggo ogc goc Leu Gly Arg Ala tgq Trp 230 cac 000 gag cao His Pro Giu His ttC Phe 235 gtt tgc gga ggo Val Cys Giy Gly too aco goo otg Ser Thr Ala Leu ggo ago ago tto Gly Ser Ser Phe gag aag gat gga Glu Lys Asn Gly goc coo Ala Pro 255 tto tgc coo Phe Cys Pro tgo aac cag Cys Asn Gin 275 gag Giu 260 tgc tao ttt gag Cys Tyr Phe Glu ogo Arg 265 tto tog ooa aga Phe Ser Pro Arg tgt ggo tto Cys Giy Phe 270 ggc aot oac Gly Thr His 0cc ato oga oac Pro Ile Arg His aag Lys 280 atg gtg aco goo Met Val Thr Ala tgg cao Trp His 290 cca gag oat ttc Pro Giu His Phe tgo gto agt tgo Cys Val Ser Cys ggg Gly 300 gag ccc ttc gga Glu Pro Phe Gly ga t Asp 305 gag ggt ttc cac Glu Gly Phe His gag Glu 310 ogc gag ggo cgc Arg Giu Gly Arg tac tgc ogc cgg Tyr Cys Arg Arg 912 960 1008 tto otg cag otg Phe Leu Gin Leu tto Phe 325 goo cog ogo tgc Ala Pro Arg Cys ggc tgc cag ggc Gly Cys Gin Gly coo ato Pro Ile 335 cig gat aac tac atc tcg gcg ctc agc ctg Ctc tgg cac ccg gac tgt 1056 Leu Asp Asri 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 Giu 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 Giu Gly Arg Pro Leu Cys Giu 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 Gin Giu Arg Ala Gly Lys 420 425 430 ccc tac tgc cag ccc tgc ttc ctg aag ctc ttc ggc tga. 1335 Pro Tyr Cys Gin Pro Cys Phe Leu Lys Leu Phe Gly 435 440 445 <210> 4 <211> 444 <212> PRT <213> Homo sapien <400> 4 Met Pro Arg Ser Gly Ala Pro Lys Giu Arg Pro Ala Giu Pro Leu Thr 1 5 10 is Pro Pro Pro Ser Tyr Gly His Gin Pro Thr Gly Gin Ser Gly Glu Ser 25 Ser Gly Ala Ser Giy Asp Lys Asp His Leu Tyr Ser Thr Val Cys Lys 40 Pro Arg Ser Pro Lys Pro Ala Ala Pro Ala Ala Pro Pro Phe Ser Ser s0 55 Ser Ser Giy Val Leu Giy Thr Gly Leu Cys Glu Leu Asp Arg Leu Leu 70 75 Gin Giu Leu Asn Ala Thr Gin Phe Asn Ile Thr Asp Giu Ile Met Ser 90 Gin Phe Pro Ser Ser Lys Val Ala Ser Gly Giu Gin Lys Glu Asp Gin 100 Ser Leu Met 2.45 Gly Ser Leu Gly Ala 225 Ser Phe Cys Trp Asp 305 Phe Leu Phe Giu Pro 130 Al a Pro Pro Leu Leu 210 Leu Thr Cys Asn Hi s 290 Glu Leu Asp Val1 *Asp 115 Lys Ser Thr Pro Gin 195 Cys Gly Ala Pro Gin 275 Pro Gly Gin Asn Cys 355 Lys Ala Leu Gi n Giu 180 Ser Gly Arg Leu Glu 260 Pro Giu Phe Leu Tyr 340 Arg Lys Ser Pro Pro 1.65 Pro Asp Ser Al a Gly 245 Cys Ile His His Phe 325 Ile Glu Arg Al a Asp 2.50 Pro Thr Leu Cys Trp 230 Gly Tyr Arg Phe Glu 310 Ala Ser Cys Pro Thr 2.35 Phe Val1 Al a Ser Asn 22.5 His Ser Phe His Cys 295 Arg Pro Al a Phe Ser 2.20 Ser Arg Val1 Lys Arg 200 Lys Pro Ser Giu Lys 280 Cys Giu Arg Leu Al a 360 105 Leu Al a Val1 Ser Gly 185 Arg Pro Giu Phe Arg 265 Met Val Gly Cys Ser 345 Pro 2.10 Pro Thr Gin Ser 2.70 Ser Gly Ile His Phe 250 Phe Val S er Arg Gin 330 Leu Phe Ser Leu Asn 155 Thr Leu V al Ala Phe 235 Giu Ser Thr Cys Pro 32.5 Gly Leu Ser Ser Giu 140 His Asn Asp Pro Gly 220 Val Lys Pro Ala Gly 300 Tyr Cys Trp Gly Pro 125 Leu Leu Glu Thr Thr 205 Gin Cys Asp Arg Leu 285 Glu Cys Gin His Gly 365 Ser Asp Pro Gly Met 190 Gin Val Gly Gly Cys 270 Gly Pro Arg Gly Pro 350 Ser Pro Arg Ala S er 175 Leu Ala Val Gly Ala 255 Gly Thr Phe Arg Pro 335 Asp Phe Gly Leu Ser 2.60 Pro Gly Lys Thr Cys 240 Pro Phe His Gly Asp 320 Ile Cys Phe Glu His Glu Gly Arg Pro Leu Cys Glu Asn His Phe His Ala ArgArg 370 375 380 Gly Ser Leu Cys Pro Thr Cys Gly Leu Pro Val Thr Gly Arg Cys VaJl 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 Gin Glu Arg Ala Gly Lys 420 425 430 Pro Tyr Cys Gin Pro Cys Phe Leu Lys Leu Phe Gly 435 440 <210> <211> 1566 <212> DNA <213> Homo sapien <220> <221> CDS <222> (25)..(675) <220> <221> 3'U'rR <222> (676)..(1566) <220> <221> 51UTR <222> <400> ggcgcttctg gaaggaacgc cgcg atg gct gcg cag gga, gag ccc cag gtc 51 Met Ala Ala Gin Gly Glu Pro Gin Val 1 S. cag ttc aaa ctt gta ttg gtt gqt gat ggt ggt act gga, aaa. acg acc 99 Gin Phe Lys Leu Val Leu Val Gly Asp Gly Gly Thr Gly Lys Thr Thr 15 20 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 35 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 50 *cct att aag ttc Pro Ile Lys Phe aat gta tgg gac aca gcc ggc cag gag aaa ttc ggt Asn Val Trp Asp Thr Ala Gly Gin Giu Lys Phe Gly gga G ly t tt Phe aga Arg aac Asn ttc Phe aac Asn gga Gly 170 gaa Giu gag Giu ct~g Leu ga t Asp gat Asp aaa Lys ca c His tac Tyr is s gac Asp gt t Val gtt Val aga Arg gta Val C tg Leu gtg Val Arg 140 aac Asn cct Pro gic Val gct Ala gat Asp aca Thr gta Val gat Asp 125 aag Lys ttt Phe aac Asn aig Met cag Gin ggc Giy tcg Ser cga Arg 110 att Ile aag Lys gaa Glu ttg Leu gac Asp 3.90 aca Thr tat Tyr aga Arg .95 gtg Val aag Lys aat Asn aag Lys gaa Giu 175 cca Pro act Thr tat atc caa Tyr Ile Gin gtt act tac Val Thr Tyr tgt gaa aac Cys Glu Asn gac agg aaa Asp Arg Lys 130 ctt cag tac Leu Gin Tyr 145 Ccc ttc ctc Pro Phe Leu ttt gtt gcc Phe Val Ala gct ttg gca Ala Leu Ala get ctc ccg Ala Leu Pro 210 gCC Ala aag Lys atc Ile 115 gtg Val tac Tyr tgg Trp atg Met gca Ala 195 gat Asp cag Gin aa t Asn 100 ccc Pro aag Lys gac Asp ctt Leu cet Pro iso cag Gin gag Glu Asp Asp Asp Leu 205 gaatgaagct agcggtgcag tgtgggatgc ttcattgttt tataagactg ttcccattcc ggagcccagc cgtgtgtgcc tgaaggagat ggacctgcat ctgcagtcac ttttctttag gtcagaagtc acctcattat gagtgggctt atttagctgt atcacaatat aatcagaata. tagttttata tatctagcta cggagtgaat t tggacgcag t cagtggtga aagttgtatt 12 ggcagctgtc ctgtgatgtc agcggaacat gtgctttatc gtggcagttt aaaaaataac ttgattcctt gagtttcata aatcttgttt gttactgtca tcaaatatct. aagcaagtga 735 795 855 915 975 1035 actcatccct tgtttataaa tgttaatatt tgaattgcct tccctatgtt tatttttgta cagtattcta tttggttaga cagtgtttgc tccaccttca aatctgtgac ctgtcagaat taatattttc ttttatggca aacatcagtg gatggcagga cactttftgt ttgaatgtta 'z210> 6 <211> 216 <212> PRT <213> Homio sapien tagcatttgg tgCttttatc catttgagcc agggttacat tat tggctag aaaaatgtgg aaagtaatca ggt tgggaat gatgcttagt aaaccactaa acttaatttg atgtcacaca ggtgtaaata gtagggtcac tttgtacata tgttttaatg tcttgctgtt gtgaagttga agtagggaag aaatctattg aactgatgat ttagtgcagt ctagggaagc tcaaatagat tagaacctca aaaaataatt tacgcaagcc ttttatgcca ggttaatttc gacaggtcag taagctaaag acttgctcaa attttaaggg aacaggatgg acaaattttg 1095 1155 1215, 1275 1335 1395 1455 1515 1566 <400> 6 Met Ala I Gly Asp Ala Gin Gly Gly Giy Giu Pro Gin Val 5 Thr Gly Lys Thr Thr 25 Lys Lys Tyr Val Ala Gin Phe Lys Leu 10 Phe Val Lys Arg Thr Leu Gly Val Pro Ile Lys Phe Val Leu Val His Leu.Thr Glu Val His Asn Val Ti-p Gly Glu Phe Pro Leu Val Giu Phe His Thr Gly so Asp Thr Ala Gly Gin Phe Gly Gly Ile Leu Asp Asp Gly Tyr Gin Ala Gin Ile Ile Met Val Thr Ser Arg Val Thr Tyr Lys Glu Asn Ile 115 Asn 100 Pro Pro Asn Trp Asp Leu Val Ile Val Leu Asn Lys Val Arg Val Cys 110 Ile Lys Asp Lys Asn Leu Arg Lys Val Lys Ala Lys Ser Ile Val Phe His Gin 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 Lau 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 Gin Tyr Giu His Asp Leu Giu Val Ala Gin Thr Thr Ala 195 200 205 Leu Pro Asp Glu Asp Asp Asp Leu 210 215 <210> 7 <211> 4839 <212> DNA <213> Homo sapien <220> <221> CDS <222> (138) (2924) <400> 7 tccggttttt ctcaggggac gttgaaatta tttttgtaac gggagtcggg agaggacggg gcgtgccccg cgtgcgcgcg cgtcgtcctc coggcgctc 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 acc gcc gcc gct gcc gcC gcg gaa ccc ccg gca ccg ccg ccg cog ccc 218 Thr Ala Ala Ala Ala Ala Ala Glu Pro Pro Ala Pro Pro Pro Pro Pro 20 oct cot gag gag gao oca. gag cag gao agc ggc ccg gag gao ctg cot 266 Pro Pro Giu Glu Asp Pro Glu Gin Asp Ser Gly Pro Glu Asp Leu Pro 35 ctc gtc agg ctt gag ttt gaa gaa aca. gaa. gaa cot gat ttt act gca 314 Leu Val Arg Leu Giu Phe Glu Glu Thr Glu Glu Pro Asp Phe Thr Ala so tta tgt cag aaa tta aag ata oca gat cat gtc aga gag aga. got tgg 362 Leu Cys Gin Lys Leu Lys Ile Pro Asp His Val Arg Glu Arg Ala Trp 65 70 tta act tgg gag aaa gtt tca tct gtg gat gga gta ttg gga Leu Thr Trp Giu Lys Val. Ser Ser Val Asp Gly Val Leu Gly ggt tat Gly Tyr att caa aag Ile Gin Lys aaa aag Lys Lys gaa ctg tgg Giu Leu Trp gga Gly 100 atc tgt atc ttt att gca gca Ile Cys Ile Phe Ile Ala Ala 105 gtt gac cta gat gag atg tcg Vai Asp Leu Asp Giu Met Ser act ttt act gag Thr Phe Thr Glu cag aaa aac Gin Lys Asn ata gaa Ile Giu 125 atc agt gtc cat Ile Ser Val His ttc ttt aac tta Phe Phe Asn Leu aaa gaa att gat Lys Giu Ile Asp acc Thr 140 agt acc aaa gtt Ser Thr Lys Val gat aat gct atg tca Asp Asn Ala Met Ser 145 ctc ttc agc aaa ttg Leu Phe Ser Lys Leu 165 ctg ttg aag aag Leu Leu Lys Lys gat gta ttg ttt Asp Val Leu Phe gaa agg aca tgt Giu Arg Thr Cys gaa ctt Giu Leu 170 ata. tat ttg Ile Tyr Leu gca ttg gtg Ala Leu Val 190 caa ccc agc agt Gin Pro Ser Ser ata tct act gaa Ile Ser Thr Glu ata aat tct Ile Asn Ser 185 gct aaa ggg Aia Lys Gly cta aaa gtt tct Leu Lys Val Ser atc aca ttt tta Ile Thr Phe Leu ta Leu 200 gaa gta Glu Vai 205 tta caa atg gaa Leu Gin Met Giu gat ctg gtg att Asp Leu Vai Ile ttt cag tta atg Phe Gin Leu Met cta Leu 220 tgt gtc ctt. gac Cys Vai Leu Asp tat Tyr 225 ttt att aaa, ctc Phe Ile Lys Leu tca Ser 230 cct ccc atg ttg Pro Pro Met Leu ctc Leu -235 aaa. gaa cca tat Lys Giu Pro Tyr aaa Lys 240 aca gct gtt ata ccc att aat ggt tca Thr Ala Val Ile Pro Ile Asn Gly Ser cct cga Pro Arg 250 aca ccc agg cga ggt cag aac agg Thr Pro Arg Arg Gly Gin Asn Arg 255 agt Ser 260 gca cgg ata gca aaa caa cta Aia Arg Ile Ala Lys Gin Leu 265 gaa. aat gat aca Giu Asn Asp Thr 270 aga att att Arg Ile Ile gaa Giu 275 gtt ctc tgt aaa Val Leu Cys Lys cat gaa tgt His Giu Cys aat ata Asn Ile 285 gat gag gtg aaa aat gtt tat ttc aaa Asp GJlu Val. Lys Asn Val Tyr Phe Lys 290 aat Asn 295 ttt ata cct ttt Phe Ile Pro Phe 1034 aat tct ctt gga Asn Ser Leu Gly ctt Leu 305 gta aca tct aat Val Thr Ser Asn gga Gly 310 ctt cca gag gtt Leu Pro Glu Val 1082 aat ctt tct aaa Asn Leu Ser Lys tac gaa gaa att Tyr Giu Glu Ile ctt aaa aat aaa Leu Lys Asn Lys gat cta Asp Leu 330 1130 gat gca aga Asp Ala Arg tta Leu 335 ttt ttg gat cat Phe Leu Asp His aaa act ctt cag Lys Thr Leu Gin act gat tct Thr Asp Ser 345 aac ctt gat Asn Leu Asp 1178 ata gac agt ttt gaa aca cag Ile Asp Ser Phe Giu Thr Gin aca cca cga aaa Thr Pro Arg Lys 1226 gaa gag Giu Glu 365 gtg aat gta att Val Asn Val Ile cca cac act cca Pro His Thr Pro agg act gtt atg Arg Thr Val Met 1274 aac Asn 380 act atc caa caa Thr Ile Gin Gin tta Leu 385 atg atg att tta Met Met Ile Leu tca gca agt gat Ser Ala Ser Asp 1322 cct tca gaa aat ctg att tcc tat ttt Pro Ser Glu Asn Leu Ile Ser Tyr Phe aac tgc aca gtg Asn Cys Thr Val aat cca Asn Pro 410 1370 aaa gaa agt Lys Giu Ser gag aaa ttt Glu Lys Phe 430 ctg aaa aga gtg Leu Lys Arg Val aag Lys 420 gat ata. gga tac Asp Ile Gly Tyr atc ttt aaa. Ile Phe Lys 425 att gga tca le Gly Ser 141i8 gct aaa gct gtg Ala Lys Ala Val gga Giy 435 cag ggt tgt gtc Gin Gly Cys Val 1466 cag cga Gin Arg 445 tac aaa ctt gga Tyr Lys Leu Gly cgc ttg tat tac Arg Leu Tyr Tyr cga Arg 455 gta atg gaa tC Val Met Glu Ser 1514 atg Met 460 ctt aaa tca Leu Lys Ser gaa gaa Giu Glu 465 gaa cga tta tcc Glu Arg Leu Ser att Ile 470 caa aat ttt agc Gin Asn Phe Ser 1562 ctt ctg aat gac Leu Leu Asn Asp att ttt cat atg Ile Phe His Met tta ttg gcg tgc Leu Leu Ala Cys gct ctt Ala Leu 490 1610 gag gtt gta Giu Val Val tet gga aca Ser Gly Thr 510 gcc aca tat age aga agt aca tct cag Ala Thr Tyr Ser Arg Ser Thr Ser Gin 500 aat ctt gat Asn Leu Asp 505 ctt aat tta Leu Asn Leu 1658 gat ttg tet ttc Asp Leu Ser Phe eca Pro 515 tgg att ctg aat Trp, Ile Leu Asn 1706 aaa gcc Lys Ala 525 ttt gat ttt tac Phe Asp Phe Tyr gtg ate gaa agt Val Ile Glu Ser ttt ate aaa gea gaa Phe Ile Lys Ala Giu 535 gaa ega tgt gaa eat Glu Arg Cys Glu His 555 1754 1802 gge Gly 540 aae ttg aca aga Asn Leu Thr Arg atg ata aaa eat Met Ile Lys His ega, ate atg gaa Arg Ile Met Giu ett. gca. tgg etc Leu Ala Trp, Leu gat tea cct tta Asp Ser Pro Leu ttt gat Phe Asp 570 1850 ctt att, aaa Leu Ile Lys tct get tgt Ser Ala Cys 590 tca aag gac cga gaa gga cca act gat Ser Lys Asp Arg Giu Giy Pro Thr Asp 580 cac ett gaa His Leu Giu 585 act gca gca Thr Ala Ala 1898 ect ctt aat ctt Pro Leu Asn Leu ect Pro 595 etc cag aat aat Leu Gin Asn Asn 1946 gat atg Asp Met 605 tat ctt tct. ect, Tyr Leu Ser Pro gta Val 610 aga tet eca aag Arg Ser Pro Lys aaa ggt tea act Lys Giy Ser Thr acg Thr 620 cgt gta. aat tet Arg Val Asn Ser gca aat gca gag Ala Asn Ala Giu aca Thr 630 caa gca ace tea Gin Ala Thr Ser 1994 2042 2090 ttc cag aec cag Phe Gin Thr Gin cca ttg aaa tct Pro Leu Lys Ser tet ctt tea ctg Ser Leu Ser Leu ttt tat Phe Tyr 650 aaa aaa gtg Lys Lys Val ege ctt etg Arg Leu Leu 670 egg eta gee tat Arg Leu Ala Tyr etc Leu 660 egg eta aat aca Arg Leu Asn Thr ctt tgt gaa Leu Cys Giu 665 tgg ace ctt Trp Thr Leu 2138 tet gag cac eca gaa tta. gaa eat ate Ser Glu His Pro Giu Leu Giu His Ile ate Ile 680 2186 2234 ttc eag Phe Gin 685 eac ace ctg cag His Thr Leu Gin aat Asn 690 gag tat gaa etc Giu Tyr Glu Leu aga gac agg cat Arg Asp Arg His ttg gac caa att atg atg tgt too atg tat Leu 700 Asp Gin Ile Met Met Cys Ser Met Tyr ggc ata tgc aaa gtg aag Gly Ile Cys Lys Val Lys 710 715 2282 aat ata gac ctt Asn Ile Asp Leu ttc aaa atc att Phe Lys Ile Ile aca gca tao aag Thr Ala Tyr Lys gat Ott Asp Leu 730 2330 cot cat gct Pro His Ala gag tat gat Giu Tyr Asp 750 cag gag aca ttc Gin Giu Thr Phe cgt gtt ttg atc Arg Val Leu Ile aaa gaa gag Lys Giu Giu 745 atg cag aga Met Gin Arg 2378 tot att ata gta Ser Ile Ile Val tat aac tog gtc Tyr Asn Ser Val 2426 ctg. aaa Leu Lys- 765 aca aat att ttg Thr Asn Ile Leu cag Gin 770 tat got tco acc Tyr Ala Ser -'i agg Arg 775 000 oct aco ttg Pro Pro Thr Leu 2474 tca Ser 780 cca ata oct cac Pro Ile Pro His cot cga agc Oct Pro Arg Ser Pro aag ttt oct agt Lys Phe.Pro Ser 2522 ccc tta cgg att Pro Leu Arg Ile gga ggg aac ato Gly Gly Asn Ile att tca ccacotg le Ser Pro Leu aag agt Lys Ser 810 act coa Thr Pro 2570 2618 oca tat aaa Pro Tyr Lys aga toa aga Arg Ser Arg 830 gaa ggt otg Giu Gly Leu oca. aca aaa. Pro Thr Lys ato tta gta toa Ile Leu Val Ser att Ile 835 ggt gaa toa tto Giy Giu Ser Phe act tot gag Thr Ser Glu 2666 aag ttc Lys Phe 845 aaa aga Lys Arg cag aaa ata aat Gin Lys Ile Asn oag Gin 850 ago Ser atg gta tgt aao Met Val Cys Asn ago Ser 855 oca Pro gao ogt gtg oto Asp Arg Val Leu 2714 2762 agt got gaa Ser Ala Giu aac cot cot Asn Pro Pro ctg aaa aaa Leu Lys Lys ogo ttt gat att Arg Phe Asp Ile gga toa gat gaa Gly Ser Asp Giu gat gga agt aaa Asp Gly Ser Lys cat oto His Leu 890 2810 oca gga gag Pro Giy Giu too aaa ttt oag cag aaa, Ser Lys Phe Gin Gin Lys 895 900 ctg gca, gaa atg Leu Ala Glu Met act tot act Thr Ser Thr 905 2858 cga aca cga atg caa aag cag aaa atg aat gat agc atg gat acc tca 2906 Arg Thr Arg Met Gin Lys Gin Lys Met Asn Asp Ser Met Asp Thr Ser 910 915 920 aac aag gaa gag aaa tga ggatctcagg accttggtgg acactgtgta 2954 Asn Lys Giu Giu Lys 925 cacctctgga tggccacact tgggtgattc ttgtgtaaat tgctgtgctt gtctgactac tttatatgta ttccaaatgc aatggatatt ctattggaat agctggaagc cagaatgtaa ttattgatag ctagtacttg tatatcccaa tttactccat tctaattgca ggaatggtac tttttaagtc attacaagta aaaagcttca ctaaaggtgt ttcattgtct taatatcttc ctaagccact cctgccattt tatggatagt tttgccttct tattttttta aatttgattg attagaaatt ctgatatact aaagtataac aagaacttac tactet tggt aaaaagtaaa gtgcactttc aaacagactg tctcaaaatt atgtcttcca agtatggtct atcaagggtc aattaaaaca atttaaacta ctcacagatg agctcttttt tgaaatgtta aaaaagttgt aagaatggcc tttgtagcat atttaacatg actgcccatt agaaaaaaat gtgtgcttgt catatgatac tgattatttt ttttatacca gtgttctgcc taatgtttct ttaattatag attctgccct tgtatctttt aacactggca attatgggtt gctgcattag tcttgtgtga tgactgtata gtggatataa gtcattgtta agcagat tgt ctagagtggg ataggtgatg aacaccctta caccaaaatt tactaatttt tttataaaat tatcatacta cttcatccaa ttcagatcac agatcttagg gggtcctgaa gagccttaat ccttaatttg gaactggcaa tgttcaaagc aggcattaat aaaaagaggc ttaacttatt actttcccag aatgtgcaga tttatacaag ttCcttcc agtcctgata tttgctcttg gaaaatgtgt atcctgaAct acacattaga tttgctttta ctgaaacaga cttatgtttt tgaatttata tatagaggac gaattaagat ttttttttca ggaaggtttg ttgtctattt cacattattt gtttctatct gcttctcccc tagagatgct gttctgttta tgcaattgtt attgaaaatc aaagtaaaat acccaggcct tttttattaa cctatctatc cttctgcaaa ttttatttta attaaataaa tttcatacct taaatgagga aagtacccat cctaacacag acaaattaat tagagatttg tgttttctct atcttttatt ctagtccaaa gattttgtgc tcccctacac gtaacttaaa 3014 3074 3134 3194 3254 3314 3374 3434 3494 3554 3614 3674 3734 3794 3854 3914 3974 4034 4094 4154 4214 4274 ataggggata tttaaggtag aattattttt aaaaagaaat taagtttcaa ac.ttactatt Ctccatttca tcattgtttc ggcttactat ttctgggtct aggaacttca gagatcgtgt attgcttttt tgtattggtt aattaatagt ttgtctattt tgtgttcttt gtattaagta agataactct cctagtatct <210> 8 <211> 928 <212> PRT <213> Homo sapien <400> 8 cttcagctag ctggtcttgt ttgacagtta tgcatgaata tttgctacta attgagattt aaaactgtac taaaataaat cactaatgtt catcc cttttaggaa tagaaaacaa ttttgataac tcatacaaat agttcacatt cttaaataat atttaaaatt tagttgttaa ct ctttt ctg aatcactttg aattttattt aatgacacta cagttagctt agaattagtg gcttcagata gctatgttac gagtcttaat tctaggagaa tctaactcag tgtgctcatt gaaaacttga ttaggtcaag ccagaatttt ttattgcttt tattttctac ggtCtgatgt gatagataga 4334 4394 4454 4514 45.74 4634 4694 4754 4814 4839 Met Pro Pro Lys Thr Pro Arg Lys.Thr Ala Ala Thr Ala Ala Ala Ala Ala Ala Glu Pro Pro Ala Pro Pro Giu Gln Asp Ser Gly Pro 3S Phe Giu Glu Thr Giu Giu Pro Pro Pro 2S Giu Asp Pro Pro Pro Leu Pro Leu Glu Glu Asp Arg Leu Giu Gin Lys Leu Phe Thr Ala Lys Ile Pro Asp His Glu Arg Ala Val Trp, Tyr Thr Trp, Glu Ser Ser Val Asp? as Gly Val Leu Gly Ile Gin Lys Lys Lys Glu Leu Trp Met Ser Phe Uis Gly Ile 100 Cys Ile Phe Ala Val Asp Leu Asp Giu 110 Ile Ser Val Thr Phe Thr Giu Leu 120 Gin Lys Asn Ile His Asp 145 Leu Pro Val Giu Tyr 225 Thr Gin Ile Lys Leu 305 Tyr Leu Thr Ile Leu 385 Lys 130 Asn Phe Ser Ser Asp 210 Phe Al a Asn Ile Asn 290 Val Giu Asp Gin Pro 370 Met Phe Ala Ser Ser Trp 195 Asp Ile Vai Arg Glu 275 Val Thr Glu His Arg 355 Pro Met Phe Met Lys Ser 180 Ile Leu Lys Ile Ser 260 Val Tyr Ser Ile Asp 340 Thr His Ile Asn Ser Leu 165 Ile Thr Val Leu Pro 245 Ala Leu Phe Asn Tyr 325 Lys Pro Thr Leu Leu Arg 150 Giu Ser Phe Ile Ser 230 Ile Arg Cys Lys Gly 310 Leu Thr Arg Pro Asn 390 Leu 135 Leu Arg Thr Leu Ser 215 Pro Asn Ile Lys Asn 295 Leu Lys Leu Lys Val 375 S er Lys Leu Thr Giu Leu 200 Phe Pro Gly Ala Giu 280 Phe Pro Asn Gin Ser 360 Arg Ala Giu Lys Cys Ile 185 Ala Gin Met Ser Lys 265 His Ile Glu Lys Thr 345 Asn Thr Ser Ile Lys Giu 170 Asri Lys Leu Leu Pro 250, Gin Giu Pro Val Asp 330 Asp Leu Val Asp Asp Tyr 155 Leu Ser Gly Met Leu 235 Arg Leu Cys Phe Giu 315 Leu Ser Asp Met Gin 395 Thr 140 Asp Ile Al a Giu Leu 220 Lys Thr Giu Asn Met 300 Asn Asp Ile Giu Asn 380 Pro Ser Val1 Tyr Leu Val 205 Cys Giu Pro Asn Ile 285 Asn Leu Ala Asp Giu 365 Thr Ser Thr Leu Leu Val1 190 Leu Val Pro Arg Asp 270 Asp Ser Scr Arg Ser 350 Val Ile Glu Lys Phe Thr 175 Leu Gin Leu Tyr Arg 255 Thr Giu Leu Lys Leu 335 Phe Asn Gin Asn Ile Ser Tyr Phe Asn Asn Cys Thr Vai Asn Pro Lys Giu Ser Ile Leu 'i un 410 415 Lys Al a Gly Giu 465 Ile Arg Val Val Giy 435 Val Arg 450 Giu Arg Phe His Lys 420 Gin Leu Leu Met Asp Ile Gly Tyr Gly Tyr Ser Ser Cys Tyr Ile 470 Leu Val1 Arg 455 Gin Leu Glu 440 Val Asn Aila 485 Thr Ser Tyr Glu 545 Leu Lys Asn Pro Phe Lys 530 Met Ala Asp Leu Val *Ser Pro 515 Val Ile Trp Arg Pro 595 Arg Arg 500 Trp Ile Lys Leu Glu 580 Leu Ser Ser Thr Ser Gin Ile Glu His Ser 565 Gly Gin Pro Leu Ser Leu 550 Asp Pro Asn Lys Asn Val 520 Phe Ile 535 GJlu Arg Ser Pro Thr Asp Asn His 600 Lys Lys C 615 Gin Ala TI Ile Phe Lys Glu 425 Ile Gly Ser Gin Met Giu Ser Met 460 Phe Ser Lys Leu 475 Cys Ala Leu Giu 490 Asn Leu Asp Ser 505 Leu Asn Leu Lys Lys Ala Glu Gly 540 Cys Giu His Arg 555 Leu Phe Asp Leu 570 iis Leu Glu Ser? 585 E'hr Ala Ala Asp to 6 ;ly Ser Thr Thr A 620 'hr Ser Ala Phe G Lys Arg 445 Leu Leu Val Gly Ala 525 Asn Ile Ile let ~rg Phe 430 Tyr Lys Asn Val Thr 510 Phe Leu Met Lys Cys 590 Tyr Val Ala Lys Ser Asp Met 495 Asp Asp Thr Giu Gin 575 Pro Leu ksn Lys Leu Glu Asn 480 Ala Leu Phe Arg Ser 560 Ser Leu Ser Ser 610 Thr 625 Pro Leu Ala Asn Ala Glu Thr 630 in Thr Gin Lys Leu Ala Lys Tyr Ser Thr 645 Leu Arg 660 Ser Leu Ser Leu Asn Thr Leu Leu 665 Phe 650 Cys Tyr Glu Lys Arg His Pro Giu Leu Giu His Ile Ile Trp, Thr Leu Phe Gin His Thr Leu 675 r Gin Asn Giu Tyr Giu Let 690 Met 705 Phe Giu Ile Leu Ile 785 Gly Giu Val Asn Gly 865 Gly Cys Lys Thr Vai Gin 770 Pro Giy Giy Ser Glm 850 Ser 3er Ser Ile Phe Phe 755 Tyr Arg Asn Leu Ile 835 Met Asn Asp Met Ile Lys 740 Tyr Ala Ser Ile Pro 820 Gly Vai Pro 3lu Tyx Val 725 Arg Asn Ser Pro Tyr 805 Thr Glu Cys Pro Ala 710 *Thr Vai Ser Thr 790 Ile Pro Ser Asn Lys 870 Asp Met 695 Ile Aila Leu Vai Arg 775 Lys Ser Thr Phe Ser 855 Pro 31y *Arg *Cys Tyr Sle Phe 760 Pro Phe Pro Lys Giy 840 Asp Leu Ser Asr Lys Lys Lys 745 Met Pro Pro Leu Met 825 Thr Arg Lys Arc Val Asp 730 Giu Gin Thr Ser Lys 810 Thr Ser Vai Lys His *Lys 715 Leu Giu Arg Leu Ser 795 Ser Pro Giu Leu Leu 875 Leu Leu 700 Asn Pro Giu Leu Ser 780 Pro Pro Arg Lys Lys 860 Pro IAsp Ile His Tyr Lys 765 Pro Leu Tyr Ser Phe 845 Arg Phe Giy Gin Asp Al a Asp 750 Thr Ile Arg Lys Arg 830 Gin Ser Asp .lu Sle Leu Vai 735 Ser Asn Pro Ile. Ile Ile Lys Aia Ile Met Lys 720 Gin Ile Ile His Pro 800 Ser Leu Ile Giu Glu 880 885 890 895 Phe Lys Gin Lys Lys 900 Met Leu Asn Giu Met Ser Met 920 Thr 905 Asp Thr Lys 925 <210> 9 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide <400> 9 ttctgtagtt taattttctg aacctttggc <210> <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide <400> tcagccgaag agcttcagga agcaggg 27 <210> 11 <211> 32 <212> PRT <213> Homo sapien <220> <221> VARIANT <222> (3) <220> <221> VARIANT <222> (13) <220> <221> VARIANT <222> <220> <221> VARIANT <222> (17) .(18) <220> <221> VARIANT <222> (20) .(21) <220> <221>' VARIANT <222> <220> <221> VARIANT <z222> <400> 11 Cys Xaa Xaa Cys Xaa Xa-a Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa His 1 5 10 1 Xaa Xaa Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys 25 <210> 12 <211> <212> PRT <213> Homo sapien <220> <221> VARIANT <222> (3) <220> <221> VARIANT <222> <220> <221> VARIANT <222> <220> <221> VARIANT <222> (25) (26.) <220> <221> VARIANT <222> <220> <221> VARIANT <222> <220> <221> VARIANT <222> (48) (49) <400> 12 Cys Xaa xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 is Xaa Xaa Xaa Xaa His Xaa Xaa Cys Xaa Xaa Cys Xaa Xaa Cys Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 40 Xaa Cys