AU771564B2 - Androgen receptor coactivators - Google Patents

Description

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.

20 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 S* 25 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 30 different receptors recognize the same DNA sequence. For Poo**: example, the androgen receptor the glucocorticoid receptor and the progesterone receptor (PR) all WO 00/04152 PCT/US99/16122 recognize the same sequence but activate different transcription activities. Some have speculated that accessory factors may selectively interact with the androgen receptor to determine the specificity of gene activation by the androgen receptor.

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 j 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 (Crawford, Br. J.

Urology 70: suppl. i, 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, A1BI 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 efficacy of drugs in the treatment of prostate cancers.

Brief Summary of the Invention There is described herein 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 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 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 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.

According to a first embodiment of the invention there is provided an isolated polynucleotide comprising the sequence set forth in SEQ ID NO:1.

25 According to a second embodiment of the invention there is provided 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 ARA54 polypeptide set forth in SEQ ID NO: 2.

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

According to a fourth embodiment of the invention there is provided an isolated polynucleotide encoding the ARA54 polypeptide set forth in SEQ ID NO:2.

According to a fifth embodiment of the invention there is provided a genetic construct comprising a nucleic acid sequence encoding the ARA54 polypeptide of SEQ ID NO:2 operably linked to a heterologous promoter.

(R:\LIBZZ]061 11. FEB.'2004 14:36 SPRUSON FERGUSON NO. 6213 P. 4 4 According to a sixth embodiment of the invention there is provided an isolated ARA54 polypeptide comprising amino acids 361-471 of SEQ ID NO: 2 and a RING finger motif, wherein the ARA54 polypeptide is capable of enhancing the transcription activity of an androgen receptor or a progesterone receptor.

s According to a seventh embodiment of the invention there is provided an isolated ARA54 polypeptide comprising amino acids 361-471 of SEQ ID NO: 2 and a RING finger, wherein the ARA54 polypeptide is capable of enhancing the transcription activity of a mutant androgen receptor or a mutant progesterone receptor, causing an enhancement of the transcription activity of the mutant androgen receptor to or the mutant progesterone receptor.

According to an eighth embodiment of the invention there is provided an isolated ARA54 polypeptide comprising amino acids 361-471 of SEQ ID NO: 2, wherein the ARA54 polypeptide is capable of inhibiting the transcription activity of an androgen receptor or a progesterone receptor.

According to a ninth embodiment of the invention there is provided an isolated ARA54 polypeptide comprising a sequence corresponding to amino acids 361-471 of SEQ ID NO: 2, wherein the ARA54 polypeptide is capable of inhibiting the transcription activity of a mutant androgen receptor or a mutant progesterone receptor.

According to a tenth embodiment of the invention there is provided a method of 20 screening molecules for the ability to modulate androgen receptor activity comprising *::the steps of: a) cotransforming a suitable eukaryotic cell that expresses AR with a genetic construct, wherein the genetic construct comprises a promoter functional in a eukaryotic cell operably connected to a polynucleotide comprising a sequence that encodes an ARA54 polypeptide, and a construct comprising at least a portion of an expressible androgen receptor sequence; b) culturing the cells in the presence of a candidate molecule; and c) assaying the transcriptional activity induced by the ARA54 S polypeptide; wherein an increase or decrease in androgen receptor activity relative to the ARA54 polypeptide and AR without the molecule indicates modulation of androgen receptor activity.

According to an eleventh embodiment of the invention there is provided a S: method of screening for molecules that modulate androgen receptor activity comprising the steps of: a) incubating a compound with a cell, wherein the cell is engineered to express ARA54, and b) assaying for androgen receptor activity; wherein an increase or decrease in androgen receptor activity relative to androgen receptor activity in the presence of ARAS4 without the compound indicates modulation.

[R:\ALIZZI I e l.doc KO COMS ID No: SMBI-00613183 Received by IP Australia: Time 14:44 Date 2004-02-11 '11. F EB."2004 14 :36 SPRUSON FERGUSON NO. 6 213 P. 4a According to a twelfth embodiment of the invention there is provided a method of screening for molecules that modulate progesterone receptor activity comprising the steps of: a) incubating a compound with a cell, wherein the cell is engineered to express AR.A54, and b) assaying for progesterone receptor activity; where in an increase or decrease in progesterone receptor activity relative to progesterone receptor a ctivity in the presence of ARA54 without the compound indicates modulation.

According to a thirteenth embodiment of the invention there is provided a method of screening for molecules that modulate mutant androgen receptor activity comprising the steps of: a) incubating a compound with a cell, wherein the cell is engineered to express ARA54, and b) assaying for mutant androgen receptor activity; wherein an increase or decrease in mutant androgen receptor activity relative to mutant androgen receptor activity in the presence of ARA54 without the compound indicates modulation.

According to a fourteenth embodiment of the invention there is provided a method of screening for molecules that modulate androgen receptor activity comprising the steps of: a) incubating a compound with an ARAS4-androgen'receptor complex, and b) assaying whether the compound decreases the amount of the ARA5 4androgen receptor complex.

According to a fifteenth embodiment of the invention there is provided a method of screening molecules that modulate progesterone receptor activity comprising the steps of: a)-incubating a compound with an ARA54-progesterone receptor complex, and b) assaying whether the compound decreases the, amount of the ARA54progesterone receptor complex.

According to a sixteenth embodiment of the invention there is provided a genetic construct comprising a nucleic acid encoding the polypeptide of any one of the fifth to ninth embodiments.

According to a seventeenth embodiment of the invention, there is provided a method of making a polypeptide. according to any one of the fifth to ninth embodiments comprising transforming a cell with a nucleic acid encoding the polypeptide according to any one of the fifth to ninth embodiments.

According to an eighteenth embodiment of the invention there is provided a product produced by the process of the seventeenth embodiment.

According to a nineteenth embodiment of the invention there is provided a method of screening for molecules that modulate androgen receptor coactivation by AIRA54 comprising the steps of: a) incubating a compound with a cell, wherein the cell comprises ER UafZZJIdt54doc:KO COMS ID No: SMBI-0061 3183 Received by IP Australia: Time 14:44 Date 2004-02-11 6. FEB. 2004 14:57 SPRUSON FERGUSON NO.5816 P. 4b ARAS4, b) incubating a compound with a cell, wherein the cell lacks ARAS4, and c) assaying for androgen receptor activity, wherein an increase or decrease in androgen receptor activity relative to androgen receptor activity in the presence of ARA54 without the compound, and relative to androgen receptor activity in the absence of ARA54 with the compound, indicates modulation.

According to a twentieth embodiment of the invention there is provided a method of screening for molecules that modulate progesterone receptor coactivation by ARA54 comprising the steps of: a) incubating a compound with a cell, wherein the cell comprises ARA54, b) incubating a compound with a cell, wherein the cell lacks ARA54, and c) assaying for progesterone receptor activity, wherein an increase or decrease in progesterone, receptor activity relative to progesterone receptor activity in the presence of ARA54 without the compound, and relative to progesterone receptor activity in the absence of ARA54 with the compound, indicates modulation.

According to a twenty-first embodiment of the invention there is provided a method of screening for molecules that modulate mutant androgen receptor coactivation by ARA54 comprising the steps of: a) incubating a compound with a cell, *00*00wherein the cell comprises ARAS4, b) incubating a compound with a cell, wherein the cell lacks ARA54, and c) assaying for mutant androgen receptor activity; wherein an increase or decrease in mutant androgen receptor activity relative to androgen receptor activity in the presence of ARA54 without the compound, and relative to mutant androgen receptor activity in the absence of ARA54 with the compound indicates modulation.

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.

Other objects, features, and advantages of the present invention will become apparent upon reading the specification and claims.

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

Detailed Description of the Invention Transactivation of genes by the androgen receptor is a complicated systemn that involves many different coactivators. It is not currently known just how many factors are involved in androgen receptor-mediated regulation of gene expression. The identification and/or characterization of four androgen receptor coactivators is COMS ID No: SMBI-00607571 Received by IP Australia: Time 15:03 Date 2004-02-06 8FEB. 2 0 04 14 5 SPRUSON FERGUSON NO. 58 10 P. 16 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 s 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 o0 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 15 ARA54 polypeptide, an ARA55 polypeptide, an ARA24 polypeptide and an Rb polypeptide.

The present invention includes a method for screening .j.

o o oo oooo oooo ooooo oO o lK'ULIJBZ61Z B domr COMS ID No: SMBI-00607571 Received by IP Australia: Time 15:03 Date 2004-02-06 WO 00/04152 PCT/US99/16122 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; culturing 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, WO 00/04152 PCT/US99/16122 respectively. The putative amino acid sequences of ARA54 and ARA55 are shown in SEQ ID NO:2 and SEQ ID NO:4, respectively. Also provided are the DNA and amino acid sequences of ARA24 (SEQ ID NO:5 and SEQ ID NO:6, respectively) and Rb (SEQ ID NO:7 and SEQ ID NO:8, respectively). These coactivators were further characterized as detailed below. It is expected that some minor variations from SEQ ID NOs:1-8 associated with nucleotide additions, deletions, and mutations, whether naturally occurring or introduced in vitro, will not affect coactivation by the expression product or polypeptide.

Briefly, ARA54 is a 54 kDa protein that interacts with AR in an androgen-dependent manner. Coexpression of ARA54 and AR in a mammalian two-hybrid system demonstrated that reporter gene activity was enhanced in an 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 (10-7-10 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 (10-9-10 7 M) and HF (10- 7 10 5 and ARA54 o1 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 25 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 a o [R:\LIBZZ]06185.doc:mrr WO 00/04152 PCT/US99/16122 (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- 9 M 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- WO 00/04152 PCT/US99/16122 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 WO 00/04152 PCT/US99/16122 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.

11 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, to 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.

pSG5 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 25 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) Sfrom wtAR, mARt877s (Dr. S.P. Balk, o o o [R:UBZZ]06185.d.:.~i WO 00/04152 PCT/US99/16122 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 Pglobulin 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 -12- WO 00/04152 PCT/US99/16122 vector. 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 LED, 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 3-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- WO 00/04152 PCT/US99/16122 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°C for 1 min, 5 cycles of 94 0 C for 5 sec-72 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

CX,._CXHX

2

CX

2

CX

6 17 CXC (SEQ ID NO: 11), -14- WO 00/04152 PCTIUS99/16122 a domain conserved among several human transcriptional factor or proto-oncogeny proteins, including BRCA1, RING1,- PML and MEL-18 (Miki et al., Science 266:66-71 (1994); Borden et al., EMBO J. 14:1532-1541 (1995); Lovering et al., Proc. Natl. Acad. Sci. USA 90:2112-2116 (1993); Blake et al., Oncogene 6: 653-657 (1991); Ishida et al, Gene 129:249-255 (1993)). In addition, ARA54 also contains a second cysteine-rich motif which has a B box like structure located at 43 amino acids downstream from the RING finger motif. However, ARA54 differs from members of the RING finger-B-box family in that it lacks a predicted 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

CX

16 23

HX

2

CX

2

CX

2

CX

16 21

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 WO 00/04152 PCT/US99/16122 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 (25ug) 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 2 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. [-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, ImM PMSF, aprotinin, leupeptin, pepstatin, 0.25% Na-deoxycholate, 0.25% gelatin) and rocked at 4°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 -16- WO 00/04152 PCT/US99/16122 by 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-Rb.

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

Biol. Chem. 272:12738-12746, 1997). Approximately 2 Ag 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 NaCi, 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- WO 00/04152 PCT/US99/16122 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,ug/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 TGFP, respectively. A P-galactosidase expression plasmid, pCMV--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- WO 00/04152 PCT/US99/16122 ARA54 in the presence of 10- 8 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 (109-10 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 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, 0o exhibited differential specificity for ligands. In the absence of ARA54, Art877a responded to E2 (10--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 25 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 o oo 9 [R:\LIBZZ]06185.doc: rr WO 00/04152 PCT/US99/16122 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,_ 444 251-444 of SEQ ID NO:4) did not enhance CAT activity.

Cotransfection of PC3 cells, which contain endogenous with ARA55 251 444 AR and MMTV-CAT reporter in the presence of 10 nM DHT demonstrated dramatically reduced AR transcriptional activity relative to cells transfected with AR and MMTV-CAT alone. These results demonstrate that 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 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 -21- WO 00/04152 PCT/US99/16122 at a 1:4.5 ratio, however, increases AR transcriptional activity at 10-8-10 7 M for E2 or 10- 6 to 10i 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 TGF((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 TGFP treatment (5 ng/ml) could induce ARA55 mRNA by 2-fold in PC3 cells. In the same cells, TGFP 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 TGF-induced AR transcriptional activity will be an interesting question to investigate. The unexpected discovery that TGF 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 -22- WO 00/04152 PCT/US99/16122 inducing 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 -23- WO 00/04152 PCT/US99/16122 correlates with their affinity. These results suggest that ARA24/Ran could achieve differential transactivation of AR, with ARs having different poly-Q length could existing in a single cell or cell system. 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 Ag) 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 -24- WO 00/04152 PCT/US99/16122 between 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 7 o 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 AM HF.

WO 00/04152 PCT/US99/16122 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 AM 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 -26- WO 00/04152 PCT/US99/16122 several 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- WO 00/04152 PCT/US99/16122 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- EDITORIAL NOTE APPLICATION NUMBER 51069/99 The following Sequence Listing pages 1 to 26 are part of the description. The claims pages follow on pages "29" to "33".

WO 00/04152 PCT/US99/16122 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> <222> <223> <220> <221> <222> <223> misc feature (697) (834) Coding sequence and form a cystein-rich misc feature (964) (1089) Coding sequence and polypeptide region which may RING finger motif.

polypeptide region for a cystein-rich B box like structure.

WO 00/04152 WO 0004152PCT/US99/I 6122 <400> 1 ggtctctggtcCtcccctctc tgagcactct gaggtcctt atg tcg tca gaa gat Met Ser Ser Glu Asp cga gaa gct cag Arg Giu Ala Gin gat gaa ttg ctg gcc ctg gca agt att Asp Glu Leu Leu Ala Leu Ala Ser Ile tac gat Tyr Asp gga gat gaa Gly Asp Giu atc tat ttg Ile Tyr Leu aga aaa gca gag tct gtc caa ggt gga Arg Lys Aia Giu Ser Val Gin Gly Gly gaa acc agg Giu Thr Arg agc ggc aat Ser Gly Asn gat ttg cca cag Asp Leu Pro Gin ttc aag ata ttt Phe Lys Ile Phe gtg Val1 tca aat Ser Asn gag tgt ctc cag aat agt ggc ttt gaa Giu Cys Leu Gin Asn Ser Gly Phe Giu acc att tgc ttt Thr Ile Cys Phe 246 ctg Leu 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 cct Ser Ser Pro Pro ttc aca ctt agt Phe Thr Leu Ser aaa tgg ctg tca Lys Trp Leu Ser cca act Pro Thr 100 cag cta tct Gin Leu Ser cgt ggc agc Arg Gly Ser 120 cta tgc aag cac Leu Cys Lys His tta Leu 110 gac aac cta tgg Asp Asn Leu Trp gaa gaa cac Giu Giu His 115 aag gaa gag Lys Giu Giu gtg gtc ctg ttt Vai Vai Leu Plie tgg atg caa ttt Trp Met Gin Phe ctt Leu 130 acc cta Thr Leu 135 gca tac ttg aat Ala Tyr Leu Asn gtc tct cct ttt Val 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 gct tct ccc aac Ala Ser Pro Asn gag cta gat ttt gga gga gct gct gga Giu Leu Asp Phe Gly Gly Ala Ala Gly 170 tct Ser 175 gat gta gac caa Asp Val Asp Gin gag gaa Giu Giu 180 WO 00/04152 WO 0004152PCT/US99/I 6122 att gtg gat gag aga gca gtg cag Ile Val Asp Giu Arg Ala Val Gin 185 gat Asp 190 gtg gaa tca ctg Val Giu Ser Leu tca aat ctg Ser Asn Leu 195 aaa tgc ttt Lys Cys Phe atc cag gaa Ile Gin Giu 200 atc ttg gac ttt Ile Leu Asp Phe caa gct cag cag Gin Ala Gin Gin at~a Ile 210 aat agt Asn Ser 215 aaa ttg ttc ctg Lys Leu Phe Leu tgC Cys 220 agt atc tgt ttc Ser Ile Cys Phe gag aag ctg ggt Glu Lys Leu Gly agt Ser 230 gaa tgc atg tac Giu Cys Met Tyr ttg gag tgc agg Leu Giu Cys Arg gtg tac tgc aaa Val Tyr Cys Lys gcC Al a 245 tgt ctg aag gac Cys Leu Lys Asp ttt gaa atc cag Phe Giu Ile Gin aga gat ggc cag Arg Asp Gly Gin gtt caa Val Gin 260 tgc ctc aac Cys Leu Asn cag gtc aaa Gin Val Lys 280 tgC Cys 265 cca gaa cca aag Pro Glu Pro Lys tgC Cys 270 cct tcg gtg gcc Pro Ser Val Ala act cct ggt Thr Pro Gly 275 tat gac cgc Tyr Asp Arg gag tta gtg gaa Giu Leu Val Giu gca Ala 285 gag tta ttt gcc Glu Leu Phe Ala ctt ctc Leu Leu 295 ctc cag tcc tcc Leu Gin Ser Ser gac ctg atg gca Asp Leu Met Ala gat Asp 305 gtg gtg tac tgc Val Val Tyr Cys cgg ccg tgC tgC Arg Pro Cys Cys cag Gin 315 ctg cct gtg atg Leu Pro Val Met gaa cct ggc tgc Glu Pro Giy Cys 966 1014 1062 atg ggt atc tgc Met Gly Ile Cys tcC Ser 330 agc tgc aat ttt Ser Cys Asn Phe gcc Ala 335 ttc tgt act ttg Phe Cys Thr Leu tgc agg Cys Arg 340 ttg acc tac Leu Thr Tyr atg gac tta Met Asp Leu 360 ctt ttg gat Leu Leu Asp 375 ggg gtc tcc cca Giy Val Ser Pro tgt Cys 350 aag gtg act gca Lys Val Thr Ala gag aaa tta Giu Lys Leu 355 aat aaa aga Ash Lys Arg cga aat gaa tac Arg Ash Glu Tyr caa gcg gat gag Gin Ala Asp Giu gct Ala 370 1110 1158 1206 caa agg tat Gin Arg Tyr aag aga gtg att Lys Arg Val Ile cag Gin 385 aag gca ctg gaa Lys Ala Leu Glu WO 00/04152 PCT/US99/I 6122 gag atg gaa agt aag gag tgg cta gag aag aac tca aag agc tgc cca 1254 Giu Met Giu Ser Lys Glu Trp Leu Glu Lys Asn Ser Lys Ser Cys Pro 390 395 400 405 tgt tgt gga act ccc ata gag aaa tta gao gga tgt aac aag atg 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 cct tac aaa cat ttc aat gac cct ggt tca cca tgt 1398 Ser Arg Ala Asn Pro Tyr Lys His Phe Asn Asp Pro Gly Ser Pro Cys 440 445 450 ttt aac cgg ctg ttt tat got gtg gat gtt gac gac gat att tgg gaa 1446 Phe Asn Arg Leu Phe Tyr Ala Vai Asp Val Asp Asp Asp Ile Trp Glu 455 460 465 gat gag gta gaa gac tag ttaactactg ctcaagatat, ttaactactg 1494 Asp Glu Val Glu Asp 470 475 ctcaagatat ggaagtggat tgtttttccc taatcttccg tcaagtacac aaagtaactt 1554 tgcgggatat ttagggtact attcattcac tcttcctgcg tagaagatat. ggaagaacga 1614 ggtttatatt ttcatgtggt. actactgaag aaggtgcatt gatacatttt taaatgtaag 1674 ttgagaaaaa tttataagcc aaaggttcag aaaattaaac tacagaa 1721 <210> 2 <211> 474 <212> PRT <213> Homno sapien <400> 2 Met Ser Ser Giu Asp Arg Giu Ala Gin Glu Asp Giu Leu Leu Ala Leu 1 5 10 Ala Ser Ile Tyr Asp Gly Asp Glu Phe Arg Lys Ala Giu Ser Val Gin 25 Gly Giy Giu Thr Arg Ile Tyr Leu Asp Leu Pro Gin Asn Phe Lys Ile 40 Phe Val Ser Gly Asn Ser Asn Giu Cys Leu Gin Asn Ser Giy Phe Giu 55 WO 00/04 152 Tyr Thr Ile Pro Asp Tyr Trp Leu Ser Leu Trp Giu 115 Phe Leu Lys 130 Giu Leu Lys 145 Ala Ser Pro Val Asp Gin Ser Leu Ser 195 Gin Ile Lys 210 Cys Giu Lys 225 Val Tyr Cys Asp Giy Gin Val Ala Thr 275 Ala Arg Tyr 290 Asp Val Val 305 Glu Pro Gly PCT[US99/16122 Cys Pro Pro 100 Giu Giu Ile Asn Glu 180 Asn Cys Leu Lys Val 260 Pro Asp Tyr Cys Phe Ser Thr His Giu Gly Thr 165 Glu Leu Phe Gly Ala 245 Gin Gly Arg Cys Thr 325 Leu 70 Ser Gin Arg Thr Ser 150 Giu Ile Ile Asn Ser 230 Cys Cys Gin Leu Pro 310 Met Pro Ser Leu Gly Leu 135 Gin Leu Val Gin Ser 215 Glu Leu Leu Val Leu 295 Arg Gly Pro Pro Ser Ser 120 Ala Lys Asp Asp Giu 200 Lys Cys Lys Asn Lys 280 Leu Pro Ile Leu Vai Pro Ser 90 Ala Leu 105 Val Val Tyr Leu Lys Val Phe Gly 170 Giu Arg 185 Ile Leu Leu Phe Met Tyr Asp Tyr 250 Cys Pro 265 Glu Leu Gin Ser Cys Cys Cys Ser 330 Leu 75 Phe Cys Leu Asn Gin 155 Gly Ala Asp Leu Phe 235 Phe Giu Val Ser Gin 315 Ser Asn Thr Lys Phe Ile 140 Arg Ala Val Phe Cys 220 Leu Glu Pro Glu Leu 300 Leu Cys Phe Leu His Ala 125 Val Arg Al a Gin Asp 205 Ser Giu Ile Lys Ala 285 Asp Pro Asn Giu Ser Leu 110 Trp Ser Thr Gly Asp 190 Gin Ile Cys Gin Cys 270 Glu Leu Val Phe Leu Gly Asp Met Pro Al a Ser 175 Val Ala Cys Arg Ile 255 Pro Leu Met Met Ala 335 Pro Lys Asn Gin Phe Gin iS0 Asp Giu Gin Phe His 240 Arg Ser Phe Ala Gin 320 Phe WO 00/04152 Cys Thr Leu Cys Arg Leu Thr Tyr His Gly Val Ser Pro Cys 340 345 350 Thr Ala Glu Lys Leu Met Asp Leu Arg Asn Glu Tyr Leu Gin 355 360 365 Glu Ala Asn Lys Arg Leu Leu Asp Gin Arg Tyr Gly Lys Arg 370 375 380 Gin Lys Ala Leu Glu Glu Met Glu Ser Lys Glu Trp Leu Glu 385 390 395 Ser Lys Ser Cys Pro Cys Cys Gly Thr Pro Ile Glu Lys Leu 405 410 Cys Asn Lys Met Thr Cys Thr Gly Cys Met Gin Tyr Phe Cys 420 425 430 Cys Met Gly Ser Leu Ser Arg Ala Asn Pro Tyr Lys His Phe 435 440 445 Pro Gly Ser Pro Cys Phe Asn Arg Leu Phe Tyr Ala Val Asp 450 455 460 Asp Asp Ile Trp Glu Asp Glu Val Glu 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> miscfeature <222> (631) (783) <223> Coding sequence and polypeptide region for a cystein rich LIM motif PCT/US99/16122 Lys Ala Val Lys Asp 415 Trp Asn Val Val Asp Ile Asn 400 Gly Ile Asp Asp WO 00/04152 PCT/US99/16122 <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> misc_feature (808)..(996) Coding sequence and polypeptide region for a cystein rich LIM motif misc_feature (985)..(1137) Coding sequence and polypeptide region for a cystein rich LIM motif miscfeature (1162) (1314) Coding sequence and polypeptide region for a cystein rich LIM motif <400> 3 atg cca Met Pro 1 agg tca ggg get Arg Ser Gly Ala 5 ccc aaa gag Pro Lys Glu cct gcg gag cct Pro Ala Glu Pro ctc acc Leu Thr cct ccc cca Pro Pro Pro tea gga gcc Ser Gly Ala tat ggc cac cag Tyr Gly His Gin aca ggg cag tct Thr Gly Gin Ser ggg gag tct Gly Glu Ser gta tgc aag Val Cys Lys tcg ggg gac aag Ser Gly Asp Lys gac Asp 40 cac ctg tac age His Leu Tyr Ser acg Thr cct cgg Pro Arg tec cca aag cct Ser Pro Lys Pro gcc cog gcc gcc Ala Pro Ala Ala cca ttc tcc tct Pro Phe Ser Ser tecc Ser age 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 act cag ttc aac Thr Gin Phe Asn ate Ile aca gat gaa atc Thr Asp Glu Ile atg tct Met Ser cag ttc cca Gin Phe Pro ago aag gtg get Ser Lys Val Ala gga gag cag aag Gly Glu Gin Lys gag gac cag Glu Asp Gin 110 tct cct ggc Ser Pro Gly tct gaa gat aag aaa aga ccc Ser Glu Asp Lys Lys Arg Pro 115 ago Ser 120 etc cct tec age Leu Pro Ser Ser ccg Pro 125 WO 00/04152 WO 0004152PCT/US99/16122 ctc cca Leu Pro 130 aag gct tct gcc acc tca gcc act ctg Lys Ala Ser Ala Thr Ser Ala Thr Leu 135 ctg gat aga ctg Leu Asp Arg Leu atg Met 145 gcc tca ctc cct Ala Ser Leu Pro gac Asp 150 ttc cgc gtt caa Phe Arg Val Gin cat ctt cca gcc His Leu Pro Ala tct Ser 160 ggg cca act cag Gly Pro Thr Gln ccg gtg gtg agc Pro Val Val Ser tcc Ser 170 aca aat gag ggc Thr Asn Giu Gly tcc cca Ser Pro 175 tcc cca cca Ser Pro Pro ctg ctg cag Leu Leu Gin 195 ccg act gca aag Pro Thr Ala Lys ggc Gly 185 cgg Arg agc cta gac acc Ser Leu Asp Thr atg ctg ggg Met Leu Gly 190 cag gcc aaa Gin Ala Lys gac ctc agc Asp Leu Ser ggt gtt ccc Giy Val Pro acc Thr 205 ggc ctc Gly Leu 210 tgt ggc tcc tgc Cys Gly Ser Cys aaa cct att gct Lys Pro Ile Ala ggg Gly- 220 caa gtg gtg acg Gin Val Val Thr gct Ala 225 tcc Ser ctg ggc cgc gcc Leu Gly Arg Ala acc gcc ctg gga Thr Ala Leu Gly 245 cac ccc gag cac His Pro Giu His ttc Phe 235 gtt tgc gga ggc Val Cys Gly Gly ggc agc agc ttc Gly Ser Ser Phe gag aag gat gga Glu Lys Asp Gly gcc ccc Ala Pro 255 ttc tgc ccc Phe Cys Pro tgc aac cag Cys Asn Gin 275 gag Glu 260 tgc tac ttt gag Cys Tyr Phe Giu ttc tcg cca aga Phe Ser Pro Arg tgt ggc ttc Cys Gly Phe 270 ggc act cac Gly Thr His ccc atc cga cac Pro Ile Arg His atg gtg acc gcc Met Val Thr Ala ttg Leu 285 tgg cac Trp, His 290 cca. gag cat ttc Pro Glu His Phe tgc gtc agt tgc Cys Val Ser Cys gag ccc ttc gga Giu Pro Phe Gly gat Asp 305 gag ggt ttc cac Glu Gly Phe His gag Glu 310 cgc gag ggc cgc Arg Glu Gly Arg ccc Pro 315 tac tgc cgc cgg Tyr Cys Arg Arg gac Asp 320 912 960 1008 ttc ctg cag ctg Phe Leu Gin Leu gcc ccg cgc tgc Ala Pro Arg Cys cag Gin 330 ggc tgc cag ggc Gly Cys Gin Gly ccc atc Pro Ile 335 WO 00/04152 WO 00/4 152PCT/US99/16122 ctg gat aac tac atc tcg gcg ctc agc ctg ctc tgg cac ccg gac tgt 1056 Leu Asp Asn Tyr Ile Ser Ala Leu Ser Leu Leu Trp His Pro Asp Cys 340 345 350 ttc gtc tgc agg gaa tgc ttc gcg ccc ttc tcg gga ggc agc ttt ttc 1104 Phe Val Cys Arg Glu Cys Phe Ala Pro Phe Ser Gly Gly Ser Phe Phe 355 360 365 gag cac gag ggc cgc ccg ttg tgc gag aac cac ttc cac gca cga cgc 1152 Giu His Giu Giy Arg Pro Leu Cys Glu Asn His Phe His Ala Arg Arg 370 375 380 ggc tcg ctg tgc ccc acg tgt ggc ctc cct gtg acc ggc cgc tgc gtg 1200 Gly Ser Leu Cys Pro Thr Cys Gly Leu Pro Val Thr Gly Arg Cys Val.

385 390 395 400 tcg gcc ctg ggt cgc cgc ttc cac ccg gac cac ttc gca tgc acc ttc 1248 Ser Ala Leu Gly Arg Arg Phe His Pro Asp His Phe Ala Cys Thr Phe 405 410 415 tgo ctg cgc ccg ctc acc aag ggg toc 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 Giy Ala Pro Lys Giu Arg Pro Ala Giu Pro Leu Thr 1 5 10 Pro Pro Pro Ser Tyr Gly His Gin Pro Thr Gly Gin Ser Gly Giu Ser 25 Ser Gly Ala Ser Gly 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 55 Ser Ser Gly Val Leu Gly Thr Gly Leu Cys Giu Leu Asp Arg Leu Leu 70 75 Gin Giu Leu Asn Ala Thr Gin Phe Asn Ile Thr Asp Glu Ile Met Ser 90 9 WO 00/04152 PCT/US99/16122 Gin Phe Pro Ser Ser Lys Val Ala Ser Gly Glu Gln Lys Glu Asp Gin 100 105 110 Ser Glu Asp Lys Lys Arg Pro Ser Leu Pro Ser Ser Pro Ser Pro Gly 115 120 125 Leu Pro Lys Ala Ser Ala Thr Ser Ala Thr Leu Glu Leu Asp Arg Leu 130 135 140 Met Ala Ser Leu Pro Asp Phe Arg Val Gln Asn His Leu Pro Ala Ser 145 150 155 160 Gly Pro Thr Gln Pro Pro Val Val Ser Ser Thr Asn Glu Gly Ser Pro 165 170 175 Ser Pro Pro Glu Pro Thr Ala Lys Gly Ser Leu Asp Thr Met Leu Gly 180 185 190 Leu Leu Gin Ser Asp Leu Ser Arg Arg Gly Val Pro Thr Gin Ala Lys 195 200 205 Gly Leu Cys Gly Ser Cys Asn Lys Pro Ile Ala Gly Gin Val Val Thr 210 215 220 Ala Leu Gly Arg Ala Trp His Pro Glu His Phe Val Cys Gly Gly Cys 225 230 235 240 Ser Thr Ala Leu Gly Gly Ser Ser Phe Phe Glu Lys Asp Gly Ala Pro 245 250 255 Phe Cys Pro Glu Cys Tyr Phe Glu Arg Phe Ser Pro Arg Cys Gly Phe 260 265 270 Cys Asn Gin Pro Ile Arg His Lys Met Val Thr Ala Leu Gly Thr His 275 280 285 Trp His Pro Glu His Phe Cys Cys Val Ser Cys Gly Glu Pro Phe Gly 290 295 300 Asp Glu Gly Phe His Glu Arg Glu Gly Arg Pro Tyr Cys Arg Arg Asp 305 310 315 320 Phe Leu Gin Leu Phe Ala Pro Arg Cys Gin Gly Cys Gin Gly Pro Ile 325 330 335 Leu Asp Asn Tyr Ile Ser Ala Leu Ser Leu Leu Trp His Pro Asp Cys 340 345 350 Phe Val Cys Arg Glu Cys Phe Ala Pro Phe Ser Gly Gly Ser Phe Phe 355 360 365 WO 00/04152 PCTIUS99/1 6122 Glu His Glu Gly Arg Pro Leu Cys Glu Asn His Phe His Ala Arg Arg 370 375 380 Gly Ser Leu Cys Pro Thr Cys Gly Leu Pro Val Thr Gly Arg Cys Val 385 390 395 400 Ser Ala Leu Gly Arg Arg Phe His Pro Asp His Phe Ala Cys Thr Phe 405 410 415 Cys Leu Arg Pro Leu Thr Lys Gly Ser Phe 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> 31UTrR <222> (676) (1566) <22 0> <221> 5 1 TTR <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 cag ttc aaa ctt gta ttg gtt ggt 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 Giu 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 WO 00/04152 WO 0004152PCT/1US99/1 6122 cct att aag ttc Pro Ile Lys Phe gga ctg aga gat Gly Leu Arg Asp aat gta tgg gac aca gcc ggc cag gag aaa ttc ggt Asn Vai Trp Asp Thr Aia Giy Gin Giu Lys Phe Gly ggc tat Gly Tyr tat Tyr 80 caa gcc cag Gin Ala Gin tgt Cys atc ata atg Ile Ile Met ttt gat Phe Asp gta aca tcg Vai Thr Ser aga Arg aga Arg gtg Val gtt act tac aag Vai Thr Tyr Lys gtg cct aac tgg Vai Pro Asn Trp gat ctg gta Asp Leu Vai cga Arg att Ile tgt gaa aac Cys Giu Asn att gtg ttg Ile Val Leu tgt ggc Cys Gly 120 aac aaa gtg Asn Lys Val ttc cac cga Phe His Arg 140 aac tac aac Asn Tyr Asn aag gac agg Lys Asp Arg aag gcg aaa Lys Ala Lys aag aat ctt.

Lys Asn Leu cag Gin 145 ttc Phe tac gac att- Tyr Asp Ile tct Ser 150 agg Axg tcc att gtc Ser Ile Val 135 gcc aaa agt Aia Lys Ser aag ctc att Lys Leu Ile 243 291 ~339 387 435 483 531 579 627 675 ttt gaa aag Phe Giu Lys ctc tgg ctt Leu Trp Leu 155 gga gac Gly Asp cct aac ttg Pro Asn Leu 170 gaa Glu gaa Giu 175 cca Pro gtt gcc atg Val Ala Met ctc gcc cca Leu Ala Pro gtt gtc atg Val Val Met gct, ttg gca Ala Leu Ala tat gag cac Tyr Giu His gac tta Asp Leu 200 ctg tga Leu gag gtt gct. cag aca act gct ctc ccg gat gag gat gat gac Giu Val Ala Gin Thr Thr Ala Leu Pro Asp Glu Asp Asp Asp 205 210 215 gaatgaagct agcggtgcag tgtgggatgc ttcattgttt tataagactg ttcccattcc ggagc ccagc cgtgtgtgcc tgaaggagat ggacctgcat ctgcagtcac ttttctttag gtcagaagtc tagttttata acctcattat tatctagcta gagtgggctt cggagtgaat atttagctgt ttggacgcag atcacaatat tcagtggtga aatcagaata aagttgtatt ggcagctgtc ctgtgatgtc agcggaacat gtgctttatc gtggcagttt aaaaaataac ttgattcctt gagtttcata aatcttgttt gttactgtca tcaaatatct aagcaagtga 735 795 855 915 975 1035 WO 00/04152 WO 0004152PCT/US9916122 actcatccct tgttaatatt tccctatgtt cagtattcta cagtgtttgc aatctgtgac taatattttc aacatcagtg cactttttgt tgtttataaa tgaattgcct tatttttgta tttggttaga tccaccttca ctgtcagaat ttttatggca gatggcagga ttgaatgtta t agca t ttgg tgCttttatc catttgagcc agggttacat tat tggc tag aaaaatgtgg aaagtaatca ggttgggaat 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 9 1095 1155 1215 1275 1335 1395 1455 1515 1566 <210> 6 '<211> 216 <212> PRT <213> Homo sapie <400> 6 Met Ala Ala Gin 1 Gly Asp Gly Giy Gly Glu Pro Gin Val Gin Phe Lys Leu Val Leu Val 5 10 Thr Gly Lys Thr Thr Phe Val Lys Arg His Leu Thr 25 Lys Lys Tyr Val Ala Thr Leu Gly Val Giu Val His Glu Gly Giu Phe Pro Leu Val so Phe His Thr Gly Pro Ile Lys Phe Asn Val Trp Asp Ile Thr Ala Giy Gin Phe Gly Gly Arg Asp Gly Tyr Gin Ala Gin Cys Val Ala Ile Ile Met Asp Val Thr Ser Thr Tyr Lys Glu Asn Ile 115 Arg Lys Val 130 Pro Asn Trp Asp Leu Val Arg Val Val Cys Lys Asp Pro Ile Val Leu Lys Ala Lys Ser 135 Asn Lys Val Asp 125 Lys Val Phe His Lys Asn Leu WO 00/04152 PCT/US99/1 6122 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 Leu Glu Phe 165 170 175 Val Ala Met Pro Ala Leu Ala Pro Pro Glu Val Val Met Asp Pro Ala 180 185 190 Leu Ala Ala Gin Tyr Glu His Asp Leu Glu 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 cccggcgctc ctccacagct cgctggctcc 120 cgccgcggaa aggcgtc atg ccg ccc aaa. acc ccc cga aaa acg gcc gcc 170 Met Pro Pro Lys Thr Pro Arg Lys Thr Ala Ala 1 5 acc gcc gcC gct gcc gcc gcg gaa. ccc ccg gca ccg ccg ccg ccg ccc 218 Thr Ala Ala Ala Ala Ala Ala Glu Pro Pro Ala Pro Pro Pro Pro Pro 20 cct Oct gag gag gac cca gag cag gac agc ggc ccg gag gac ctg cct 266 Pro Pro Glu 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 Glu Phe Glu Glu Thr Glu Glu Pro Asp Phe Thr Ala 50 tta tgt cag aaa tta aag ata cca gat cat gtc aga gag aga gct tgg 362 Leu Cys Gin Lys Leu Lys Ile Pro Asp His Val Arg Glu Arg Ala Trp, 65 70 WO 00/04152 WO 0004152PCT/JS99/1 6122 tta act tgg gag Leu Thr Trp Giu gtt tca tct gtg Val Ser Ser Val gga gta ttg gga Gly Val Leu Gly ggt tat Gly Tyr att caa aag Ile Gin Lys aag gaa ctg tgg Lys Giu Leu Trp atc tgt atc ttt att gca gca Ile Cys Ile Phe Ile Aia Ala 105 gtt gac cta gat gag atg tcg Val Asp Leu Asp Glu Met Ser 110 ata gaa atc agt gtc cat aaa Ile Giu Ile Ser Val His Lys 125 130 act ttt act gag Thr Phe Thr Glu cta Leu 120 cag aaa aac Gin Lys Asn ttc ttt aac tta Cta aaa gaa att gat Phe Phe Asn Leu Leu Lys Giu Ile Asp acc Thr 140 agt acc aaa gtt Ser Thr Lys Val aat gct atg tca aga ctg ttg aag aag Asn Ala Met Ser Arg Leu Leu Lys Lys 150 tat Tyr 155 gat gta ttg ttt Asp Val Leu Phe gca Ala 160 ctc ttc agc aaa Leu Phe Ser Lys ttg Leu 165 gaa agg aca. tgt Glu Arg Thr Cys gaa ctt Glu Leu 170 ata. tat ttg Ile Tyr Leu gca ttg gtg Ala Leu Val 190 aca Thr 175 caa ccc agc agt Gin Pro Ser Ser ata tct act gaa.

Ile Ser Thr Giu ata aat tct Ile Asn Ser 185 gct aaa ggg Ala Lys Gly 698 746 cta aaa gtt tct, Leu Lys Val Ser atc aca ttt tta Ile Thr Phe Leu tta Leu 200 gaa gta Glu Val 205 tta caa atg gaa.

Leu Gin Met Giu gat ctg gtg att tca ttt cag tta atg Asp Leu Val Ile Ser Phe Gin Leu Met 215 794 cta.

Leu 220 tgt gtc ctt gac Cys Val Leu Asp tat Tyr 225 ttt att aaa ctc Phe Ile Lys Leu cct ccc atg ttg Pro Pro Met Leu ctc Leu 235 842 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 245 cct cga Pro Arg 250 aca. ccc agg Thr Pro Arg gaa, aat gat Giu Asn Asp 270 cga Arg 255 ggt. cag aac agg Gly Gin Asn Arg gca cgg ata gca Ala Arg Ile Ala aaa caa cta Lys Gin Leu 265 cat gaa tgt His Glu Cys 938 986 aca aga att att Thr Arg Ile Ile gtt ctc tgt aaa Val Leu Cys Lys gaa Glu 280 WO 00/04152 WO 0004152PCT[US99/1 6122 aat ata Asn Ile 285 gat gag gtg aaa Asp Giu Val. Lys aat Asn 290 gtt tat ttc aaa Val Tyr Phe Lys aat Asn 295 ttt ata cct ttt Phe Ile Pro Phe atg Met 300 aat tct ctt gga Asn Ser Leu Giy gta aca tct aat Vai Thr Ser Asn cit cca gag gt Leu Pro Giu Val 1034 1082 1130 1178 aat ctt tct aaa Asn Leu Ser Lys cga Arg 320 tac gaa gaa at Tyr Giu Giu Ile tat Tyr 325 cit aaa aat aaa Leu Lys Asn Lys gat cta Asp Leu 330 gat gca aga Asp Ala Arg ata gac agt Ile Asp Ser 350 tta Leu 335 ttt tig gat cat Phe Leu Asp His aaa act cit cag act gat tct Lys Thr Leu Gin Thr Asp Ser 345 ttt gaa aca cag Phe Giu Thr Gin aca cca cga aaa Thr Pro Arg Lys agt Ser 360 aac ctt g~t Asn Leu Asp 1226 gaa gag Giu Giu 365 gtg aat gta att Val Asn Vai Ile cca cac act cca Pro His Thr Pro agg act git atg Arg Thr Vai Met act atc caa caa Thr Ile Gin Gin tta Leu 385 atg atg att tta Met Met Ile Leu aat Asn 390 tca gca agt gat Ser Ala Ser Asp caa Gin 395 1274 1322 1370 CCt tca gaa aat Pro Ser Giu Asn ctg Leu 400 att tcc tat tt Ile Ser Tyr Phe aac Asn 405 aac igc aca gtg Asn Cys Thr Vai aat cca Asn Pro 410 aaa gaa agt Lys Giu Ser gag aaa tt Giu Lys Phe 430 ctg aaa aga gig Leu Lys Arg Vai gat ata gga tac Asp Ile Gly Tyr atc ttt aaa Ile Phe Lys 425 ait gga tca Ile Giy Ser 1418 gct aaa gct. gtg gga cag ggt tgt gic Ala Lys Aia Vai Giy Gin Gly Cys Vai gaa Giu 440 cag cga Gin Arg 445 tac aaa ctt gga Tyr Lys Leu Gly cgc ttg tat tac Arg Leu Tyr Tyr cga Arg 455 gia atg gaa tcc Val Met Giu Ser 1466 1514 1562 1610 atg Met 460 ctt aaa tca gaa Leu Lys Ser Giu gaa Giu 465 gaa cga tia icc Giu Arg Leu Ser ati Ile 470 caa aat ttt agc Gin Asn Phe Ser aaa Lys 475 cti cig aai gac Leu Leu Asn Asp aac Asn 480 att ttt cat atg Ile Phe His Met tci Ser 485 tia ttg gcg tgc Leu Leu Ala Cys gct ct Aia Leu 490 WO 00/04152 WO 0004152PCT/US99/I 6122 gag gtt gta atg gcc aca tat agc aga Giu Vai Val tct gga aca Ser Gly Thr 510 Ala Thr Tyr Ser agt aca tct cag Ser Thr Ser Gin aat ctt gat Asn Leu Asp 505 ctt aat tta Leu Asn Leu 1658 gat ttg tct ttc cca tgg att ctg aat Asp Leu Ser Phe Pro Trp Ile Leu Asn aaa gcc Lys Ala 525 ttt gat ttt tac Phe Asp Phe Tyr gtg atc gaa agt Val Ile Giu Ser ttt Phe 535 atc aaa gca gaa Ile Lys Ala Glu 1706 1754 1802 ggc Giy 540 aac ttg aca aga Asn Leu Thr Arg atg ata aaa cat Met Ile Lys His gaa cga tgt gaa cat Giu Arg Cys Giu His cga atc atg gaa Arg Ile Met Giu ctt gca tgg ctc Leu Ala Trp Leu tca Ser 565 gat tca cct tta Asp Ser Pro Leu ttt gat Phe Asp 570 1850 ctt att. aaa Leu Ile Lys tct gct tgt Ser Ala Cys 590 tca aag gac cga Ser Lys Asp Arg gaa Giu 580 gga cca act gat Gly Pro Thr Asp cac ctt gaa His Leu Giu 585 act gca gca Thr Ala Ala 1898 1946 cct ctt aat ctt.

Pro Leu Asn Leu ctc cag aat aat Leu Gin Asn Asn cac His 600 gat atg Asp Met 605 tat ctt tct cct, Tyr Leu Ser Pro gta Val 610 aga tct cca aag Arg Ser Pro Lys aaa Lys 615 aaa. ggt tca act Lys Gly Ser Thr acg Thr 620 cgt gta aat tct Arg Val Asn Ser act Thr 625 gca aat gca gag Ala Asn Ala Giu caa gca acc tca Gin Ala Thr Ser 1994 2042 2090 ttc cag acc cag Phe Gin Thr Gin aag Lys 640 cca ttg aaa tct Pro Leu Lys Ser tct ctt tca ctg Ser Leu Ser Leu ttt tat Phe Tyr 650 aaa, aaa gtg Lys Lys Val cgc ctt ctg Arg Leu Leu 670 tat Tyr 655 cgg cta gcc tat Arg Leu Ala Tyr ctc Leu 660 cgg cta aat aca Arg Leu Asn Thr ctt tgt gaa Leu Cys Giu 665 tgg acc ctt Trp Thr Leu 2138 2186 tct gag cac cca Ser Glu His Pro tta gaa cat atc Leu Giu His Ile atc Ile 680 ttc cag Phe Gin 685 cac acc ctg cag His Thr Leu Gin aat Asn 690 gag tat gaa ctc Giu Tyr Glu Leu atg Met 695 aga gac agg cat Arg Asp Arg His 2234 WO 00/04152 WO 0004152PCT[US99/16122 t tg Leu 700 gac caa att atg Asp Gin Ile Met tgt tcc atg tat Cys Ser Met Tyr ata tgc aaa gtg Ile Cys Lys Val 2282 2330 aat ata gac ctt Asn Ile Asp Leu ttc aaa atc att Phe Lys Ile Ile aca gca tac aag Thr Aia Tyr Lys gat ctt Asp Leu 730 cct cat gct Pro His Aia gag tat gat Giu Tyr Asp 750 cag gag aca ttc Gin Giu Thr Phe aaa Lys 740 cgt gtt ttg atc Arg Val Leu Ile aaa gaa gag Lys Glu Giu 745 atg cag aga Met Gin Arg 2378 2426 tct att ata gta Ser Ile Ile Val tat aac tcg gtc Tyr Asn Ser Vai ttc Phe 760 ctg aaa Leu Lys 765 aca aat att ttg Thr Asn Ile Leu cag Gin 770 tat gct. tcc acc Tyr Ala Ser -ir agg Arg 775 ccc cct acc ttg Pro Pro Thr Leu tca Ser 780 cca ata cct cac Pro Ile Pro His cct cga agc Cct Pro Arg Ser Pro aag. ttt cct agt.

Lys Phe Pro Ser tca Ser 795 2474 2522 2570 2618 ccc tta cgg att Pro Leu Arg Ile gga ggg aac atc Giy Gly Asn Ile att tca ccc ctg Ile Ser Pro Leu aag agt Lys Ser 810 act cca Thr Pro cca tat aaa Pro Tyr Lys aga tca aga.

Arg Ser Arg 830 gaa ggt ctg Giu Gly Leu cca aca aaa Pro Thr Lys atc tta gta tca Ile Leu Vai Ser att Ile 835 ggt gaa tca ttc Giy Giu Ser Phe ggg Giy 840 act tct gag Thr Ser Giu 2666 aag ttc cag aaa ata aat Lys Phe Gin Lys Ile Asn atg gta tgt aac Met Vai Cys Asn agc Ser 855 c ca Pro gac cgt gtg ctc Asp Arg Vai Leu 2714 2762 agt gct gaa Ser Ala Giu aac cct, oct Asn Pro Pro ctg aaa aaa Leu Lys Lys cgc ttt gat att gaa gga tca gat gaa Arg Phe Asp Ile Giu Giy Ser Asp Glu 880 gat gga. agt aaa Asp Giy Ser Lys cat ctc His Leu 890 2810 cca gga gag Pro Giy Giu tcc Ser 895 aaa ttt cag cag Lys Phe Gin Gin ctg gca gaa at~g Leu Ala Giu Met act tct act Thr Ser Thr 905 2858 WO 00/04152 PCTIUS99/16122 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 Glu Glu Lys 925 cacctctgga tggccacatt 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 tactcttggt 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.

agcagattgt ctagagtggg ataggtgatg aacaccctta.

caccaaaatt tactaatttt tttataaaat tatcatacta cttcatccaa.

ttcagatcac agatcttagg gggtcctgaa gagccttaat ccttaatttg gaactggcaa tgttcaaagc aggcattaat aaaaagaggc ttaacttatt actttcccag aatgtgcaga tttatacaag ttcCtcttcc 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 aagt acc cat 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 WO 00/04152 WO 0004152PCTIUS99/1 6122 ataggggata tttaaggtag aattattttt aaaaagaaat taagtttcaa acttactatt ctccatttca tcattgtttc ggcttactat ttctgggtct aggaacttca gagatcgtgt attgcttttt tgtattggtt aattaatagt ttgtctattt tgtgttcttt gtattaagta agataactct cctagtatct <210> 8 <211> 928 <212> PRT <213> Homo sapien Ct tcagc tag ctggtcttgt ttgacagtta tgcatgaata tttgctacta at tgaga tt t aaaactgtac taaaataaat cactaatgtt catcc cttttaggaa tagaaaacaa ttttgataac tcatacaaat agttcacatt cttaaataat atttaaaatt tagttgttaa ctcttttctg aatcactttg aattttattt aatgacacta cagttagttt agaattagtg gcttcagata gctatgttac gagtcttaat tctaggagaa tctaactcag tgtgctcatt gaaaacttga t taggtcaag ccagaatttt ttattgcttt tattttctac ggtctgatgt gatagataga 4334 4394 4454 4514 4574 4634 4694 4754 4814 4839 <400> 8 Met Pro Pro Lys Thr Pro Pro Arg Lys Thr Ala Ala Thr Ala Ala Ala Ala Ala Ala Glu Pro Glu Gin Phe Glu Glu Ala Pro Pro Pro Pro Pro Ser Gly Pro Glu 40 Asp Leu Pro Leu Val Cys Glu Glu Asp Arg Leu Glu Gin Lys Leu Thr Giu Glu Phe Thr Ala Lys Ile Leu Leu Pro Asp His Giu Arg Ala Thr Trp Glu Val Ser Ser Val Asp Ile Val Leu Gly Ile Gin Lys Lys Lys Asp Giu Giu Leu Trp Met Ser Phe Giy 100 Thr Cys Ile Phe Ala Val Asp Phe Thr Giu Leu Gin Lys Asn Ile Giu Ile Ser Val 120 125 WO 00/04152 WO 0004152PCT/US99/16122 His Lys Phe Phe Asn Leu Leu Lys Glu Ile Asp Thr Ser Thr Lys Val 130 135 140 Asp Asn Ala Met Ser Arg Leu Leu Lys Lys Tyr Asp Val Leu Phe Ala 145 150 155 160 Leu Phe Ser Lys Leu Giu Arg Thr Cys Giu Leu Ile Tyr Leu Thr Gin 165 170 175 Pro Ser Ser Ser Ile Ser Thr Giu Ile Asn Ser Ala Leu Val Leu Lys 180 185 190 Val Ser Trp Ile Thr Phe Leu Leu Ala Lys Gly Giu Val Leu Gin Met 195 200 205 Giu Asp Asp Leu Val Ile Ser Phe Gin Leu Met Leu Cys Val Leu Asp 210 215 220 Tyr Phe Ilie Lys Leu Ser Pro Pro Met Leu Leu Lys Giu Pro Tyr Lys 225 230 235 240 Thr Ala Val Ile Pro Ile Asn Giy Ser Pro Arg Thr Pro Arg Arg Gly 245 250 255 Gin Asn Arg Ser Ala Arg Ile Ala Lys Gin Leu Giu Asn Asp Thr Arg 260 265 270 Ile Ile Giu Val Leu Cys Lys Giu His Giu Cys Asn Ile Asp Glu Val 275 280 285 Lys Asn Val Tyr Phe Lys Asn Phe Ile Pro Phe Met Asn Ser Leu Gly 290 295 300 Leu Val Thr Ser Asn Giy Leu Pro Giu Val Glu Asn Leu Ser Lys Arg 305 310 315 320 Tyr Giu Giu Ile Tyr Leu Lys Asn Lys Asp Leu Asp Ala Arg Leu Phe 325 330 335 Leu Asp His Asp Lys Thr Leu Gin Thr Asp Ser Ile Asp Ser Phe Giu 340 345 350 Thr Gin Arg Thr Pro Arg Lys Ser Asn Leu Asp Giu Giu Val Asn Val 355 360 365 Ile Pro Pro His Thr Pro Val Arg Thr Val Met Asn Thr Ile Gin Gin 370 375 380 Leu Met Met Ile Leu Asn Ser Ala Ser Asp Gin Pro Ser Glu Asn Leu 385 390 395 400 WO 00/04152 Ile Ser Tyr Phe Asn Asn Cys Thr Val Asn Pro Lys Glu Ser Ile Leu 405 410 415 PCT[US99/I 6122 435 Gly Glu 465 Ile Thr Ser Tyr Giu 545 Leu Lys Asn Pro Thr 625 Pro Leu Val 450 Glu Phe Tyr Phe Lys 530 Met Ala Asp Leu Val 610 zeu Arg Arg His Ser Pro 515 Val Ile Trp Arg Pro 595 Arg Asn Lys Tyri Lyi 42( Gir Leu Leu Met Arg 500 Trp Ile Lys Leu Giu 580 Leu Ser k1a Ser zeu 660 Asp 1Gly ITyr Ser Ser 485 Ser Ile Giu His Ser 565 Gly Gin Pro Giu *j Thr s 645 Arg I Ile Cys Tyr Ile 470 Leu Thr Leu S er Leu 550 Asp Pro ksn .sys Lhr ;30 er ~eu Gly Tyr Val Glu 440 Arg Val 455 Gin Asn Leu Ala Ser Gin Asn Val 520 Phe Ile 535 Glu Arg Ser Pro Thr Asp Asn His 600 Lys Lys 615 Gin Ala Leu Ser Asn Thr Ile 425 Ile Met Phe Cys Asn 505 Leu Lys Cys Leu His 585 Thr

G

1 y rhr Lieu 665 Phe *Gly Giu Ser Ala 490 Leu Asn Ala Giu Phe 570 Leu Ala Ser Ser1 Phe 650 CysC Ly Sez Ser Lys 475 Leu Asp Leu Glu His 555 Asp Glu 1 .la rhr k.la.

~35 Eyr liu Glu Gin Met 460 Leu Glu Ser Lys Gly 540 Arg Leu Ser Asp Thr 620 Phe Lys Arg Lys Arg 445 Leu Leu Val Gly Ala 525 Asn Ile Ile Al a Met 605 Arg Gin Lys Leu Phe 430 Tyr Lys Asn Val Thr 510 Phe Leu Met Lys Cys 590 Tyr Val Thr Val Leu 670 Ala Lys Ser Asp Met 495 Asp Asp Thr Giu Gin 575 Pro Leu A.sn .ln ryr 655 3er Lys Leu Giu Asn 480 Al a Leu Phe Arg Ser 560 Ser Leu Ser Ser Lys 640 Arg Glu WO 00/04152PCUS/162 PCTIUS99/16122 His Pro Giu Leu Giu His Ile Ile Trp Thr Leu Phe Gin His Thr Le Gin Met 705 Phe Giu Ile Leu Ile 785 Giy Giu Val Asn Giy 865 Giy Phe Asn 690 Cys Lys Thr Val Gin 770 Pro Giy Gly Ser Gin 850 Ser Ser Gln 675 Giu Ser Ile Phe Phe 755 Tyr Arg Asn Leu Ile 835 Met Asn Asp Gin Tyr Met Ile Lys 740 Tyr Ala Ser Ile Pro 820 Giy Vai Pro Giu Lys 900 Giu Tyr Vai 725 Arg Asn Ser Pro Tyr 805 Thr Giu Cys Pro Ala 885 Leu Leu Giy 710 Thr Vai Ser Thr Tyr 790 Ile Pro Ser Asn Lys 870 Asp Ala Met 695 Ile Ala Leu Val Arg 775 Lys Ser Thr Phe Ser 855 Pro Giy Glu 680 Arg Cys Tyr Ile Phe 760 Pro Phe Pro Lys Giy 840 Asp Leu Ser Met *Asp Lys Lys Lys 745 Met Pro Pro Leu Met 825 Thr Arg Lys Lys Thr 905 Arg Vai Asp 730 Giu Gin Thr Ser Lys 810 Thr Ser Vai Lys His 890 Ser His Lys 715 Leu Giu Arg Leu Ser 795 Ser Pro Giu Leu Leu 875 Leu Thr Leu 700 Asn Pro Giu Leu Ser 780 Pro Pro Arg Lys Lys 860 Arg Pro Arg 685 Asp Ile His Tyr Lys 765 Pro Leu Tyr Ser Phe 845 Arg Phe Giy Thr Gin Asp Aila Asp 750 Thr Ile Arg Lys Arg 830 Gin Ser Asp Giu Arg 910 Ile Leu Vai 735 Ser Asn Pro Ile Ile 815 Ile Lys Ala Ile Ser 895 Met Met Lys 720 Gin Ile Ile His Pro 800 Ser Leu Ile Giu Giu 880 Lys Gin Lys Gin Lys Met Asn Asp Ser Met Asp Thr Ser Asn Lys Giu Giu Lys WO 00/04152 PCT/US99/16122 <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> <220> <221> VARIANT <222> (13) <220> <221> VARIANT <222> <220> <221> VARIANT <222> <220> <221> VARIANT <222> (20) (21) WO 00/04152 WO 00/41 52PCTIUS99/1 6122 <220> <221> VARIANTr <:222> <:220> <:221> VARIANT <222> <400> 11 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa 1 Xaa Xaa Cys Xaa Xaa Cys Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Cys Xaa His Xaa Xaa Xaa Cys Xaa Xaa Cys ':210> 12 <211> <212> PRT <213> Homo sapien <220> <221> VARIANT <222> <220> <221> VARIANT <222> <220> <221> VARIANT <222> (23) <220> <221> VARIANT <222> (26) <:220> <221> VARIANT <222> <:220> <221> VARIANT <222> <220> <:221> VARIANT <222> ':400> 12 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 WO 00/04 152 PCTIUS99/1 6122 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 3S 40 Xaa Cys

Claims (45)

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

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 ARA54 polypeptide set forth in SEQ ID NO: 2.

3. The isolated polynucleotide of claim 2 wherein the protein coding region comprises a sequence set forth in SEQ ID NO:1.

4. A eukaryotic host cell comprising the isolated polynucleotide of claim 2. to 5. A eukaryotic host cell according to claim 4, substantially as hereinbefore described with reference to any one of the Examples.

6. An isolated polynucleotide encoding the ARA54 polypeptide set forth in SEQ ID NO:2.

7. A genetic construct comprising a nucleic acid sequence encoding the ARA54 polypeptide of SEQ ID NO:2 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. An isolated ARA54 polypeptide comprising amino acids 361-471 of SEQ ID NO: 2 and a RING finger motif, wherein the ARA54 polypeptide is capable of enhancing the transcription activity of an androgen receptor or a progesterone receptor.

10. The ARA54 polypeptide of claim 9, wherein the ARA54 polypeptide is capable of enhancing the transcription activity of an androgen receptor or a progesterone receptor at least 2 fold. 25 11. The ARA54 polypeptide of claim 9, wherein the ARA54 polypeptide is capable of enhancing the transcription activity of an androgen receptor at least 6 fold.

12. The ARA54 polypeptide of claim 9, wherein the ARA54 polypeptide is capable of enhancing the transcription activity of a progesterone receptor at least fold.

13. The ARA54 polypeptide of claim 9, wherein the enhancement comprises an interaction between dihydrotestosterone (DHT) and the androgen receptor or the progesterone receptor.

14. An isolated ARA54 polypeptide comprising amino acids 361-471 of SEQ ID NO: 2 and a RING finger, wherein the ARA54 polypeptide is capable of enhancing the transcription activity of a mutant androgen receptor or a mutant progesterone [R:\LIBZZ]0618 sdoc mrT SI I receptor, causing an enhancement of the transcription activity of the mutant androgen receptor or the mutant progesterone receptor. The ARA54 polypeptide of claim 14, wherein the enhancement comprises an interaction between 17 estradiol (E2) and the mutant androgen receptor or the mutant progesterone receptor.

16. The ARA54 polypeptide of claim 14, wherein the enhancement comprises an interaction between hydroxyflutamide (HF) and the mutant androgen receptor or the mutant progesterone receptor.

17. The ARA54 polypeptide of claim 14, wherein the enhancement comprises to an interaction between dihydrotestosterone (DHT) and the mutant androgen receptor.

18. An isolated ARA54 polypeptide comprising amino acids 361-471 of SEQ ID NO: 2 and a RING finger motif according to claim 9 or 14, substantially as hereinbefore described with reference to any one of the Examples.

19. An isolated ARA54 polypeptide comprising amino acids 361-471 of SEQ ID NO: 2, wherein the ARA54 polypeptide is capable of inhibiting the transcription activity of an androgen receptor or a progesterone receptor. The ARA54 polypeptide of claim 19, wherein the ARA54 polypeptide is capable of inhibiting the transcription activity of an androgen receptor or a progesterone receptor up to 70 percent.

21. An isolated ARA54 polypeptide comprising a sequence corresponding to amino acids 361-471 of SEQ ID NO: 2, wherein the ARA54 polypeptide is capable of inhibiting the transcription activity of a mutant androgen receptor or a mutant progesterone receptor.

22. The ARA54 polypeptide of claim 19, wherein the inhibition comprises an 25 interaction between 17 estradiol (E2) and the mutant androgen receptor.

23. The ARA54 polypeptide of claim 19, wherein the ARA54 polypeptide is capable of inhibiting the transcription activity of an androgen receptor up to percent.

24. An isolated ARA54 polypeptide according to claim 19 or 21, substantially as hereinbefore described with reference to any one of the Examples. o*

25. A method of screening molecules for the ability to modulate androgen receptor activity comprising the steps of: a) cotransforming a suitable eukaryotic cell that expresses AR with a genetic construct, wherein the genetic construct comprises a promoter functional in a eukaryotic cell operably connected to a polynucleotide comprising a sequence that encodes an ARA54 polypeptide, and a construct (R:\LIBZZ]06185.doc:mrr f1. FEB. 2004 14:37 SPRUSON FERGUSON NO. 6213 P. 6 31 comprising at least a portion of an expressible androgen receptor sequence; b) culturing the cells in the presence of a candidate molecule; and c) assaying the transcriptional activity induced by the ARA54 polypeptide; wherein an increase or decrease in androgen receptor activity relative to the ARA54 polypeptide and AR without the molecule indicates modulation of androgen receptor activity.

26. A method of screening for molecules that modulate androgen receptor activity comprising the steps of: a) incubating a compound with a cell, wherein the cell is engineered to express ARA54, and b) assaying for androgen receptor activity; wherein an increase or decrease in androgen receptor activity relative to androgen receptor activity in the presence of ARA54 without the compound indicates modulation.

27. The method of claim 26, wherein the cell further comprises DHT.

28. The method of claim 26 or 27, further comprising identifying compounds that inhibit androgen receptor activity. Is 29. A method of screening for molecules that modulate progesterone receptor activity comprising the steps of: a) incubating a compound with a cell, wherein the cell is engineered to express ARA54, and b) assaying for progesterone receptor activity; wherein an increase or decrease in progesterone receptor activity relative to progesterone receptor activity in the presence of ARA54 without the compound indicates modulation. The method of claim 29, wherein the cell further comprises DHT.

31. The method of claim 29 or claim 30, further comprising identifying compounds that inhibit progesterone receptor activity.

32. The method of claim 28 or 31 further comprising synthesizing the compound.

33. A method of screening for molecules that modulate mutant androgen receptor activity comprising the steps of: a) incubating a compound with a cell, wherein the cell is engineered to express ARA54, and b) assaying for mutant androgen receptor activity; wherein an increase or decrease in mutant androgen receptor activity relative to mutant androgen receptor activity in the presence of ARA54 without the compound indicates modulation.

34. The method of claim 33, wherein the mutant androgen receptor is Art877s. The method of claim 33, wherein the mutant androgen receptor is Are708k.

36. The method of claim 33, wherein the cell further comprises E2. (;\UsZZM G 115.doc:KOa COMS ID No: SMBI-00613183 Received by IP Australia: Time 14:44 Date 2004-02-11 .11. FEB.. 2004 14:37 SPRUSON FERGUSON NO. 6213 P. 7 32

37. The method of claim 33, wherein the cell further comprises HF.

38. A method of screening for molecules that modulate androgen receptor activity comprising the steps of: a) incubating a compound with an ARA54-androgen receptor complex, and b) assaying whether the compound decreases the amount of the s ARA54-androgen receptor complex.

39. A method of screening molecules that modulate progesterone receptor activity comprising the steps of: a) incubating a compound with an ARA54- progesterone receptor complex, and b) assaying whether the compound decreases the amount of the ARA54-progesterone receptor complex.

40. A method of screening molecules according to any one of claims 25 to 39, wherein said ARA54 polypeptide is an ARA54 polypeptide, substantially as hereinbefore described with reference to any one of the Examples.

41. A genetic construct comprising a nucleic acid encoding the polypeptide of anyone of claims 9 to 24. is 42. A method of making a polypeptide according to any one of claims 9 to 24 comprising transforming a cell with a nucleic acid encoding the polypeptide according to any one of claims 9 to 24.

43. A product produced by the process of claim 42.

44. A method of screening for molecules that modulate androgen receptor coactivation by ARA54 comprising the steps of: a) incubating a compound with a cell, wherein the cell comprises ARA54, b) incubating a compound with a cell, wherein the cell lacks ARA54, and c) assaying for androgen receptor activity; wherein an increase or decrease in androgen receptor activity relative to androgen receptor activity in the presence of ARA54 without the compound, and relative to androgen receptor activity in 2s the absence of ARA54 with the compound, indicates modulation. The method of claim 44, wherein the cell of one or both of step and step further comprises DHT.

46. The method of claim 44 or 45, further comprising identifying compounds that :inhibit androgen receptor activity, 30 47. A method of screening for molecules that modulate progesterone receptor coactivation by ARA54 comprising the steps of: a) incubating a compound with a cell, wherein the cell comprises ARA54, b) incubating a compound with a cell, wherein the cell lacks ARA54, and c) assaying for progesterone receptor activity; wherein an increase or decrease in progesterone receptor activity relative to progesterone receptor activity in RAJR,7,ZIORIS.&1c0 COMS ID No: SMBI-00613183 Received by IP Australia: Time 14:44 Date 2004-02-11 '11. FEB.2004 14: 37 SPRUSON FERGUSON NO. 62 13 P. 8 33 the presence of ARA54 without the compound, and relative to progesterone receptor activity in the absence of ARA54 with the compound, indicates modulation.

48. The method of claim 47, wherein the cell of one or both of step and step fiuther comprises DHT.

49. The method of claim 47 or 48, further comprising identifying compounds that inhibit progesterone activity. The method of claim 46 or 49 further comprising synthesizing the compound.

51. A method of screening for molecules that modulate mutant androgen receptor coactivation by ARA54 comprising the steps of: a) incubating a compound 1o with a cell, wherein the cell comprises ARA54, b) incubating a compound with a cell, wherein the cell lacks ARAS4, and c) assaying for mutant androgen receptor activity; wherein an increase or decrease in mutant andirogen receptor activity relative to androgen receptor activity in the presence of ARA54 without the compound, and relative to mutant androgen receptor activity in the absence of ARA54 with the compound indicates modulation.

52. The method of claim 5 1, wherein the mutant androgen receptor is ArtS77s.

53. The method of claim 51, wherein the mutant androgen receptor is ArMOMk.

54. The method of claim 51, wherein the cell of one or both of step and .2 step further comprises E2. step 5. The method of claim 51, wherein the cell of one or both of step and stp(b) further comprises HP. *56. The method of claim 33 or claim 51, further comprising identifying *compounds that modulate mutant androgen receptor activity.

57. The method of claim 38, further comprising identifying compounds that modulate androgen receptor activity.

58. The method of claim 56 or 57 further comprising synthesizing the compound. A method according to any one of claims 25 to 58, substantially as hereinbefore described with reference to any one of the examples. Dated 11 February, 2004 University of Rochester Patent Attorneys for the Applicant/Nominated Person SPRUSOCN FERGUSON 1(03 COMS ID No: SMBI-0081 3183 Received by IP Australia: Time 14:44 Date 2004-02-11