The Molecular Mechanisms of Coactivator Utilization in Ligand-dependent Transactivation by the Androgen Receptor

Abstract

Androgens drive sex differentiation, bone and muscle development, and promote growth of hormone-dependent cancers by binding the nuclear androgen receptor (AR), which recruits coactivators to responsive genes. Most nuclear receptors recruit steroid receptor coactivators (SRCs) to their ligand binding domain (LBD) using a leucine-rich motif (LXXLL). AR is believed to recruit unique coactivators to its LBD using an aromatic-rich motif (FXXLF) while recruiting SRCs to its N-terminal domain (NTD) through an alternate mechanism. Here, we report that the AR-LBD interacts with both FXXLF motifs and a subset of LXXLL motifs and that contacts with these LXXLL motifs are both necessary and sufficient for SRC-mediated AR regulation of transcription. Crystal structures of the activated AR in complex with both recruitment motifs reveal that side chains unique to the AR-LBD rearrange to bind either the bulky FXXLF motifs or the more compact LXXLL motifs and that AR utilizes subsidiary contacts with LXXLL flanking sequences to discriminate between LXXLL motifs. Androgens drive sex differentiation, bone and muscle development, and promote growth of hormone-dependent cancers by binding the nuclear androgen receptor (AR), which recruits coactivators to responsive genes. Most nuclear receptors recruit steroid receptor coactivators (SRCs) to their ligand binding domain (LBD) using a leucine-rich motif (LXXLL). AR is believed to recruit unique coactivators to its LBD using an aromatic-rich motif (FXXLF) while recruiting SRCs to its N-terminal domain (NTD) through an alternate mechanism. Here, we report that the AR-LBD interacts with both FXXLF motifs and a subset of LXXLL motifs and that contacts with these LXXLL motifs are both necessary and sufficient for SRC-mediated AR regulation of transcription. Crystal structures of the activated AR in complex with both recruitment motifs reveal that side chains unique to the AR-LBD rearrange to bind either the bulky FXXLF motifs or the more compact LXXLL motifs and that AR utilizes subsidiary contacts with LXXLL flanking sequences to discriminate between LXXLL motifs. The cellular effects of the hormone 5-α-dihydrotestosterone (DHT) 1The abbreviations used are: DHT, 5-α-dihydrotestosterone; AR, androgen receptor; NR, nuclear receptor; Fmoc, N-(9-fluorenyl)methoxycarbonyl; SRC, steroid receptor coactivator; LBD, ligand binding domain; NTD, N-terminal domain; MMTV, mouse mammary tumor virus; LTR, long terminal repeat; GST, glutathione S-transferase; DBD, DNA binding domain; CMV, cytomegalovirus; TR, thyroid receptor; AF, activation function. are mediated by the androgen receptor (AR), a member of the nuclear hormone receptor superfamily (1Lee H.J. Chang C. Cell Mol. Life Sci. 2003; 60: 1613-1622Crossref PubMed Scopus (139) Google Scholar). AR is absolutely required for normal male development, plays a variety of important roles in metabolism and homeostasis in adult men and women (2Liu P.Y. Death A.K. Handelsman D.J. Endocr. Rev. 2003; 24: 313-340Crossref PubMed Scopus (631) Google Scholar, 3Legros J.J. Charlier C. Bouillon G. Plomteux G. Ann. Endocrinol. (Paris). 2003; 64: 136PubMed Google Scholar), and is required for prostate cancer growth. Consequently, AR is a major target for pharmaceutical development and the recognized target for existing prostate cancer therapies, including androgen withdrawal and antiandrogens (1Lee H.J. Chang C. Cell Mol. Life Sci. 2003; 60: 1613-1622Crossref PubMed Scopus (139) Google Scholar, 4Gregory C.W. He B. Johnson R.T. Ford O.H. Mohler J.L. French F.S. Wilson E.M. Cancer Res. 2001; 61: 4315-4319PubMed Google Scholar, 5Culig Z. Klocker H. Bartsch G. Hobisch A. Endocr. Relat. Cancer. 2002; 9: 155-170Crossref PubMed Scopus (201) Google Scholar, 6Santos A.F. Huang H. Tindall D.J. Steroids. 2004; 69: 79-85Crossref PubMed Scopus (63) Google Scholar). It is nonetheless desirable to obtain new antiandrogens that spare patients from harmful side-effects and inhibit AR action in secondary hormone-resistant prostate cancer, where AR action becomes sensitized to low levels of androgens or existing antiandrogens (6Santos A.F. Huang H. Tindall D.J. Steroids. 2004; 69: 79-85Crossref PubMed Scopus (63) Google Scholar, 7Balk S.P. Urology. 2002; 60 (discussion 138-139): 132-138Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Improved understanding of AR signaling pathways will facilitate development of these compounds. Like most nuclear receptors (NRs), AR activity depends on interactions with members of the steroid receptor coactivator (SRC) family (1Lee H.J. Chang C. Cell Mol. Life Sci. 2003; 60: 1613-1622Crossref PubMed Scopus (139) Google Scholar, 8Ding X.F. Anderson C.M. Ma H. Hong H. Uht R.M. Kushner P.J. Stallcup M.R. Mol. Endocrinol. 1998; 12: 302-313Crossref PubMed Google Scholar, 9Berrevoets C.A. Doesburg P. Steketee K. Trapman J. Brinkmann A.O. Mol. Endocrinol. 1998; 12: 1172-1183Crossref PubMed Google Scholar). Several lines of evidence indicate that AR contacts with SRCs are important in prostate cancer. First, androgens promote SRC recruitment to the androgen-regulated prostate-specific antigen promoter, and this event is inhibited by the antiandrogen flutamide (10Shang Y. Myers M. Brown M. Mol. Cell. 2002; 9: 601-610Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar). Second, exogenous SRC2 (GRIP1/TIF2) promotes the androgen-dependent progression from the G1 to S phase in LNCaP prostate tumor cells, in a manner that requires specific AR contact (10Shang Y. Myers M. Brown M. Mol. Cell. 2002; 9: 601-610Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar). Third, SRCs often become expressed at high levels in prostate cancers (5Culig Z. Klocker H. Bartsch G. Hobisch A. Endocr. Relat. Cancer. 2002; 9: 155-170Crossref PubMed Scopus (201) Google Scholar). Finally, AR contacts with SRCs mediate hormone-independent AR signaling in conditions that resemble secondary prostate cancer (11Gregory C.W. Fei X. Ponguta L.A. He B. Bill H.M. French F.S. Wilson E.M. J. Biol. Chem. 2004; 279: 7119-7130Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 12Blaszczyk N. Masri B.A. Mawji N.R. Ueda T. McAlinden G. Duncan C.P. Bruchovsky N. Schweikert H.U. Schnabel D. Jones E.C. Sadar M.D. Clin. Cancer Res. 2004; 10: 1860-1869Crossref PubMed Scopus (55) Google Scholar). Thus, strategies to inhibit AR contacts with SRCs could be useful in blocking prostate cancer cell growth. For many NRs, overall transcriptional activity stems mostly from the hormone-dependent activation function (AF-2) within the NRs ligand binding domain (LBD), and involves interaction between a conserved hydrophobic cleft on the surface of the LBD and short leucine-rich hydrophobic motifs (NR boxes, consensus LXXLL motif) reiterated within each SRC (13Needham M. Raines S. McPheat J. Stacey C. Ellston J. Hoare S. Parker M. J. Steroid Biochem. Mol. Biol. 2000; 72: 35-46Crossref PubMed Scopus (72) Google Scholar, 14Leo C. Chen J.D. Gene (Amst.). 2000; 245: 1-11Crossref PubMed Scopus (442) Google Scholar). In contrast, current models of AR action suggest that AR activity stems from a potent hormone-independent activation function, AF-1, within the N-terminal domain (NTD) of the AR and emphasize the role of contacts between NTD and glutamine-rich sequences within the SRC C terminus in SRC recruitment (15Alen P. Claessens F. Schoenmakers E. Swinnen J.V. Verhoeven G. Rombauts W. Peeters B. Mol. Endocrinol. 1999; 13: 117-128Crossref PubMed Scopus (117) Google Scholar, 16He B. Lee L.W. Minges J.T. Wilson E.M. J. Biol. Chem. 2002; 277: 25631-25639Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 17Ma H. Hong H. Huang S.M. Irvine R.A. Webb P. Kushner P.J. Coetzee G.A. Stallcup M.R. Mol. Cell. Biol. 1999; 19: 6164-6173Crossref PubMed Scopus (215) Google Scholar, 18Christiaens V. Bevan C.L. Callewaert L. Haelens A. Verrijdt G. Rombauts W. Claessens F. J. Biol. Chem. 2002; 277: 49230-49237Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 19Powell S.M. Christiaens V. Voulgaraki D. Waxman J. Claessens F. Bevan C.L. Endocr. Relat. Cancer. 2004; 11: 117-130Crossref PubMed Scopus (65) Google Scholar). The AR-LBD is proposed to bind LXXLL motifs weakly and, instead, bind preferentially to aromatic-rich motifs that are found within the AR NTD (FQNLF and WHTLF) and AR-specific coactivators such as ARA70 (16He B. Lee L.W. Minges J.T. Wilson E.M. J. Biol. Chem. 2002; 277: 25631-25639Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 20He B. Minges J.T. Lee L.W. Wilson E.M. J. Biol. Chem. 2002; 277: 10226-10235Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 21He B. Wilson E.M. Mol. Genet. Metab. 2002; 75: 293-298Crossref PubMed Scopus (79) Google Scholar, 22Zhou Z.X. He B. Hall S.H. Wilson E.M. French F.S. Mol. Endocrinol. 2002; 16: 287-300Crossref PubMed Scopus (43) Google Scholar, 23He B. Kemppainen J.A. Wilson E.M. J. Biol. Chem. 2000; 275: 22986-22994Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar). The intramolecular interactions between the LBD and the NTD FQNLF motif promote formation of head to tail dimers (N-C interaction), which render the AF-2 surface unavailable for direct cofactor contacts (21He B. Wilson E.M. Mol. Genet. Metab. 2002; 75: 293-298Crossref PubMed Scopus (79) Google Scholar). Together, the notion that AR AF-2 binds coactivators weakly, and the fact that it will be occluded by the N-C to the that AR AF-2 an role in SRC lines of evidence suggest that AR AF-2 to coactivator recruitment in First, the N-C interaction is required for AR activity at including of prostate-specific and at including of the and the mouse mammary tumor terminal (16He B. Lee L.W. Minges J.T. Wilson E.M. J. Biol. Chem. 2002; 277: 25631-25639Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Thus, AF-2 be for coactivator contacts in Second, of AR AF-2 sequences within target coactivators AR (16He B. Lee L.W. Minges J.T. Wilson E.M. J. Biol. Chem. 2002; 277: 25631-25639Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 19Powell S.M. Christiaens V. Voulgaraki D. Waxman J. Claessens F. Bevan C.L. Endocr. Relat. Cancer. 2004; 11: 117-130Crossref PubMed Scopus (65) Google Scholar, 20He B. Minges J.T. Lee L.W. Wilson E.M. J. Biol. Chem. 2002; 277: 10226-10235Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Thus, of FXXLF motifs within AR-specific coactivators such as ARA70 their to with AR and AF-2 the notion that AR AF-2 contacts with LXXLL motifs are of SRC LXXLL motifs AR SRCs are AR NTD FQNLF and motifs are or AR at such as the It is important to the overall of AR to and the of these to strategies to inhibit AR activity in prostate cancer. In this we AR AF-2 interactions with target that AR AF-2 binds FXXLF that AR AF-2 binds a subset of SRC LXXLL motifs with and, that the LXXLL motifs are required to mediate AR AF-2 Crystal structures of AR-LBD in complex with FXXLF and LXXLL reveal the for these coactivator binding and suggest new to and expressed in and to using a of P. M. C. S. N. S. P. A. S. M. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). cell at or to high at in with AR-LBD expressed by with for at and cell by and an using a by of the with the from in that we for the of which AR in of with the motif of C where C is is is and is The sequences of the from and The in using in with in The using and in by the through a of and the The from the with and using a by used to the The to the N-terminal the using and using using a in the for AR-LBD from to in binding ligand in of to of in of and The to for using on an for each in using and the by to the and AR expressed in a AR-LBD or AR-LBD expressed in E. as a and to glutathione to the by of by with of or AR in and to a of The at for in of binding The in by using and by Cell and and in steroid and For and in and of of AR or and of at and to and for at with androgen or and by of of For with AR, the the to For with LBD, and the of a and using the and to the and of and ARA70 and AR-LBD by at for with of and by using from The complex with the or and of the and to these to new necessary to that the of either or to a a to AR-LBD at for either or to a new of for The using and using the at the at the and to and of ARA70 with AR-LBD and a at the to and The and using the J. T. Sci. S. A. 2004; PubMed Scopus Google Scholar). for AR-LBD structures using and from Biol. 1998; PubMed Scopus Google Scholar). The the that the for the the of the the for more flanking of the The for the as a short of the as an as such such on the AR-LBD and as an including the in the of for phase for each of the of the a of the and to more that in the using the using the of the and with using the target of and R.A. A. Scopus Google Scholar). Several of and using in structures with that most of the the most or The for and of each of the be found in for and used of of most The of in a new The structures with the and the and AR AF-2 and with the of AR AF-2 coactivator we binding of the AR-LBD to a of from including both SRCs and AR specific coactivators are to bind to NRs with to the coactivator Stallcup M.R. Kushner P.J. J.D. 1998; 12: PubMed Scopus Google Scholar). AR-LBD to with of the an LXXLL motif the of AR-LBD with FXXLF sequences in ARA70 and the AR NTD (21He B. Wilson E.M. 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Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, F. J.D. Ann. N. Y. Sci. PubMed Scopus Google Scholar, J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). SRC2 and we the of SRC2 to AR AF-2 and for LXXLL motifs in this a the AR-LBD to the DNA binding function androgen-dependent transcriptional activity on a in cell and of SRC2 AR AF-2 activity AR AF-2 activity more potent that of AR in and in the of and as potent as that by and which bind a of SRCs from signaling in cells, the effects of and AF-2 are in cells, and AF-2 in T. T. J. F. Mol. Endocrinol. 2000; PubMed Scopus Google Scholar, J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Thus, are with the notion that AR AF-2 is potent T. T. J. F. Mol. Endocrinol. 2000; PubMed Scopus Google Scholar, J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus (69) Google and the notion that AR AF-2 or of to a for and to AR AF-2 both in and in In contrast, of SRC2 is required to mediate AF-2 in with of the of SRC2 for and with X.F. Anderson C.M. Ma H. Hong H. Uht R.M. Kushner P.J. Stallcup M.R. Mol. Endocrinol. 1998; 12: 302-313Crossref PubMed Google Scholar, Stallcup M.R. Kushner P.J. J.D. 1998; 12: PubMed Scopus Google Scholar). each SRC to activity of which binds the SRCs at a and in a manner that is of X.F. Anderson C.M. Ma H. Hong H. Uht R.M. Kushner P.J. Stallcup M.R. Mol. Endocrinol. 1998; 12: 302-313Crossref PubMed Google Scholar). Thus, the that AR of SRC2 a role in the of SRC2 to AR For these we an the N-C interaction is for AR activity at this promoter, and cells, AR AF-2 activity is in this cell Here, of AR signaling the H. Hong H. Huang S.M. Irvine R.A. Webb P. Kushner P.J. Coetzee G.A. Stallcup M.R. Mol. Cell. Biol. 1999; 19: 6164-6173Crossref PubMed Scopus (215) Google Scholar, 18Christiaens V. Bevan C.L. Callewaert L. Haelens A. Verrijdt G. Rombauts W. Claessens F. J. Biol. Chem. 2002; 277: 49230-49237Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 19Powell S.M. Christiaens V. Voulgaraki D. Waxman J. Claessens F. Bevan C.L. Endocr. Relat. Cancer. 2004; 11: 117-130Crossref PubMed Scopus (65) Google Scholar, C.L. Hoare S. Claessens F. Parker Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar). In of the action Thus, is between the of for AR and their for in the of the AR-LBD and of AR-LBD in with the for AR to SRC2 and AR binds aromatic-rich coactivator and a subset of SRC boxes, we structures of the AR-LBD in complex with and by with and ARA70 bind as a short the hydrophobic cleft by the the low to bind to AR-LBD through an that could be as an of the structures that the of the AR AF-2 to bind to both LXXLL and FXXLF motifs. The AR-LBD in complex with the to of this are in the and the interaction of hydrophobic surface from both that hydrophobic motif binds in the manner as in NRs with LXXLL coactivator motifs A.K. D. L. Kushner P.J. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, A.K. D. J.T. J.A. Biol. 2002; 9: Google Scholar, D.J. J.T. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, H. 2002; 9: Full Text PDF PubMed Scopus Google Scholar). The from the are and the from are The hydrophobic motif of the to a short that binds in the by and The LBD interacts with the hydrophobic of the by the side chains of the LXXLL motif and The side of is within the and contacts with the side chains of and The side of is within the and contacts with the side chains of and The side of the contacts with the side chains of and The LBD in hydrophobic contacts with the are and and and and the of and and The of and from the with which is conserved in NRs and the of a that the to on the of of AR surface B. Wilson E.M. Mol. Cell. Biol. 2003; PubMed Scopus Google Scholar), and with a D.J. J.T. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), the to the conserved on the in hydrophobic contacts with and the to the of the is is The in to in its at with on the AR surface that a to on the of a D.J. J.T. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). the in the current for N-terminal to the hydrophobic motif and for more to the in complex with a LBD to The AR-LBD in complex with the to of this are in the and the interaction of hydrophobic surface from both that hydrophobic motif binds in the manner as for The LBD in hydrophobic contacts with the are and and and the of and is and with in this complex the role the interaction is in the in this for at the terminus that to of the of AR, and this is the that such are in an are in a The AR-LBD complex with the the the fact that the of to the that for the more to and that its for AR-LBD is It to that The to the and as a short a more to a could be and as the In the at the of the and at this from the within the of the and the to the for and the is and as a and at these is and for the of the the is in the the side chains in of hydrophobic surface in of hydrophobic surface from both In the the side of is within the and contacts with the side chains of and The side of contacts with the side of The side of the contacts with the side of The LBD in hydrophobic contacts with the are and and the of The interacts with through its In the interacts with through its to the conserved in either could be to for N-terminal that are the and interactions of the ARA70 FXXLF with the AR surface more the binding in structures of of SRC LXXLL motifs with and A.K. D. L. Kushner P.J. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, A.K. D. J.T. J.A. Biol. 2002; 9: Google Scholar, D.J. J.T. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, H. 2002; 9: Full Text PDF PubMed Scopus Google Scholar). The of side chains (FXXLF) that the hydrophobic of the coactivator a In at either of the and the The interaction is in the binding of this coactivator and its The AR-LBD in and of structures is that the that the AR-LBD on and on with FXXLF and LXXLL that absolutely required for SRC structures that is with the of the FXXLF that of the LXXLL in of the AR-LBD in complex with FXXLF and LXXLL from E. H. Biol. 2004; PubMed Scopus Google Scholar). the between the for in AR activity and its of contact with the LXXLL motif of in the we the effects of a of on AR AF-2 activity in and binding in a within the inhibited AR AF-2 activity and the recruitment of The of the normal at by of a and the to of the B. Wilson E.M. Mol. Cell. Biol. 2003; PubMed Scopus Google Scholar, T. T. J. F. Mol. Endocrinol. 2000; PubMed Scopus Google Scholar). that or at and AF-2 in the of SRC2 C.A. Doesburg P. Steketee K. Trapman J. Brinkmann A.O. Mol. Endocrinol. 1998; 12: 1172-1183Crossref PubMed Google Scholar). effects recruitment of SRC2 and a on recruitment of the AR is in with the effects of the on recruitment of cell that these in transcriptional activity to of the AR-LBD Thus, the is for binding required for ARA70 the for this in both of In this we the binding of AR AF-2 to a of target motifs within AR the of these and AR AF-2 binds to motifs. that AR AF-2 FXXLF motifs from the AR NTD and ARA70 with that AR binds LXXLL and with The that AR AF-2 binds to LXXLL motifs is lines of evidence the of these Thus, expressed AR-LBD binds SRC2 as with AF-2 and AR AF-1, and these interactions are AR AF-2 and in a manner that is by SRC2 and and Finally, SRC2 LXXLL motifs required for of at at the Thus, AR AF-2 binds FXXLF important contacts with a subset of LXXLL motifs. AR the to in an manner to the of AR AF-2 for target coactivator we the structures of the AR-LBD in complex with an FXXLF motif from ARA70 and both high and low AR motifs. structures indicate that the ARA70 FXXLF motif a to of in complex with of of each of the with each and with structures of AR in the of an reveal a of the AF-2 surface that the of AR to the bulky hydrophobic side chains of the FXXLF motifs. of and the and the binding of the ARA70 in of AR-LBD in complex with FXXLF and LXXLL from E. H. Biol. 2004; PubMed Scopus Google Scholar). these is unique the and conserved at an within the receptor Thus, the of the unique of the AR AF-2 surface to bind motifs with bulky hydrophobic side Crystal structures of AR-LBD in complex with and suggest an alternate for the of AR AF-2 to discriminate between LXXLL motifs. The and LXXLL by to the ARA70 FXXLF motif and a variety of in complex with a variety of are by in the this the of most AR binding to most LXXLL motifs. for the high of in the the motif interacts with on the receptor In these of the are in and are in AR-LBD structures with the which binds the AR-LBD with and the binding of AR AF-2 to Thus, AR between cofactor motifs by contacts of the LXXLL the ARA70 is of are in it that discriminate between target motifs by that the hydrophobic LXXLL Stallcup M.R. Kushner P.J. J.D. 1998; 12: PubMed Scopus Google Scholar, E.M. S. A. J. J. R.T. N. 1998; 12: PubMed Scopus Google Scholar), the of a for this AR AF-2 the to in transcriptional activation in the of of AR AF-2 action are The N-C interaction is required for AR action at a variety of androgen-regulated including of prostate-specific such as and that AF-2 intramolecular interactions in these that AR AF-2 could in coactivator binding in including in the AR specific coactivators that FXXLF in conditions of SRC2 and at that resemble the The for AR AF-2 in growth of prostate cancer it is to that SRC2 of the androgen-dependent G1 to S in LNCaP prostate tumor is the of the SRC2 binds and of the SRC2 C terminus binds AR (10Shang Y. Myers M. Brown M. Mol. Cell. 2002; 9: 601-610Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar). AR AF-2 contacts with SRC LXXLL motifs will to be for cell In AR a potent AF-2 that the by binding FXXLF motifs and LXXLL motifs. The receptor the coactivator binding as NRs, by a cleft that interaction with in to The of the AR surface to rearrange to with FXXLF motifs is unique and a of function to interactions in the Most NRs are to bulky side chains in the binding of the and the of AR development of more complex the NTD and the of of Most the new function at the of a of to with AR AF-2 interactions with SRCs are to be in of prostate cancer. M. and for useful and and for in and with