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The transcriptional activity of the androgen recep- E2K) show reduced transcriptional responses to
tor (AR) is modulated by interactions with coregu- ART-27, whereas their response to the p160 class
latory molecules. It has been proposed that aber- of coactivators was not diminished. Relative to the
rant interactions between AR and its coregulators wild-type receptor, less ART-27 protein associated
may contribute to diseases related to AR activity, with the AR E2K substitution, consistent with re-
such as prostate cancer and androgen insensitivity duced transcriptional response. Surprisingly, more
syndrome (AIS); however, evidence linking abnor- ART-27 associated with AR P340L, despite the fact
mal receptor-cofactor interactions to disease is that the mutation decreased transcriptional activa-
scant. ART-27 is a recently identified AR N-terminal tion in response to ART-27. Our findings suggest
coactivator that is associated with AR-mediated that aberrant AR-coactivator association inter-
growth inhibition. Here we analyze a number of feres with normal ART-27 coactivator function, re-
naturally occurring AR mutations identified in pros- sulting in suppression of AR activity, and may con-
tate cancer and AIS for their ability to affect AR tribute to the pathogenesis of diseases related to
response to ART-27. Although the vast majority of alterations in AR activity, such as prostate cancer
AR mutations appeared capable of increased acti- and AIS. (Molecular Endocrinology 19: 2273–2282,
vation in response to ART-27, an AR mutation iden- 2005)
tified in prostate cancer (AR P340L) and AIS (AR
2273
2274 Mol Endocrinol, September 2005, 19(9):2273–2282 Li et al. • AR Mutants Affecting ART-27 Activity
includes proteins that function in transcriptional rally occurring AR N-terminal mutations identified in
regulation. prostate cancer and AIS for their effect on the AR
We have also shown that in normal adult human transcriptional response to ART-27. We focused on
prostate, ART-27 protein is expressed in luminal epi- AR N-terminal mutations because ART-27 has been
thelial cells, in contrast to the stroma, where ART-27 is shown to bind exclusively to this region of the receptor
not expressed (12). During prostate development in (10). Eleven AR mutations, spanning amino acids
humans, ART-27 is expressed in differentiated luminal 2–491, were made in the N terminus. Of these muta-
epithelial cells but is not detected in undifferentiated tions, five were mutations identified in human AIS
epithelial cell precursors, suggesting a role for ART-27 (E2K, Q194R, N233K, L255P, and G491S), and six
in AR-mediated growth suppression and differentia- were mutations from individuals with prostate cancer
tion. Consistent with a growth-suppressive function, (K180R, E198G, M266T, P269S, S334P, and P340L)
G491S, did not appear to significantly affect AR activ- receptor variants are stabilized in the presence of
ity relative to the wild-type receptor. Immunoblot anal- R1881 and that some variability in AR protein expres-
ysis revealed that both wild-type AR and all of the sion is also observed among the mutant receptors
2276 Mol Endocrinol, September 2005, 19(9):2273–2282 Li et al. • AR Mutants Affecting ART-27 Activity
ditions that preserve the interaction between AR and indicate that this mutant shows a diminished interac-
ART-27. AR immunoprecipitates were analyzed by im- tion and transcriptional response to ART-27. Because
munoblotting with an HA antibody specific for the the AR E2K mutation is located outside of the ART-27
HA-tagged ART-27. As seen in Fig. 5A, ART-27 was binding region (Fig. 1A) and induces a local conforma-
coimmunoprecipitated with both wild-type AR, AR tional change (Fig. 1C), we suggest that the change in
E2K, and AR P340L. About half the level of ART-27 conformation affects the global architecture of the re-
was detected in association with AR E2K as compared ceptor, which reduces ART-27 binding (Fig. 5). The
with the wild-type receptor. Thus, the AR E2K alter- unexpected finding that the AR E2K displays an en-
ation reduces interaction with ART-27. In contrast, AR hanced transcriptional response to SRC-1 is consis-
P340L showed an increase in its association with ART- tent with the notion that the E2K mutation affects
27, despite its inability to enhance AR transcription. To global AR conformation. Such changes in the AR re-
ART-27 is detected only when the prostate gland has MATERIALS AND METHODS
proceeded from a solid mass of undifferentiated cells
to a stage at which differentiated luminal epithelial Plasmid Construction
cells are evident (12). In light of these findings, we
suggest that ART-27 plays a role in suppressing pros- The AR mutants were generated by QuikChange Site-Di-
rected Mutagenesis system (Stratagene, La Jolla, CA) using
tate cancer development by contributing to the main- the oligonucleotides described in supplemental Table 1,
tenance of a program of AR-mediated differentiation. which is published as supplemental data on The Endocrine
Thus, the AR P340L mutant would facilitate prostate Society’s Journals Online web site at http://mend.endojournals.
cancer progression by preventing the normal “growth- org and the wild-type pcDNA3:hAR expression plasmid as
the template. All mutations were confirmed by DNA sequenc-
suppressive” function of ART-27. This may represent a ing. The p160 expression vectors, pcDNA3-GRIP-1 and
tor gene mutations in prostate cancer. Clin Cancer Res 29. Li P, Yu X, Ge K, Melamed J, Roeder RG, Wang Z 2002
7:1273–1281 Heterogeneous expression and functions of androgen
14. Gottlieb B, Pinsky L, Beitel LK, Trifiro M 1999 Androgen receptor co-factors in primary prostate cancer. Am J
insensitivity. Am J Med Genet 89:210–217 Pathol 161:1467–1474
15. Brinkmann AO 2001 Molecular basis of androgen insen- 30. Adachi M, Takayanagi R, Tomura A, Imasaki K, Kato S,
sitivity. Mol Cell Endocrinol 179:105–109 Goto K, Yanase T, Ikuyama S, Nawata H 2000 Androgen-
16. Frishman D, Argos P 1997 Seventy-five percent accuracy insensitivity syndrome as a possible coactivator disease.
in protein secondary structure prediction. Proteins 27: N Engl J Med 343:856–862
329–335 31. Reid J, Betney R, Watt K, McEwan IJ 2003 The androgen
17. Frishman D, Argos P 1996 Incorporation of non-local receptor transactivation domain: the interplay between
interactions in protein secondary structure prediction protein conformation and protein-protein interactions.
from the amino acid sequence. Protein Eng 9:133–142 Biochem Soc Trans 31:1042–1046
18. Reid J, Kelly SM, Watt K, Price NC, McEwan IJ 2002 32. Chen CD, Welsbie DS, Tran C, Baek SH, Chen R, Ves-
Molecular Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost
professional society serving the endocrine community.