BACKGROUND. The androgen receptor (AR) is a ligand-dependent transcription factor that mediates androgenic hormone action in cells. We recently demonstrated the involvement of phosphoinositide 3-OH kinase (PI3K) p110beta in AR transactivation and gene expression. In this study, we determined the upstream signals that lead to PI3K/p110beta activation and AR transactivation after androgen stimulation. METHODS. Human prostate cancer LAPC-4 and 22Rv1 cell lines were used for the experiments. AR transactivation was assessed using an androgen responsive element-driven luciferase (ARE-LUC) assay. Cell proliferation was examined using BrdU incorporation and MTT assays. Target genes were silenced using small interfering RNA (siRNA) approach. Gene expression was evaluated at the mRNA level (real-time RT-PCR) and protein level (Western blot). PI3K kinase activities were measured using immunoprecipitation-based in vitro kinase assay. The AR DNA-binding activity was determined using chromatin-immunoprecipitation (ChIP) assay. RESULTS. First, at the cellular plasma membrane, disrupting the integrity of caveolae microdomain with methyl-beta-cyclodextrin (M-beta-CD) abolished androgen-induced AR transactivation and gene expression. Then, knocking down caveolae structural proteins caveolin-1 or -2 with the gene-specific siRNAs significantly reduced androgen-induced AR transactivation. Next, silencing G alpha(s) and G alpha(12) genes but not other G-proteins blocked androgen-induced AR transactivation and cell proliferation. Consistently, overexpression of G alpha s or G alpha(12) active mutants enhanced androgen-induced AR transactivation, of which G alpha(s) active mutant sensitized the AR to castration-level of androgen (R1881). Most interestingly, knocking down G alpha(s) but not G alpha(12) subunit significantly suppressed androgen-stimulated PI3K p110beta activation. However, ChIP analysis revealed that both G alpha(s) or G alpha(12) subunits are involved in androgen-induced AR interaction with the AR target gene PSA promoter region. CONCLUSION. These data suggest that caveolae-associated G-protein alpha subunits are involved in AR transactivation by modulating the activities of different PI3K isoforms. Prostate 71: 1276-1286, 2011. (C) 2011 Wiley-Liss, Inc.