Evidence for GPRC6A sensing extracellular androgens. A, RT-PCR analysis showing that GPRC6A and AR transcripts are endogenously expressed in 22Rv1 cells, but not in untransfected HEK-293 cells. HEK-293 cells stably transfected with a GPRC6A expression construct display abundant levels of GPRC6A message. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used as a control for RNA abundance. Data are representative of at least three independent experiments. B, dose-dependent effects of testosterone and R1881 to stimulate GPRC6A. Both testosterone and R1881 stimulated ERK activation in HEK-293 cells transfected with GPRC6A cDNA (upper panel), whereas non-transfected HEK-293 cells (middle panel) and HEK-293 cells transfected with CaSR cDNA, which encodes a related GPCR (lower panel), showed no response to exogenously added androgen ligands. C, time course of R1881 activation of ERK phosphorylation. D, extracellular calcium is required for GPRC6A sensing testosterone. At medium calcium concentrations of 0.5 mm, testosterone failed to activate GPRC6A. E, GPRC6A is activated by extracellular testosterone. The cell impermeable testosterone/BSA dose-dependently stimulated GPRC6A-mediated activation of phospho-ERK in HEK-293 cells transfected with GPRC6A (right panel) but not in untransfected HEK-293 cells (left panel). F, effects of testosterone/BSA in 22Rv1 human prostate cancer cells. 22Rv1 cells expressing GPRC6A endogenously also exhibited a dose-dependent stimulation of ERK in response to testosterone/BSA. Representative blots are shown, and the results were verified in at least three independent experiments.

Testosterone binding to GPRC6A. A, fluorescent image showing surface binding of testosterone/BSA/FITC in HEK-293 cells transfected with GPRC6A, but not in untransfected HEK-293 cells. As control, we also found that BSA/FITC did not bind to the cell surface of HEK-293 cells transfected with GPRC6A. The nuclei were stained by DAP1. B, radioligand binding studies on membranes from nontransfected HEK-293 cells and HEK-293 cells transfected with GPRC6A using [³H]testosterone. Depicted is a saturation-binding curve showing cpm of maximum specific binding to [³H]testosterone that is a cpm of the membrane isolated from HEK-293 cells transfected with GPRC6A cDNA subtracted from the cpm of membrane isolated from untransfected HEK-293 cells. C, competition curve of testosterone binding to GPRC6A. Binding is expressed as cpm of maximum specific binding to [³H]testosterone. See “Experimental Procedures” for details.

GPRC6A mediates the non-genomic effects of androgens ex vivo. A, GPRC6A is expressed in testis and bone marrow derived from wild-type mice but not in GPRC6A^−/− mice by RT-PCR. B, BMSCs derived from the male GPRC6A^−/− mice exhibited reduced ability to activate ERK in response to testosterone (80 nm). C, overexpression of hGPRC6A siRNA-202 and siRNA-514 inhibited GPRC6A mRNA expression in 22Rv1 cells. D and E, effect of GPRC6A knockdown in 22Rv1 prostate cancer cells. siRNAs mediated knockdown of GPRC6A-inhibited testosterone, calcium and osteocalcin stimulated phospho-ERK activation (D) and cell proliferation (E) in 22Rv1 cells. G6PDH, glyceraldehyde-3-phosphate dehydrogenase.

GPRC6A deficiency altered androgen responsiveness of seminal vesicles and LH secretion. A, GPRC6A is expressed in seminal vesicle derived from wild-type mice but not in GPRC6A^−/− mice by RT-PCR. B and C, the gross size (B) and the weight of seminal vesicle (C) were markedly reduced from wild-type and GPRC6A^−/− mice following castration (ORX) compared with sham operated controls. Testosterone replacement fully restored the size and appearance of sham wild-type orchiectomized mice, whereas GPRC6A^−/− orchiectomized mice had incomplete responses to exogenously administered testosterone. D, changes in serum LH (left panel) and FSH (right panel) 2 h after administration of testosterone immunoprecipitation at a dose of 200 mg/kg. Values sharing the same superscript are not significantly different at p < 0.05. G6PDH, glyceraldehyde-3-phosphate dehydrogenase.

Cross-talk between GPRC6A and AR signaling pathways. A, dose-dependent effects of the synthetic androgen R1881 on GPRC6A-mediated activation of SRE-luciferase activity. Studies were performed in HEK-293 cells transfected with GPRC6A (closed circles) or empty plasmid (open squares), and the SRE-luciferase reporter construct. The presence of GPRC6A imparts a dose-dependent activation of SRE-luciferase activity in the presence of 1 mm calcium. The values depicted represent the mean ± S.E. luciferase activity in a minimum of three separate experiments. B, AR does not modulate GPRC6A signaling. Dose-dependent effects of calcium on GPRC6A-mediated activation of SRE-luciferase in HEK-293 cells transfected with GPRC6A (closed circles), hAR (closed triangles), or both (closed squares) activity. C, GPRC6A attenuates AR signaling. Dose-dependent effects of R1881 on AR-mediated activation of ARE-luciferase in HEK-293 cells transfected with GPRC6A (closed circles), hAR (closed triangles), or both (closed squares) activity. The presence of GPRC6A attenuates the effects of R1881 to stimulate canonical nuclear receptor pathways. Values represent the mean ± S.E. of at least three experiments. The asterisk indicates a significant difference (p < 0.05) between GPRC6A or hAR versus both GPRC6A and hAR transfected groups.

Use of inhibitors to evaluate signaling transduction pathways activated by GPRC6A. A, Gα_i coupling of GPRC6A. Testosterone-stimulated GPRC6A-mediated activation of phospho-ERK was specifically blocked by 100 ng/ml of pertussis toxin (PTx). B, involvement of Src, PI 3-kinase, and MEK in GPRC6A signaling. Testosterone-stimulated GPRC6A-mediated activation of phospho-ERK was also inhibited by MEK inhibitor PD89059 (10 μm), PI3K inhibitor Ly294002 (50 μm), and Src inhibitor PP-1 (10 μm) (left panel). The inhibitors of PD89059, Ly294002, and PP-1 had nonspecific effects on activities of phospho-ERK in HEK-293 cells stably transfected with GPRC6A without testosterone stimulation (right panel). C, R1881 stimulated GPRC6A activation of SRE-luciferase was also inhibited by PD89059, the PKC inhibitor Ro31-8220, and Src inhibitor PP-1. HEK-293 cells were co-transfected with pcDNA3.GPRC6A and SRE-luciferase reporter gene plasmid. The values depicted represent the mean ± S.E. of luciferase activity in a minimum of three separate experiments. D and E, time course of R1881 activation of Raf-1 and p-Src. R1881 stimulated GPRC6A-mediated non-genomic activation of intercellular phospho-Raf-1 (D) and phospho-Src (E).

Resistance to non-genomic effects of androgens in organs from GPRC6A^−/− mice. Testosterone (200 mg/kg) or PBS vehicle was injected intraperitoneally into wild-type and GPRC6A^−/− male mice. A and B, Western blot analysis of ERK phosphorylation in bone marrow (A) and testis (B) 20 min after injection of testosterone shows loss of testosterone-mediated ERK activation in GPRC6A null tissues. C and D, RT-PCR analysis of the Egr-1 mRNA abundance in bone marrow (C) and testis (D) 60 min after intraperitoneal injection of testosterone also shows loss of testosterone-mediated activation in GPRC6A null tissues. G6PDH, glyceraldehyde-3-phosphate dehydrogenase.