TY - JOUR T1 - Human GPRC6A mediates testosterone-induced ERK and mTORC1 signaling in prostate cancer cells JF - Molecular Pharmacology JO - Mol Pharmacol DO - 10.1124/mol.118.115014 SP - mol.118.115014 AU - Ruisong Ye AU - Min Pi AU - Mohammed M Nooh AU - Suleiman W Bahouth AU - L. Darryl Quarles Y1 - 2019/01/01 UR - http://molpharm.aspetjournals.org/content/early/2019/03/21/mol.118.115014.abstract N2 - GPRC6A is activated by testosterone and modulates prostate cancer (PCa) progression. Most humans have a GPRC6A variant that contains a recently evolved KGKY insertion/deletion in the third intracellular loop (ICL3) (designated GPRC6AICL3_KGKY) that replaces the ancestral KGRKLP sequence (GPRC6AICL3_RKLP) present in all other species. In vitro assays purport that human GPRC6AICL3_KGKY is retained intracellularly and lacks function. These findings contrast with ligand-dependent activation and coupling to mTORC1 signaling of endogenous human GPRC6AICL3_KGKY in PC-3 prostate cancer cells. To understand these discrepant results, we expressed mouse (mGPRC6AICL3_KGRKLP), human (hGPRC6AICL3_KGKY) and "humanized" mouse (mGPRC6AICL3_KGKY) GPRC6A into HEK-293 cells. Our results demonstrate that the mGPRC6AICL3_KGRKLP acts as a classical GPCR that is expressed at the cell membrane and internalizes in response to ligand activation by testosterone. In contrast, hGPRC6AICL3_KGKY and "humanized" mouse mGPRC6AICL3_KGKY are retained intracellularly in ligand naive cells, yet exhibit β-arrestin dependent signaling responses, ERK and S6K phosphorylation in response to testosterone, indicating that hGPRC6AICL3_KGKY is functional. Indeed, testosterone stimulates time- and dose-dependent activation of ERK, AKT and mTORC1 signaling in wild-type PC-3 cells that express endogenous GPRC6AICL3_KGKY. In addition, testosterone stimulates GPRC6A-dependent cell proliferation in wild-type PC-3 cells and inhibits autophagy by activating mTORC1 effectors 4E-BP1 and ULK1. Testosterone activation of GPRC6A has the obligate requirement for calcium in the incubation media. In contrast, in GPRC6A deficient cells, the effect of testosterone to activate down-stream signaling is abolished, indicating that human GPRC6A is required for mediating the effects of testosterone on cell proliferation and autophagy. ER -