Human GPRC6A Mediates Testosterone-Induced Mitogen-Activated Protein Kinases and mTORC1 Signaling in Prostate Cancer Cells

G protein-coupled receptor family C group 6 member A (GPRC6A) is activated by testosterone and modulates prostate cancer progression. Most humans have a GPRC6A variant that contains a recently evolved KGKY insertion/deletion in the third intracellular loop (ICL3) (designated as GPRC6A(ICL3_KGKY)that replaces the ancestral KGRKLP sequence (GPRC6A(ICL3_RKLP)) present in all other species. In vitro assays purport that human GPRC6A(ICL3_KGKY) is retained intracellularly and lacks function. These findings contrast with ligand-dependent activation and coupling to mammalian target of rapamycin complex 1 (mTORC1) signaling of endogenous human GPRC6A(ICL3_KGKY) in PC-3 cells. To understand these discrepant results, we expressed mouse (m G P RC 6A ICL3 ) KGRKLP , human (hGPRC6A(I)(L3)(_KGKY)) and humanized mouse (nnGPRC6A(ICL3_KGKY)) GPRC6A into human embryonic kidney 293 cells. Our results demonstrate that mGPRC6A(ICL3_KGRKLP) acts as a classic G protein-coupled receptor, which is expressed at the cell membrane and internalizes in response to ligand activation by testosterone. In contrast, hGPRC6A(ICL3_KGKY)and humanized mouse nnGPRC6A(ICL3_KGKY)are retained intracellularly in ligand naive cells, yet exhibit beta-arrestin-dependent signaling responses, mitogen-activated protein kinase [i.e., extracellular signal-regulated kinase (ERK)], and p70S6 kinase phosphoVation in response to testosterone, indicating that hGPRC6A(ICL3_KGKY) is functional. Indeed, testosterone stimulates time- and dose-dependent activation of ERK, protein kinase B, and mTORC1 signaling in wild-type PC-3 cells that express endogenous GPRC6A(ICL3_KGKY). In addition, testosterone stimulates GPRC6A-dependent cell proliferation in wild-type PC-3 cells and inhibits autophagy by activating mTORC1 effectors eukaryotic translation initiation factor 4E binding protein 1 and Unc-51 like autophagy activating kinase 1. 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 downstream signaling is abolished, indicating that human GPRC6A is required for mediating the effects of testosterone on cell proliferation and autophagy.