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) that replaces the ancestral KGRKLP sequence (GPRC6A) present in all other species. In vitro assays purport that human GPRC6A 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 in PC-3 cells. To understand these discrepant results, we expressed mouse (mGPRC6A), human (hGPRC6A), and humanized mouse (mGPRC6A) GPRC6A into human embryonic kidney 293 cells. Our results demonstrate that mGPRC6A 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 and humanized mouse mGPRC6A are retained intracellularly in ligand naive cells, yet exhibit -arrestin-dependent signaling responses, mitogen-activated protein kinase [i.e., extracellular signal-regulated kinase (ERK)], and p70S6 kinase phosphorylation in response to testosterone, indicating that hGPRC6A 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 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.