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

• Published in: Molecular pharmacology 2019-05, Vol.95 (5), p.563-572
• Main Authors: Ye, Ruisong, Pi, Min, Nooh, Mohammed M., Bahout, Suleiman W., Quarles, L. Darryl
• Format: Article
• Language: English
• Subjects: Animals; Autophagy - drug effects; Cell Line, Tumor; Cell Membrane - drug effects; Cell Membrane - metabolism; Cell Proliferation - drug effects; Extracellular Signal-Regulated MAP Kinases - metabolism; HEK293 Cells; Humans; Ligands; Male; Mechanistic Target of Rapamycin Complex 1 - metabolism; Mice; Mitogen-Activated Protein Kinases - metabolism; PC-3 Cells; Prostatic Neoplasms - metabolism; Proto-Oncogene Proteins c-akt - metabolism; Receptors, G-Protein-Coupled - metabolism; Ribosomal Protein S6 Kinases, 70-kDa - metabolism; Signal Transduction - drug effects; Testosterone - pharmacology
• ISSN: 0026-895X; 1521-0111
• DOI: 10.1124/mol.118.115014

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 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 mammalian target of rapamycin complex 1 (mTORC1) signaling of endogenous human GPRC6AICL3_KGKY in PC-3 cells. To understand these discrepant results, we expressed mouse (mGPRC6AICL3_KGRKLP), human (hGPRC6AICL3_KGKY), and humanized mouse (mGPRC6AICL3_KGKY) GPRC6A into human embryonic kidney 293 cells. Our results demonstrate that mGPRC6AICL3_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, hGPRC6AICL3_KGKY and humanized mouse mGPRC6AICL3_KGKY are retained intracellularly in ligand naive cells, yet exhibit β-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 hGPRC6AICL3_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 GPRC6AICL3_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.