The G protein-coupled receptor GPRC6A is a nutrient receptor, promiscuously activated by L-amino acids in mouse and human. However, published data is conflicting about the ability of the receptor to respond to other ligands, such as testosterone, its analogues, and the bone-derived osteocalcin, where there are species differences. Reports have shown that whilst the murine receptor is successfully expressed at the cell membrane, the human GPRC6A (hGPRC6A) remains intracellularly retained. Receptor activity-modifying proteins (RAMPs) are small accessory proteins known to influence surface trafficking and ligand affinity when complexed with a GPCR. We hypothesised that hGPRC6A interacts with RAMPs thereby aiding in its forward trafficking and signalling capabilities. Here we show that association of the hGPRC6A receptor with RAMP1, allows the receptor to traffic to the cell surface and respond to additional ligands to which it is insensitive to in the absence of RAMP1. Specifically, we demonstrate using FRET and ELISA that transfection of hGPRC6A-expressing cells with RAMP1 (but neither RAMP2, nor RAMP3) changes the location of the GPRC6A within cells, so that there is cell surface localisation. This leads to the activation of intracellular calcium mobilisation by testosterone [pEC50 6.63±0.28] and DHEA [pEC50 6.61±0.34], as well as the L-amino acids L-Orn [pEC50 6.23±0.35], L-Arg [pEC50 6.01±0.33], L-Lys [pEC50 6.52±0.38], although not osteocalcin. In order to determine the functional significance of these findings, we have explored the role of hGPRC6A and RAMP1 in prostate cancer cells in vitro and in vivo. It is already known that knockout of GPRC6A reduces PC-3 xenograft tumour growth in mice and that SNPs in GPRC6A reduces disease severity in men, while GPRC6A knockout in PC-3 cells also reduces tumour growth. We show that the absence of RAMP1 in hGPRC6A-expressing PC-3 prostate cancer cells reduces viability by 41% (p<0.0001), colony formation 89% (p<0.0001) and other markers of tumorigenesis, and the effects of GPRC6A agonists and antagonists is blunted in those cells, compared with GPRC6A/RAMP1-expressing cells. Taken together, our data are consistent with a requirement for RAMP1 for full functionality of GPRC6A in humans, and displays distinctly different profile in mice. Additionally, our data may provide a novel target for research into treatments for hormone refractory prostate cancer.
