Pharmacological characterization of mouse GPRC6A, an L-α-amino-acid receptor modulated by divalent cations

Background and purpose: GPRC6A is a novel member of family C of G protein- coupled receptors with so far unknown function. We have recently described both human and mouse GPRC6A as receptors for L-alpha-amino acids. To date, functional characterization of wild- type GPRC6A has been impaired by the lack of activity in quantitative functional assays. The aim of this study was thus to develop such an assay and extend the pharmacological characterization of GPRC6A. Experimental approach: We have engineered a novel cell- based inositol phosphate turnover assay for wild- type mouse GPRC6A based on transient co-expression with the promiscuous G(alpha qG66D) protein, known to increase receptor signalling sensitivity. This assay allowed for measurements of L-alpha-amino acid potencies. Furthermore, in combination with an assay measuring inward currents at Ca2+-activated chloride channels in Xenopus oocytes, the divalent cation-sensing ability of the receptor was examined. Key results: Using our novel assay, we demonstrate that the basic L-alpha-amino acids ornithine, lysine, and arginine are the most potent agonists at wild- type mouse GPRC6A. Using two different assay systems, we show that divalent cations do not activate the G(q) signalling pathway of mouse GPRC6A per se but positively modulate the amino-acid response. Conclusions and Implications: This is the first reported assay for a wild- type GPRC6A successfully applied for quantitative pharmacological characterization of amino acid and divalent cation responses at mouse GPRC6A. The assay enables further search for GPRC6A ligands such as allosteric modulators, which may provide essential information about the physiological function of GPRC6A.