Cloning and characterization of a Family C orphan G‐protein coupled receptor

Abstract

Members of the family C receptors within the G-protein coupled receptor superfamily include the metabotropic glutamate receptors, GABA B receptors, the calcium-sensing receptor (CaSR), the V2R pheromone receptors, the T1R taste receptors, and a small group of uncharacterized orphan receptors. We have cloned and studied the mouse GPRC6A family C orphan receptor. The open reading frame codes for a protein with highest sequence identity to the fish 5.24 odorant receptor and the mammalian CaSR. The gene structure shows a striking resemblance to that of the CaSR. Results from RT-PCR analyses showed that mouse GPRC6A mRNA is expressed in mouse brain, skeletal muscle, heart, lung, spleen, kidney, liver, and in the early stage mouse embryo. Immunocytochemical analysis of the cloned mouse GPRC6A cDNA expressed in human embryonic kidney 293 cells demonstrated that GPRC6A was present on the plasma membrane, as well as in the endoplasmic reticulum and nuclear envelope membranes of transfected cells. A chimeric cDNA construct in which the extracellular ligand binding domain of the fish 5.24 amino acidactivated odorant receptor was ligated to the complementary downstream sequence of the mouse GPRC6A receptor indicated that GPRC6A is coupled to phosphoinositol turnover and release of intracellular calcium. Further studies with mouse GPRC6A expressed in Xenopus laevis ooctyes demonstrated that this receptor possesses a pharmacological profile resembling that of the fish 5.24 odorant receptor. These findings suggest that GPRC6A may function as the receptor component of a novel cellular transmitter system in mammals.

Genetic Variations in the Human G Protein-coupled Receptor Class C, Group 6, Member A (GPRC6A) Control Cell Surface Expression and Function

“…The starting point of the present study was the surprising observation that recombinantly expressed h6A is retained intracellularly in contrast to mouse, rat, and goldfish analogs (2,3,6). Our previous analyses of h6A/5.24 chimeric analyses uncovered that the lack of surface expression was due to a motif located in the 7TM domain and/or the C-terminal region of h6A (3).…”

“…Other groups later confirmed these agonists on mouse GPRC6A (m6A) using various experimental systems (2,7,8). Divalent cations such as Ca 2ϩ and Mg 2ϩ have also been suggested to function either as positive allosteric modulators (2,9) or as direct, endogenous agonists on m6A (10,11). The steroid testosterone and the peptide osteocalcin have also been suggested as endogenous agonists on m6A (12)(13)(14); however, we and others have been unable to confirm this (6,11).…”

“…These studies, together with recent evidence (5) support the existence of GPRC6A as a dimer on the cell surface. However, surprisingly, h6A has been shown to be retained intracellularly and thus does not respond to agonists, which contrasts findings for the mouse, rat, and goldfish orthologs (2,3,6). To deorphanize h6A, we generated chimeras between the human and goldfish GPRC6A orthologs.…”

“…Using these chimeras it was shown that h6A is a promiscuous L-␣-amino acid receptor, most potently activated by the basic amino acids L-arginine, L-lysine, and L-ornithine (3). Other groups later confirmed these agonists on mouse GPRC6A (m6A) using various experimental systems (2,7,8). Divalent cations such as Ca 2ϩ and Mg 2ϩ have also been suggested to function either as positive allosteric modulators (2,9) or as direct, endogenous agonists on m6A (10,11).…”

“…A pivotal class of proteins, specialized to carry out such signal transduction, is the G proteincoupled receptors (GPCRs), 2 a family that includes ϳ800 subtypes in humans. The GPCR class C, group 6, member A (GPRC6A) belongs to the non-olfactory GPCRs as part of the class C receptors.…”