Delineation of the GPRC6A Receptor Signaling Pathways Using a Mammalian Cell Line Stably Expressing the Receptor

Abstract

The GPRC6A receptor is a recently "deorphanized" class C G protein-coupled receptor. We and others have shown that this receptor is coactivated by basic L-a-amino acids and divalent cations, whereas other groups have also suggested osteocalcin and testosterone to be agonists. Likewise, the GPRC6A receptor has been suggested to couple to multiple G protein classes albeit via indirect methods. Thus, the exact ligand preferences and signaling pathways are yet to be elucidated. In the present study, we generated a Chinese hamster ovary (CHO) cell line that stably expresses mouse GPRC6A. In an effort to establish fully the signaling properties of the receptor, we tested representatives of four previously reported GPRC6A agonist classes for activity in the G q , G s , G i , and extracellular-signal regulated kinase signaling pathways. Our results confirm that GPRC6A is activated by basic L-a-amino acids and divalent cations, and for the first time, we conclusively show that these responses are mediated through the G q pathway. We were not able to confirm previously published data demonstrating G i -and G s -mediated signaling; neither could we detect agonistic activity of testosterone and osteocalcin. Generation of the stable CHO cell line with robust receptor responsiveness and optimization of the highly sensitive homogeneous time resolved fluorescence technology allow fast assessment of G q activation without previous manipulations like cotransfection of mutated G proteins. This cell-based assay system for GPRC6A is thus useful in highthroughput screening for novel pharmacological tool compounds, which are necessary to unravel the physiologic function of the receptor.

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

“…We therefore tested the 20 proteinogenic L-␣-amino acids in addition to L-ornithine (3) in one fixed concentration and selected the 10 most potent agonists for generation of full concentration-response curves. We found that L-ornithine, L-arginine, and L-lysine were the most potent agonists on h6A_KGRKLP (Table 2) in accordance with previous results on m6A (6,11).…”

“…Because m6A, in our hands, signals via the G q pathway (11), we then tested the functional importance of ICL3 and the C-terminal tail in an IP-One assay, which measures the accumulation of D-myo-inositol monophosphate (IP 1 ) as a consequence of G q activation (5). We have previously shown that the co-transfection with G␣ q (G66D) boosts the m6A response (5, 9) without changing the pharmacological profile of the m6A receptor (10,11). We found that mouse ICL3 is essential for functionality of the receptor when cells were stimulated with L-ornithine, whereas the C-terminal part of m6A had no positive effect on the functionality of the chimeras (Fig.…”

“…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).…”

The GPRC6A receptor displays constitutive internalization and sorting to the slow recycling pathway

“…(GPCR) belonging to class C. It is activated by basic L-␣-amino acids and divalent cations, which trigger coupling to the G q protein and thereby an increase in the intracellular levels of the second messengers inositol trisphosphate and Ca 2ϩ (1)(2)(3)(4)(5)(6)(7)(8). Other ligands and signaling pathways have been reported (3, 9 -13); however Rueda et al (8) and we (7) have not been able to confirm these findings.…”

Assessing the Role of Gαq/11 in Cellular Responses: An Analysis of Investigative Tools

“…LOrn IP1 and Ca+2 responses of transfected CHO cells was mediated through Gαq activation, both these pathways were inhibited by UBO-QIC [14].The PLC inhibitor, U73122, was used to further demonstrate that L-Orn-induced IP1reponse was mediated through Gαq signalling. In TRPV4 transfected HEK cells, UBO-QIC abolished PAR-2 mediated intracellular calcium release when compared to control and non-transfected HEK cells, however UBO-QIC had no effect on the extracellular calcium influx through TRPV4 ion channels, thus showing that PAR-2 coupling to TRPV4 is not mediated by Gαq signalling [15].…”