Known regulators of nitric oxide synthase and arginase are agonists at the human G‐protein‐coupled receptor GPRC6A

Abstract

1 GPRC6A is a novel family C G-protein-coupled receptor (GPCR) with so far unknown physiological function. It was the aim of our study to further characterize the ligand preferences of the receptor and elucidate structural requirements for activity. 2 We have previously generated a functional chimeric receptor construct, h6A/5.24, containing the ligand-binding amino-terminal domain of the human GPRC6A and the seven-transmembrane domain and carboxy terminus of the homologous goldfish receptor 5.24. Based on knowledge that this chimera prefers basic L-a-amino acids such as arginine, lysine and ornithine, we searched for commercially available analogues of these and other L-a-amino acids, and tested them for activity in a fluorescence-based calcium assay. The majority of the tested compounds are involved in the regulation of nitric oxide synthase (NOS) and arginase enzymes. Altogether we profiled 30 different analogues. 3 We found that a structurally wide range of L-a-amino-acid analogues of both arginine, lysine, and ornithine are agonists at h6A/5.24, whereas no antagonists were identified. From the profiling it is concluded that L-a-amino acids containing a highly basic side chain confer the highest activity, although the most potent compound was only twice as potent as L-arginine, which has a EC 50 value of 23.5 mM. 4 The reported agonism of NOS-and arginase-active compounds at GPRC6A has obvious implications in interpretation of experiments involving the NOS and arginase systems, and interfering effects at GPRC6A should be regarded of relevance, especially as the physiological function of the receptor is not yet understood.

Hypoxia Inducible Factor Signaling Modulates Susceptibility to Mycobacterial Infection via a Nitric Oxide Dependent Mechanism

“…The iNOS inhibitor L-NIL (N6-(1-iminoethyl)-L-lysine, dihydrochloride, Tocris Bioscience) was used at a 200 µM concentration [38]. DMSO solvent controls were used at corresponding concentrations for each treatment.…”

The orphan G protein-coupled receptor GPR139 is activated by the peptides: Adrenocorticotropic hormone (ACTH), α-, and β-melanocyte stimulating hormone (α-MSH, and β-MSH), and the conserved core motif HFRW

“…However, the potencies of l -Trp and l -Phe on GPR139 are modest (EC 50 values of 220 μM and 320 μM, respectively), and interestingly, GPR139 is also activated by di-peptides comprised of aromatic amino acids (Isberg et al., 2014). This contrasts with other amino acids receptors, such as GPRC6A, which require an intact α-amino acid moiety for agonist activity (Christiansen et al., 2006). This has led us to hypothesize that GPR139 is not simply an aromatic amino acid sensing receptor, but could also be activated by peptides.…”

The rat GPRC6A: Cloning and characterization

“…Functional characterization of GPRC6A 3.6.1. Amino acid sensitivity Initial pharmacological quantitative data on GPRC6A was all obtained using the h6A/5.24 chimeric receptor (Wellendorph et al, 2005;Christiansen et al, 2006). To properly investigate the signalling propensities of GPRC6A and to explore potential allosteric modulation in the 7TM domain, it is important to develop a wild-type-based assay.…”