CaS

The calcium-sensing receptor is a member of family C (or family 3) of the G protein-coupled receptor superfamily. Unlike other members of this family (e.g., mGlus, GABAB receptors), the calcium-sensing receptor is encoded by a single gene and has only one known biologically active splice variant. It is expressed in the parathyroid chief cells and various cells along the kidney tubules, where it plays crucial roles in extracellular calcium homeostasis by sensing minute changes in extracellular calcium and eliciting changes in cellular function that normalize the level of extracellular calcium. It is also expressed in numerous other cells types (e.g. neurons, oligodendrocytes, keratinocytes), where its physiological importance remains to be established. In addition to sensing calcium ions, it senses several other physiologically relevant biochemical species and modalities, including magnesium, polyamines, L-amino acids, ionic strength and pH. It couples via various G proteins to inhibition of adenylate cyclase and activation of PLC, PLA2, PLD. It also activates, through mechanisms not fully elucidated, Rho, the epidermal growth factor receptor, ERK1/2, p38 MAPK, JNK, PI3-K and PI4-K. Through its respective second messenger pathways, the receptor regulates numerous biological processes, including hormonal secretion, gene expression, cellular proliferation and differentiation, and the activities of ion transporters and ion and water channels. The expression of the calcium-sensing receptor gene can be regulated by extracellular calcium itself and 1,25-dihydroxyvitamin D, actions that likely play a role in mineral ion homeostasis.

GPRC₆

Human and mouse GPRC6A was identified by bioinformatics searches of genomic and expressed sequence tag (EST) databases and subsequently cloned by polymerase chain reaction (PCR). GPRC6A bears the highest resemblance with an odorant goldfish 5.24 receptor (45% amino acid identity) and slightly lower resemblance with the calcium sensing receptor (34%), the taste receptor T1R1 (28%) and the metabotropic glutamate receptor subtype 1 (mGluR1, 24%). Like these homologous receptors, GPRC6A contain a large extracellular amino-terminal domain (ATD), a cystein-rich region and a 7-transmembrane domain (7TM). Site-directed mutagenesis has demonstrated that the endogenous agonists bind to the ATD.

Analysis of amino acid sequence alignments and a homology model of the ATD (based on the X-ray crystallographic structure of mGlu₁) revealed that the amino acids in the receptor that bind the glycine part of L-Glu in mGlu₁ was conserved whereas the amino acids that bind the negatively charged sidechain of L-Glu was not conserved in GPRC6A. This lead to the conclusion that the natural ligand most likely would be an amino acid but probably not L-Glu. The high resemblance with 5.24, which is activated by almost all natural L-alpha-amino acids, albeit with varying potency, lead to the hypothesis that the natural agonist could be a basic amino acid, which was confirmed by modeling.

Human GPRC6A is not expressed on the cell surface in tsA cells and a surface expressed chimeric receptor consisting of the ATD of GPRC6A and the 7TM of 5.24 was thus used for deorphanization. The chimeric receptor was expressed in Xenopus oocytes where activation lead to calcium-activated chloride currents. Wild-type mouse GPRC6A is surface expressed and it was shown that this receptor activates the G_αq pathway and leads to calcium-activated chloride currents. It was demonstrated that GPRC6A, like 5.24, is a promiscuous L-alpha-amino acid receptor with preference for the basic amino-acids L-Arg, L-Lys and L-ornithine.

In order to obtain qualitative pharmacology, the chimeric receptor was transiently expressed in tsA cells followed by measurement of intracellular calcium levels using a fluorescent probe. In order to resensitize the receptor the cells were pre-incubated in amino-acid free buffer for 2 x 2 hrs before assay. This lead to robust responses and revealed the following rank-order of potency: L-Arg > L-Orn ≥ L-Lys = L-Ala ≥ Gly = L-Cit > L-Gln = L-Ser ≥ L-Met.

At the present time, the physiological function of GPRC6A remains unknown.