Several members of the C family of GPCRs have dual amino acid and calcium-sensing properties due to conserved binding sites in their large extracellular domains. In the current study we show that GPRC6A, an orphan receptor recently shown to sense amino acids (40
), has conserved binding sites for calcium (i.e.
Ser-149 and Ser-171) and an amino acid (Thr-172) in its large extracellular domain and a conserved consensus calcimimetic binding site (), suggesting that this receptor may also dually sense both extracellular calcium and amino acids as well as calcimimetics. Indeed, we found that GPRC6A is capable of responding to extracellular cations, including calcium as well as the calcimimetic NPS 568, which was previously thought to be specific for CASR ( and ). These findings suggest that GPRC6A is a unique receptor with similarities to both CASR and amino acid sensing mGluRs and is the second member of this family shown to be a target for calcimimetics.
There are important differences, however, between the extracellular cation-sensing properties of GPRC6A and CASR. For example, the apparent affinity for extracellular calcium appears to be lower for GPRC6A compared with CASR. In this regard, calcium in doses of up to 40 mm
was necessary to maximally activate GPRC6A, whereas the CASR is maximally activated by lower calcium concentrations (). In addition, using intracellular calcium to detect GPRC6A, we consistently failed to see evidence for activation by calcium at concentrations of 5 mm
, which we have shown will activate CASR (). This suggests that the affinity of GPRC6A for calcium might be lower than for CASR. In contrast, using ERK or SRE-luciferase to detect GPRC6A activation, we observed activation by 5 mm
extracellular calcium but not 1 mm
calcium unless osteocalcin was present (Figs.3and4). Cautionisneeded, however, in estimating the affinity of receptors in vitro
, since this might be influenced by the level of receptor expression, the cell context, and the sensitivity of the method used to detect receptor activation. Indeed, millimolar levels of calcium are required to activate CASR transfected into heterologous cells in vitro
, whereas this receptor is sensitive to 0.1 mm
changes in ionized calcium in vivo
. The ligand specificity of GPRC6A is also different from CASR. Whereas GPRC6A shares with CASR ligands such as magnesium, strontium, and gadolinium, the trivalent cation aluminum, which activates GPRC6A at μm
concentrations, is only a weak agonist for CASR (45
). Finally, osteocalcin in the presence of a threshold concentration of calcium can stimulate GPRC6A, but these conditions inhibit calcium-mediated CASR activation (). These concentrations of osteocalcin necessary to activate GPRC6A are well with the physiological concentrations found in normal serum. Thus, GPRC6A is evolutionarily linked to CASR but has distinct apparent affinities for extracellular cations and differences in ligand specificity.
The expression of GPRC6A in bone and osteoblasts () raises the possibility that GPRC6A may account for the novel calcium-sensing response reported in bone and osteoblasts derived from CASR knock-out mice (41
). The ability of GPR6A to respond to aluminum, which is capable of stimulating de novo
bone formation under certain experimental conditions (64
), and the ability of osteocalcin to stimulate GPRC6A in the presence of calcium qualify this receptor as a candidate for Ob. CASR, which is also activated by aluminum (65
) and osteocalcin in combination with calcium (). Moreover, calcimimetics, which activate the putative osteoblastic cation-sensing receptor (66
), also activate GPRC6A (). Other characteristics of GPRC6A, however, differ from the putative Ob. CASR. In this regard, magnesium, which activates GPRC6A, is not a ligand for Ob. CASR. In addition, Ob. CASR appears to be coupled to SRE-luciferase activity via pathways not linked to RhoA (10
), whereas GPRC6A is coupled to RhoA-dependent pathways in HEK-293 cells. Whether some of these discrepancies can be explained by the differences between evaluating a transfected receptor in HEK-293 cells and endogenous receptor osteoblasts remains to be established. Regardless, our data raise the novel possibility that calcium and osteocalcin released from bone resorption may act in concert to stimulate osteoblast-mediated bone formation through the activation of GPRC6A.
At present we have insufficient data to determine the physiologically relevant ligand for GPRC6A or to establish whether it is a primary amino acid-sensing, calcium-sensing, or osteocalcin-sensing receptor. Other members of the C family of GPCRs, such as mGluRs and GABA receptors, also are stimulated by extracellular calcium in vitro
, but their physiological ligands are not calcium (29
). Conversely, l
-amino acids are capable of stimulating CASR, but calcium, rather than amino acids, is the most important physiologically relevant ligand for CASR. Additional information is needed to determine whether calcium and/or other cations are the physiological ligands for GPRC6A or whether its ability to sense calcium represents a secondary property common to all members of the family C GPCRs. At present, mutations of GPRC6A, which is located on human chromosome 6 and mouse chromosome 10, have not been implicated in any human diseases. Therefore, mouse genetic approaches will be necessary to elucidate the physiological function of this receptor and to establish whether it has a role in regulating calcium homeostasis. Efforts are currently under way to characterize a GPRC6A null mouse model.
Our findings differ from other recent studies of GPRC6A, which failed to find an effect of extracellular calcium to activate the receptor transfected into Xenopus
). These discrepancies might be explained by difference in cell type, the sensitivity of the method used to assess receptor activation, or the use of insufficient concentrations of extracellular calcium. These studies also did not evaluate the effects of other cations, which we have shown to activate GRPC6A transfected into HEK-293 cells. It is also important to note that we used a full-length GPRC6A cDNA construct used in our studies, whereas others examined GPRC6A function using a using a chimeric receptor generated using the extracellular domain of GPRC6A and transmembrane domain and COOH terminus of the homologous goldfish 5.24 receptor (67
). Also, prior studies of the human GPRC6A activation were confounded by the inability to achieve cell surface expression in mammalian cell lines (41
). On the other hand, others have demonstrated trafficking of mouse GPRC6A to the cell surface membrane (40
). We have confirmed cell surface expression of GPRC6A (), and the functional responses to the cell-impermeable aluminum and gadolinium of our transfected human GPRC6A cDNA are consistent with its cell surface expression. In addition, β-arrestins, which uncouple the receptors from their cognate G-protein-mediated signaling (56
), also was effective in inhibiting GPRC6A activation (). Additional data indicate that GPRC6A is coupled to Gαi
. Evidence for a role Gαi
include the effects of pertussis toxin (PT
) and RGS4 to inhibit GPRC6A activation (). There is also evidence of involvement of Gαq
. In this regard RGS2, which has GTPase-activating effects for Gαq
subunits () dominant negative Gαq
-(305–355) (), all inhibited GPRC6A signaling. Thus, GPRC6A appears to be a cell surface-expressed GPCR that is coupled to Gαi
. It is not clear, however, why we were unable to demonstrate an effect of GPRC6A activation to increase intracellular calcium unless HEK-293 cells were transfected with the chimeric Gαq/i
construct, which allows Gαi
-coupled receptors to activate phosphatidylinositol-phospholipase C (55
). A similar paradox exists for Ob. CASR, which appears to be both Gαi
, but does not lead to increments in intracellular calcium (10
). Also, the activation of the rabbit PGF2αreceptor, which is coupled to Gαi
, failed to increase intracellular calcium in HEK-293 cells (68
), suggesting that activation of Gαq
is not always sufficient for stimulating signal transduction pathway required for increasing intracellular calcium.
Regardless, the similar response to amino acids, extracellular calcium, and calcimimetics by the related calcium-sensing receptor homolog, CASR, along with the conserved residues between GPRC6A and CASR that are responsible for cation and calcimimetic responses suggest that CASR and GPRC6A have overlapping functions. Whether GPRC6A is the functionally important extracellular cation-sensing receptor in bone or other tissues and explains the GPCR response to extracellular cations found in CASR deficient tissues is speculative. However, the bone microenvironment, which consists of high extracellular calcium concentrations and bone-specific extracellular matrix calcium binding proteins such as osteocalcin, likely imposes unique requirements for osteoblast sensing of extracellular calcium, possibly through a novel, low calcium affinity receptor. Additional studies are warranted to investigate the functional role of GPRC6A in osteoblasts and the role of GPRC6A in regulating bone formation as well as its function in other tissues, such as skeletal muscle, adipose tissue, and kidney, where it is also expressed. Moreover, the response of GPRC6A to calcimimetics indicates that it may be possible to develop specific ligands or allosteric modulators to target GPRC6A and to develop therapeutic agents that regulate receptor function in tissues where it is of physiological importance.