Identification of Serine-875 as an Inhibitory Phosphorylation Site in the Calcium-Sensing Receptor

The calcium-sensing receptor (CaS) is the principal controller of extracellular calcium (Ca ) homeostasis and is inhibited and by protein kinase C (PKC)-mediated phosphorylation at CaR However, PKC inhibition enhances signalling even in CaSs lacking Thr-888, suggesting that an additional inhibitory site exists. An apparently equivalent PKC regulatory site in metabotropic glutamate receptor-5 (Ser-839) aligns not with CaS but instead with CaS, not previously considered a PKC site. CaS (non-phosphorylatable) exhibited significantly enhanced Ca sensitivity of both intracellular Ca mobilisation and extracellular signal-regulated kinase 1/2 (ERK1/2) activation, whereas the phosphomimetic CaS mutant exhibited a loss of function. The CaS double mutant exhibited even greater Ca sensitivity than CaS alone, a response no longer enhanced by PKC inhibition. Finally, when expressed in CaS lacking its extracellular domain, the CaS double mutation elicited maximal activation even under control conditions, but remained sensitive to negative allosteric modulation (NPS-2143) or Ca removal. Therefore, we have now identified CaS as the missing PKC phosphorylation site that, together with CaS, shapes the CaS signalling that underpins Ca homeostasis. Together with the inactive form of the CaS's extracellular domain, these sites attenuate Ca sensitivity to attain appropriate physiological Ca sensing. SIGNIFICANCE STATEMENT: The calcium-sensing receptor (CaS) controls whole body calcium homeostasis by regulating parathyroid hormone secretion and renal calcium reabsorption. CaS function can be inhibited upon phosphorylation by protein kinase C (PKC), principally but not exclusively at Thr-888. Here we provide evidence that Ser-875, a previously unrecognised PKC site in CaS, is an additional inhibitory phosphorylation site for the receptor.