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 (Ca2+o) homeostasis and is inhibited in vitro and in vivo by protein kinase C (PKC)-mediated phosphorylation at CaRT888. 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 CaST888 but instead with CaSS875, not previously considered a PKC site. CaSS875A (non-phosphorylatable) exhibited significantly enhanced Ca2+o sensitivity of both intracellular Ca2+ mobilisation and extracellular signal-regulated kinase 1/2 (ERK1/2) activation, whereas the phosphomimetic CaSS875D mutant exhibited a loss of function. The CaSS875A/T888A double mutant exhibited even greater Ca2+o sensitivity than CaST888 alone, a response no longer enhanced by PKC inhibition. Finally, when expressed in CaS lacking its extracellular domain, the CaSS875A/T888A double mutation elicited maximal activation even under control conditions, but remained sensitive to negative allosteric modulation (NPS-2143) or Ca2+o removal. Therefore, we have now identified CaSS875 as the missing PKC phosphorylation site that, together with CaST888, shapes the CaS signalling that underpins Ca2+o homeostasis. Together with the inactive form of the CaS9s extracellular domain, these sites attenuate Ca2+o sensitivity to attain appropriate physiological Ca2+o 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.