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 CaS^T888. 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^T888 but instead with CaS^S875, not previously considered a PKC site. CaS^S875A (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 CaS_S875D mutant exhibited a loss of function. The CaS^S875A/T888A double mutant exhibited even greater Ca²⁺_o sensitivity than CaS^T888A alone, a response no longer enhanced by PKC inhibition. Finally, when expressed in CaS lacking its extracellular domain, the CaS^S875A/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 CaS^S875 as the missing PKC phosphorylation site that, together with CaS^T888, shapes the CaS signalling that underpins Ca²⁺_o homeostasis. Together with the inactive form of the CaS’s extracellular domain, these sites attenuate Ca²⁺_o sensitivity to attain appropriate physiological Ca²⁺_o sensing.