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 signaling 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, which was not previously considered to be a PKC site. CaS^S875A (nonphosphorylatable) exhibited significantly enhanced Ca²⁺ _o sensitivity of both intracellular Ca²⁺ mobilization and extracellular signal-regulated kinase 1/2 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 [N-(2-hydroxy-3-(2-cyano-3-chlorophenoxy)propyl)-1,1-dimethyl-2-(2-nephthyl)ethylamine] 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 signaling that underpins Ca²⁺ _o homeostasis. Together with the inactive form of the CaS extracellular domain, these sites attenuate Ca²⁺ _o sensitivity to attain appropriate physiologic Ca²⁺ _o sensing. SIGNIFICANCE STATEMENT: Serine-875 represents the missing inhibitory PKC phosphorlyation site in CaS that in tandem with Thr-888 controls receptor activity.