Identification of serine-875 as an inhibitory phosphorylation site in the calcium-sensing receptor

Abstract

The calcium-sensing receptor (CaS) is the principal controller of extracellular calcium (Ca2o1) homeostasis and is inhibited in vitro and in vivo by protein kinase C (PKC)-mediated phosphorylation at CaST888. 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 CaST888 but instead with CaSS875, which was not previously considered to be a PKC site. CaSS875A (nonphosphorylatable) exhibited significantly enhanced Ca2o1 sensitivity of both intracellular Ca21 mobilization and extracellular signal-regulated kinase 1/2 activation, whereas the phosphomimetic CaSS875D mutant exhibited a loss of function. The CaSS875A/T888A double mutant exhibited even greater Ca2o1 sensitivity than CaST888A 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 [N-(2-hydroxy-3-(2-cyano-3-chlorophenoxy)propyl)-1,1-dimethyl-2-(2-nephthyl)ethylamine] or Ca2o1 removal. Therefore, we have now identified CaSS875 as the missing PKC phosphorylation site that, together with CaST888, shapes the CaS signaling that underpins Ca2o1 homeostasis. Together with the inactive form of the CaS extracellular domain, these sites attenuate Ca2o1 sensitivity to attain appropriate physiologic Ca2o1 sensing.