PROTEIN-KINASE-C PHOSPHORYLATION OF THREONINE AT POSITION-888 IN CA-0(2-SENSING RECEPTOR (CAR) INHIBITS COUPLING TO CA2+ STORE RELEASE())

Previous studies in parathyroid cells, which express the G protein-cou pled, extracellular calcium-sensing receptor (CaR), showed that activa tion of protein kinase C (PKC) blunts high extracellular calcium (Ca-o (2+))-evoked stimulation of phospholipase C and the associated increas es in cytosolic calcium (Ca-i(2+)), suggesting that PKC may directly m odulate the coupling of the CaR to intracellular signaling systems, In this study, we examined the role of PKC in regulating the coupling of the CaR to Ca-i(2+) dynamics in fura-2-loaded human embryonic kidney cells (HEK293 cells) transiently transfected with the human parathyroi d CaR, We demonstrate that several PKC activators exert inhibitory eff ects on CaR-mediated increases in Ca-i(2+) due to release of Ca2+ from intracellular stores. Consistent with the effect being mediated by ac tivation of PKC, the inhibitory effect of PRC activators on Ca2+ relea se can be blocked by a PKC inhibitor. The use of site-directed mutagen esis reveals that threonine at amino acid position 888 is the major PK C site that mediates the inhibitory effect of PKC activators on Ca2+ m obilization, The effect of PKC activation can be maximally blocked by mutating three PKC sites (Thr(888), Ser(895), and Ser(915)) or all fiv e PKC sites. In vitro phosphorylation shows that Thr(888) is readily p hosphorylated by PKC, Our results suggest that phosphorylation of the CaR, is the molecular basis for the previously described effect of PKC activation on Ca-o(2+)-evoked changes in Ca-i(2+) dynamics in parathy roid cells.