Sustained intracellular calcium oscillations induced by activation of the extracellular calcium sensing receptor do not require receptor phosphorylation by protein kinase C

Abstract

Activation of the calcium sensing receptor (CaR) by small increments of extracellular calcium (Ca2+e), phenylalkylamines (calcimimetics) or aromatic amino acids induces intracellular calcium (Ca2+i) oscillations. Phosphorylation / dephosphorylation of G protein-coupled receptors by protein kinase C (PKC) can represent a feedback mechanism necessary for the initiation and maintenance of Ca2+i oscillations. However, the importance of PKC-mediated phosphorylation / dephosphorylation of CaR is debated. Whereas addition of PKC inhibitors did not affect the initiation of CaR-mediated Ca2+i oscillations (Breitwieser & Gama 2001, Am J Physiol 280: C1412–21), mutation of the major PKC phosphorylation site appeared to eliminate the Ca2+i oscillatory behaviour (Young et al. 2002, J Biol Chem 277: 46871–46876). We utilized heterologous expression in HEK-293 cells to clarify whether alanine mutations in all five predicted PKC phosphorylation sites of CaR would affect the initiation and maintenance of Ca2+i oscillations. We find that small increments of Ca2+e induce Ca2+i oscillations in cells transfected with the mutant receptors, however at lower [Ca2+]e compared to wildtype CaR and over a narrow range. Abolishing all PKC sites did not affect the maintenance of Ca2+i oscillations. Desensitization of CaR appeared to be independent of receptor phosphorylation by PKC. Depletion of thapsigargin-sensitive Ca2+i stores in the presence of [Ca2+]e above the EC50 of mutant and wildtype CaR respectively, or the PKC inhibitors negatively affected the maintenance of Ca2+i oscillations and potentiated receptor desensitization. PKC activation by phorbol esters eliminated the highly cooperative Ca2+e dependent activation of CaR as well as Ca2+i oscillations. We conclude that initiation and maintenance of Ca2+i oscillations are not dependent on PKC-mediated phosphorylation / dephosphorylation of CaR. Receptor cooperativity as well as the content of thapsigargin-sensitive Ca2+i stores are critical determinants for initiation and maintenance of Ca2+i oscillations.