Protein kinase C-mediated phosphorylation of the calcium-sensing receptor is stimulated by receptor activation and attenuated by calyculin-sensitive phosphatase activity.

SL Davies, A Ozawa, WD McCormick, MM Dvorak, DT Ward

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The agonist sensitivity of the calcium-sensing receptor (CaR) can be altered by protein kinase C (PKC), with CaR residue Thr888 contributing significantly to this effect. To determine whether CaRT888 is a substrate for PKC and whether receptor activation modulates such phosphorylation, a phospho-specific antibody against this residue was raised (CaRpT888). In HEK-293 cells stably expressing CaR (CaR-HEK), but not in cells expressing the mutant receptor CaRT888A, phorbol ester (PMA) treatment increased CaRpT888 immunoreactivity as observed by immunoblotting and immunofluorescence. Raising extracellular Ca2+ concentration from 0.5 to 2.5 mM increased CaRT888 phosphorylation, an effect that was potentiated stereoselectively by the calcimimetic NPS R-467. These responses were mimicked by 5 mM extracellular Ca2+ and abolished by the calcilytic NPS-89636 and also by PKC inhibition or chronic PMA pretreatment. Whereas CaRT888A did exhibit increased apparent agonist sensitivity, by converting intracellular Ca2+ (Ca2+i) oscillations to sustained plateau responses in some cells, we still observed Ca2+i oscillations in a significant number of cells. This suggests that CaRT888 contributes significantly to CaR regulation but is not the exclusive determinant of CaR-induced Ca2+i oscillations. Finally, dephosphorylation of CaRT888 was blocked by the protein phosphatase 1/2A inhibitor calyculin, a treatment that also inhibited Ca2+i oscillations. In addition, calyculin/PMA cotreatment increased CaRT888 phosphorylation in bovine parathyroid cells. Therefore, CaRT888 is a substrate for receptor-induced, PKC-mediated feedback phosphorylation and can be dephosphorylated by a calyculin-sensitive phosphatase.
Original languageEnglish
Pages (from-to)15048
Number of pages15056
JournalThe Journal of Biological Chemistry
Early online date21 Mar 2007
Publication statusPublished - May 2007


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