Protein kinase C-mediated phosphorylation of the calcium-sensing receptor is stimulated by receptor activation and attenuated by calyculin-sensitive phosphatase activity.
The agonist sensitivity of the calcium-sensing receptor (CaR) can be altered by protein kinase C (PKC), with CaR residue Thr(888) contributing significantly to this effect. To determine whether CaR(T888) is a substrate for PKC and whether receptor activation modulates such phosphorylation, a phospho-specific antibody against this residue was raised (CaR(pT888)). In HEK-293 cells stably expressing CaR (CaR-HEK), but not in cells expressing the mutant receptor CaR(T888A), phorbol ester (PMA) treatment increased CaR(pT888) immunoreactivity as observed by immunoblotting and immunofluorescence. Raising extracellular Ca(2+) concentration from 0.5 to 2.5 mM increased CaR(T888) phosphorylation, an effect that was potentiated stereoselectively by the calcimimetic NPS R-467. These responses were mimicked by 5 mM extracellular Ca(2+) and abolished by the calcilytic NPS-89636 and also by PKC inhibition or chronic PMA pretreatment. Whereas CaR(T888A) did exhibit increased apparent agonist sensitivity, by converting intracellular Ca(2+) (Ca(2+)(i)) oscillations to sustained plateau responses in some cells, we still observed Ca(2+)(i) oscillations in a significant number of cells. This suggests that CaR(T888) contributes significantly to CaR regulation but is not the exclusive determinant of CaR-induced Ca(2+)(i) oscillations. Finally, dephosphorylation of CaR(T888) was blocked by the protein phosphatase 1/2A inhibitor calyculin, a treatment that also inhibited Ca(2+)(i) oscillations. In addition, calyculin/PMA cotreatment increased CaR(T888) phosphorylation in bovine parathyroid cells. Therefore, CaR(T888) is a substrate for receptor-induced, PKC-mediated feedback phosphorylation and can be dephosphorylated by a calyculin-sensitive phosphatase.
Davies
Sarah L
SL
Faculty of Life Sciences, University of Manchester, Manchester M13 9PL, United Kingdom.
Ozawa
Ai
A
McCormick
Wanda D
WD
Dvorak
Melita M
MM
Ward
Donald T
DT
D000814:Aniline Compounds / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects* / Q000235:genetics; D002273:Carcinogens / Q000494:pharmacology; D002417:Cattle; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015870:Gene Expression; D006801:Humans; D009154:Mutation; D010280:Parathyroid Glands / Q000166:cytology / Q000201:enzymology*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D054645:Protein Phosphatase 1; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000235:genetics; D044169:Receptors, Calcium-Sensing / Q000235:genetics / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000814:Aniline Compounds; D002273:Carcinogens; D004791:Enzyme Inhibitors; C111654:NPS R-467; D044169:Receptors, Calcium-Sensing; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium