title: Ca2+-stimulated Ca2+ oscillations produced by the Ca2+-sensing receptor require negative feedback by protein kinase C.
abstract: We examined the role of protein kinase C (PKC) in the mechanism and regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations elicited by an increase in the extracellular concentration of Ca(2+) ([Ca(2+)](e)) in human embryonic kidney 293 cells expressing the Ca(2+)-sensing receptor (CaR). Exposure to the PKC inhibitors bisindolylmaleimide I (GF I) or Ro-31-8220 converted oscillatory responses to transient, non-oscillatory responses, significantly reducing the percentage of cells that showed [Ca(2+)](i) oscillations but without decreasing the overall response to increase in [Ca(2+)](e). Exposure to 100 nm phorbol 12,13-dibutyrate, a direct activator of PKC, eliminated [Ca(2+)](i) oscillations. Addition of phorbol 12,13-dibutyrate at lower concentrations (3 and 10 nm) did not eliminate the oscillations but greatly reduced their frequency in a dose-dependent manner. Co-expression of CaR with constitutively active mutants of PKC (either epsilon or beta(1) isoforms) also reduced [Ca(2+)](i) oscillation frequency. Expression of a mutant CaR in which the major PKC phosphorylation site is altered by substitution of alanine for threonine (T888A) eliminated oscillatory behavior, producing [Ca(2+)](i) responses almost identical to those produced by the wild type CaR exposed to PKC inhibitors. These results support a model in which phosphorylation of the CaR at the inhibitory threonine 888 by PKC provides the negative feedback needed to cause [Ca(2+)](i) oscillations mediated by this receptor.
mesh_terms: D000409:Alanine / Q000737:chemistry; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002273:Carcinogens; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D009154:Mutation; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000502:physiology*; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D014162:Transfection
chemical_list: D002273:Carcinogens; D004791:Enzyme Inhibitors; D007211:Indoles; D020033:Protein Isoforms; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface; D013912:Threonine; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D000409:Alanine; D002118:Calcium; C064758:Ro 31-8220