Intracellular Ca2+ oscillations produced by the Ca2+-sensing receptor are mediated by negative feedback by protein kinase C at Thr888

OSVALDO REY; JAMES SINNETT-SMITH; STEVEN YOUNG; ENRIQUE ROZENGURT

Congreso; Digestive Diseases Week; 2013

AGA

he extracellular Ca2+-sensing receptor (CaR), a member of the C family of heptahelical GPCRs, is expressed in many tissues and organs not directly involved in the control of Ca2+ homeostasis, including intestinal epithelial cells. Our recent studies indicate that CaR function negatively regulates the proliferation of colonic epithelial cells (J. Biol. Chem. 287,1158-1167, 2012). Consequently, the signaling mechanisms triggered via CaR activation are attracting intense attention. Studies in individual living cells have shown that intracellular Ca2+ concentration ([Ca2+]i) oscillates upon stimulation of CaR by an elevation in extracellular Ca2+ concentration ([Ca2+]e). Here, we reexamined the notion that PKC-mediated phosphorylation of the CaR at Thr888 is necessary and sufficient for generating [Ca2+]i oscillations in response to an increase in [Ca2+]e. Results: To identify the mechanism(s) underlying [Ca2+]e-evoked [Ca2+]i oscillations via CaR, we analyzed the pattern of [Ca2+]i response in 1335 individual HEK-293 cells transfected with the human CaR, an extensively used model system in studies of CaR regulation. An increase in [Ca2+]e from 1.5 mM to 3 mM produced a rapid elevation in [Ca2+]i followed by striking oscillatory fluctuations in [Ca2+]i that did not return to baseline values, a pattern known as sinusoidal oscillations. This pattern was observed in 70% of the cell population. Most other cells (27%) displayed a rapid peak and plateau response. To determine the role of PKC in the generation of [Ca2+]i oscillations, cells were exposed to increasing concentrations (0.5-5 ?M) of the preferential PKC inhibitor Ro-31-8220 prior to stimulation by extracellular Ca2+. Ro-31-8220 at 3-5 ?M completely eliminated the [Ca2+]e-evoked [Ca2+]i oscillations and transformed the pattern to a peak and sustained plateau response. Treatment with increasing concentrations of other PKC inhibitors, including GFI or Go6983 produced an identical response. Similarly, treatment with GFI eliminated [Ca2+]e-evoked [Ca2+]i oscillations in colon-derived SW-480 cells expressing the CaR. To test further the role of PKC in the generation of oscillations, we expressed a CaR mutant in which the major PKC phosphorylation site, Thr888, was convereted to alanine (CaRT888A). None of the cells expressing CaRT888A (n= 225) showed [Ca2+]i oscillations after CaR activation. The [Ca2+]i response consisted of a rapid rise in [Ca2+]i followed by a sustained phase of elevated [Ca2+]i. Conclusion: Based on single cell analysis, we substantiate the notion that sinusoidal [Ca2+]i oscillations induced by activation of the CaR result from periodic negative feedback involving PKC-mediated phosphorylation of the CaR at Thr888.