Increased receptor stimulation elicits differential calcium-sensing receptor(T888) dephosphorylation.

The calcium-sensing receptor (CaR) elicits oscillatory Ca2+i mobilization associated with dynamic, inhibitory protein kinase C-mediated phosphorylation of CaRT888. While modest CaR stimulation elicits Ca2+i oscillations, greater stimulation either increases oscillation frequency or elicits sustained responses by an unknown mechanism. Here, moderate CaR stimulation (2.5 mM Ca2+o, 10 min) increased CaRT888 phosphorylation (160-kDa mature receptor) 5-fold in CaR stably transfected HEK-293 cells, whereas 3–5 mM Ca2+o treatments were without apparent effect. Treatment with 2 mM Ca2+o caused sustained CaRT888 phosphorylation (≥20 min) and oscillatory Ca2+i mobilization. However, 5 mM Ca2+o increased CaRT888 phosphorylation only briefly while eliciting sustained Ca2+i mobilization, suggesting that greater CaR activation induces rapid CaRT888 dephosphorylation, thus permitting sustained Ca2+i responses. Indeed, 5 mM Ca2+o stimulated protein phosphatase 2A activity and induced CaRT888 dephosphorylation following acute phorbol ester pretreatment, the latter effect being mimicked by CaR-positive allosteric modulators (NPS-R467 and L-Phe). Finally, the phosphatase inhibitor calyculin-A reversed CaR-induced inhibition of parathyroid hormone secretion from bovine parathyroid slices and normal human parathyroid cells, demonstrating the physiological importance of phosphorylation status on parathyroid function. Therefore, high Ca2+o-stimulated protein kinase C acts in concert with high Ca2+o-induced phosphatase activity to generate and maintain CaR-induced Ca2+i oscillations via the dynamic phosphorylation and dephosphorylation of CaRT888.