A Carboxyl-terminal Domain Controls the Cooperativity for Extracellular Ca2+ Activation of the Human Calcium Sensing Receptor

Abstract

Calcium sensing receptors are part of a growing G protein-coupled receptor family, which includes metabotropic glutamate, gamma-aminoisobutyric acid, and pheromone receptors. The distinctive structural features of this family include large extracellular domains that bind agonist and large intracellular, carboxyl-terminal domains of as yet undefined function(s). We have explored the contribution(s) of the carboxyl terminus of the human calcium sensing receptor (CaR) by assessing extracellular Ca2+-mediated changes in intracellular Ca2+ in individual HEK-293 cells transfected with CaR clones. In-frame fusion of EGFP to the carboxyl terminus of CaR had no effect on either the dose response for extracellular Ca2+ activation or CaR desensitization. Carboxyl-terminal truncations, fused in-frame with EGFP (CaRDelta1024-EGFP, CaRDelta908-EGFP, CaRDelta886-EGFP, and CaRDelta868-EGFP), were assessed for alterations in Ca2+-dependent activation or desensitization. Significant effects on the dose-response relation for extracellular Ca2+ were observed only for the CaRDelta868 truncation, which exhibited a decreased affinity for extracellular Ca2+ and a decrease in the apparent cooperativity for Ca2+-dependent activation. The alterations in extracellular Ca2+ affinity and cooperativity observed with CaRDelta868 were recapitulated by a point mutation, T876D, in the full-length CaR-EGFP background. All truncations with wild type dose-response relations exhibited desensitization time courses that were comparable to the full-length CaR, whereas the CaRDelta868 receptor desensitized completely after two exposures to 10 mM Ca2+. Interestingly, the CaR point mutation T876D exhibited desensitization comparable to wild type CaR, suggesting that this mutation specifically modifies CaR cooperativity. In conclusion, these studies suggest that amino acid residues between 868 and 886 are critical to the apparent cooperativity of Ca2+-mediated activation of G proteins and to CaR desensitization.