A CARBOXYL-TERMINAL DOMAIN CONTROLS THE COOPERATIVITY FOR EXTRACELLULAR CA2- A STUDY WITH RECEPTOR GREEN FLUORESCENT PROTEIN FUSIONS( ACTIVATION OF THE HUMAN CALCIUM SENSING RECEPTOR )

Calcium sensing receptors are part of a growing G protein coupled rece ptor family, which includes metabotropic glutamate, gamma-aminoisobuty ric 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 ter minus of the human calcium sensing receptor (CaR) by assessing extrace llular Ca2+-mediated changes in intracellular Ca2+ in individual HEK-2 93 cells transfected with CaR clones. In-frame fusion of EGFP to the c arboxyl terminus of CaR had no effect on either the dose response for extracellular Ca2+ activation or CaR desensitization. Carboxyl-termina l truncations, fused in-frame with EGFP (CaR Delta 1024-EGFP, CaR Delt a 908-EGFP, CaR Delta 886-EGFP, and CaR Delta 868-EGFP), were assessed for alterations in Ca2+-dependent activation or desensitization. Sign ificant effects on the dose-response relation for extracellular Ca2+ w ere observed only for the CaR Delta 868 truncation, which exhibited a decreased affinity for extracellular Ca2+ and a decrease in the appare nt cooperativity for Ca2+-dependent activation. The alterations in ext racellular Ca2+ affinity and cooperativity observed with CaR Delta 868 were recapitulated by a point mutation, T876D, in the full-length CaR -EGFP background. All truncations with wild type dose-response relatio ns exhibited desensitization time courses that were comparable to the full-length CaR, whereas the CaR Delta 868 receptor desensitized compl etely 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+-mediat ed activation of G proteins and to CaR desensitization.