A carboxyl-terminal domain controls the cooperativity for extracellular Ca²⁺ activation of the human calcium sensing receptor: A study with receptor-green fluorescent protein fusions

Calcium sensing receptors are part of a growing G protein-coupled receptor family, which includes metabotropic glutamate, γ-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 (CaRΔ1024-EGFP, CaRA908-EGFP, CaRΔA886-EGFP, and CaRΔ868-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 CaRΔ868 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 CaRΔ868 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 CaRΔ868 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 eooperativity of Ca²⁺-mediated activation of G proteins and to CaR desensitization.