Identification of acidic residues in the extracellular loops of the seven-transmembrane domain of the human Ca²⁺ receptor critical for response to Ca²⁺ and a positive allosteric modulator

We investigated the role of the eight acidic residues in the extracellular loops (exo-loops) of the seven-transmembrane domain of the human Ca²⁺ receptor (hCaR) in receptor activation by Ca²⁺ and in response to a positive allosteric modulator, NPS R-568. Both in the context of the full-length receptor and of a truncated receptor lacking the extracellular domain (Rho-C-hCaR), we mutated each acidic residue to alanine, singly and in combination, and tested the effect on expression of the receptor, on activation by Ca²⁺, and on NPS R-568 augmentation of sensitivity to Ca²⁺. Of the eight acidic residues, mutation of any of three in exo-loop 2, Asp⁷⁵⁸, Glu⁷⁵⁹, and Glu⁷⁶⁷, increased the sensitivity of both the full-length hCaR and of Rho-C-hCaR to activation by Ca²⁺. Mutation of all five acidic residues in exo-loop 2, whether in the full-length receptor or in Rho-C-hCaR, impaired cell surface expression of the mutant receptor and thereby largely abolished response to Ca²⁺. Mutation of Glu⁸³⁷ in exo-loop 3 to alanine did not alter Ca²⁺ sensitivity of the full-length receptor, but in both the latter context and in Rho-C-hCaR, alanine substitution of Glu⁸³⁷ drastically reduced sensitivity to NPS R-568. Our data point to a key role of three specific acidic residues in exo-loop 2 in hCaR activation and to Glu⁸³⁷ at the junction between exo-loop 3 and transmembrane helix seven in response to NPS R-568. We speculate on the basis of these results that the three acidic residues we identified in exo-loop 2 help maintain an inactive conformation of the seven-transmembrane domain of the hCaR.