Functional importance of the Ala(116)-Pro(136) region in the calcium-sensing receptor - Constitutive activity and inverse agonism in a family C G-protein-coupled receptor

The calcium-sensing receptor (CaR) belongs to family C of the G-protein-cou pled receptor superfamily. To date 14 activating mutations in Call showing increased sensitivity to Ca2+ have been identified in humans with autosomal dominant hypocalcemia. Four of these activating mutations are found in the Ala(116)-Pro(136) region of CaR, indicating that this part of the receptor is particularly sensitive to mutation-induced activation. This region was subjected to random saturation mutagenesis, and 219 mutant receptor clones were isolated and screened pharmacologically in a high throughput screening assay. Selected mutants were characterized further in an inositol phosphat e assay. The vast majority of the mutants tested displayed an increased aff inity for Ca2+. Furthermore, 21 of the mutants showed increased basal activ ity in the absence of agonist. This constitutive activity was not diminishe d when the mutations were transferred to a chimeric receptor Ca/la consisti ng of the amino-terminal domain of the CaR and the 7 transmembrane and intr acellular domains of the metabotropic glutamate receptor mGluR1a. CPCCOEt, a noncompetitive antagonist acting at the 7 transmembrane domain of mGluR1a , suppressed the elevated basal response of the constitutively activated Ca lla mutants demonstrating inverse agonist activity of CPCCOEt Taken togethe r, our results demonstrate that the Ala(116)-Pro(136) region is of key impo rtance for the maintenance of the inactive conformation of CaR.