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

• Published in: The Journal of biological chemistry 2000-09, Vol.275 (38), p.29547
• Main Authors: Jensen, A A, Spalding, T A, Burstein, E S, Sheppard, P O, O'Hara, P J, Brann, M R, Krogsgaard-Larsen, P, Bräuner-Osborne, H
• Format: Article
• Language: English
• Subjects: 3T3 Cells; Amino Acid Sequence; Animals; Base Sequence; GTP-Binding Proteins - metabolism; Mice; Molecular Sequence Data; Mutagenesis; Protein Conformation; Receptors, Calcium-Sensing; Receptors, Cell Surface - chemistry; Receptors, Cell Surface - genetics; Receptors, Cell Surface - metabolism; Signal Transduction; Structure-Activity Relationship
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M910023199

The calcium-sensing receptor (CaR) belongs to family C of the G-protein-coupled receptor superfamily. To date 14 activating mutations in CaR showing increased sensitivity to Ca(2+) 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 phosphate assay. The vast majority of the mutants tested displayed an increased affinity for Ca(2+). Furthermore, 21 of the mutants showed increased basal activity in the absence of agonist. This constitutive activity was not diminished when the mutations were transferred to a chimeric receptor Ca/1a consisting of the amino-terminal domain of the CaR and the 7 transmembrane and intracellular 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/1a mutants demonstrating inverse agonist activity of CPCCOEt. Taken together, our results demonstrate that the Ala(116)-Pro(136) region is of key importance for the maintenance of the inactive conformation of CaR.