title: 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.
abstract: 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.
authors.affiliation: NeuroScience PharmaBiotec Research Centre, Department of Medicinal Chemistry, The Royal Danish School of Pharmacy, 2 Universitetsparken, DK-2100 Copenhagen, Denmark.
authors.forename: A A
authors.initials: AA
authors.lastname: Jensen
authors.forename: T A
authors.initials: TA
authors.lastname: Spalding
authors.forename: E S
authors.initials: ES
authors.lastname: Burstein
authors.forename: P O
authors.initials: PO
authors.lastname: Sheppard
authors.forename: P J
authors.initials: PJ
authors.lastname: O'Hara
authors.forename: M R
authors.initials: MR
authors.lastname: Brann
authors.forename: P
authors.initials: P
authors.lastname: Krogsgaard-Larsen
authors.forename: H
authors.initials: H
authors.lastname: Bräuner-Osborne
mesh_terms: D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019204:GTP-Binding Proteins / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011487:Protein Conformation; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013329:Structure-Activity Relationship
chemical_list: D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface; D019204:GTP-Binding Proteins