Functional Importance of the Ala116–Pro136 Region in the Calcium-sensing Receptor

• Published in: The Journal of biological chemistry 2000-09, Vol.275 (38), p.29547
• Main Authors: Anders A. Jensen, Tracy A. Spalding, Ethan S. Burstein, Paul O. Sheppard, Patrick J. O'Hara, Mark R. Brann, Povl Krogsgaard-Larsen, Hans Bräuner-Osborne
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• 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.