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J. Biol. Chem., Vol. 275, Issue 38, 29547-29555, September 22, 2000

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Functional Importance of the Ala¹¹⁶-Pro¹³⁶ Region in the Calcium-sensing Receptor
CONSTITUTIVE ACTIVITY AND INVERSE AGONISM IN A FAMILY C G-PROTEIN-COUPLED RECEPTOR^*

Anders A. Jensen, Tracy A. Spalding^§, Ethan S. Burstein^§, Paul O. Sheppard^¶, Patrick J. O'Hara^¶, Mark R. Brann^§, Povl Krogsgaard-Larsen, and Hans Bräuner-Osborne

From the  NeuroScience PharmaBiotec Research Centre, Department of Medicinal Chemistry, The Royal Danish School of Pharmacy, 2 Universitetsparken, DK-2100 Copenhagen, Denmark, ^§ ACADIA Pharmaceuticals Inc., San Diego, California, 92121, and the ^¶ ZymoGenetics Inc., Seattle, Washington 98102

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 Ca²⁺ have been identified in humans with autosomal dominant hypocalcemia. Four of these activating mutations are found in the Ala¹¹⁶-Pro¹³⁶ 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 Ca²⁺. 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 Ala¹¹⁶-Pro¹³⁶ region is of key importance for the maintenance of the inactive conformation of CaR.

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