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J. Biol. Chem., Vol. 275, Issue 38, 29547-29555, September 22, 2000
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, and
From the 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.
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
To whom correspondence should be addressed: Dept. of Medicinal
Chemistry, Royal Danish School of Pharmacy, 2 Universitetsparken, DK-2100 Copenhagen, Denmark. Tel.: 45-3530-6518; Fax: 45-3530-6040; E-mail: hbo@dfh.dk.
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