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The Agonist-binding Domain of the Calcium-sensing Receptor Is Located at the Amino-terminal Domain

The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that displays 19–25% sequence identity to the γ-aminobutyric acid type B (GABA B ) and metabotropic glutamate (mGlu) receptors. All three groups of receptors have a large amino-terminal domain (ATD), which for the mGlu receptors h...

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Published in:The Journal of biological chemistry 1999-06, Vol.274 (26), p.18382-18386
Main Authors: Bräuner-Osborne, Hans, Jensen, Anders A., Sheppard, Paul O., O'Hara, Patrick, Krogsgaard-Larsen, Povl
Format: Article
Language:English
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Summary:The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that displays 19–25% sequence identity to the γ-aminobutyric acid type B (GABA B ) and metabotropic glutamate (mGlu) receptors. All three groups of receptors have a large amino-terminal domain (ATD), which for the mGlu receptors has been shown to bind the endogenous agonist. To investigate whether the agonist-binding domain of the CaR also is located in the ATD, we constructed a chimeric receptor named Ca/1a consisting of the ATD of CaR and the seven transmembrane region and C terminus of mGlu 1a . The Ca/1a receptor stimulated inositol phosphate production when exposed to the cationic agonists Ca 2+ , Mg 2+ , and Ba 2+ in transiently transfected tsA cells (a transformed HEK 293 cell line). The pharmacological profile of Ca/1a (EC 50 values of 3.3, 2.6, and 3.9 m m for these cations, respectively) was very similar to that of the wild-type CaR (EC 50 values of 3.2, 4.7, and 4.1 m m , respectively). For the mGlu 1a receptor, it has been shown that Ser-165 and Thr-188, which are located in the ATD, are involved in the agonist binding. An alignment of CaR with the mGlu receptors showed that these two amino acid residues have been conserved in CaR as Ser-147 and Ser-170, respectively. Each of these residues was mutated to alanines and tested pharmacologically using the endogenous agonist Ca 2+ . CaR-S147A showed an impaired function as compared with wild-type CaR both with respect to potency of Ca 2+ (4-fold increase in EC 50 ) and maximal response (79% of wild-type response). CaR-S170A showed no significant response to Ca 2+ even at 50 m m concentration. In contrast, each of the two adjacent mutations, S169A and S171A, resulted in pharmacological profiles almost identical to that of the wild-type receptor. These data demonstrate that Ser-170 and to some extent Ser-147 are involved in the Ca 2+ activation of the CaR, and taken together, our results reveal a close resemblance of the activation mechanism between the CaR and the mGlu receptors.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.274.26.18382