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 dis plays 19-25% sequence identity to the gamma-aminobutyric acid type B (GABA, ) and metabotropic glutamate (mGlu) receptors, All three groups of receptor s have a large amino-terminal domain (ATD), which for the mGlu receptors ha s been shown to bind the endogenous agonist, To investigate whether the ago nist-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 t ransmembrane region and C terminus of mGlu(1a). The Ca/1a receptor stimulat ed inositol phosphate production when exposed to the cationic agonists Ca2, Mg2+, and Ba2+ in transiently transfected tsA cells (a transformed HEK 29 3 cell line). The pharmacological profile of Ca/1a (EC50 values of 3.3, 2.6 , and 3.9 mM for these cations, respectively) was very similar to that of t he wild-type CaR (EC50 values of 3.2, 4.7, and 4.1 mM, respectively). For t he 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 C aR with the mGlu receptors showed that these two amino acid residues have b een conserved in CaR as Ser-147 and Ser-170, respectively. Each of these re sidues was mutated to alanines and tested pharmacologically using the endog enous agonist Ca2+. CaR-S147A showed an impaired function as compared with wild-type CaR both with respect to potency of Ca2+ (4-fold increase in EC50 ) and maximal response (79% of wild-type response). CaR-S170A showed no sig nificant response to Ca2+ even at 50 mM concentration. In contrast, each of the two adjacent mutations, S169A and S171A, resulted in pharmacological p rofiles almost identical to that of the wild-type receptor. These data demo nstrate that Ser-170 and to some extent Ser-147 are involved in the Ca2+ ac tivation of the CaR, and taken together, our results reveal a close resembl ance of the activation mechanism between the CaR and the mGlu receptors.