Abstract
The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that displays 19-25% sequence identity to the gamma-aminobutyric acid type B (GABAB) 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 mGlu1a. The Ca/1a receptor stimulated inositol phosphate production when exposed to the cationic agonists Ca2+, Mg2+, and Ba2+ in transiently transfected tsA cells (a transformed HEK 293 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 the wild-type CaR (EC50 values of 3.2, 4.7, and 4.1 mM, respectively). For the mGlu1a 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 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 significant response to Ca2+ even at 50 mM 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 Ca2+ activation of the CaR, and taken together, our results reveal a close resemblance of the activation mechanism between the CaR and the mGlu receptors.
Highlights
The cloning of the gene coding for the calcium-sensing receptor (CaR1 [1, 2]) has revealed that it together with the ␥-ami
In contrast to the metabotropic glutamate (mGlu) receptors very little is known about the site of action of agonists on the CaR, and the aim of the present study was to investigate whether the results of structure-function studies on the mGlu receptors could be applied to the CaR
The amino-terminal domain (ATD) of mGlu receptors has previously been subjected to structure-function studies, which have disclosed that for mGlu1 the endogenous agonist glutamate binds to Ser-165 and Thr188 in the ATD [9]
Summary
The cloning of the gene coding for the calcium-sensing receptor (CaR1 [1, 2]) has revealed that it together with the ␥-ami-. We have shown that the ATD of mGlu1a is related to bacterial periplasmic binding proteins (PBP), and based on the crystal structure of the Leu/Ile/Val binding protein (LIVBP) we developed a molecular model of the glutamate-binding domain of the mGlu1a receptor [9] As predicted by this model, we have shown that glutamate binding was significantly reduced by mutations of Ser-165 and/or Thr-188 in mGlu1a, which align with Ser-79 and Thr-102, respectively, which bind the amino acid ligand in LIVBP [9, 10]. By use of chimeric receptors, it was shown that this modulatory site is situated in the ATD, and point mutations located the site of action to Ser-166 in mGlu1a [15] It is at present unclear whether Ca2ϩ acts as a direct agonist [15] or a coagonist [16], it is interesting to note the pharmacological similarity to the CaR. In contrast to the mGlu receptors very little is known about the site of action of agonists on the CaR, and the aim of the present study was to investigate whether the results of structure-function studies on the mGlu receptors could be applied to the CaR
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
