Abstract
The Ca(2+)-sensing receptor (CaSR) belongs to the class III G-protein-coupled receptors (GPCRs), which include receptors for pheromones, amino acids, sweeteners, and the neurotransmitters glutamate and gamma-aminobutyric acid (GABA). These receptors are characterized by a long extracellular amino-terminal domain called a Venus flytrap module (VFTM) containing the ligand binding pocket. To elucidate the molecular determinants implicated in Ca(2+) recognition by the CaSR VFTM, we developed a homology model of the human CaSR VFTM from the x-ray structure of the metabotropic glutamate receptor type 1 (mGluR1), and a phylogenetic analysis of 14 class III GPCR VFTMs. We identified critical amino acids delineating a Ca(2+) binding pocket predicted to be adjacent to, but distinct from, a cavity reminiscent of the binding site described for amino acids in mGluRs, GABA-B receptor, and GPRC6a. Most interestingly, these Ca(2+)-contacting residues are well conserved within class III GPCR VFTMs. Our model was validated by mutational and functional analysis, including the characterization of activating and inactivating mutations affecting a single amino acid, Glu-297, located within the proposed Ca(2+) binding pocket of the CaSR and associated with autosomal dominant hypocalcemia and familial hypocalciuric hypercalcemia, respectively, genetic diseases characterized by perturbations in Ca(2+) homeostasis. Altogether, these data define a Ca(2+) binding pocket within the CaSR VFTM that may be conserved in several other class III GPCRs, thereby providing a molecular basis for extracellular Ca(2+) sensing by these receptors.
Highlights
The maximal stimulation by mutant receptors is expressed as a percent of that observed for the WT receptor in the same experiment
Our model shows that the CaSR presents a binding cavity between the two lobes of the amino-terminal domain, which is reminiscent of an amino acid-binding site but of smaller dimensions than that observed for the mGluR1 (47 versus 68 Å3, respectively) (Fig. 2, B and C)
The functional characterization of the E297D mutation identified in the family described above indicated that this receptor shows a leftshifted concentration-response curve to Ca2ϩ compared with the WT receptor (EC50 ϭ 2.70 Ϯ 0.30 mM versus 4.30 Ϯ 0.20 mM, mean Ϯ S.E., n ϭ 4, p Ͻ 0.001) (Fig. 3A and TABLE TWO)
Summary
Alignment of 19 amino acids lining the orthosteric binding site of 14 human class III GPCRs Here we report a natural activating mutation affecting Glu-297 (boldface) associated with ADH. 37918 JOURNAL OF BIOLOGICAL CHEMISTRY ence of Ca2ϩ and within a box of 23,844 TIP3P water molecules, placed automatically with the leap module of AMBER 8.0. Neighbor-joining Tree—19 residues lining the binding pocket of glutamate in the mGluR1 structure [3] were extracted from 14 human class III GPCRs, concatenated into ungapped sequences, and were used to calculate a distance matrix with the MEGA2 software [29]. A neighborjoining tree was calculated out of 100 bootstrap replicas using the ␥ correction for estimating pairwise protein distances
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