Abstract
A homology model for the human calcium sensing receptor (hCaR) transmembrane domain utilizing bovine rhodopsin (bRho) structural information was derived and tested by docking the allosteric antagonist, NPS 2143, followed by mutagenesis of predicted contact sites. Mutation of residues Phe-668 (helix II), Arg-680, or Phe-684 (helix III) to Ala (or Val or Leu) and Glu-837 (helix VII) to Ile (or Gln) reduced the inhibitory effects of NPS 2143 on [Ca2+]i responses. The calcimimetic NPS R-568 increases the potency of Ca2+ in functional assays of CaR. Mutations at Phe-668, Phe-684, or Glu-837 attenuated the effects of this compound, but mutations at Arg-680 had no effect. In all cases, mutant CaRs responded normally to Ca2+ or phenylalanine, which act at distinct site(s). Discrimination by the Arg-680 mutant is consistent with the structural differences between NPS 2143, which contains an alkyl bridge hydroxyl group, and NPS R-568, which does not. The homology model of the CaR transmembrane domain robustly accounts for binding of both an allosteric antagonist and agonist, which share a common site, and provides a basis for the development of more specific and/or potent allosteric modulators of CaR. These studies suggest that the bRho backbone can be used as a starting point for homology modeling of even distantly related G protein-coupled receptors and provide a rational framework for investigation of the contributions of the transmembrane domain to CaR function.
Highlights
The calcium sensing receptor (CaR)1 is a member of family C of the G protein-coupled receptor (GPCR) superfamily, which
Chimeras between CaR and metabotropic glutamate receptors (mGluRs) have shown that the phenylalkylamine binding site is localized to the transmembrane domain of CaR (13–15), and recent studies have suggested that negatively charged residues within the e2 and e3 loops may contribute to binding of NPS R-568 (15, 16)
Sequence Alignment of human calcium sensing receptor (hCaR) with Bovine Rhodopsin—The alignment shown in Table I is the result of an iterative process following several rounds of model building and experimental verification or falsification
Summary
CaR, calcium sensing receptor; hCaR, human CaR; GPCR, G protein-coupled receptor; mGluR, metabotropic glutamate receptor; GABABR, ␥-aminobutyric acid (type B) receptor; bRho, bovine rhodopsin; TM, transmembrane; GFP, green fluorescent protein; EGFP, enhanced GFP; IP, immunoprecipitation; ELISA, enzyme-linked immunosorbent assay; wt, wild type. CaR activity (in the presence of Ca2ϩ) is allosterically modulated by amino acids (6), small peptides (7, 8), as well as a family of structurally related phenylalkylamines (9, 10). The putative binding site(s) for amino acids and potentially for peptides (including poly-L-arginine, protamine, -amyloid) have been identified within the extracellular agonist binding domain by homology with metabotropic glutamate receptors, and have been localized to a triple serine motif, Ser-169 –171 (12). In addition to providing a basis for the development of more specific and/or potent allosteric modulators of CaR, these studies provide an example of the generalizability of the bRho backbone as a starting point for homology modeling of even distantly related GPCRs
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