Identification of Acidic Residues in the Extracellular Loops of the Seven-transmembrane Domain of the Human Ca2+ Receptor Critical for Response to Ca2+ and a Positive Allosteric Modulator

Abstract

We investigated the role of the eight acidic residues in the extracellular loops (exo-loops) of the seven-transmembrane domain of the human Ca 2؉ receptor (hCaR) in receptor activation by Ca 2؉ and in response to a positive allosteric modulator, NPS R-568. Both in the context of the full-length receptor and of a truncated receptor lacking the extracellular domain (Rho-C-hCaR), we mutated each acidic residue to alanine, singly and in combination, and tested the effect on expression of the receptor, on activation by Ca 2؉ , and on NPS R-568 augmentation of sensitivity to Ca 2؉ . Of the eight acidic residues, mutation of any of three in exo-loop 2, Asp 758 , Glu 759 , and Glu 767 , increased the sensitivity of both the full-length hCaR and of Rho-C-hCaR to activation by Ca 2؉ . Mutation of all five acidic residues in exo-loop 2, whether in the full-length receptor or in Rho-C-hCaR, impaired cell surface expression of the mutant receptor and thereby largely abolished response to Ca 2؉ . Mutation of Glu 837 in exo-loop 3 to alanine did not alter Ca 2؉ sensitivity of the full-length receptor, but in both the latter context and in Rho-C-hCaR, alanine substitution of Glu 837 drastically reduced sensitivity to NPS R-568. Our data point to a key role of three specific acidic residues in exo-loop 2 in hCaR activation and to Glu 837 at the junction between exo-loop 3 and transmembrane helix seven in response to NPS R-568. We speculate on the basis of these results that the three acidic residues we identified in exo-loop 2 help maintain an inactive conformation of the seven-transmembrane domain of the hCaR.The G protein-coupled [Ca 2ϩ ] o receptor (CaR) 1 plays a central role in the regulation of [Ca 2ϩ ] o homeostasis (1, 2). [Ca 2ϩ ] o activates the CaR in the parathyroid, thereby inhibiting parathyroid hormone secretion, and in the kidney, causing increased urinary calcium excretion. The physiological importance of the CaR in determining the level at which [Ca 2ϩ ] o is set in vivo has been documented by the identification of inactivating mutations in the CaR gene as the cause of familial hypocalciuric hypercalcemia and activating mutations as the cause of autosomal dominant hypocalcemia (3, 4). Naturally occurring CaR mutations identified in subjects with autosomal dominant hypocalcemia generally cause increased CaR sensitivity to [Ca 2ϩ ] o rather than causing constitutive activation (5).The CaR belongs to a unique subfamily, family 3, of G protein-coupled receptors (GPCR) with an unusually large N-terminal, extracellular domain (ECD) comprised of Venus's-flytrap (VFT) and cysteine-rich domains, in addition to the seventransmembrane domain (7TM) characteristic of all GPCR (6). Studies with chimeric family 3 GPCR (7-10) and the threedimensional structure of the metabotropic glutamate type 1 receptor (mGluR1) determined by x-ray crystallography (11) show that the VFT is the site of agonist binding in family 3 GPCR. The precise site(s) for binding to the CaR, however, have not been identified.The activation of family ...

Positive and Negative Allosteric Modulators of the Ca2+-sensing Receptor Interact within Overlapping but Not Identical Binding Sites in the Transmembrane Domain

“…No attempt was made to model the CaSR in its activated form bound to Calindol or NPS R-568, because of the lack of an adequate three-dimensional template (33). However, our data emphasizes the crucial role of Glu-837 in anchoring NPS R-568, as previously observed (17,19), and of Calindol, presumably through a salt bridge with the protonated secondary amine of the two compounds as we previously proposed for the calcilytics. Our study also underscores the role of Ile-841 in anchoring the two calcimimetics as indicated by the lack of Ca 2ϩ potentiation of IP response by both compounds in the mutants.…”

“…The subsequent conformational change that presumably occurs throughout the cysteine-rich region affects the seven TM regions leading to receptor activation. Interestingly, three acidic residues located in the extracellular loop 2 of the CaSR have been demonstrated to maintain an inactive conformation of the seven TM regions, further highlighting the complex processes of CaSR activation (17). However, the precise molecular mechanisms involved in such activation are not known.…”

The Venus Fly Trap Domain of the Extracellular Ca2+-sensing Receptor Is Required for l-Amino Acid Sensing

“…Headless CaR Construct-A headless CaR deletion mutant elsewhere referred to as Rho-C-CaR (9 -11) or T903-Rhoc (12) containing 20 amino acids of the N terminus of rhodopsin and amino acids 600 -903 of the hCaR was constructed in pcDNA3.1 using PCR as described previously (9,11). Briefly, the sense primer was 100 nucleotides long, corresponding to the nucleotide sequence of the N-terminal 20-amino acid signal peptide of bovine rhodopsin and nucleotide positions 1797-1832 of the hCaR (GenBank TM 20759).…”

Modeling and Mutagenesis of the Binding Site of Calhex 231, a Novel Negative Allosteric Modulator of the Extracellular Ca2+-sensing Receptor

“…Position 3.36 has also been shown to play a pivotal role in antagonist binding to several different GPCRs. Last, Glu-837 7.39 is a key residue of various unrelated GPCRs for anchoring competitive antagonists and is implicated in the recognition of a reference calcimimetic in the CaSR (21). Our results concerning the E837A mutant are in agreement with those obtained by Hu et al (21) showing that this mutant is expressed at levels comparable to those of the WT receptor when transfected in HEK293 cells, and its sensitivity to Ca 2ϩ is not altered despite a lower maximal response.…”