Abstract
We previously demonstrated that the human calcium-sensing receptor (CaR) is allosterically activated byl-amino acids (Conigrave, A. D., Quinn, S. J., and Brown, E. M. (2000)Proc. Natl. Acad. Sci. U. S. A.97, 4814–4819). However, the domain-based location of amino acid binding has been uncertain. We now show that the Venus Fly Trap (VFT) domain of CaR, but none of its other major domains, is required for amino acid sensing. Several constructs were informative when expressed in HEK293 cells. First, the wild-type CaR exhibited allosteric activation byl-amino acids as previously observed. Second, two CaR-mGlu chimeric receptor constructs that retained the VFT domain of CaR, one containing the extracellular Cys-rich region of CaR and the other containing the Cys-rich region of the rat metabotropic glutamate type-1 (mGlu-1) receptor, together with the rat mGlu-1 transmembrane region and C-terminal tail, retained amino acid sensing. Third, a CaR lacking residues 1–599 of the N-terminal extracellular head but retaining an intact CaR transmembrane region and a functional but truncated C terminus (headless-T903 CaR) failed to respond tol-amino acids but retained responsiveness to the type-II calcimimetic NPS R-467. Finally, a T903 CaR control that retained an intact N terminus also retainedl-amino acid sensing. Taken together, the data indicate that the VFT domain of CaR is necessary forl-amino acid sensing and are consistent with the hypothesis that the VFT domain is the site ofl-amino acid binding. The findings support the concept that the mGlu-1 amino acid binding site forl-glutamate is conserved as anl-amino acid binding site in its homolog, the CaR.
Highlights
The extracellular Ca2ϩ-sensing receptor (CaR)[1] plays a key role in the regulation of whole body calcium metabolism
Effects of L-Amino Acids and NPS R-467 on calciumsensing receptor (CaR)/metabotropic glutamate type-1 (mGlu-1) Chimeric Receptors Composed of mGlu-1 Transmembrane Domains—Two chimeric receptor constructs that were composed of an intact CaR Venus Fly Trap (VFT) domain and mGlu-1 transmembrane domain were stably expressed in HEK293 cells and functionally active in Fura-2-loaded cells
The current work has demonstrated that the VFT domain of CaR alone is required for L-amino acid sensing
Summary
CaR, calcium-sensing receptor; mGlu-1, metabotropic glutamate type-1; VFT, Venus fly trap; T903 CaR, CaR truncated after residue 903. In an analysis of CaR mutants, differential effects of the type-II calcimimetic NPS R-467, whose binding site lies in the transmembrane region of the receptor, and the aromatic amino acid L-Phe indicated that their binding sites were distinct (6). We have evaluated the domain-based requirements for amino acid sensing using chimeric receptors devised by domain swapping between two receptor homologs, the human CaR, which responds to Ca2ϩ and aromatic and aliphatic amino acids, and the rat mGlu-1, which responds to L-glutamate. Functional analysis of three key chimeric receptor constructs (listed as VFT/Cys-rich region/transmembrane domainϩC-tail), including CaR/CaR/mGlu-1, CaR/mGlu-1/ mGlu-1, and Ϫ/Ϫ/T903 CaR constructs together with two controls, the wild-type CaR and T903 CaR, indicates that the VFT domain alone is required for amino acid sensing. The VFT domain of CaR is the likely site of L-amino acid binding
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