title: A double mutation in the extracellular Ca2+-sensing receptor's venus flytrap domain that selectively disables L-amino acid sensing.
abstract: The extracellular Ca(2+)-sensing receptor is activated allosterically by l-amino acids, and recent molecular analysis indicates that amino acids are likely to bind in the receptor's Venus flytrap domain. In the current study we set out to identify residues in the VFT domain that specifically support amino acid binding and/or amino acid-dependent receptor activation. Herein we describe two mutations of the Ca(2+)-sensing receptor (CaR) Venus Flytrap domain, T145A and S170T, that specifically impair amino acid sensing, leaving Ca2+ sensing intact, as determined by receptor-dependent activation of intracellular Ca2+ mobilization in fura-2-loaded HEK293 cells. With respect to the wild-type CaR, T145A and S170T exhibited reduced sensitivity to l-Phe, and T145A also exhibited markedly impaired l/d selectivity. When combined, the double mutant T145A/S170T exhibited normal or near-normal sensitivity to extracellular Ca2+ but was resistant to l-Phe at concentrations up to 100 mm. We conclude that T145A/S170T selectively disables l-amino acid sensing and that the Ca2+ and l-amino acid-sensing functions of the CaR can be dissociated.
authors.affiliation: School of Molecular and Microbial Biosciences, University of Sydney, New South Wales 2006, Australia and Department of Endocrinology, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia.
authors.forename: Hee-Chang
authors.initials: HC
authors.lastname: Mun
authors.forename: Emma L
authors.initials: EL
authors.lastname: Culverston
authors.forename: Alison H
authors.initials: AH
authors.lastname: Franks
authors.forename: Charles A
authors.initials: CA
authors.lastname: Collyer
authors.forename: Roderick J
authors.initials: RJ
authors.lastname: Clifton-Bligh
authors.forename: Arthur D
authors.initials: AD
authors.lastname: Conigrave
mesh_terms: D000495:Allosteric Site; D000596:Amino Acids / Q000737:chemistry*; D049148:Aminoacylation; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D004252:DNA Mutational Analysis; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D044169:Receptors, Calcium-Sensing / Q000737:chemistry* / Q000235:genetics*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014162:Transfection
chemical_list: D000596:Amino Acids; D044169:Receptors, Calcium-Sensing; D010649:Phenylalanine; D002118:Calcium