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A Double Mutation in the Extracellular Ca2+-sensing Receptor's Venus Flytrap Domain That Selectively Disables l-Amino Acid Sensing
- Source :
- Journal of Biological Chemistry. 280:29067-29072
- Publication Year :
- 2005
- Publisher :
- Elsevier BV, 2005.
-
Abstract
- The extracellular Ca2+-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 Ca2+-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.
- Subjects :
- Cytoplasm
Time Factors
Phenylalanine
Blotting, Western
DNA Mutational Analysis
Transfection
Sensitivity and Specificity
Biochemistry
Cell Line
Extracellular
Humans
Venus flytrap
Aminoacylation
Amino Acids
Receptor
Molecular Biology
chemistry.chemical_classification
biology
Chemistry
HEK 293 cells
Cell Biology
biology.organism_classification
Protein Structure, Tertiary
Amino acid
Kinetics
Mutation
Domain (ring theory)
Mutagenesis, Site-Directed
Biophysics
Calcium
Amino acid binding
Receptors, Calcium-Sensing
Allosteric Site
Intracellular
Protein Binding
Subjects
Details
- ISSN :
- 00219258
- Volume :
- 280
- Database :
- OpenAIRE
- Journal :
- Journal of Biological Chemistry
- Accession number :
- edsair.doi.dedup.....ea3d2d1ef9639086a4408c2ba3598179