Effects of pH and temperature on photoaffinity labeling of Family B G protein-coupled receptors |
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Authors: | Maoqing Dong Laurence J. Miller |
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Affiliation: | aDepartment of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, 13400 East Shea Boulevard, Scottsdale, AZ 85259, United States |
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Abstract: | The efficiency of covalent labeling of a receptor by a photolabile analogue of its natural ligand is dependent on the spatial approximation of the probe and its target. Systematic application of intrinsic photoaffinity labeling to the secretin receptor, a prototypic Family B G protein-coupled receptor, demonstrated reduced efficiency of labeling for amino-terminal and mid-region sites of labeling relative to carboxyl-terminal sites. Reduction of pH from 7.4 to 5.5 and reduction of temperature from 25 °C to 4 °C improved the efficiency of covalent labeling of the receptor with these probes. This correlated with sites of labeling at the interface between the receptor amino terminus and the receptor core, a region containing histidine residues that have their ionization affected in this pH range. Application to the calcitonin receptor, another Family B G protein-coupled receptor, yielded analogous results. These results support the consistent mode of docking peptide ligands to this group of receptors. |
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Keywords: | G protein-coupled receptors Secretin receptor Calcitonin receptor Photoaffinity labeling |
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