Effects of pH and temperature on photoaffinity labeling of Family B G protein-coupled receptors.
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 degrees C to 4 degrees 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.
Dong
Maoqing
M
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, 13400 East Shea Boulevard, Scottsdale, AZ 85259, USA.
Miller
Laurence J
LJ
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D019920:Photoaffinity Labels; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D019920:Photoaffinity Labels; D043562:Receptors, G-Protein-Coupled