AN APPROACH TO THE STUDY OF G-PROTEIN-COUPLED RECEPTOR KINASES - AN IN VITRO-PURIFIED MEMBRANE ASSAY REVEALS DIFFERENTIAL RECEPTOR SPECIFICITY AND REGULATION BY G-BETA-GAMMA SUBUNITS

Phosphorylation of GTP-binding-regulatory (G)-protein-coupled receptor s by specific G-protein-coupled receptor kinases (GRKs) is a major mec hanism responsible for agonist-mediated desensitization of signal tran sduction processes. However, to date, studies of the specificity of th ese enzymes have been hampered by the difficulty of preparing the puri fied and reconstituted receptor preparations required as substrates. H ere we describe an approach that obviates this problem by utilizing hi ghly purified membrane preparations from Sf9 and 293 cells overexpress ing G-protein-coupled receptors. We use this technique to demonstrate specificity of several GRKs with respect to both receptor substrates a nd the enhancing effects of G-protein beta gamma subunits on phosphory lation. Enriched membrane preparations of the beta(2)- and alpha(2)-C2 (-)adrenergic receptors (ARs, where (a)lpha(2)-C-2-AR refers to the AR whose gene is located on human chromosome 2) prepared by sucrose dens ity gradient centrifugation from Sf9 or 293 cells contain the receptor at 100-300 pmol/mg of protein and serve as efficient substrates for a gonist-dependent phosphorylation by beta-AR kinase 1 (GRK2), beta-AR k inase 2 (GRK3), or GRK5. Stoichiometries of agonist-mediated phosphory lation of the receptors by GRK2 (beta-AR kinase 1), in the absence and presence of G beta gamma, are 1 and 3 mol/mol, respectively. The rate of phosphorylation of the membrane receptors is 3 times faster than t hat of purified and reconstituted receptors. While phosphorylation of the beta(2)-AR by GRK2, -3, and -5 is similar, the activity of GRK2 an d -3 is enhanced by G beta gamma whereas that of GRK5 is not. In contr ast, whereas GRK2 and -3 efficiently phosphorylate alpha(2)-C-2-AR, GR K5 is quite weak, The availability of a simple direct phosphorylation assay applicable to any cloned G-protein-coupled receptor should great ly facilitate elucidation of the mechanisms of regulation of these rec eptors by the expanding family of GRKs.