EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF THE G-PROTEIN-COUPLED RECEPTOR KINASE GRK5

G protein-coupled receptor kinases (GRKs) such as rhodopsin kinase and the beta-adrenergic receptor kinase (betaARK) play an important role in agonist-specific phosphorylation and desensitization of G protein-c oupled receptors. GRK5 is a recently identified member of the GRK fami ly that has greater homology with rhodopsin kinase than with PARK. To further characterize the activity of GRK5, it has been overexpressed i n Sf9 insect cells and purified by successive chromatography on S-Seph arose and Mono S columns. GRK5 phosphorylates the beta2-adrenergic rec eptor (beta2AR), m2 muscarinic cholinergic receptor, and rhodopsin in an agonist-dependent manner to maximal stoichiometries of approximatel y 2.5,1.5, and 1 mol of phosphate/mol of receptor, respectively, with K(m) values of approximately 0.5 muM for the beta2Ar, approximately mu M for rhodopsin, and approximately 24 muM for ATP. Peptide phosphoryla tion studies suggest that in contrast to betaARK and rhodopsin kinase, GRK5 preferentially phosphorylates nonacidic peptides with a K(m) of approximately 1.5 mM. Heparin and dextran sulfate were found to be pot ent inhibitors of GRK5 with IC50 values of approximately 1 nM, thereby being at least 150-fold more potent on GRK5 than on betaARK. GRK5 can also be activated by polycations, with 10 mM polylysine promoting an approximately 2.6-fold activation. Overall, these studies demonstrate that GRK5 has unique properties that distinguish it from other members of the GRK family and that likely play an important role in modulatin g its mechanism of action.