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pubmed:abstractText |
G protein-coupled receptor kinases (GRKs) specifically phosphorylate activated G protein-coupled receptors. While the X-ray crystal structures of several GRKs have been determined, the mechanism of interaction of GRK with GPCRs is currently unknown. To further characterize the role of the GRK2 amino terminus in receptor interaction and phosphorylation, we generated a series of point mutations within the first 10 amino acids of GRK2 and tested their ability to phosphorylate receptor and nonreceptor substrates. Although all mutants exhibited some impairment in receptor phosphorylation, three of the mutants, D3K, L4A, and D10A, were the most severely affected. Using the beta2-adrenergic receptor and rhodopsin as receptor substrates and tubulin as a nonreceptor substrate, we demonstrated that the kinase activity toward the receptors was severely decreased in the mutants, while they fully retained their ability to phosphorylate tubulin. Moreover, the amino-terminal mutants were able to bind to the receptor but, in contrast to wild-type GRK2, were not activated by receptor binding. A synthetic peptide containing residues 1-14 of GRK2 served as a noncompetitive inhibitor of receptor phosphorylation by GRK2, while a comparable peptide from GRK5 had no effect on GRK2 activity. Secondary structure prediction and circular dichroism suggest that the GRK2 amino-terminal peptide forms an amphipathic alpha-helix. Taken together, we propose a mechanism whereby the extreme amino terminus of GRK2 forms an intramolecular interaction that selectively enhances the catalytic activity of the kinase toward receptor substrates.
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