A surface of the kinase domain critical for the allosteric activation of G protein-coupled receptor kinases

Abstract

G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate activated GPCRs and initiate their desensitization. Many prior studies suggest that activated GPCRs dock to an allosteric site on the GRKs and thereby stimulate kinase activity. The extreme N-terminal region of GRKs is clearly involved in this process, but its role is not understood. Using our recent structure of bovine GRK1 as a guide, we generated mutants of solvent-exposed residues in the GRK1 kinase domain that are conserved among GRKs but not in the extended protein kinase A, G, and C family and evaluated their catalytic activity. Mutation of select residues in strands β1 and β3 of the kinase small lobe, αD of the kinase large lobe, and the protein kinase A, G, and C kinase C-tail greatly impaired receptor phosphorylation. The most dramatic effect was observed for mutation of an invariant arginine on the β1-strand (∼1000-fold decrease in kcat/Km). These residues form a continuous surface that is uniquely available in GRKs for protein-protein interactions. Surprisingly, these mutants, as well as a 19-amino acid N-terminal truncation of GRK1, also show decreased catalytic efficiency for peptide substrates, although to a lesser extent than for receptor phosphorylation. Our data suggest that the N-terminal region and the newly identified surface interact and stabilize the closed, active conformation of the kinase domain. Receptor binding is proposed to promote this interaction, thereby enhancing GRK activity. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.