Background: Activation of GRK2 requires interaction with agonist-occupied GPCRs.Results: Residues on the GRK2 N terminus and kinase domain extension collaborate to create a GPCR docking site.Conclusion: Three GRK subfamilies use similar determinants to create the putative docking site, but subtle differences may dictate selectivity.Significance: Mapping the GRK-GPCR interface is required to understand the mechanism and specificity of GRK activation, and, therefore, the regulation of GPCRs.

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G protein‐coupled receptor (GPCR) kinases (GRKs) were identified by their ability to phosphorylate activated GPCRs. They constitute a branch of the AGC kinase superfamily and are activated by hormone‐treated receptors. Their mechanism of activation is largely unknown; thus, we initiated a study to identify GRK2 residues involved in interactions with their substrate receptors. Our study focused on the kinase large lobe and an extension of the kinase domain known as the C‐tail. Four substitutions, all within or adjacent to the C‐tail, resulted a decrease in the ability of GRK2 to phosphorylate activated GPCRs, rhodopsin and the beta‐adrenergic receptor. The mutant exhibiting the most dramatic impairment, V477D, also showed significant defects in phosphorylation of non‐receptor substrates. V477D had 12‐fold lower turnover number and was resistant to activation by agonist‐treated beta‐adrenergic receptor. Therefore, Val477 and other residues in the C‐tail are expected to play a role in the activation of GRK2 by GPCRs. Current studies focus on other regions of GRK2 required for receptor binding and activation. NSF Grants MCB0315888 and MCB0744739 to R.S.‐M. and NIH Grants HL086865 and HL071818 and American Heart Association Scientist Development Grant 0235273N to J.J.G.T.

G protein‐coupled receptor (GPCR) kinases (GRKs) phosphorylate agonist‐activated GPCRs to initiate receptor desensitization, but the mechanism of kinase domain activation is unclear. Because agonist‐activated receptors dramatically stimulate the catalytic activity of GRKs, we sought to identify GRK2 residues located outside the active site that play a role in receptor interaction. Previous work suggested that active site tether (AST) residues of the kinase carboxyl‐tail extension are required for GRK activation, and that N‐terminal helix and AST interaction is important for receptor phosphorylation. We therefore carried out systematic mutagenesis of residues 3–18 and the AST of GRK2, and characterized these mutants using in vitro rhodopsin and peptide phosphorylation, GRK activation, intact cell β2‐adrenergic receptor phosphorylation, and cell based α2‐adrenergic receptor recruitment assays. Our results suggest that the N‐terminus and AST, both intrinsically disordered in the inactive kinase, play important roles in formation of the interaction site with activated receptors. Funding: NSF (RSM), NIH (JT), Canadian Institute for Health Research (MB), and FRSQ & Groupe de Recherche Universitaire sur le Médicament (AB).