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.

G protein-coupled receptors (GPCRs), which regulate a vast number of eukaryotic processes, are desensitized by various mechanisms but, most importantly, by the GPCR kinases (GRKs). Ever since GRKs were first identified, investigators have sought to determine which structural features of GRKs are used to select for the agonist-bound states of GPCRs and how this binding event in turn enhances GRK catalytic activity. Despite a wealth of molecular information from high-resolution crystal structures of GRKs, the mechanisms driving activation have remained elusive, in part because the GRK N-terminus and active site tether region, previously proposed to serve as a receptor docking site and to be key to kinase domain closure, are often disordered or adopt inconsistent conformations. However, two recent studies have implicated other regions of GRKs as being involved in direct interactions with active GPCRs. Atomic resolution structures of GPCR–GRK complexes would help refine these models but are, so far, lacking. Here, we assess three distinct models for how GRKs recognize activated GPCRs, discuss limitations in the approaches used to generate them, and then experimentally test a hypothetical GPCR interaction site in GRK2 suggested by the two newest models.

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).

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