Role of the GRK2 extreme amino terminus and active site tether in forming G protein‐coupled receptor docking site

Abstract

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).