The phosphorylation of agonist-occupied G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) functions to turn off G-protein signaling and turn on arrestin-mediated signaling. While a structural understanding of GPCR/G-protein and GPCR/arrestin complexes has emerged in recent years, the molecular architecture of a GPCR/GRK complex remains poorly defined. We used a comprehensive integrated approach of cross-linking, hydrogen-deuterium exchange mass spectrometry, electron microscopy, mutagenesis, molecular dynamics simulations and computational docking to analyze GRK5 interaction with the β 2-adrenergic receptor (β 2AR). These studies revealed a dynamic mechanism of complex formation that involves large conformational changes in the GRK5 RH/catalytic domain interface upon receptor binding. These changes facilitate contacts between intracellular loops 2 and 3 and the C-terminus of the β 2AR with the GRK5 RH bundle subdomain, membrane-binding surface and kinase catalytic cleft, respectively. These studies significantly contribute to our understanding of the mechanism by which GRKs regulate the function of activated GPCRs.
Biophysical analysis of a G protein-coupled receptor (GPCR) complex with a GPCR kinase reveals significant conformational changes in the kinase that are essential for effective receptor phosphorylation.