Molecular Medicine Israel

Structural and Functional Analysis of a β2-Adrenergic Receptor Complex with GRK5


  • GRK5-β2AR binding is enhanced by receptor and kinase ligands and acidic lipids
  • GRK5 binding to the β2AR involves a multi-site interaction
  • Receptor binding triggers substantial conformational changes in GRK5
  • RH/catalytic domain separation in GRK5 is essential for receptor phosphorylation


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 (MS), electron microscopy, mutagenesis, molecular dynamics simulations, and computational docking to analyze GRK5 interaction with the β2-adrenergic receptor (β2AR). 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 β2AR 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.


G-protein-coupled receptors (GPCRs) regulate the activity of numerous effector molecules and play an essential role in coordinating the ability of cells to rapidly respond to their environment (Lefkowitz, 2007). Agonist binding to a GPCR activates heterotrimeric G proteins, which mediate downstream signaling and ultimately a physiological response. GPCR signaling is dynamic and undergoes rapid regulation by GPCR kinases (GRKs), which specifically phosphorylate activated GPCRs, and arrestins, which bind to GRK-phosphorylated GPCRs to promote receptor desensitization and endocytosis as well as arrestin-mediated signaling (Figure 1A). While significant structural and dynamic insight on GPCR interaction with G proteins (Rasmussen et al., 2011) and arrestins (Kang et al., 2015) has been gained in recent years, we still know little about how GRKs target activated GPCRs.

The GRK family includes seven mammalian members across three sub-families: GRK1 (GRK1 and 7), GRK2 (GRK2 and 3), and GRK4 (GRK4, 5, and 6). Significant insight into GRK function has come from X-ray crystallography, and structures for GRK1, GRK2, GRK4, GRK5, and GRK6 have been reported. These structures reveal that the Regulator of G-protein signaling homology (RH) and catalytic domains have extensive contacts with each other and help to hold the kinase in an inactive open conformation (Figure 1B). Recent studies suggest that an N-terminal α-helical domain may regulate catalytic domain closure and that this process may be regulated by receptor binding (Pao et al., 2009, Boguth et al., 2010, Huang et al., 2011). Indeed, this domain has been observed in some crystal forms of GRK1 (Huang et al., 2011) and GRK6 (Boguth et al., 2010) and appears to stabilize catalytic domain closure and activation. While these studies have provided significant insight into how GRKs might function, we currently know little about the critical regions that mediate GRK interaction with GPCRs or how this interaction ultimately regulates GRK activation. Here, we utilize the β2AR and GRK5 as a model system to characterize the mechanisms by which GRKs phosphorylate activated GPCRs.

Sign up for our Newsletter