The Role of Gβγ and Domain Interfaces in the Activation of G Protein-Coupled Receptor Kinase 2†
mediaposted on 10.05.2005, 00:00 by David T. Lodowski, Jennifer F. Barnhill, Robyn M. Pyskadlo, Rodolfo Ghirlando, Rachel Sterne-Marr, John J. G. Tesmer
In response to extracellular signals, G protein-coupled receptors (GPCRs) catalyze guanine nucleotide exchange on Gα subunits, enabling both activated Gα and Gβγ subunits to target downstream effector enzymes. One target of Gβγ is G protein-coupled receptor kinase 2 (GRK2), an enzyme that initiates homologous desensitization by phosphorylating activated GPCRs. GRK2 consists of three distinct domains: an RGS homology (RH) domain, a protein kinase domain, and a pleckstrin homology (PH) domain, through which it binds Gβγ. The crystal structure of the GRK2−Gβγ complex revealed that the domains of GRK2 are intimately associated and left open the possibility for allosteric regulation by Gβγ. In this paper, we report the 4.5 Å structure of GRK2, which shows that the binding of Gβγ does not induce large domain rearrangements in GRK2, although small rotations of the RH and PH domains relative to the kinase domain are evident. Mutation of residues within the larger domain interfaces of GRK2 generally leads to diminished expression and activity, suggesting that these interfaces are important for stability and remain intact upon activation of GRK2. Geranylgeranylated Gβγ, but not a soluble mutant of Gβγ, protects GRK2 from clostripain digestion at a site within its kinase domain that is 80 Å away from the Gβγ binding site. Equilibrium ultracentrifugation experiments indicate that neither abnormally large detergent micelles nor protein oligomerization can account for the observed protection. The Gβγ-mediated binding of GRK2 to CHAPS micelles or lipid bilayers therefore appears to rigidify the kinase domain, perhaps by encouraging stable contacts between the RH and kinase domains.