Abstract
Heterotrimeric guanine nucleotide-binding proteins (G proteins) transmit signals from membrane bound G protein-coupled receptors (GPCRs) to intracellular effector proteins. The G(q) subfamily of Galpha subunits couples GPCR activation to the enzymatic activity of phospholipase C-beta (PLC-beta). Regulators of G protein signaling (RGS) proteins bind to activated Galpha subunits, including Galpha(q), and regulate Galpha signaling by acting as GTPase activating proteins (GAPs), increasing the rate of the intrinsic GTPase activity, or by acting as effector antagonists for Galpha subunits. GPCR kinases (GRKs) phosphorylate agonist-bound receptors in the first step of receptor desensitization. The amino termini of all GRKs contain an RGS homology (RH) domain, and binding of the GRK2 RH domain to Galpha(q) attenuates PLC-beta activity. The RH domain of GRK2 interacts with Galpha(q/11) through a novel Galpha binding surface termed the "C" site. Here, molecular modeling of the Galpha(q).GRK2 complex and site-directed mutagenesis of Galpha(q) were used to identify residues in Galpha(q) that interact with GRK2. The model identifies Pro(185) in Switch I of Galpha(q) as being at the crux of the interface, and mutation of this residue to lysine disrupts Galpha(q) binding to the GRK2-RH domain. Switch III also appears to play a role in GRK2 binding because the mutations Galpha(q)-V240A, Galpha(q)-D243A, both residues within Switch III, and Galpha(q)-Q152A, a residue that structurally supports Switch III, are defective in binding GRK2. Furthermore, GRK2-mediated inhibition of Galpha(q)-Q152A-R183C-stimulated inositol phosphate release is reduced in comparison to Galpha(q)-R183C. Interestingly, the model also predicts that residues in the helical domain of Galpha(q) interact with GRK2. In fact, the mutants Galpha(q)-K77A, Galpha(q)-L78D, Galpha(q)-Q81A, and Galpha(q)-R92A have reduced binding to the GRK2-RH domain. Finally, although the mutant Galpha(q)-T187K has greatly reduced binding to RGS2 and RGS4, it has little to no effect on binding to GRK2. Thus the RH domain A and C sites for Galpha(q) interaction rely on contacts with distinct regions and different Switch I residues in Galpha(q).
Highlights
Proteins) transmit signals from membrane bound G protein-coupled receptors (GPCRs) to intracellular effector proteins
The C site of the RGS homology (RH) domain may bind residues on G␣q that are distinct from those that interact with regulators of G protein signaling (RGS) proteins such as RGS2 and RGS4
To predict which G␣q residues could interact with the RH domain of GRK2, a homology model of G␣q was manually docked with the RH domain from the GRK2-G␥ crystal structure (14) by imposing several specific constraints required by prior biochemical and structural analyses
Summary
Materials—HEK-293 cells were from American Type Culture Collection (CRL-1573). FuGENE 6 transfection reagent was from Roche Molecular Biochemicals. Inositol Phosphate Production Assay—HEK-293 cells were transfected with 0.1 g of EE G␣qRC or EE G␣q RC mutant constructs, 0.2 g of myc-His-tagged G1, 0.1 g of G␥2, the indicated amounts of GRK2K220R or RGS2, and pcDNA3 up to a total of 1 g of DNA. GST-GRK2-(45–178), GST-RGS2, and GST-RGS4 Interaction Assays—HEK-293 cells were transfected in 6-cm dishes with 2.0 g of G␣q or mutant G␣q cDNA, 0.2 g of myc-His-tagged G1, 0.1 g of G␥2, and. 20 l of each pull-down sample was subjected to 12% SDS-PAGE and transferred to polyvinylidene difluoride, which was probed with 2 g/ml EE monoclonal antibody followed by horseradish peroxidase-conjugated secondary antibody (1:10,000 dilution). To allow comparison of severely defective G␣q-binding mutants, a more sensitive assay was developed by using 20 g/ml fusion protein and 500 g/ml bovine brain extract protein in the pull-down assays
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
