Characterization of the GRK2 binding site of Galphaq.
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).
Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, 233 S. 10th St., Philadelphia, PA 19107, USA.
Peter W
PW
Day
John J G
JJ
Tesmer
Rachel
R
Sterne-Marr
Leslie C
LC
Freeman
Jeffrey L
JL
Benovic
Philip B
PB
Wedegaertner
D001665:Binding Sites; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D054769:G-Protein-Coupled Receptor Kinase 2; D020558:GTP Phosphohydrolases / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007295:Inositol Phosphates / Q000737:chemistry; D007527:Isoenzymes / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D054799:Phospholipase C beta; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism; D051552:beta-Adrenergic Receptor Kinases
D007295:Inositol Phosphates; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D017868:Cyclic AMP-Dependent Protein Kinases; C496567:GRK2 protein, human; D051552:beta-Adrenergic Receptor Kinases; D054769:G-Protein-Coupled Receptor Kinase 2; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D020558:GTP Phosphohydrolases; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D008239:Lysine