G protein-coupled receptor Kinase 2/G alpha q/11 interaction. A novel surface on a regulator of G protein signaling homology domain for binding G alpha subunits.
G protein-coupled receptors (GPCRs) transduce cellular signals from hormones, neurotransmitters, light, and odorants by activating heterotrimeric guanine nucleotide-binding (G) proteins. For many GPCRs, short term regulation is initiated by agonist-dependent phosphorylation by GPCR kinases (GRKs), such as GRK2, resulting in G protein/receptor uncoupling. GRK2 also regulates signaling by binding G alpha(q/ll) and inhibiting G alpha(q) stimulation of the effector phospholipase C beta. The binding site for G alpha(q/ll) resides within the amino-terminal domain of GRK2, which is homologous to the regulator of G protein signaling (RGS) family of proteins. To map the Galpha(q/ll) binding site on GRK2, we carried out site-directed mutagenesis of the RGS homology (RH) domain and identified eight residues, which when mutated, alter binding to G alpha(q/ll). These mutations do not alter the ability of full-length GRK2 to phosphorylate rhodopsin, an activity that also requires the amino-terminal domain. Mutations causing G alpha(q/ll) binding defects impair recruitment to the plasma membrane by activated G alpha(q) and regulation of G alpha(q)-stimulated phospholipase C beta activity when introduced into full-length GRK2. Two different protein interaction sites have previously been identified on RH domains. The G alpha binding sites on RGS4 and RGS9, called the "A" site, is localized to the loops between helices alpha 3 and alpha 4, alpha 5 and alpha 6, and alpha 7 and alpha 8. The adenomatous polyposis coli (APC) binding site of axin involves residues on alpha helices 3, 4, and 5 (the "B" site) of its RH domain. We demonstrate that the G alpha(q/ll) binding site on the GRK2 RH domain is distinct from the "A" and "B" sites and maps primarily to the COOH terminus of its alpha 5 helix. We suggest that this novel protein interaction site on an RH domain be designated the "C" site.
Sterne-Marr
Rachel
R
Biology Department, Siena College, Loudonville, New York 12211, USA. sternemarr@siena.edu
Tesmer
John J G
JJ
Day
Peter W
PW
Stracquatanio
RoseAnn P
RP
Cilente
Jill-Ann E
JA
O'Connor
Katharine E
KE
Pronin
Alexey N
AN
Benovic
Jeffrey L
JL
Wedegaertner
Philip B
PB
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D019556:COS Cells; D002417:Cattle; D002460:Cell Line; D002522:Chlorocebus aethiops; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D005982:Glutathione Transferase / Q000235:genetics; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007294:Inositol / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D051552:beta-Adrenergic Receptor Kinases
D011993:Recombinant Fusion Proteins; D007294:Inositol; D005982:Glutathione Transferase; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins