The binding site for the beta gamma subunits of heterotrimeric G proteins on the beta-adrenergic receptor kinase.
The beta gamma subunits of heterotrimeric G proteins play important roles in regulating receptor-stimulated signal transduction processes. Recently appreciated among these is their role in the signaling events that lead to the phosphorylation and subsequent desensitization of muscarinic cholinergic (Haga, K., and Haga, T. (1992) J. Biol. Chem. 267, 2222-2227) and beta-adrenergic (Pitcher, J. A., Inglese, J., Higgins, J. B., Arriza, J. L., Casey, P. J., Kim, C., Benovic, J. L., Kwatra, M. M., Caron, M. G., and Lefkowitz, R. J. (1992) Science 257, 1264-1267) receptors. Beta gamma mediates the membrane targeting of the beta-adrenergic receptor kinase (beta ARK), in response to receptor activation, through a specific beta ARK-beta gamma interaction. This process utilizes the membrane-anchoring properties of the isoprenylated gamma subunit of beta gamma. In the present study, we have employed three distinct approaches to identify the region within the carboxyl terminus of beta ARK which binds beta gamma and thereby results in membrane translocation. We studied the ability of beta gamma to enhance the enzymatic activity of a series of truncated mutants of bovine beta ARK1, the ability of glutathione S-transferase fusion proteins containing various lengths of the carboxyl terminus of beta ARK to bind beta gamma subunits, and the ability of synthetic peptides comprised of beta ARK sequences to inhibit beta gamma activation of beta ARK1. We find that the minimal beta gamma binding domain of beta ARK is localized to a 125-amino acid residue stretch, the distal end of which is located 19 residues from the carboxyl terminus. A single 28-mer peptide (Trp643 to Ser670) derived from this sequence effectively inhibited beta gamma activation of beta ARK1, with an IC50 of 76 microM. The identification of this "beta gamma binding domain" on beta ARK and the development of peptide inhibitors provide important tools for the study of G protein-coupled receptor desensitization, as well as for the investigation of beta gamma activation of other G protein-effector systems.
Koch
W J
WJ
Howard Hughes Medical Institute, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
Inglese
J
J
Stone
W C
WC
Lefkowitz
R J
RJ
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008715:Methionine / Q000378:metabolism; D016296:Mutagenesis; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion; D015398:Signal Transduction; D013462:Sulfur Radioisotopes; D014162:Transfection; D051552:beta-Adrenergic Receptor Kinases
D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D013462:Sulfur Radioisotopes; D008715:Methionine; D005982:Glutathione Transferase; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D019204:GTP-Binding Proteins