Mapping of the mastoparan-binding site on G proteins. Cross-linking of [125I-Tyr3,Cys11]mastoparan to Go.
Mastoparan (MP) activates GTP-binding regulatory proteins (G proteins) by promoting GDP/GTP exchange through a mechanism similar to that of G protein-coupled receptors (Higashijima, T., Burnier, J., and Ross, E. M. (1990) J. Biol. Chem. 265, 14176-14186). [Tyr3, Cys11]MP was synthesized and shown to have regulatory activity similar to that of mastoparan when assayed in the presence of dithiothreitol (DTT). Activation by [Tyr3,Cys11]MP in the absence of DTT was complex in its kinetics, concentration dependence, and dependence on detergents. [125I-Tyr3,Cys11]MP bound covalently to the alpha subunit of G proteins. Cross-linking was blocked by mastoparan or [Tyr3,Cys11]MP. Cross-linking was enhanced by the addition of beta gamma subunits, but no cross-linking to beta gamma subunits was observed. Cross-linking was inhibited by incubation of Go with guanosine 5'-O-(thiotriphosphate) and Mg2+ and was reversed by incubation with DTT or 2-mercaptoethanol. Stoichiometry of labeling was consistent with the cross-linking of one molecule of [125I-Tyr3,Cys11]MP/alpha subunit, and CNBr hydrolysis of the [125I-Tyr3,Cys11]MP-alpha o adduct yielded one major labeled peptide fragment of approximately 6 kDa. Amino acid sequencing of this CNBr fragment prepared from recombinant alpha o showed that cross-linking occurred at Cys3. No alpha o sequence was obtained from the same fragment prepared from bovine brain alpha o, which is blocked by a myristoyl group at Gly2. Regulation of Go by MP was eliminated by tryptic proteolysis of the amino-terminal region. These observations suggest that the amino-terminal region of G protein alpha subunits contributes to the mastoparan-binding site, which may also be the receptor-binding site, and is involved in regulation of nucleotide exchange.
Higashijima
T
T
Department of Pharmacology, Southwestern Graduate School of Biomedical Sciences, University of Texas Southwestern Medical Center, Dallas 75235-9041.
Ross
E M
EM
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D006868:Hydrolysis; D036341:Intercellular Signaling Peptides and Proteins; D010449:Peptide Mapping; D010455:Peptides; D014357:Trypsin; D014862:Wasp Venoms / Q000378:metabolism*
D000596:Amino Acids; D003432:Cross-Linking Reagents; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D014862:Wasp Venoms; C037500:mastoparan; D014357:Trypsin; D019204:GTP-Binding Proteins; D003488:Cyanogen Bromide