Mapping of the Mastoparan - binding Site on G Proteins CROSS - LINKING OF [ 12 " I - Tyr 3 , Cys " ] MASTOPARAN

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, 1417614186). [Tyr", Cys"]MP was synthesized and shown to have regulatory activity similar to that of mastoparan when assayed in the presence of dithiothreitol (DTT). Activation by [ T ~ r ~ , c y s ~ ~ ] M P in the absence of DTT was complex in its kinetics, concentration dependence, and dependence on detergents. [lZ5ITyr3,Cys"]MP bound covalently to the a subunit of G proteins. Cross-linking was blocked by mastoparan or [Tyr3,Cys"]MP. Cross-linking was enhanced by the addition of /3r subunits, but no cross-linking to 8-y 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 [ 1251-Tyr3,Cy~11]MP/~ subunit, and CNBr hydrolysis of the ['z51-Tyr3,Cys"]MP-ao adduct yielded one major labeled peptide fragment of -6 kDa. Amino acid sequencing of this CNBr fragment prepared from recombinant a. showed that cross-linking occurred at Cys3. No a, sequence was obtained from the same fragment prepared from bovine brain ao, which is blocked by a myristoyl group at Gly'. Regulation of Go by MP was eliminated by tryptic proteolysis of the amino-terminal region. These observations suggest that the aminoterminal region of G protein a subunits contributes to the mastoparan-binding site, which may also be the receptor-binding site, and is involved in regulation of nucleotide exchange.