Mapping of the mastoparan-binding site on G proteins: Cross-linking of [¹²⁵I-Tyr³,Cys¹¹]mastoparan to G_a

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 a subunit of G proteins. Cross-linking was blocked by mastoparan or [Tyr³,Cys¹¹]MP. Cross-linking was enhanced by the addition of βγ subunits, but no cross-linking to βγ subunits was observed. Cross-linking was inhibited by incubation of G_o 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/α subunit, and CNBr hydrolysis of the [¹²⁵I-Tyr³,Cys¹¹]MP-α_o adduct yielded one major labeled peptide fragment of ∼6 kDa. Amino acid sequencing of this CNBr fragment prepared from recombinant α_o showed that cross-linking occurred at Cys³. No α_o sequence was obtained from the same fragment prepared from bovine brain α_o, which is blocked by a myristoyl group at Gly². Regulation of G_o by MP was eliminated by tryptic proteolysis of the amino-terminal region. These observations suggest that the aminoterminal region of G protein α subunits contributes to the mastoparan-binding site, which may also be the receptor-binding site, and is involved in regulation of nucleotide exchange.