Regulation of Gi and G,, by Mastoparan, Related Amphiphilic Peptides, and Hydrophobic Amines

Mastoparan (MP), a cationic, amphiphilic tetradecapeptide, stimuIates guanine nucleotide exchange by GTP-binding regulatory proteins (G proteins) in a manner similar to that of G protein-coupled receptors. 1) MP stimulated exchange by isolated G protein a subunits and @y trimers. Relative stimuIation was greater with aj3~ trimers and fly subunits could increase net MP-stimuIated activity. 2) MP action was enhanced by reconstitution of trimeric G protein into phospholipid vesicles. Hill coefficients for activation were 24. The membrane-bound a-helical conformation of MP appeared to be the activating species. 3) MP blocked the ability of G0 to increase the affinity of muscarinic receptors for agonist ligands, suggesting that MP and the receptor may compete for a common binding site on GO. 4) MP stimulated steady state GTPase activity at cl pM Mg*+ and stimulated the dissociation of both GDP and guanosine 5’-O-(3-thiotriphosphate) at <l nM Mg2+. Millimolar Mg2+ blocked the stimulatory effect of MP. Both high and low affinity Mg2+ binding sites are on the a subunit. 5) Increasing the amphiphilicity or hydrophobicity of MP enhanced its regulatory activity more than 2-fold and lowered the EC60 more than lo-fold. Several natural amphiphilic peptides also displayed modest stimulatory activity. 6) Benzalkonium chIoride competitively antagonized the stimulation of Gi by MP but potentIy stimulated nucleotide exchange on GO. Because cationic, amphiphilic sequences on the cytoplasmic faces of receptors are required for G protein regulation, these findings suggest that nucleotide exchange on G proteins is regulated by the presentation of multiple cationic structures on the inner face of the plasma membrane.