Regulation of G_i and G_o by mastoparan, related amphiphilic peptides, and hydrophobic amines: Mechanism and structural determinants of activity

Mastoparan (MP), a cationic, amphiphilic tetradecapeptide, stimulates guanine nucleotide exchange by GTP-binding regulatory proteins (G proteins) m a manner similar to that of G protein-coupled receptors. 1) MP stimulated exchange by isolated G protein α subunits and αβγ trimers. Relative stimulation was greater with αβγ trimers and βγ subunits could increase net MP-stimulated activity. 2) MP action was enhanced by reconstitution of trimeric G protein into phospholipid vesicles. Hill coefficients for activation were 2-4 The membrane-bound α-helical conformation of MP appeared to be the activating species. 3) MP blocked the ability of G_o to increase the affinity of muscarinic receptors for agonist ligands, suggesting that MP and the receptor may compete for a common binding site on G_o. 4) MP stimulated steady state GTPase activity at <1 μM Mg²⁺ and stimulated the dissociation of both GDP and guanosine 5′-O-(3-thiotriphosphate) at <1 nM Mg²⁺. Millimolar Mg²⁺ blocked the stimulatory effect of MP. Both high and low affinity Mg²⁺ binding sites are on the α subunit. 5) Increasing the amphiphilicity or hydrophobicity of MP enhanced its regulatory activity more than 2-fold and lowered the EC₅₀ more than 10-fold. Several natural amphiphihc peptides also displayed modest stimulatory activity 6) Benzalkonium chloride competitively antagonized the stimulation of G_i by MP but potently stimulated nucleotide exchange on G_o. 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.