Regulation of Gi and Go 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) in a manner similar to that of G protein-coupled receptors. 1) MP stimulated exchange by isolated G protein alpha subunits and alpha beta gamma trimers. Relative stimulation was greater with alpha beta gamma trimers and beta gamma 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 alpha-helical conformation of MP appeared to be the activating species. 3) MP blocked the ability of Go 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 less than 1 microM Mg2+ and stimulated the dissociation of both GDP and guanosine 5'-O-(3-thiotriphosphate) at less than 1 nM Mg2+. Millimolar Mg2+ blocked the stimulatory effect of MP. Both high and low affinity Mg2+ binding sites are on the alpha 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 amphiphilic peptides also displayed modest stimulatory activity. 6) Benzalkonium chloride competitively antagonized the stimulation of Gi by MP but potently 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.
Higashijima
T
T
Department of Pharmacology, Southwestern Graduate School of Biomedical Sciences, University of Texas Southwestern Medical Center, Dallas 75235-9041.
Burnier
J
J
Ross
E M
EM
D000588:Amines / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D001514:Bee Venoms / Q000494:pharmacology*; D001921:Brain / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000494:pharmacology*; D010455:Peptides; D011817:Rabbits; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship; D013552:Swine; D013873:Thionucleotides / Q000378:metabolism; D014862:Wasp Venoms / Q000138:chemical synthesis / Q000494:pharmacology*
D000588:Amines; D001514:Bee Venoms; D036341:Intercellular Signaling Peptides and Proteins; D046911:Macromolecular Substances; D009842:Oligopeptides; D010455:Peptides; D011976:Receptors, Muscarinic; D013873:Thionucleotides; D014862:Wasp Venoms; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C037500:mastoparan; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D008274:Magnesium