Mastoparan interacts with the carboxyl terminus of the alpha subunit of Gi.
Mastoparan, a peptide toxin from wasp venom, stimulates guanine nucleotide binding and hydrolysis by G proteins. To elucidate the site of mastoparan-G protein interaction, we utilized a polyclonal antibody (R16,17) directed against the carboxyl terminus of the Gi alpha subunit to develop a competitive enzyme-linked immunosorbent assay. We investigated the ability of mastoparan to influence R16,17 antibody binding to G protein alpha subunits in a purified preparation of brain Gi and in neutrophil membrane extracts. Mastoparan antagonized the ability of R16,17 to detect G protein alpha subunits with an IC50 of 15 microM in the purified preparation and with an IC50 of 1 microM for the predominant G protein population in membrane extracts. This reduction was not seen when an unrelated peptide or a peptide of similar charge composition to mastoparan was used in place of mastoparan in the assay. Additionally, antibody R16,17 blocked up to 85% of mastoparan-stimulated GTPase activity. Taken together, these data indicate that the interaction of mastoparan with G protein depends in part on the carboxyl terminus of Gi alpha. Pertussis toxin-catalyzed ADP-ribosylation of Gi alpha markedly inhibited mastoparan-stimulated GTPase activity but only slightly attenuated the ability of mastoparan to recognize G protein. These data suggest that ribosylation inhibits mastoparan-induced G protein activation by a mechanism distinct from the ability of mastoparan to physically interact with G protein. Since mastoparan is thought to mimic hormone-liganded receptors, these findings may be applicable to the mechanism of receptor-Gi protein uncoupling that results from ADP-ribosylation of the G protein.
Weingarten
R
R
Department of Immunology, Research Institute of Scripps Clinic, La Jolla, California 92037.
Ransnäs
L
L
Mueller
H
H
Sklar
L A
LA
Bokoch
G M
GM
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D000918:Antibody Specificity; D001514:Bee Venoms / Q000378:metabolism*; D001667:Binding, Competitive; D001923:Brain Chemistry; D002417:Cattle; D002462:Cell Membrane / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D009504:Neutrophils / Q000032:analysis; D010455:Peptides; D037342:Pertussis Toxin; D010566:Virulence Factors, Bordetella / Q000378:metabolism; D014862:Wasp Venoms / Q000378:metabolism* / Q000494:pharmacology
D000906:Antibodies; D001514:Bee Venoms; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D010566:Virulence Factors, Bordetella; D014862:Wasp Venoms; D000246:Adenosine Diphosphate Ribose; C037500:mastoparan; D037342:Pertussis Toxin; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins