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

Tsutomu Higashijima, Elliott M. Ross

Research output: Contribution to journalArticle

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Abstract

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 Go 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-αo adduct yielded one major labeled peptide fragment of ∼6 kDa. Amino acid sequencing of this CNBr fragment prepared from recombinant αo showed that cross-linking occurred at Cys3. No αo sequence was obtained from the same fragment prepared from bovine brain αo, which is blocked by a myristoyl group at Gly2. Regulation of Go 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.

Original languageEnglish (US)
Pages (from-to)12655-12661
Number of pages7
JournalJournal of Biological Chemistry
Volume266
Issue number19
StatePublished - 1991

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Guanosine Triphosphate
GTP-Binding Proteins
Binding Sites
Proteins
Dithiothreitol
mastoparan
Proteolysis
Peptide Fragments
Mercaptoethanol
Guanosine
Protein Sequence Analysis
Protein Subunits
Stoichiometry
Detergents
Labeling
Hydrolysis
Brain
Nucleotides
Chemical activation
Amino Acids

ASJC Scopus subject areas

  • Biochemistry

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Mapping of the mastoparan-binding site on G proteins : Cross-linking of [125I-Tyr3,Cys11]mastoparan to Ga. / Higashijima, Tsutomu; Ross, Elliott M.

In: Journal of Biological Chemistry, Vol. 266, No. 19, 1991, p. 12655-12661.

Research output: Contribution to journalArticle

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abstract = "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 Go 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-αo adduct yielded one major labeled peptide fragment of ∼6 kDa. Amino acid sequencing of this CNBr fragment prepared from recombinant αo showed that cross-linking occurred at Cys3. No αo sequence was obtained from the same fragment prepared from bovine brain αo, which is blocked by a myristoyl group at Gly2. Regulation of Go 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.",
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