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

Tsutomu Higashijima, Elliott M. Ross

Research output: Contribution to journalArticle

78 Scopus citations

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 α 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 G(o) 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-α(o) adduct yielded one major labeled peptide fragment of ~6 kDa. Amino acid sequencing of this CNBr fragment prepared from recombinant α(o) showed that cross-linking occurred at Cys3. No α(o) sequence was obtained from the same fragment prepared from bovine brain α(o), which is blocked by a myristoyl group at Gly2. Regulation of G(o) by MP was eliminated by tryptic proteolysis of the amino-terminal region. These observations suggest that the amino-terminal 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 - Sep 23 1991

    Fingerprint

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this