Gα Minigenes Expressing C-terminal Peptides Serve as Specific Inhibitors of Thrombin-mediated Endothelial Activation

Annette Gilchrist, Jurgen F. Vanhauwe, Anli Li, Tarita O. Thomas, Tatyana Voyno-Yasenetskaya, Heidi E. Hamm*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

87 Scopus citations

Abstract

The C termini of G protein α subunits are critical for binding to their cognate receptors, and peptides corresponding to the C terminus can serve as competitive inhibitors of G protein-coupled receptor-G protein interactions. This interface is quite specific as a single amino acid difference annuls the ability of a Gαi peptide to bind the A1 adenosine receptor (Gilchrist, A., Mazzoni, M., Dineen, B., Dice, A., Linden, J., Dunwiddie, T., and Hamm, H. E. (1998) J. Biol. Chem. 273, 14912-14919). Recently, we demonstrated that a plasmid minigene vector encoding the C-terminal sequence of Gαi could specifically inhibit downstream responses to agonist stimulation of the muscarinic M2 receptor (Gilchrist, A., Bunemann, M., Li, A., Hosey, M. M., and H. E. Hamm (1999) J. Biol. Chem. 274, 6610-6616). To selectively antagonize G protein signal transduction events and determine which G protein underlies a given thrombin-induced response, we generated minigene vectors that encode the C-terminal sequence for each family of Gα subunits. Minigene vectors expressing Gα C-terminal peptides (Gαi, Gα q, Gα12, and Gα13) or the control mini-gene vector, which expresses the Gαi peptide in random order (GiR), were systematically introduced into a human microvascular endothelial cell line. The C-terminal peptides serve as competitive inhibitors presumably by blocking the site on the G protein-coupled receptor that normally binds the G protein. Our results not only confirm that each G protein can control certain signaling events, they emphasize the specificity of the G protein-coupled receptor-G protein interface. In addition, the C-terminal Gα minigenes appear to be a powerful tool for dissecting out the G protein that mediates a given physiological function following thrombin activation.

Original languageEnglish (US)
Pages (from-to)25672-25679
Number of pages8
JournalJournal of Biological Chemistry
Volume276
Issue number28
DOIs
StatePublished - Jul 13 2001

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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