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

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 Gα_i 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 Gα_i 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 (Gα_i, Gα _q, Gα₁₂, and Gα₁₃) or the control mini-gene vector, which expresses the Gα_i peptide in random order (G_iR), 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.