GTP-Binding Protein Fragment, G alpha

Subtype-specific binding of azidoanilido-GTP by purified G protein alpha subunits

Azidoanilido-GTP (AA-GTP), a hydrolysis-resistant, photoreactive GTP analog, is becoming an increasingly popular tool for identifying activation of specific G proteins by receptors within native plasma membranes. Despite the use of AA-GTP as an affinity probe, surprisingly little is known regarding the ability of various G protein alpha subunits to bind this analog. To directly address this issue, we compared the ability of four purified G protein alpha subunits (Go, Gi2, Gs, and Gz) to bind AA-GTP with their ability to bind GTP gamma S, a GTP analog commonly used to characterize the GTP-binding properties of G proteins. All four G alpha subunits tested bound AA-GTP in a manner distinct from their binding of GTP gamma S. One of these proteins, Gs alpha, required millimolar levels of free Mg2+ for significant binding of AA-GTP, while Go alpha and Gi alpha 2 displayed peak AA-GTP binding at approximately 100 microM free Mg2+. The fourth G alpha subunit, Gz, bound AA-GTP very poorly relative to GTP gamma S regardless of the magnesium concentration. These results indicate that individual G protein alpha subunits differ markedly in their ability to bind AA-GTP. Use of AA-GTP to identify specific G protein-receptor interactions must therefore take into account the varied abilities of G alpha subunits to bind this analog.

G alpha COOH-terminal minigene vectors dissect heterotrimeric G protein signaling

The COOH-termini of heterotrimeric guanine nucleotide-binding protein (G protein) alpha subunits (Galpha) are critical for both binding to their cognate G protein-coupled receptors (GPCRs) and determining specificity. Additionally, synthetic peptides corresponding to the COOH-terminus can serve as competitive inhibitors of receptor-G protein interactions, presumably by blocking the site on the GPCR that normally binds the G protein. To selectively antagonize G protein signal transduction events, we have generated minigene vectors that encode 14 unique COOH-terminal sequence for the 16 Galpha subunits. Minigene vectors expressing Galpha COOH-terminal peptides, or the control minigene vector, which expresses the inhibitory Galpha subunit (G(i)) peptide in random order, can be systematically introduced into cells by transfection and used to determine which G protein underlies a given GPCR-mediated response. Because Galpha COOH-terminal minigene vectors selectively block signal transduction through a given G protein, they are a powerful tool for dissecting out which G protein mediates a given biochemical or physiological function. This also provides a novel strategy for exploring the coupling mechanisms of receptors that interact with multiple G proteins, as well as for teasing out the downstream responses mediated by a specific G protein.

Alpha helix content of G protein alpha subunit is decreased upon activation by receptor mimetics

To elucidate the mechanism whereby liganded receptor molecules enhance nucleotide exchange of GTP-binding regulatory proteins (G proteins), changes in the secondary structure of the recombinant Gi1 alpha subunit (Gi1alpha) upon binding with receptor mimetics, compound 48/80 and mastoparan, were analyzed by circular dichroism spectroscopy. Compound 48/80 enhanced the initial rate of GTPgammaS binding to soluble Gi1alpha 2.6-fold with an EC50 of 30 microg/ml. With the same EC50, the mimetic decreased the magnitude of ellipticity, which is ascribed to a reduction in alpha helix content of the Gi1alpha by 7%. Likewise, mastoparan also enhanced the rate of GTPgammaS binding by 3.0-fold and decreased the magnitude of ellipticity of Gi1alpha similar to compound 48/80. In corresponding experiments using a K349P-Gi1alpha, a Gi1alpha counterpart of the unc mutant in Gsalpha in which Pro was substituted for Lys349, enhancement of the GTPgammaS binding rate by both activators was quite small. In addition, compound 48/80 showed a negligible effect on the circular dichroism spectrum of the mutant. On the other hand, a proteolytic fragment of Gi1alpha lacking the N-terminal 29 residues was activated and showed decreased ellipticity upon interaction with the compound, as did the wild-type Gi1alpha. Taken together, our results strongly suggest that the activator-induced unwinding of the alpha helix of the G protein alpha subunit is mechanically coupled to the enhanced release of bound GDP from the alpha subunit.

Binding of G alpha(o) N terminus is responsible for the voltage-resistant inhibition of alpha(1A) (P/Q-type, Ca(v)2.1) Ca(2+) channels

G-protein-mediated inhibition of presynaptic voltage-dependent Ca(2+) channels is comprised of voltage-dependent and -resistant components. The former is caused by a direct interaction of Ca(2+) channel alpha(1) subunits with G beta gamma, whereas the latter has not been characterized well. Here, we show that the N terminus of G alpha(o) is critical for the interaction with the C terminus of the alpha(1A) channel subunit, and that the binding induces the voltage-resistant inhibition. An alpha(1A) C-terminal peptide, an antiserum raised against G alpha(o) N terminus, and a G alpha(o) N-terminal peptide all attenuated the voltage-resistant inhibition of alpha(1A) currents. Furthermore, the N terminus of G alpha(o) bound to the C terminus of alpha(1A) in vitro, which was prevented either by the alpha(1A) channel C-terminal or G alpha(o) N-terminal peptide. Although the C-terminal domain of the alpha(1B) channel showed similar ability in the binding with G alpha(o) N terminus, the above mentioned treatments were ineffective in the alpha(1B) channel current. These findings demonstrate that the voltage-resistant inhibition of the P/Q-type, alpha(1A) channel is caused by the interaction between the C-terminal domain of Ca(2+) channel alpha(1A) subunit and the N-terminal region of G alpha(o).