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, Gi(2), 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 mariner distinct from their binding of GTP gamma S. One of these proteins, Gs alpha, required millimolar levels of flee Mg2+ for significant binding of AA-GTP, while Goa and Gi alpha(2) displayed peak AA-GTP binding at approximately 100 mu M 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 or 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.