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.
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Department of Molecular Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710.
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Casey
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D000345:Affinity Labels / Q000138:chemical synthesis / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000138:chemical synthesis / Q000378:metabolism*; D002417:Cattle; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D066298:In Vitro Techniques; D008274:Magnesium / Q000502:physiology; D010446:Peptide Fragments / Q000378:metabolism; D010777:Photochemistry; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D010446:Peptide Fragments; D011994:Recombinant Proteins; C031907:GTP gamma-4-azidoanilide; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D008274:Magnesium