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.
Gilchrist
Annette
A
cue BIOtech, Inc., 303 East Chicago Avenue, Ward 17-171, Chicago, IL 60611, USA. annette@cuebiotech.com
Li
Anli
A
Hamm
Heidi E
HE
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics*; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics*; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D010446:Peptide Fragments / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics*
D010446:Peptide Fragments; D020962:Heterotrimeric GTP-Binding Proteins