G
COOH-Terminal Minigene Vectors Dissect Heterotrimeric G Protein Signaling
Annette Gilchrist1,
Anli Li2, and
Heidi E Hamm3*
cue BIOtech, Inc., 303 East Chicago Avenue, Ward 17-171, Chicago, IL 60611, USA. E-mail: annette{at}cuebiotech.com
Department of Pharmacology, Vanderbilt University Medical Center, 442 Robinson Research Building, Nashville, TN 37232, USA. E-mail: Anli.Li{at}mcmail.vanderbilt.edu
Heidi E. Hamm, Department of Pharmacology, Vanderbilt University Medical Center, 442 Robinson Research Building, Nashville, TN 37232, USA. E-mail: heidi.hamm{at}mcmail.vanderbilt.edu
Abstract:
The COOH-termini of heterotrimeric guanine nucleotide-binding protein (G protein) 
subunits (G) 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 G subunits. Minigene vectors expressing G COOH-terminal peptides, or the control minigene vector, which expresses the inhibitory G subunit (Gi) 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 G 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.
*Corresponding author: Telephone, 615-343-3533; fax, 615-343-1084; e-mail, heidi.hamm{at}mcmail.vanderbilt.edu
