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Sci. STKE, 5 February 2002
Vol. 2002, Issue 118, p. pl1
[DOI: 10.1126/stke.2002.118.pl1]

PROTOCOLS

Gα 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) α subunits (Gα) 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 Gα subunits. Minigene vectors expressing Gα COOH-terminal peptides, or the control minigene vector, which expresses the inhibitory Gα 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 Gα 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

Citation: A. Gilchrist, A. Li, H. E. Hamm, Gα COOH-Terminal Minigene Vectors Dissect Heterotrimeric G Protein Signaling. Sci. STKE 2002, pl1 (2002).

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