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J Biol Chem, Vol. 274, Issue 10, 6610-6616, March 5, 1999

A Dominant-Negative Strategy for Studying Roles of G Proteins in Vivo

Annette Gilchrist^§¶, Moritz Bünemann^§, Anli Li, M. Marlene Hosey^§, and Heidi E. Hamm^§¶

From the  Institute for Neuroscience, ^§ Department of Molecular Pharmacology and Biochemistry, Northwestern University, Chicago, Illinois 60611 and ^¶ Department of Pharmacology, University of Illinois, Chicago, Illinois 60612

G proteins play a critical role in transducing a large variety of signals into intracellular responses. Increasingly, there is evidence that G proteins may play other roles as well. Dominant-negative constructs of the  subunit of G proteins would be useful in studying the roles of G proteins in a variety of processes, but the currently available dominant-negative constructs, which target Mg²⁺-binding sites, are rather leaky. A variety of studies have implicated the carboxyl terminus of G protein  subunits in both mediating receptor-G protein interaction and in receptor selectivity. Thus we have made minigene plasmid constructs that encode oligonucleotide sequences corresponding to the carboxyl-terminal undecapeptide of G_i, G_q, or G_s. To determine whether overexpression of the carboxyl-terminal peptide would block cellular responses, we used as a test system the activation of the M₂ muscarinic receptor activated K⁺ channels in HEK 293 cells. The minigenes were transiently transfected along with G protein-regulated inwardly rectifying K⁺ channels (GIRK) into HEK 293 cells that stably express the M₂ muscarinic receptor. The presence of the G_i carboxyl-terminal peptide results in specific inhibition of GIRK activity in response to agonist stimulation of the M₂ muscarinic receptor. The G_i minigene construct completely blocks agonist-mediated M₂ mAChR K⁺ channel response whereas the control minigene constructs (empty vector, pcDNA3.1, and the G carboxyl peptide in random order, pcDNA-G_iR) had no effect on agonist-mediated M₂ muscarinic receptor GIRK response. The inhibitory effects of the G_i minigene construct were specific because overexpression of peptides corresponding to the carboxyl terminus of G_q or G_s had no effect on M₂ muscarinic receptor stimulation of the K⁺ channel.

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