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

• Published in: The Journal of biological chemistry Vol. 274; no. 10; pp. 6610 - 6616
• Main Authors: Gilchrist, A, Bünemann, M, Li, A, Hosey, M M, Hamm, H E
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 05.03.1999
• Subjects: Cell Line; Gene Expression Regulation; Genes, Dominant; GTP-Binding Proteins - genetics; GTP-Binding Proteins - metabolism; Humans; Plasmids; Potassium Channels - genetics; Potassium Channels - metabolism; Receptors, Muscarinic - genetics; Receptors, Muscarinic - metabolism; Transfection - methods
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.10.6610

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 α 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 Mg 2+ -binding sites, are rather leaky. A variety of studies have implicated the carboxyl terminus of G protein α 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 Gα i , Gα q , or Gα s . To determine whether overexpression of the carboxyl-terminal peptide would block cellular responses, we used as a test system the activation of the M 2 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 M 2 muscarinic receptor. The presence of the Gα i carboxyl-terminal peptide results in specific inhibition of GIRK activity in response to agonist stimulation of the M 2 muscarinic receptor. The Gα i minigene construct completely blocks agonist-mediated M 2 mAChR K + channel response whereas the control minigene constructs (empty vector, pcDNA3.1, and the Gα carboxyl peptide in random order, pcDNA-Gα i R) had no effect on agonist-mediated M 2 muscarinic receptor GIRK response. The inhibitory effects of the Gα i minigene construct were specific because overexpression of peptides corresponding to the carboxyl terminus of Gα q or Gα s had no effect on M 2 muscarinic receptor stimulation of the K + channel.