A dominant-negative strategy for studying roles of G proteins in vivo

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 ct 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 alpha 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 alpha(i), G alpha(q), Or G alpha(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 HER 293 cells that stably express the M-2 muscarinic receptor. The presence of the G alpha(i) carboxyl-terminal peptide results in specific inhibition of GIRK activity in response to agonist stimulation of the M-2 muscarinic receptor. The G alpha(i) minigene construct completely blocks agonist-mediated M-2 mAChR K+ channel response whereas the control minigene constructs (empty vector, pcDNA3,1, and the G alpha carboxyl peptide in random order, pcDNA-G alpha(i)R) had no effect on agonist-mediated M-2 muscarinic receptor GIRK response. The inhibitory effects of the G alpha i minigene construct were specific because overexpression of peptides corresponding to the carboxyl terminus of G alpha(q) Or G alpha(s) had no effect on M-2 muscarinic receptor stimulation of the K+ channel.