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 alpha 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 Galphai, Galphaq, or Galphas. 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 Galphai carboxyl-terminal peptide results in specific inhibition of GIRK activity in response to agonist stimulation of the M2 muscarinic receptor. The Galphai minigene construct completely blocks agonist-mediated M2 mAChR K+ channel response whereas the control minigene constructs (empty vector, pcDNA3.1, and the Galpha carboxyl peptide in random order, pcDNA-GalphaiR) had no effect on agonist-mediated M2 muscarinic receptor GIRK response. The inhibitory effects of the Galphai minigene construct were specific because overexpression of peptides corresponding to the carboxyl terminus of Galphaq or Galphas had no effect on M2 muscarinic receptor stimulation of the K+ channel.
Gilchrist
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A
Institute for Neuroscience, Northwestern University, Chicago, Illinois 60611, USA.
Bünemann
M
M
Li
A
A
Hosey
M M
MM
Hamm
H E
HE
D002460:Cell Line; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D005786:Gene Expression Regulation; D005799:Genes, Dominant; D006801:Humans; D010957:Plasmids; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000379:methods
D015221:Potassium Channels; D011976:Receptors, Muscarinic; D019204:GTP-Binding Proteins