Аннотация
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.
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