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An experimental strategy to probe Gq contribution to signal transduction in living cells.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2021 Jan-Jun; Vol. 296, pp. 100472. Date of Electronic Publication: 2021 Feb 25. - Publication Year :
- 2021
-
Abstract
- Heterotrimeric G protein subunits Gαq and Gα11 are inhibited by two cyclic depsipeptides, FR900359 (FR) and YM-254890 (YM), both of which are being used widely to implicate Gq/11 proteins in the regulation of diverse biological processes. An emerging major research question therefore is whether the cellular effects of both inhibitors are on-target, that is, mediated via specific inhibition of Gq/11 proteins, or off-target, that is, the result of nonspecific interactions with other proteins. Here we introduce a versatile experimental strategy to discriminate between these possibilities. We developed a Gαq variant with preserved catalytic activity, but refractory to FR/YM inhibition. A minimum of two amino acid changes were required and sufficient to achieve complete inhibitor resistance. We characterized the novel mutant in HEK293 cells depleted by CRISPR-Cas9 of endogenous Gαq and Gα11 to ensure precise control over the Gα-dependent cellular signaling route. Using a battery of cellular outcomes with known and concealed Gq contribution, we found that FR/YM specifically inhibited cellular signals after Gαq introduction via transient transfection. Conversely, both inhibitors were inert across all assays in cells expressing the drug-resistant variant. These findings eliminate the possibility that inhibition of non-Gq proteins contributes to the cellular effects of the two depsipeptides. We conclude that combined application of FR or YM along with the drug-resistant Gαq variant is a powerful in vitro strategy to discern on-target Gq against off-target non-Gq action. Consequently, it should be of high value for uncovering Gq input to complex biological processes with high accuracy and the requisite specificity.<br />Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article.<br /> (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)
- Subjects :
- Depsipeptides pharmacology
GTP-Binding Protein alpha Subunits metabolism
GTP-Binding Protein alpha Subunits, Gq-G11 metabolism
HEK293 Cells
Heterotrimeric GTP-Binding Proteins metabolism
Heterotrimeric GTP-Binding Proteins physiology
Humans
Peptides, Cyclic pharmacology
Signal Transduction drug effects
GTP-Binding Protein alpha Subunits physiology
GTP-Binding Protein alpha Subunits, Gq-G11 physiology
Signal Transduction physiology
Subjects
Details
- Language :
- English
- ISSN :
- 1083-351X
- Volume :
- 296
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 33639168
- Full Text :
- https://doi.org/10.1016/j.jbc.2021.100472