Abstract
Activator of G-protein signaling 3 (AGS3) is an accessory protein that functions to regulate the activation status of heterotrimeric G-protein subunits. To date, however, the downstream signaling pathways regulated by AGS3 remain to be fully elucidated, particularly in renal epithelial cells. In the present study, normal rat kidney (NRK-52E) proximal tubular epithelial cells were genetically modified to regulate the expression of AGS3 to investigate its role on MAPK and mTOR signaling to control epithelial cell number. Knockdown of endogenous AGS3 protein was associated with a reduced phosphorylated form of ERK5 and increased apoptosis as determined by elevated cleaved caspase-3. In the presence of the ERK5 inhibitor, BIX02189, a significant 2-fold change (P < 0.05) in G2/M transition state was detected compared to control conditions. Neither of the other MAPK, ERK1/2 or p38 MAPK, nor another pro-survival pathway, mTOR, was significantly altered by the changes in AGS3 protein levels in the renal epithelial cells. The selective ERK5 inhibitor, BIX02189, was found to dose-dependently reduce NRK cell number by up to 41% (P < 0.05) compared to control cells. In summary, these findings demonstrated that cell viability was regulated by AGS3 and was associated with ERK5 activation in renal epithelial cells.
Highlights
Accessory proteins are involved in the regulation of signal processing through their unique interactions with individual subunits in the heterotrimeric G-protein complex [1]
Activator of G-protein signaling 3 (AGS3)-deficient NRK cells were denoted as NRK-AGS3sh there was a significant reduction (P < 0.05) in and the endogenous AGS3 protein levels
NRK cells transduced with a control shRNA To confirm whether selective inhibition of ERK5 (NRK-Ctrl) were used as a comparative cell line to dem- activity would reduce cell numbers, genetically modionstrate that the effect of lentiviral vector integra- fied NRK cells expressing either control tion had no impact on AGS3 expression or normal cell or AGS3 shRNA were counted in the presence and absence of a selective ERK5 inhibitor, BIX02189, at 3 and 10 mM over a 72 hour period (n = 3–6 values per time point; Figure 3)
Summary
Accessory proteins are involved in the regulation of signal processing through their unique interactions with individual subunits in the heterotrimeric G-protein complex [1]. Accessory proteins have the ability to control the strength, efficiency, time of activation, and specificity of the signaling output. AGS proteins can alter the stability and availability of individual Gα and Gβγ subunits to control the rate of formation for the heterotrimeric Gαβγ. In some cases, this can affect the ability of cell surface G-protein coupled receptors to transmit extracellular signals into the cell [5,6,7]
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