Kidney Disease Center

Milwaukee, WI, United States

Kidney Disease Center

Milwaukee, WI, United States
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Chahdi A.,Kidney Disease Center | Sorokin A.,Kidney Disease Center
Biochemical and Biophysical Research Communications | Year: 2010

Endothelin-1 (ET-1) is a potent mitogen that transmits signals through its cognate G protein-coupled receptors to stimulate extracellular signal-regulated kinase Erk1/2. Endothelin-1 receptors (ET-Rs) are known to interact with caveolin-1 and co-localize in caveolae which integrate different receptor and signaling proteins. We have recently shown that β1Pix binds specifically to ET-Rs. Here, we show that β1Pix binding to caveolin-1 is dependent on heterotrimeric G proteins activation state. β1Pix interaction with different G proteins is increased in the presence of the G protein activator AMF. Moreover, extraction of cholesterol with methyl-β-cyclodextrin disrupts the binding of β1Pix to Gαq, Gα12 and phospho-Erk1/2 but not the binding of β1Pix to Gβ1. The disruption of β1Pix dimerization strongly reduced the binding of caveolin-1, Gαq and Gα12. Constitutively active mutants of Gαq and Gα12 increased Cdc42 activation when co-expressed with β1Pix but not in the presence of β1Pix dimerization deficient mutant β1PixΔ (602-611). ET-1 stimulation increased the binding of phosphorylated Erk1/2 to β1Pix but not to β1PixΔ (602-611). RGS3 decreased ET-1-induced Cdc42 activation. These results strongly suggest that the activation of ET-Rs leads to the compartmentalization and the binding of Gαq to β1Pix in caveolae, where dimeric β1Pix acts as platform to facilitate the binding and the activation of Erk1/2. © 2009 Elsevier Inc. All rights reserved.


Chahdi A.,Kidney Disease Center | Sorokin A.,Kidney Disease Center
Cellular Signalling | Year: 2010

Endothelin-1 (ET-1) is a vasoconstrictor peptide known to be a potent mitogen for glomerular mesangial cells. We have shown that ET-1 stimulates the adaptor protein p66Shc through Rac/Cdc42 guanine nucleotide exchange factor β1Pix. In this study, we demonstrate that ET-1-induced serine phosphorylation of p66Shc is mediated through Gαi3. Pertussis toxin treatment of cells induced a significant decrease in the interaction between β1Pix and ETA-R, and an increase in the binding of Gαi3 and Gβ1 to β1Pix. Activation of heterotrimeric G proteins by AlF4 - resulted in an increase of Gαi3 binding to β1Pix, which was significantly disrupted in cells expressing β1Pix dimerization deficient mutant, β1PixΔ (602-611). In cells expressing β1PixΔ (602-611), ET-1-induced p66Shc activation was also significantly decreased. Specific inhibition of EGF receptor by AG1478 blocked ET-1-induced p66Shc activation and the binding of p66Shc and Gαi3 to β1Pix. Inhibition of Erk1/2 blocked p66Shc activation induced by ET-1. Altogether, our results indicate that ET-1 activates p66Shc through EGF receptor transactivation, leading to the activation of Gαi3, β1Pix and Erk1/2. © 2009 Elsevier Inc. All rights reserved.

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