The role of β1Pix/caveolin-1 interaction in endothelin signaling through Gα subunits

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 beta(1)Pix binds specifically to ET-Rs. Here, we show that beta(1)Pix binding to caveolin-1 is dependent on heterotrimeric G proteins activation state. beta(1)Pix interaction with different G proteins is increased in the presence of the G protein activator AMF. Moreover, extraction of cholesterol with methyl-beta-cyclodextrin disrupts the binding of beta(1)Pix to G alpha(q), G alpha(12) and phospho-Erk1/2 but not the binding of beta(1)Pix to G(beta 1). The disruption of beta(1)Pix dimerization strongly reduced the binding of caveolin-1, G alpha(q) and G alpha(12). Constitutively active mutants of G alpha(q) and G alpha(12) increased Cdc42 activation when co-expressed with beta(1)Pix but not in the presence of beta(1)Pix dimerization deficient mutant beta(1)Pix Delta (602-611). ET-1 stimulation increased the binding of phosphorylated Erk1/2 to beta(1)Pix but not to beta(1)Pix Delta (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 G(Xq to beta(1)Pix in caveolae, where dimeric beta(1)Pix acts as platform to facilitate the binding and the activation of Erk1/2. (C) 2009 Elsevier Inc. All rights reserved.