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 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.