Endothelin 1 stimulates β₁pix-dependent activation of Cdc42 through the G_sα pathway

β₁Pix (PAK-interacting exchange factor) is a recently identified guanine nucleotide exchange factor (GEF) for the Rho family small G protein Cdc42/Rac. On stimulation with extracellular signals, GEFs induce the exchange of guanosine diphosphate to guanosine triphosphate, resulting in the activation of the small guanosine 5C-triphosphatases. This activation enables the signal to propagate to downstream effectors. Herein, we show that G _sα stimulation by cholera toxin increased Cdc42 activation by endothelin-1 (ET-1), whereas pertussis toxin had no effect. H-89, a protein kinase A (PKA) inhibitor, strongly inhibited Cdc42 activation by ET-1. Moreover, the overexpression of β₁Pix enhanced ET-1-induced Cdc42 activation. The essential role of β₁Pix in ET-1-induced Cdc42 activation was evidenced by the blocking of Cdc42 activation in cells expressing β₁Pix mutant lacking the ability to bind PAK (β₁Pix SH3m[W43K]) or mutant lacking GEF activity (β₁PixΔDH). The overexpression of mutant lacking the pleckstrin homology domain β₁PixΔPH, which is unable to bind phospholipids, had no effect on Cdc42 activation. These results demonstrate that β₁Pix, along with PKA, plays a crucial role in the regulation of Cdc42 activation by ET-1.