Endothelin-1 (ET-1) induces cell proliferation and differentiation through multiple G-protein-linked signaling systems, including p21 ras activation. Whereas p21 ras activation and desensitization by receptor tyrosine kinases have been extensively investigated, the kinetics of p21 ras activation induced by engagement of G-protein-coupled receptors remains to be fully elucidated. In the present study we show that ET-1 induces a biphasic activation of p21 ras in rat glomerular mesangial cells. The first peak of activation of p21 ras , at 2-5 min, is mediated by immediate association of phosphorylated Shc with the guanosine exchange factor Sos1 via the adaptor protein Grb2. This initial activation of p21 ras results in activation of the extracellular signal-regulated kinase (ERK) cascade. We demonstrate that ET-1 signaling elicits a negative feedback mechanism, modulating p21 ras activity through ERKdependent Sos1 phosphorylation, findings which were confirmed using an adenovirus MEK construct. Subsequent to p21 ras and ERK deactivation, Sos1 reverts to the non-phosphorylated condition, enabling it to bind again to the Grb2/Shc complex, which is stabilized by persistent Shc phosphorylation. However, the resulting secondary activation of p21 ras at 30 min does not lead to ERK activation, correlating with intensive, ET-1-induced expression of MAP kinase phosphatase-1, but does result in increased p21 ras -associated phosphatidylinositol 3-kinase activity. Our data provide evidence that ET-1-induced biphasic p21 ras activation causes sequential stimulation of divergent downstream signaling pathways.