While the mitogen-activated protein kinase (MAPK) pathway coupled to receptor tyrosine kinases has been largely clarified, little is known about MAPK activation mediated by heterotrimeric G protein-coupled receptors. In a previous study, it has been shown that endothelin-1 (ET-1) signaling through heterotrimeric G protein-coupled receptors stimulates MAPK activity in primary cultures of astrocytes (Cazaubon, S., Parker, P. J., Strosberg, A.D., and Couraud, P.O. (1993) Biochem. J. 293, 381-386). To clarify the molecular mechanism responsible for this response, involvement of the adapter proteins, Shc and Grb2, has now been investigated. It is shown here that in these cells, ET-1 stimulates tyrosine phosphorylation of Shc, resulting in its stable association with Grb2 but not with Grb3-3, a Grb2 isoform with partially deleted SH2 domain. These results demonstrate that tyrosine-phosphorylated Shc specifically interacts with the SH2 domain of Grb2. This response was rapid and transient, showing a maximum at 10 min and declining at 60 min. Interestingly, direct activation of G proteins by fluoroaluminate mimics the ET-1 effect. In addition, a shift to a higher apparent molecular mass of Raf-1 kinase, likely reflecting its hyperphosphorylation, was also detected in ET-1-treated cells. These data strongly suggest that ET-1-induced MAPK activation is a G protein-coupled pathway that involves Shc, Grb2, and probably Raf-1. In conclusion, the Shc-Grb2 complex may be involved in the activation of the MAPK pathway, not only by several receptor tyrosine kinases but also by heterotrimeric G protein-coupled receptors, such as ET-1 receptors.