THE G(12) COUPLED THROMBIN RECEPTOR STIMULATES MITOGENESIS THROUGH THE SHC SH2 DOMAIN

Our previous studies in 1321N1 astrocytoma cells demonstrate that thro mbin stimulates Ras-dependent mitogenesis through the pertussis toxin insensitive G protein G(12). While the direct effecters of G(12) are u nknown, G alpha(12) can transform fibroblasts, utilize Ras and Rac dep endent signaling pathways and stimulate GTP loading of Ras. Here we ha ve examined the role of the Shc adaptor protein in mitogenic signaling by the thrombin receptor in 1321N1 cells. As has been reported in oth er systems, thrombin stimulation results in tyrosine phosphorylation o f Shc in 1321N1 cells. We also show that transient expression of G alp ha(12) results in tyrosine phosphorylation of Shc, thereby identifying Shc as the most proximal G(12) effector to date. In addition, we demo nstrate by microinjection that thrombin stimulated mitogenesis require s Shc and occurs specifically through the Shc SH2 domain. Expression o f the SH2 domain of Shc also inhibits G alpha(12) mediated induction o f an AP-1 dependent reporter gene demonstrating that G(12) utilizes Sh c to propagate downstream signals. Our data indicate that Shc is essen tial for stimulation of Ras dependent mitogenesis and gene expression by the G(12) coupled thrombin receptor.