The G12 coupled thrombin receptor stimulates mitogenesis through the Shc SH2 domain

Our previous studies in 1321N1 astrocytoma cells demonstrate that thrombin stimulates Ras-dependent mitogenesis through the pertussis toxin insensitive G protein G12. While the direct effectors of G12 are unknown, G12 can transform fibroblasts, utilize Ras and Rac dependent signaling pathways and stimulate GTP loading of Ras. Here we have examined the role of the Shc adaptor protein in mitogenic signaling by the thrombin receptor in 1321N1 cells. As has been reported in other systems, thrombin stimulation results in tyrosine phosphorylation of Shc in 1321N1 cells. We also show that transient expression of G alpha12 results in tyrosine phosphorylation of Shc, thereby identifying Shc as the most proximal G12 effector to date. In addition, we demonstrate by microinjection that thrombin stimulated mitogenesis requires Shc and occurs specifically through the Shc SH2 domain. Expression of the SH2 domain of Shc also inhibits G alpha12 mediated induction of an AP-1 dependent reporter gene demonstrating that G12 utilizes Shc to propagate downstream signals. Our data indicate that Shc is essential for stimulation of Ras dependent mitogenesis and gene expression by the G12 coupled thrombin receptor.