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

Abstract

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, Gα₁₂ 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 Gα₁₂ 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 Gα₁₂ 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.