Divergence in the G-protein-coupled receptor mitogenic signalling pathway at the level of Raf kinase.

While activation of tyrosine kinase growth factor receptors is accompanied by hyperphosphorylation of Raf-1, stimulation of receptors coupled to G-proteins has in some cases been shown to result in activation of a non-Raf MEKK rather than of Raf itself. Our finding (Weiss, R. H., and Nuccitelli, R. (1992) J. Biol. Chem. 267, 5608-5613) that the thrombin receptor requires tyrosine phosphorylation for its mitogenic effect in vascular smooth muscle cells led us to search for the molecules which are being tyrosine phosphorylated by this receptor. To determine whether mitogenic signalling of G-protein-coupled growth factor receptors results in tyrosine phosphorylation of Raf, we examined activation of Raf by two such receptors. Both thrombin and angiotensin II are mitogenic in NIH3T3 cells, but only thrombin causes hyperphosphorylation of Raf-1. Activation of Raf by thrombin is associated with phosphorylation of Raf-1 on tyrosine residues, whereas activation of Raf by angiotensin II does not involve significant tyrosine phosphorylation. However, Shc is tyrosine phosphorylated by both thrombin and angiotensin II. Thus, there exists a divergence in the mitogenic signalling pathways of the G-protein-coupled receptors relative to the Raf signalling cascade. While both thrombin and angiotensin II phosphorylate Shc and activate Raf catalytic activity, only thrombin phosphorylated Raf-1 on tyrosine. This signalling through disparate Raf-coupled pathways suggests one means by which the G-protein-coupled receptors may confer specificity in their signalling properties.