Shc adaptor proteins are key transducers of mitogenic signaling mediated by the G protein-coupled thrombin receptor.

Abstract

The serine protease thrombin activates G protein signaling systems that lead to Ras activation and, in certain cells, proliferation. Whereas the steps leading to Ras activation by G protein‐coupled receptors are not well defined, the mechanisms of Ras activation by receptor tyrosine kinases have recently been elucidated biochemically and genetically. The present study was undertaken to determine whether common signaling components are used by these two distinct classes of receptors. Here we report that the adaptor protein Shc, is phosphorylated on tyrosine residues following stimulation of the thrombin receptor in growth‐responsive CCL39 fibroblasts. Shc phosphorylation by thrombin or the thrombin receptor agonist peptide is maximal by 15 min and persists for > or = 2 h. Following thrombin stimulation, phosphorylated Shc is recruited to Grb2 complexes. One or more pertussis toxin‐insensitive proteins appear to mediate this effect, since (i) pertussis toxin pre‐treatment of cells does not blunt the action of thrombin and (ii) Shc phosphorylation on tyrosine can be stimulated by the muscarinic m1 receptor. Shc phosphorylation does not appear to involve protein kinase C, since the addition of 4‐beta‐phorbol‐12,13‐dibutyrate has no effect. Rather, thrombin‐induced Shc phosphorylation is enhanced in cells depleted of phorbol ester‐sensitive protein kinase C isoforms. Expression of mutant Shc proteins defective in Grb2 binding displays a dominant‐negative effect on thrombin‐stimulated p44 MAP kinase activation, gene induction and cell growth. From these data, we conclude that Shc represents a crucial point of convergence between signaling pathways activated by receptor tyrosine kinases and G protein‐coupled receptors.

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

“…In this report we present data suggesting that Shc also transduces thrombin and G 12 signals to gene expression and mitogenesis. Our results, in accordance with those of van Obberghen-Schilling's group (Chen et al, 1996), indicate that Shc is phosphorylated in response to activation of the thrombin receptor. This ®nding alone does not indicate that Shc functions in a proliferative pathway.…”

“…However, by microinjecting antibodies against phosphotyrosine or Shc, we have shown that tyrosine phosphorylation and Shc are indeed necessary for thrombin-stimulated mitogenesis in 1321N1 astrocytoma cells. These data extend those of a recent report demonstrating that a Shc mutant (Y317F), which acts as a dominant negative, inhibits thrombininduced focus formation in CCL39 cells (Chen et al, 1996). However, it should be noted that in that study the transforming e ect of thrombin was assessed in the presence of insulin, at concentrations su cient to activate the IGF-1 receptor which is expressed in ®broblasts and requires Shc for mitogenesis (Sasaoka et al, 1994).…”

“…Recently, however, several G protein-coupled receptors have been shown to stimulate tyrosine phosphorylation of Shc Cazaubon et al, 1994;Sadoshima and Izumo, 1996;Chen et al, 1996). In the case of G ilinked receptors, G bg subunits appear to mediate Shc phosphorylation, since expression of G bg subunits results in Shc phosphorylation Touhara et al, 1995), and proteins that sequester G bg subunits block Shc phosphorylation by these receptors .…”

The ras-related GTPase rac1 regulates a proliferative pathway selectively utilized by G-protein coupled receptors

“…The dbl proteins are themselves oncogenic, stimulate JNK activation and many show exchange factor activity towards the racrelated GTPases (Hart et al, 1994;Horii et al, 1994;Cerione and Zheng, 1996;Denhardt, 1996). Candidate molecules which could act upstream of these exchange factors include the src-like non-receptor tyrosine kinases, Pyk2 and the adaptor molecule shc (Dikic et al, 1996;Chen et al, 1996). Deletion of the src-like kinases Lyn, Syk, Btk and Csk abolishes muscarinic receptor activation of the MAP kinase cascade in avian Figure 4 Multiplexed assay of the e ects of ras and rac on proliferative responses to the Alpha 1B adrenergic receptor and the TrkC neurotrophin receptor.…”

Concomitant activation of MEK-1 and Rac-1 increases the proliferative potential of thyroid epithelial cells, without affecting their differentiation

“…Activation of the TSH-R, a G-protein coupled receptor, results in stimulation of adenylyl cyclase, elevation of cAMP levels and cAMP-dependent protein kinase (PKA) activation (Vassart and Dumont, 1992). Recent ®ndings suggest that Ras activation plays a role in mitogenic signal transduction induced by G-proteincoupled receptors (Alblas et al, 1993;Chen et al, 1996;Dikic et al, 1996;Sadoshima and Izumo, 1996). Indeed in thyroid cells, proliferation is dependent both on Ras as well as on PKA activation, since a dominant-interfering Ras mutant causes a signi®cant reduction of TSH-induced proliferation (Kupperman et al, 1993).…”