Receptor heterodimerization: essential mechanism for platelet-derived growth factor-induced epidermal growth factor receptor transactivation

Previous studies showed that the epidermal growth factor receptor (EGFR) can be transactivated by platelet-derived growth factor (PDGF) stimulation and that EGFR transactivation is required for PDGF-stimulated cell migration. To investigate the mechanism for cross talk between the PDGF beta receptor (PDGFbetaR) and the EGFR, we stimulated rat aortic vascular smooth muscle cells (VSMC) with 20 ng of PDGF/ml. Transactivation of the EGFR, defined by receptor tyrosine phosphorylation, occurred with the same time course as PDGFbetaR activation. Basal formation of PDGFbetaR-EGFR heterodimers was shown by coimmunoprecipitation studies, and interestingly, disruption of this receptor heterodimer abolished EGFR transactivation. Breakdown of the heterodimer was observed when VSMC were pretreated with antioxidants or with a Src family kinase inhibitor. Disruption of heterodimers decreased ERK1 and ERK2 activation by PDGF. Although PDGF-induced PDGFbetaR activation was abolished after pretreatment with 1 microM AG1295 (a specific PDGF receptor kinase inhibitor), EGFR transactivation was still observed, indicating that PDGFbetaR kinase activity is not required. In conclusion, our data demonstrate that the PDGFbetaR and the EGFR form PDGFbetaR-EGFR heterodimers basally, and we suggest that heterodimers represent a novel signaling complex which plays an important role in PDGF signal transduction.     

Sphingosine 1-phosphate transactivates c-Met as well as epidermal growth factor receptor (EGFR) in human gastric cancer cells

Receptor tyrosine kinases (RTKs) are transactivated by the stimulation of G protein-coupled receptors (GPCRs). Sphingosine 1-phosphate (S1P), a ligand of GPCR, is known as a tumor-promoting lipid, but its signaling pathways are not fully understood. We here demonstrated that S1P induces rapid and transient tyrosine phosphorylation of epidermal growth factor receptor (EGFR) and c-Met in gastric cancer cells, both of which have been proposed as prognostic markers of gastric cancers. The pathway of S1P-induced c-Met transactivation is Gi-independent and matrix metalloproteinase-independent, which differs from that of EGFR transactivation. Our results indicate that S1P acts upstream of various RTKs and thus may act as a potent stimulator of gastric cancer.     

Beta-adrenergic receptor-mediated DNA synthesis in cardiac fibroblasts is dependent on transactivation of the epidermal growth factor receptor and subsequent activation of extracellular signal-regulated kinases

Cardiac hypertrophy often leads to heart failure and is associated with abnormal myocardial adrenergic signaling. This enlargement of myocardial mass can involve not only an increase in cardiomyocyte size, but increased proliferation of cardiac fibroblasts. A potential key player in the cardiac hypertrophic response is the ERK family of MAPKs. To gain mechanistic insight into adrenergic regulation of myocardial mitogenic signaling, we examined beta-adrenergic receptor (beta-AR) stimulation of ERK activation and DNA synthesis in cultured adult rat cardiac fibroblasts, including the involvement of tyrosine kinases in this signaling pathway. Addition of the beta-AR agonist isoproterenol (ISO) to serum-starved cells induced DNA synthesis in a dose-dependent manner, and this was inhibited by selective inhibitors of the epidermal growth factor receptor (EGFR). Importantly and in agreement with the involvement of MAPKs and the EGFR in this response in cardiac fibroblasts, the EGFR inhibitor AG1478 attenuated ISO-induced ERK phosphorylation. Moreover, pretreatment with PP2, a selective inhibitor of the Src tyrosine kinase, attenuated both ISO-mediated EGFR phosphorylation and ERK activation. Furthermore, studies in these cardiac fibroblasts showed that phosphatidylinositol 3-kinase contributed to beta-AR-mediated ERK activation, but not to EGFR activation. Finally, studies using selective inhibitors of matrix metalloproteases indicated that they and heparin-bound EGF shedding were involved in beta-AR-induced ERK activation and subsequent DNA synthesis in cardiac fibroblasts. Because these cells primarily express the beta(2)-AR subtype, our findings indicate that beta(2)-AR-mediated EGFR transactivation of intracellular tyrosine kinase signaling pathways is the major signaling pathway responsible for the adrenergic stimulation of mitogenesis of cardiac fibroblasts.     

Ligand-independent and EGF receptor-supported transactivation: lessons from beta2-adrenergic receptor signalling

Transactivation of epidermal growth factor receptor (EGFR) by G protein-coupled receptors (GPCRs) is currently understood to be mediated by matrix metalloproteases (MMPs) and the release of EGF-like ligands. This ligand-mediated process also suggests that downstream of EGFR the signalling in response to GPCR ligands or EGF appears to be indistinguishable. Here we provide evidence that transactivation of EGFR by the beta2-adrenergic receptor (beta2-AR) is independent of MMPs and results in an incomplete downstream signalling involving extracellular signal-activated kinase (ERK) but not PLCgamma1 and Akt. In contrast, beta2-AR has the ability to activate PLCgamma1 when the EGFR is primed either by co-stimulation with EGF or by increased basal activity due to over-expression. In that way but not via the beta2-AR-mediated transactivation the EGFR docking sites pY992 and pY1173 may be generated which are critical for PLCgamma1. This EGFR-supported transactivation is strongly dependent on EGFR tyrosine kinase, c-Src, and the c-Src-specific EGFR tyrosine residue 845 and represents a novel paradigm of EGFR transactivation.     

Delta-opioid receptors activate ERK/MAP kinase via integrin-stimulated receptor tyrosine kinases

Integrin-mediated cell adherence to extracellular matrix proteins results in stimulation of ERK1/2 activity, a mechanism involving focal adhesion tyrosine kinases (pp125FAK, Pyk-2) and epidermal growth factor receptors (EGFRs). G protein-coupled receptors (GPCRs) may also mediate ERK1/2 activation in an integrin-dependent manner, the underlying signaling mechanism of which still remains unclear. Here we demonstrate that the δ-opioid receptor (DOR), a typical GPCR, stimulates ERK1/2 activity in HEK293 cells via integrin-mediated transactivation of EGFR function. Inhibition of integrin signaling by RGDT peptides, cytochalasin, and by keeping the cells in suspension culture both blocked [D-Ala², D-Leu⁵]enkephalin (DADLE)- and etorphine-stimulated ERK1/2 activity. Integrin-dependent ERK1/2 activation does not involve FAK/Pyk-2, because over-expression of the FAK/Pyk-2 inhibitor SOCS-3 failed to attenuate DOR signaling. Exposure of the cells to the EGFR inhibitors AG1478 and BPIQ-I blocked DOR-mediated ERK1/2 activation. Because RGDT peptides also prevented DOR-mediated EGFR activation, the present findings indicate that in HEK293 cells DOR-stimulated ERK1/2 activity is mediated by integrin-stimulated EGFRs. Further studies with the phospholipase C (PLC) inhibitors U73122 and ET-18-OCH₃ revealed that opioid-stimulated integrin activation is sensitive to PLC. In contrast, integrin-mediated transactivation of EGFR function appears to be dependent on PKC-δ, as indicated by studies with rottlerin and siRNA knock-down. A similar ERK1/2 signaling pathway was observed for NG108-15 cells, a neuronal cell line endogenously expressing the DOR. In these cells, the nerve growth factor TrkA receptor replaces the EGFR in connecting DOR-activated integrins to the Ras/Raf/ERK1/2 pathway. Together, these data describe an alternative ERK1/2 signaling pathway in which the DOR transactivates the growth factor receptor associated mitogen-activated protein kinase cascade in an integrin-dependent manner.     

When a G protein-coupled receptor does not couple to a G protein

Classically, G protein-coupled receptors (GPCRs) relay signals by directly activating heterotrimeric guanine nucleotide-binding proteins (G proteins). Increasing evidence indicates that GPCRs may also signal through G protein-independent pathways. JAK/STATs, Src-family tyrosine kinases, GRKs/beta-arrestins, and PDZ domain-containing proteins have been suggested to directly relay signals from GPCRs independent of G proteins. In addition, our laboratory recently reported that the beta(2) adrenergic receptor (beta(2)AR) could switch from G protein-coupled to G protein-independent ERK (extracellular signal-regulated kinase) activation in an agonist dosage-dependent manner. This finding provides a novel mechanism for G protein-independent GPCR signaling. This review focuses on recent progress in understanding the mechanisms by which G protein-independent GPCR signaling occurs.     

Interferon gamma-dependent transactivation of epidermal growth factor receptor

The present report provides evidence that, in A431 cells, interferon gamma (IFNgamma) induces the rapid (within 5 min), and reversible, tyrosine phosphorylation of the epidermal growth factor receptor (EGFR). IFNgamma-induced EGFR transactivation requires EGFR kinase activity, as well as activity of the Src-family tyrosine kinases and JAK2. Here, we show that IFNgamma-induced STAT1 activation in A431 and HeLa cells partially depends on the kinase activity of both EGFR and Src. Furthermore, in these cells, EGFR kinase activity is essential for IFNgamma-induced ERK1,2 activation. This study is the first to demonstrate that EGFR is implicated in IFNgamma-dependent signaling pathways.     

Src and Pyk2 mediate G-protein-coupled receptor activation of epidermal growth factor receptor (EGFR) but are not required for coupling to the mitogen-activated protein (MAP) kinase signaling cascade

The epidermal growth factor receptor (EGFR) and the non-receptor protein tyrosine kinases Src and Pyk2 have been implicated in linking a variety of G-protein-coupled receptors (GPCR) to the mitogen-activated protein (MAP) kinase signaling cascade. In this report we apply a genetic strategy using cells isolated from Src-, Pyk2-, or EGFR-deficient mice to explore the roles played by these protein tyrosine kinases in GPCR-induced activation of EGFR, Pyk2, and MAP kinase. We show that Src kinases are critical for activation of Pyk2 in response to GPCR-stimulation and that Pyk2 and Src are essential for GPCR-induced tyrosine phosphorylation of EGFR. By contrast, Pyk2, Src, and EGFR are dispensable for GPCR-induced activation of MAP kinase. Moreover, GPCR-induced MAP kinase activation is normal in fibroblasts deficient in both Src and Pyk2 (Src-/-Pyk2-/- cells) as well as in fibroblasts deficient in all three Src kinases expressed in these cells (Src-/-Yes-/-Fyn-/- cells). Finally, experiments are presented demonstrating that, upon stimulation of GPCR, activated Pyk2 forms a complex with Src, which in turn phosphorylates EGFR directly. These experiments reveal a role for Src kinases in Pyk2 activation and a role for Pyk2 and Src in tyrosine phosphorylation of EGFR following GPCR stimulation. In addition, EGFR, Src family kinases, and Pyk2 are not required for linking GPCRs with the MAP kinase signaling cascade.     

The role of G-protein coupled receptors and associated proteins in receptor tyrosine kinase signal transduction

It is well established that stimulation of G-protein coupled receptors (GPCRs) can activate signalling from receptor tyrosine kinases by a process termed transactivation. Indeed, in recent years, it has become apparent that transactivation is a general phenomenon that has been demonstrated for many unrelated GPCRs and receptor tyrosine kinases. In this case the GPCR/G-protein participation is up-stream of the receptor tyrosine kinase. Substantial research has addressed these findings but meanwhile another mechanism of cross talk has been slowly emerging. For over a decade, a growing body of evidence has demonstrated that numerous growth factors use G-proteins and attendant signalling molecules such as beta-arrestins that participate down-stream of the receptor tyrosine kinase to signal to effectors, such as p42/p44 MAPK. This review highlights this novel mechanism of cross talk between receptor tyrosine kinases and GPCRs, which is distinct from growth factor receptor transactivation by GPCRs.     

c-Src trafficking and co-localization with the EGF receptor promotes EGF ligand-independent EGF receptor activation and signaling

c-Src is a non-receptor tyrosine kinase that associates with both the plasma membrane and endosomal compartments. In many human cancers, especially breast cancer, c-Src and the EGF receptor (EGFR) are overexpressed. Dual overexpression of c-Src and EGFR correlates with a Src-dependent increase in activation of EGFR, and synergism between these two tyrosine kinases increases the mitogenic activity of EGFR. Despite extensive studies of the functional interaction between c-Src and EGFR, little is known about the interactions in the trafficking pathways for the two proteins and how that influences signaling. Given the synergism between c-Src and EGFR, and the finding that EGFR is internalized and can signal from endosomes, we hypothesized that c-Src and EGFR traffic together through the endocytic pathway. Here we use a regulatable c-SrcGFP fusion protein that is a bona fide marker for c-Src to show that c-Src undergoes constitutive macropinocytosis from the plasma membrane into endocytic compartments. The movement of c-Src was dependent on its tyrosine kinase activity. Stimulation of cells with EGF revealed that c-Src traffics into the cell with activated EGFR and that c-Src expression and kinase activity prolongs EGFR activation. Surprisingly, even in the absence of EGF addition, c-Src expression induced activation of EGFR and of EGFR-mediated downstream signaling targets ERK and Shc. These data suggest that the synergy between c-Src and EGFR also occurs as these two kinases traffic together, and that their co-localization promotes EGFR-mediated signaling.     

NADPH-oxidase-dependent reactive oxygen species mediate EGFR transactivation by FPRL1 in WKYMVm-stimulated human lung cancer cells

Cross talk between unrelated cell surface receptors, such as G-protein-coupled receptors (GPCR) and receptor tyrosine kinases (RTK), is a crucial signaling mechanism to expand the cellular communication network. We investigated the ability of the GPCR formyl peptide receptor-like 1 (FPRL1) to transactivate the RTK epidermal growth factor receptor (EGFR) in CaLu-6 cells. We observed that stimulation with WKYMVm, an FPRL1 agonist isolated by screening synthetic peptide libraries, induces EGFR tyrosine phosphorylation, p47^phox phosphorylation, NADPH-oxidase-dependent superoxide generation, and c-Src kinase activity. As a result of EGFR transactivation, phosphotyrosine residues provide docking sites for recruitment and triggering of the STAT3 pathway. WKYMVm-induced EGFR transactivation is prevented by the FPRL1-selective antagonist WRWWWW, by pertussis toxin (PTX), and by the c-Src inhibitor PP2. The critical role of NADPH-oxidase-dependent superoxide generation in this cross-talk mechanism is corroborated by the finding that apocynin or a siRNA against p22^phox prevents EGFR transactivation and c-Src kinase activity. In addition, WKYMVm promotes CaLu-6 cell growth, which is prevented by PTX and by WRWWWW. These results highlight the role of FPRL1 as a potential target of new drugs and suggest that targeting both FPRL1 and EGFR may yield superior therapeutic effects compared with targeting either receptor separately.     

Molecular mechanisms involved in epidermal growth factor receptor-mediated inhibition of dopamine D3 receptor signaling

The phenomenon wherein the signaling by a given receptor is regulated by a different class of receptors is termed transactivation or crosstalk. Crosstalk between receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs) is highly diverse and has unique functional implications because of the distinct structural features of the receptors and the signaling pathways involved. The present study used the epidermal growth factor receptor (EGFR) and dopamine D₃ receptor (D₃R), which are both associated with schizophrenia, as the model system to study crosstalk between RTKs and GPCRs. Loss-of-function approaches were used to identify the cellular components involved in the tyrosine phosphorylation of G protein-coupled receptor kinase 2 (GRK2), which is responsible for EGFR-induced regulation of the functions of D₃R. SRC proto-oncogene (Src, non-receptor tyrosine kinase), heterotrimeric G protein Gβγ subunit, and endocytosis of EGFR were involved in the tyrosine phosphorylation of GRK2. In response to EGF treatment, Src interacted with EGFR in a Gβγ-dependent manner, resulting in the endocytosis of EGFR. Internalized EGFR in the cytosol mediated Src/Gβγ-dependent tyrosine phosphorylation of GRK2. The binding of tyrosine-phosphorylated GRK2 to the T142 residue of D₃R resulted in uncoupling from G proteins, endocytosis, and lysosomal downregulation. This study identified the molecular mechanisms involved in the EGFR-mediated regulation of the functions of D₃R, which can be extended to the crosstalk between other RTKs and GPCRs.     

Transactivation of the EGF receptor mediates IGF-1-stimulated shc phosphorylation and ERK1/2 activation in COS-7 cells

The receptor for insulin-like growth factor 1 (IGF-1) mediates multiple cellular responses, including stimulation of both proliferative and anti-apoptotic pathways. We have examined the role of cross talk between the IGF-1 receptor (IGF-1R) and the epidermal growth factor receptor (EGFR) in mediating responses to IGF-1. In COS-7 cells, IGF-1 stimulation causes tyrosine phosphorylation of the IGF-1R beta subunit, the EGFR, insulin receptor substrate-1 (IRS-1), and the Shc adapter protein. Shc immunoprecipitates performed after IGF-1 stimulation contain coprecipitated EGFR, suggesting that IGF-1R activation induces the assembly of EGFR.Shc complexes. Tyrphostin AG1478, an inhibitor of the EGFR kinase, markedly attenuates IGF-1-stimulated phosphorylation of EGFR, Shc, and ERK1/2 but has no effect on phosphorylation of IGF-1R, IRS-1, and protein kinase B (Akt). Cross talk between IGF-1 and EGF receptors is mediated through an autocrine mechanism involving matrix metalloprotease-dependent release of heparin-binding EGF (HB-EGF), because IGF-1-mediated ERK activation is inhibited both by [Glu(52)]Diphtheria toxin, a specific inhibitor of HB-EGF, and the metalloprotease inhibitor 1,10-phenanthroline. These data demonstrate that IGF-1 stimulation of the IRS-1/PI3K/Akt pathway and the EGFR/Shc/ERK1/2 pathway occurs by distinct mechanisms and suggest that IGF-1-mediated "transactivation" of EGFR accounts for the majority of IGF-1-stimulated Shc phosphorylation and subsequent activation of the ERK cascade.     

Distinctive features of Trk neurotrophin receptor transactivation by G protein-coupled receptors.

Ligands for G protein-coupled receptors (GPCR) are capable of activating mitogenic receptor tyrosine kinases, in addition to the mitogen-activated protein (MAP) kinase signaling pathway and classic G protein-dependent signaling pathways involving adenylyl cyclase and phospholipase. For example, receptors for epidermal growth factor (EGF), insulin-like growth-1 and platelet-derived growth factor and can be transactivated through G protein-coupled receptors. Neurotrophins, such as NGF, BDNF and NT-3 also utilize receptor tyrosine kinases, namely TrkA, TrkB and TrkC. Recently, it has been shown that activation of Trk receptor tyrosine kinases can also occur via a G protein-coupled receptor mechanism, without involvement of neurotrophins. Adenosine and adenosine agonists can activate Trk receptor phosphorylation specifically through the seven transmembrane spanning adenosine 2A (A2A) receptor. Several features of Trk receptor transactivation are noteworthy and differ significantly from other transactivation events. Trk receptor transactivation is slower and results in a selective increase in activated Akt. Unlike the biological actions of other tyrosine kinase receptors, increased Trk receptor activity by adenosine resulted in increased cell survival. This article will discuss potential mechanisms by which adenosine can activate trophic responses through Trk tyrosine kinase receptors.     

Reactive oxygen species mediate Met receptor transactivation by G protein-coupled receptors and the epidermal growth factor receptor in human carcinoma cells

Cross-communication between the Met receptor tyrosine kinase and the epidermal growth factor receptor (EGFR) has been proposed to involve direct association of both receptors and EGFR kinase-dependent phosphorylation. Here, we demonstrate that in human hepatocellular and pancreatic carcinoma cells the Met receptor becomes tyrosine phosphorylated not only upon EGF stimulation but also in response to G protein-coupled receptor (GPCR) agonists. Whereas specific inhibition of the EGFR kinase activity blocked EGF- but not GPCR agonist-induced Met receptor transactivation, it was abrogated in the presence of a reducing agent or treatment of cells with a NADPH oxidase inhibitor. Both GPCR ligands and EGF are further shown to increase the level of reactive oxygen species within the cell. Interestingly, stimulation of the Met receptor by either GPCR agonists, EGF or its cognate ligand HGF, resulted in release of Met-associated beta-catenin and in its Met-dependent translocation into the nucleus, as analyzed by small interfering RNA-mediated knockdown of the Met receptor. Our results provide a new molecular explanation for cell surface receptor cross-talk involving the Met receptor and thereby link the wide diversity of GPCRs and the EGFR to the oncogenic potential of Met signaling in human carcinoma cells.     

Redox-dependent MAP kinase signaling by Ang II in vascular smooth muscle cells: role of receptor tyrosine kinase transactivation

We investigated the role of receptor tyrosine kinases in Ang II-stimulated generation of reactive oxygen species (ROS) and assessed whether MAP kinase signaling by Ang II is mediated via redox-sensitive pathways. Production of ROS and activation of NADPH oxidase were determined by DCFDA (dichlorodihydrofluorescein diacetate; 2 micromol/L) fluorescence and lucigenin (5 micromol/L) chemiluminescence, respectively, in rat vascular smooth muscle cells (VSMC). Phosphorylation of ERK1/2, p38MAP kinase and ERK5 was determined by immunoblotting. The role of insulin-like growth factor-1 receptor (IGF-1R) and epidermal growth factor receptor (EGFR) was assessed with the antagonists AG1024 and AG1478, respectively. ROS bioavailability was manipulated with Tiron (10(-5) mol/L), an intracellular scavenger, and diphenylene iodinium (DPI; 10(-6) mol/L), an NADPH oxidase inhibitor. Ang II stimulated NADPH oxidase activity and dose-dependently increased ROS production (p < 0.05). These actions were reduced by AG1024 and AG1478. Ang II-induced ERK1/2 phosphorylation (276% of control) was decreased by AG1478 and AG1024. Neither DPI nor tiron influenced Ang II-stimulated ERK1/2 activity. Ang II increased phosphorylation of p38 MAP kinase (204% of control) and ERK5 (278% of control). These effects were reduced by AG1024 and AG1478 and almost abolished by DPI and tiron. Thus Ang II stimulates production of NADPH-inducible ROS partially through transactivation of IGF-1R and EGFR. Inhibition of receptor tyrosine kinases and reduced ROS bioavaliability attenuated Ang II-induced phosphorylation of p38 MAP kinase and ERK5, but not of ERK1/2. These findings suggest that Ang II activates p38MAP kinase and ERK5 via redox-dependent cascades that are regulated by IGF-1R and EGFR transactivation. ERK1/2 regulation by Ang II is via redox-insensitive pathways.     

Extracellular calcium-sensing receptor transactivates the epidermal growth factor receptor by a triple-membrane-spanning signaling mechanism

Activation of the extracellular calcium-sensing receptor (CaR) stimulates mitogen-activated protein kinases to upregulate the synthesis and secretion of parathyroid hormone related peptide (PTHrP) from cells expressing the CaR heterologously or endogenously. The current experiments demonstrate that this occurs because CaR activation "transactivates" the EGF receptor (EGFR). Time dependent increases in tyrosine phosphorylation of the EGFR after addition of extracellular calcium ([Ca2+]o, 3 mM) occurred in stably CaR-transfected HEK293 cells but not in non-transfected HEK293 cells. AG1478, an EGFR kinase inhibitor, prevented the CaR-mediated increases of pERK and PTHrP release, while AG1296, a PDGFR kinase inhibitor, had no effect. Inhibitors of matrix metalloproteinase and heparin bound-EGF prevented the CaR-mediated increases of pERK and PTHrP, consistent with a "triple-membrane-spanning signaling" requirement for transactivation of the EGFR by the CaR. Proximal and distal signal transduction cascades activated by the CaR may reflect transactivation of the EGFR by the extracellular calcium-sensing receptor.     

Epidermal growth factor receptor tyrosine kinase mediates Ras activation by gonadotropin-releasing hormone

Gonadotropin releasing hormone (GnRH) contributes to the maintenance of gonadotrope function by increasing extracellular signal-regulated kinase (ERK) activity subsequent to binding to its cognate G-protein-coupled receptor. As the GnRH receptor exclusively interacts with G(q/11) proteins and as receptor expression is regulated in a beta-arrestin-independent fashion, it represents a good model to systematically dissect underlying signaling pathways. In alphaT3-1 gonadotropes endogenously expressing the GnRH receptor, GnRH challenge resulted in a rapid increase in ERK activity which was attenuated by the epidermal growth factor receptor (EGFR)-specific tyrosine kinase inhibitor AG1478. In COS-7 cells transiently expressing the human GnRH receptor, agonist-induced ERK activation was independent of free Gbetagamma subunits but could be mimicked by short-term phorbol ester treatment. Most notably, G(q/11)-induced ERK activation was sensitive to N17-Ras and to expression of the C-terminal Src kinase but also to other dominant negative mutants of signaling components localized upstream of Ras, like Shc and the EGFR. GnRH as well as phorbol esters led to Ras activation in COS-7 and alphaT3-1 cells, which was dependent on Src and EGFR tyrosine kinases, indicating that both tyrosine kinases act downstream of protein kinase C (PKC) and upstream of Ras. However, Src did not contribute to Shc tyrosine phosphorylation. GnRH or phorbol ester challenge resulted in PKC-dependent EGFR autophosphorylation. Furthermore, a 5-min phorbol ester treatment was sufficient to trigger tyrosine phosphorylation of the platelet-derived growth factor-beta receptor in L cells. Thus, in several cell systems PKC is able to stimulate Ras via activation of receptor tyrosine kinases.     

Integrins regulate the linkage between upstream and downstream events in G protein-coupled receptor signaling to mitogen-activated protein kinase

Receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs) can both activate mitogen-activated protein kinase (MAPK), a critical intermediate in the transduction of proliferative signals. Numerous observations have demonstrated that integrin-mediated cell anchorage can regulate the efficiency of signaling from RTKs to MAPK. Recently, a relationship between integrins and GPCR signaling has also emerged; however, little is understood concerning the mechanisms involved. Here, we investigate integrin regulation of GPCR signaling to MAPK, focusing on the P2Y class of GPCRs that function through activation of phospholipase Cbeta. P2Y receptor signaling to the downstream components mitogen-activated protein kinase kinase and MAPK is highly dependent on integrin-mediated cell anchorage. However, activation of upstream events, including inositol phosphate production and generation of calcium transients, is completely independent of cell anchorage. This indicates that integrins regulate the linkage between upstream and downstream events in this GPCR pathway, just as they do in some aspects of RTK signaling. However, the P2Y pathway does not involve cross-activation of a RTK, nor a role for Shc or c-Raf; thus, it is quite distinct from the classical RTK-Ras-Raf-MAPK cascade. Rather, integrin-modulated P2Y receptor stimulation of MAPK depends on calcium and on the activation of protein kinase C.     

Mig-6 is a negative regulator of the epidermal growth factor receptor signal

In contrast to signal generation and transmission, the mechanisms and molecules that negatively regulate receptor tyrosine kinase (RTK) signaling are poorly understood. Here we characterize Mig-6 as a novel negative feedback regulator of the epidermal growth factor receptor (EGFR) and potential tumor suppressor. Mig-6 was identified in a yeast two-hybrid screen with the kinase active domain of the EGFR as bait. Upon EGF stimulation Mig-6 binds to the EGFR involving a highly acidic region between amino acids 985-995. This interaction is kinase activity-dependent, but independent of tyrosine 992. Mig-6 overexpression results in reduced activation of the mitogenactivated protein kinase ERK2 in response to EGF, but not FGF or PDGF, stimulation and in enhanced receptor internalization without affecting the rate of degradation. The induction of Mig-6 mRNA expression in response to EGF, but not FGF, indicates the existence of a negative regulatory feedback loop. Consistent with these findings, a possible role as tumor suppressor is indicated by Mig-6-mediated inhibition of EGFR overexpression-induced transformation of Rati cells.