Activation of EGF receptor family members suppresses the cytotoxic effects of tumor necrosis factor-α

Tumor necrosis factor (TNF)-α has a broad range of biological activities, which depend heavily on cell type and physiological condition. In a panel of human tumor cell lines we analyzed expression of the receptor tyrosine kinases EGFR, ErbB2 and ErbB3, and the response to TNF-α. Among the cell lines tested those resistant to TNF-α were found to express high levels of either EGFR, or ErbB2 and ErbB3. In TNF-sensitive breast and cervical carcinoma cells activation of EGFR or ErbB2 by the exogenous growth factors EGF and heregulin β1 resulted in a significant increase in the number of cells surviving TNF-α treatment. In contrast, inhibition of EGFR activation in TNF-resistant breast carcinoma cells by the novel antagonistic anti-EGFR antibody 14E1 sensitized the cells to the cytotoxic effects of TNF-α. A bacterially expressed fusion protein consisting of a 14E1 single-chain (sc) Fv antibody fragment linked to human TNF-α retained TNF-α activity. This scFv(14E1)-TNF-α molecule localized specifically to EGFR on the surface of tumor cells and activated the NF-κB pathway in co-cultured T cells, as demonstrated by electrophoretic mobility shift assays. Received: 6 May 1998 / Accepted: 16 July 1998     

Activation mechanism of solubilized epidermal growth factor receptor tyrosine kinase.

Dimerization of epidermal growth factor receptor (EGFR) leads to the activation of its tyrosine kinase. To elucidate whether dimerization is responsible for activation of the intracellular tyrosine kinase domain or just plays a role in the stabilization of the active form, the activated status of wild-type EGFR moiety in the heterodimer with kinase activity-deficient mutant receptors was investigated. The kinase activity of the wild-type EGFR was partially activated by EGF in the heterodimer with intracellular domain deletion (sEGFR) or ATP binding-deficient mutant (K721A) EGFRs, while the wild-type EGFR in the heterodimer of wild-type and phosphate transfer activity-deficient mutant receptor D813N could be fully activated. After treatment with EGF, the ATP binding affinity and the V(max) of the wild-type EGFR increased. In the presence of sEGFR, a similar increase in the affinity for ATP was observed, but V(max) did not change. A two-step activation mechanism for EGFR was proposed: upon binding of EGF, the affinity for ATP increased and then, as a result of interaction between the neighboring tyrosine kinase domain, V(max) increased.     

Structural analysis of receptor tyrosine kinases

Receptor tyrosine kinases (RTKs) are single-pass transmembrane receptors that possess intrinsic cytoplasmic enzymatic activity, catalyzing the transfer of the γ-phosphate of ATP to tyrosine residues in protein substrates. RTKs are essential components of signal transduction pathways that affect cell proliferation, differentiation, migration and metabolism. Included in this large protein family are the insulin receptor and the receptors for growth factors such as epidermal growth factor, fibroblast growth factor and vascular endothelial growth factor. Receptor activation occurs through ligand binding, which facilitates receptor dimerization and autophosphorylation of specific tyrosine residues in the cytoplasmic portion. The phosphotyrosine residues either enhance receptor catalytic activity or provide docking sites for downstream signaling proteins. Over the past several years, structural studies employing X-ray crystallography have advanced our understanding of the molecular mechanisms by which RTKs recognize their ligands and are activated by dimerization and tyrosine autophosphorylation. This review will highlight the key results that have emerged from these structural studies.     

Dopamine D2 receptor stimulation of mitogen-activated protein kinases mediated by cell type-dependent transactivation of receptor tyrosine kinases

Dopamine D2 receptor activation of extracellular signal-regulated kinases (ERKs) in non-neuronal human embryonic kidney 293 cells was dependent on transactivation of the platelet-derived growth factor (PDGF) receptor, as demonstrated by the effect of the PDGF receptor inhibitors tyrphostin A9 and AG 370 on quinpirole-induced phosphorylation of ERKs and by quinpirole-induced tyrosine phosphorylation of the PDGF receptor. In contrast, ectopically expressed D2 receptor or endogenous D2-like receptor activation of ERKs in NS20Y neuroblastoma cells, which express little or no PDGF receptor, or in rat neostriatal neurons was largely dependent on transactivation of the epidermal growth factor (EGF) receptor, as demonstrated using the EGF receptor inhibitor AG 1478 and by quinpirole-induced phosphorylation of the EGF receptor. The D2 receptor agonist quinpirole enhanced the coprecipitation of D2 and EGF receptors in NS20Y cells, suggesting that D2 receptor activation induced the formation of a macromolecular signaling complex that includes both receptors. Transactivation of the EGF receptor also involved the activity of a matrix metalloproteinase. Thus, although D2 receptor stimulation of ERKs in both cell lines was decreased by inhibitors of ERK kinase, Src-family protein tyrosine kinases, and serine/threonine protein kinases, D2-like receptors activated ERKs via transactivation of the EGF receptor in NS20Y neuroblastoma cells and rat embryonic neostriatal neurons, but via transactivation of the PDGF receptor in 293 cells.     

Antibodies directed against receptor tyrosine kinases

Approximately 30 therapeutic monoclonal antibodies have already been approved for cancers and inflammatory diseases, and monoclonal antibodies continue to be one of the fastest growing classes of therapeutic molecules. Because aberrant signaling by receptor tyrosine kinases (RTKs) is a commonly observed factor in cancer, most of the subclasses of RTKs are being extensively studied as potential targets for treating malignancies. The first two RTKs that have been targeted by antibody therapy, with five currently marketed antibodies, are the growth factor receptors EGFR and HER2. However, due to systemic side effects, refractory patients and the development of drug resistance, these treatments are being challenged by emerging therapeutics. This review examines current monoclonal antibody therapies against RTKs. After an analysis of agents that have already been approved, we present an analysis of antibodies in clinical development that target RTKs. Finally, we highlight promising RTKs that are emerging as new oncological targets for antibody-based therapy.     

Physical and functional interactome atlas of human receptor tyrosine kinases

Much cell‐to‐cell communication is facilitated by cell surface receptor tyrosine kinases (RTKs). These proteins phosphorylate their downstream cytoplasmic substrates in response to stimuli such as growth factors. Despite their central roles, the functions of many RTKs are still poorly understood. To resolve the lack of systematic knowledge, we apply three complementary methods to map the molecular context and substrate profiles of RTKs. We use affinity purification coupled to mass spectrometry (AP‐MS) to characterize stable binding partners and RTK–protein complexes, proximity‐dependent biotin identification (BioID) to identify transient and proximal interactions, and an in vitro kinase assay to identify RTK substrates. To identify how kinase interactions depend on kinase activity, we also use kinase‐deficient mutants. Our data represent a comprehensive, systemic mapping of RTK interactions and substrates. This resource adds information regarding well‐studied RTKs, offers insights into the functions of less well‐studied RTKs, and highlights RTK‐RTK interactions and shared signaling pathways.     

E-cadherin-mediated adhesion inhibits ligand-dependent activation of diverse receptor tyrosine kinases

E-cadherin is an essential adhesion protein as well as a tumor suppressor that is silenced in many cancers. Its adhesion-dependent regulation of signaling has not been elucidated. We report that E-cadherin can negatively regulate, in an adhesion-dependent manner, the ligand-dependent activation of divergent classes of receptor tyrosine kinases (RTKs), by inhibiting their ligand-dependent activation in association with decreases in receptor mobility and in ligand-binding affinity. E-cadherin did not regulate a constitutively active mutant RTK (Neu*) or the ligand-dependent activation of LPA receptors or muscarinic receptors, which are two classes of G protein-coupled receptors. EGFR regulation by E-cadherin was associated with complex formation between EGFR and E-cadherin that depended on the extracellular domain of E-cadherin but was independent of beta-catenin binding or p120-catenin binding. Transfection of E-cadherin conferred negative RTK regulation to human melanoma and breast cancer lines with downregulated endogenous E-cadherin. Abrogation of E-cadherin regulation may contribute to the frequent ligand-dependent activation of RTK in tumors.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin causes an increase in protein kinases growth hepatic associated with epidermal factor receptor in the plasma membrane

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), administered to male rats at a single intraperitoneal (IP) injection dose of 25 μg/kg causes down-regulation of epidermal growth factor (EGF) receptor in the plasma membrane of rat liver which starts after two days and continues throughout the experimental period (20 days). Using monoclonal antibody to EGF receptor, it was determined that TCDD-caused EFG receptor down-regulation in the rat liver was accompanied by increased protein kinase activity. Such an increase in the protein kinase activity involves, at least in part, an activation of protein tyrosine kinase. Examination of serum samples from control and treated rats revealed no detectable difference in the level of EGF itself or EGF receptor-reacting substances (eg, hormones and other growth factors). In vivo TCDD caused early eye opening and tooth eruption and poor body weight gain and hair growth in mouse neonates similar to those observed with exogenously administered EGE The results indicate that such EGF receptor–mediated effect of TCDD has some toxocilogical significance in vivo. Although TCDD causes significant reduction in [¹²⁵I]-EGF binding in the hepatic plasma membrane in susceptible strains of mice, it has only modest effects in tolerant strains. The results are consistent with the idea that the action of TCDD on the EGF receptor is mediated through the cytosoliclnuclear TCDD receptor, which is known to be regulated by the Ah locus.     

Ligand-independent oncogenic signaling by the epidermal growth factor receptor: v-ErbB as a paradigm

Relay of information from the extracellular environment into the cell often results from a peptide growth factor binding to its cognate cell surface receptor; this event is an integral mechanism by which many cellular functions occur, including cell growth, motility, and survival. In recent years, however, this requirement for ligand binding has been shown to be surpassed by several distinct mechanisms, including cell surface receptor cross-talk (e.g., between epidermal growth factor receptor [EGFR] and G-coupled receptors), receptor-extracellular matrix interactions (e.g., EGFR: integrin complexes), and finally by structural mutations within the receptor itself. While all of these pathways result in so-called ligand-independent signaling by the EGF receptor, to date, only structural mutations in the receptor have been shown to result in qualitative changes in downstream targets of the receptor, which specifically result in oncogenic signaling, transformation, and tumorigenicity. In this review, we describe aspects of the known signaling properties of the retroviral oncogene v-ErbB as a model of ligand-independent oncogenic signaling, and compare these properties to results emerging from ongoing studies on structurally related EGF receptor mutants originally identified in human tumors. A better understanding of the signaling pathways used by these uniquely oncogenic receptor tyrosine kinase mutants may ultimately reveal new targets for the development of novel therapeutics selective for the inhibition of tumor cell growth.     

Structure and function of tyrosine kinase receptors

Over the past ten years, several growth factor receptors have been shown to be ligand-regulated tyrosine kinases. Tyrosine kinase activity is essential for signal transmission, suggesting that phosphorylation cascades may play an important role. Considerable effort has gone into understanding the structure and function of tyrosine kinase receptors in order to define their mechanisms of signal transmission. However, the protein substrates of the receptor kinases have proven to be difficult to isolate and clone. This review focuses on the receptors for insulin, epidermal growth factor, and platelet-derived growth factor. They are all tyrosine kinases, but emerging evidence suggests that they utilize multiple separate signal transduction pathways. Work carried out during the next several years should yield considerable insight into the complexity of the components which interact with these tyrosine kinase receptors to regulate cellular growth and metabolism.     

Cross-talk between the receptor tyrosine kinases Ron and epidermal growth factor receptor

Heterogeneous receptor-receptor interactions may play a role in intracellular signaling. Accordingly, the interaction of two dissimilar tyrosine kinase receptors, Ron and epidermal growth factor receptor (EGFR) was investigated. The functional interaction of Ron and EGFR in cell scatter and oncogenic transformation was investigated in vivo. Transfection of a dominant negative form of EGFR into human embryonic kidney cells stably expressing Ron (293-Ron) dramatically reduced the scatter response induced by the Ron ligand hepatocyte growth factor-like protein/macrophage stimulating protein (HGFL). The scatter response of the 293-Ron cells was also attenuated by treatment of the cells with the specific EGFR inhibitor AG 1478. Co-transfection of Ron and dominant-negative EGFR, or co-transfection of EGFR and a dominant-negative form of Ron reduced focus formation in NIH/3T3 cells. Western analysis of NIH/3T3 cells overexpressing murine Ron and expressing endogenous levels of EGFR was used to demonstrate that Ron and EGFR co-immunoprecipitate. Stimulation of the cells in vitro with the Ron ligand HGFL or with the EGFR ligand epidermal growth factor (EGF) appeared to induce phosphorylation of both receptors. Co-immunoprecipitation and phosphorylation of phosphatidyl inositol 3-kinase (PI3-K) was also observed. This novel finding of a functional and biochemical interaction between Ron and EGFR suggests that heterologous tyrosine kinase receptor interactions may play a role in cellular processes such as scatter and transformation.     

c-Kit--a hematopoietic cell essential receptor tyrosine kinase

The receptor tyrosine kinase c-Kit is expressed in hematopoietic stem and progenitor cells and in several non-hematopoietic tissues. In the hematopoietic system, c-Kit is critical for proliferation, survival and differentiation. During recent years exploration of the signalling pathways downstream of this receptor has yielded significant new insights in the field. In this review, we will summarise the c-Kit background, structure, downstream signalling and medical significance with particular focus on its role in hematopoietic progenitor cells and mast cells.     

Src-protein tyrosine kinases are required for cocaine-induced increase in the expression and function of the NMDA receptor in the ventral tegmental area

Cocaine-induced long-term potentiation of glutamatergic synapses in the ventral tegmental area (VTA) has been proposed as a key process that contributes to the development of addictive behaviors. In particular, the activation of ionotrophic glutamate NMDA receptor (NMDAR) in the VTA is critical for the initiation of cocaine sensitization. Here we show that application of cocaine both in slices and in vivo induced an increase in tyrosine phosphorylation of the NR2A, but not the NR2B subunit of the NMDAR in juvenile rats. Cocaine induced an increase in the activity of both Fyn and Src kinases, and the Src-protein tyrosine kinase (Src-PTKs) inhibitor, 4-amino-5-(4-chlorophenyl)-7-( t -butyl)pyrazolo[3,4-d]pyrimidine (PP2), abolished both cocaine-induced increase in tyrosine phosphorylation of the NR2A subunit and the increase in the expression of NR1, NR2A, and NR2B in the VTA. Moreover, cocaine-induced enhancement in NMDAR-mediated excitatory post-synaptic currents was completely abolished by PP2. Taken together, these results suggest that acute cocaine induced an increase in the expression of NMDAR subunits and enhanced tyrosine phosphorylation of NR2A-containing NMDAR through members of the Src-PTKs. This in turn, increased NMDAR-mediated currents in VTA dopamine neurons. These results provide a potential cellular mechanism by which cocaine triggers NMDAR-dependent synaptic plasticity of VTA neurons that may underlie the development of behavioral sensitization.     

Regulation of connexin43 function by activated tyrosine protein kinases

Gap junctions are specialized membrane structures that are involved in the normal functioning of numerous mammalian tissues and implicated in several human disease processes. This mini-review focuses on the regulation of gap junctions through phosphorylation of connexin43 induced by the v-Src or epidermal growth factor receptor tyrosine kinases. These tyrosine kinases markedly disrupt gap junctional communication in mammalian cells. Here, we describe work correlating the alteration of connexin43 function with the ability of the v-Src tyrosine kinase to phosphorylate connexin43 directly on two distinct tyrosine sites in mammalian cells (Y247 and Y265). We also present evidence that proline-rich regions and phosphotyrosine sites of connexin43 may mediate interactions with the SH3 and SH2 domains of v-Src. In contrast to v-Src, the activated epidermal growth factor receptor acts indirectly through activated MAP kinase which may stimulate phosphorylation of connexin43 exclusively on serine. This phosphorylation event is complex because MAP kinase phosphorylates three serine sites in connexin43 (S255, S279, and S282). These findings suggest novel interactions between connexin43, the v-Src tyrosine kinase, and activated MAP kinase that set the stage for future investigations into the regulation of gap junctions by protein phosphorylation.     

Characterisation of five novel zebrafish Eph-related receptor tyrosine kinases suggests roles in patterning the neural plate

 Eph-related receptor tyrosine kinases (RTKs) are the largest known subfamily of RTKs, comprising at least a dozen members. Expression studies suggest roles for these genes in patterning and differentiation of the nervous system, the neural crest, developing limbs and somites. Some of the recently isolated family of ligands for Eph-related RTKs have been shown to function as positional identifiers in the retinotectal system. We have previously characterised three Eph-related RTKs in the zebrafish (rtk1-3). Here we report the identification of five new zebrafish Eph-related RTKs (rtk4, rtk5, rtk6, rtk7 and rtk8) and describe their dynamic expression patterns. Based on these expression patterns, we propose that rtk4-8 play various roles in establishing territories within the developing central nervous system (CNS) and in the subsequent differentiation of defined neuronal populations. Received: 22 November 1996 / Accepted: 3 January 1997     

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.