Inactivation of neuregulin-1 by nitration

Nitration is a posttranslational modification that can compromise protein function. We hypothesized that nitration of growth factors secreted in the lung may alter their interaction with their respective receptors and modulate the normal growth and differentiation program induced by ligand-receptor interaction. We tested this hypothesis in vitro by nitration of neuregulin-1's (NRG-1) EGF-like domain and studying the effect on NRG-1's activity. Nitration of NRG-1's (nNRG-1) EGF-like domain resulted in an inability to activate its receptor, the human epidermal growth factor receptors 2 and 3 (HER2/HER3) heterodimer, as defined by loss of HER2 tyrosine phosphorylation induced by nNRG-1 in MCF-7 cells. Receptor activation was not restored with increasing nNRG-1 concentration or exposure times. nNRG-1 did not compete with NRG-1 for HER2/HER3 binding in competition assays. In addition, nNRG-1 no longer induced proliferation of the MCF-7 cell line, as MCF-7 cells exposed to nNRG-1 and NRG-1 concurrently had the same proliferation rate as that induced by NRG-1 alone. Thus nitration of NRG-1's EGF-like domain caused it to lose its ability to bind and activate its receptor with loss of ligand-induced proliferation. Posttranslational nitration of growth factors in states where reactive nitrogen species are increased may be an important means of regulating growth factor receptor effects in the lung.     

Quantitation of the Effect of ErbB2 on Epidermal Growth Factor Receptor Binding and Dimerization

The epidermal growth factor (EGF) receptor is a member of the ErbB family of receptors that also includes ErbB2, ErbB3, and ErbB4. These receptors form homo- and heterodimers in response to ligand with ErbB2 being the preferred dimerization partner. Here we use (125)I-EGF binding to quantitate the interaction of the EGF receptor with ErbB2. We show that the EGFR/ErbB2 heterodimer binds EGF with a 7-fold higher affinity than the EGFR homodimer. Because it cannot bind a second ligand, the EGFR/ErbB2 heterodimer is not subject to ligand-induced dissociation caused by the negatively cooperative binding of EGF to the second site on the EGFR homodimer. This increases the stability of the heterodimer relative to the homodimer and is associated with enhanced and prolonged EGF receptor autophosphorylation. These effects are independent of the kinase activity of ErbB2 but require back-to-back dimerization of the EGF receptor with ErbB2. Back-to-back dimerization is also required for phosphorylation of ErbB2. These findings provide a molecular explanation for the apparent preference of the EGF receptor for dimerizing with ErbB2 and suggest that the phosphorylation of ErbB2 occurs largely in the context of the EGFR/ErbB2 heterodimer, rather than through lateral phosphorylation of isolated ErbB2 subunits.     

Dynamic Analysis of the Epidermal Growth Factor (EGF) Receptor-ErbB2-ErbB3 Protein Network by Luciferase Fragment Complementation Imaging

ErbB3 is a member of the ErbB family of receptor tyrosine kinases. It is unique because it is the only member of the family whose kinase domain is defective. As a result, it is obliged to form heterodimers with other ErbB receptors to signal. In this study, we characterized the interaction of ErbB3 with the EGF receptor and ErbB2 and assessed the effects of Food and Drug Administration-approved therapeutic agents on these interactions. Our findings support the concept that ErbB3 exists in preformed clusters that can be dissociated by NRG-1β and that it interacts with other ErbB receptors in a distinctly hierarchical fashion. Our study also shows that all pairings of the EGF receptor, ErbB2, and ErbB3 form ligand-independent dimers/oligomers. The small-molecule tyrosine kinase inhibitors erlotinib and lapatinib differentially enhance the dimerization of the various ErbB receptor pairings, with the EGFR/ErbB3 heterodimer being particularly sensitive to the effects of erlotinib. The data suggest that the physiological effects of these drugs may involve not only inhibition of tyrosine kinase activity but also a dynamic restructuring of the entire network of receptors.     

Computational analysis of molecular basis of 1:1 interactions of NRG-1beta wild-type and variants with ErbB3 and ErbB4

The neuregulin/ErbB system is a growth factor/receptor cascade that has been proven to be essential in the development of the heart and the sympathetic nervous system. However, the basis of the specificity of ligand-receptor recognition remains to be elucidated. In this study, the structures of NRG-1beta/ErbB3 and NRG-1beta/ErbB4 complexes were modeled based on the available structures of the homologous proteins. The binding free energies of NRG-1beta to ErbB3 and ErbB4 were calculated using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) computational method. In addition, computational alanine-scanning mutagenesis was performed in the binding site of NRG-1beta and the difference in the binding free energies between NRG-1beta mutants and the receptors was calculated. The results specify the contribution of each residue at the interaction interfaces to the binding affinity of NRG-1beta with ErbB3 and ErbB4, identifying several important interaction residue pairs that are in agreement with previously acquired experimental data. This indicates that the presented structural models of NRG-1beta/ErbB3 and NRG-1beta/ErbB4 complexes are reliable and could be used to guide future studies, such as performing desirable mutations on NRG-1beta to increase the binding affinity and selectivity to the receptor and discovering new therapeutic agents for the treatment of heart failure.     

Epidermal growth factor and betacellulin mediate signal transduction through co-expressed ErbB2 and ErbB3 receptors.

Interleukin-3 (IL-3)-dependent murine 32D cells do not detectably express epidermal growth factor receptors (EGFRs) and do not proliferate in response to EGF, heregulin (HRG) or other known EGF-like ligands. Here, we report that EGF specifically binds to and can be crosslinked to 32D transfectants co-expressing ErbB2 and ErbB3 (32D.E2/E3), but not to transfectants expressing either ErbB2 or ErbB3 individually. [125I]EGF-crosslinked species detected in 32D. E2/E3 cells were displaced by HRG and betacellulin (BTC) but not by other EGF-like ligands that were analyzed. EGF, BTC and HRG also induced receptor tyrosine phosphorylation, activation of downstream signaling molecules and proliferation of 32D.E2/E3 cells. 32D transfectants were also generated which expressed an ErbB3-EGFR chimera alone (32D.E3-E1) or in combination with ErbB2 (32D. E2/E3-E1). While HRG stimulation of 32D.E3-E1 cells resulted in DNA synthesis and receptor phosphorylation, EGF and BTC were inactive. However, EGF and BTC were as effective as HRG in mediating signaling when ErbB2 was co-expressed with the chimera in the 32D.E2/E3-E1 transfectant. These results provide evidence that ErbB2/ErbB3 binding sites for EGF and BTC are formed by a previously undescribed mechanism that requires co-expression of two distinct receptors. Additional data utilizing MDA MB134 human breast carcinoma cells, which naturally express ErbB2 and ErbB3 in the absence of EGFRs, supported the results obtained employing 32D cells and suggest that EGF and BTC may contribute to the progression of carcinomas that co-express ErbB2 and ErbB3.     

A natural ErbB4 isoform that does not activate phosphoinositide 3-kinase mediates proliferation but not survival or chemotaxis

ErbB4 is a member of the epidermal growth factor receptor (ErbB) family that mediates cellular responses activated by neuregulins (NRG) and other epidermal growth factor-like growth factors. Two naturally occurring ErbB4 isoforms, ErbB4 CYT-1 and ErbB4 CYT-2, have previously been identified. Unlike ErbB4 CYT-1, ErbB4 CYT-2 lacks a phosphoinositide 3-kinase (PI3-K)-binding site and is incapable of activating PI3-K. We have now examined the consequences of the inability of this isoform to activate PI3-K on cell proliferation, survival, and chemotaxis in response to NRG-1beta: (i) NRG-1beta stimulated proliferation of cells expressing either ErbB4 CYT-1 or ErbB4 CYT-2. Consistent with the mitogenic responsiveness, analysis of downstream signaling showed that Shc and MAPK were phosphorylated after stimulating either isoform with NRG-1beta. (ii) NRG-1beta protected cells expressing ErbB4 CYT-1 but not cells expressing ErbB4 CYT-2 from starvation-induced apoptosis as measured by effects on cell number and 4', 6-diamidino-2-phenylindole staining. Furthermore, in cells expressing ErbB4 CYT-2, Akt, a protein kinase that mediates cell survival, was not phosphorylated. (iii) NRG-1beta stimulated chemotaxis and membrane ruffling in cells expressing ErbB4 CYT-1 but not in cells expressing ErbB4 CYT-2. In summary, ErbB4 CYT-2 can mediate proliferation but not chemotaxis or survival. These results suggest a novel mechanism by which cellular responses such as chemotaxis and survival may be regulated by the expression of alternative receptor-tyrosine kinase isoforms that differ in their coupling to PI3-K signaling.     

Role of neuregulin-1/ErbB2 signaling in endothelium-cardiomyocyte cross-talk

Neuregulin-1 (NRG-1), a cardioactive growth factor released from endothelial cells, has been shown to be indispensable for the normal function of the adult heart by binding to ErbB4 receptors on cardiomyocytes. In the present study, we have investigated to what extent ErbB2, the favored co-factor of ErbB4 for heterodimerization, participates in the cardiac effects of endothelium-derived NRG-1. In addition, in view of our previously described anti-adrenergic effects of NRG-1, we have studied which neurohormonal stimuli affect endothelial NRG-1 expression and release and how this may fit into a broader frame of cardiovascular physiology. Immunohistochemical staining of rat heart and aorta showed that NRG-1 expression was restricted to the endocardial endothelium and the cardiac microvascular endothelium (CMVE); by contrast, NRG-1 expression was absent in larger coronary arteries and veins and in aortic endothelium. In rat CMVE in culture, NRG-1 mRNA and protein expression was down-regulated by angiotensin II and phenylephrine and up-regulated by endothelin-1 and mechanical strain. CMVE-derived NRG-1 was shown to phosphorylate cardiomyocyte ErbB2, an event prevented by a 24-h preincubation of myocytes with monoclonal ErbB2 antibodies. Pretreating cardiomyocytes with these inhibitory anti-ErbB2 antibodies significantly attenuated CMVE-induced cardiomyocyte hypertrophy and abolished the protective actions of CMVE against cardiomyocyte apoptosis. Accordingly, ErbB2 signaling participated in the paracrine survival and growth controlling effects of NRG-1 on cardiomyocytes in vitro, explaining the cardiotoxicity of ErbB2 antibodies in patients. Cardiac NRG-1 synthesis occurs in endothelial cells adjacent to cardiac myocytes and is sensitive to factors related to the regulation of blood pressure.     

Growth factor-specific signaling pathway stimulation and gene expression mediated by ErbB receptors

The mechanisms by which receptor tyrosine kinases (RTKs) utilize intracellular signaling pathways to direct gene expression and cellular response remain unclear. A current question is whether different RTKs within a single cell target similar or different sets of genes. In this study we have used the ErbB receptor network to explore the relationship between RTK activation and gene expression. We profiled growth factor-stimulated signaling pathway usage and broad gene expression patterns in two human mammary tumor cell lines expressing different complements of ErbB receptors. Although the growth factors epidermal growth factor (EGF) and neuregulin (NRG) 1 similarly stimulated Erk1/2 in MDA-MB-361 cells, EGF acting through an EGF receptor/ErbB2 heterodimer preferentially stimulated protein kinase C, and NRG1beta acting through an ErbB2/ErbB3 heterodimer preferentially stimulated Akt. The two growth factors regulated partially overlapping yet distinct sets of genes in these cells. In MDA-MB-453 cells, NRG1beta acting through an ErbB2/ErbB3 heterodimer stimulated prolonged signaling of all pathways examined relative to NRG2beta acting through the same heterodimeric receptor species. Surprisingly, NRG1beta and NRG2beta also regulated partially overlapping but distinct sets of genes in these cells. These results demonstrate that the activation of different RTKs, or activation of the same RTKs with different ligands, can lead to distinct profiles of gene regulation within a single cell type. Our observations also suggest that the identity and kinetics of signaling pathway usage by RTKs may play a role in the selection of regulated genes.     

Ligand-independent regulation of ErbB4 receptor phosphorylation by activated Ras

The ErbB family of receptor tyrosine kinases regulates cell growth, differentiation and survival. Activation of the receptors is induced by specific growth factors in an autocrine, paracrine or juxtacrine manner. The activated ErbB receptors turn on a large variety of signaling cascades, including the prominent Ras-dependent signaling pathways. The activated Ras can induce secretion of growth factors such as EGF and neuregulin, which activate their respective receptors. In the present study, we demonstrate for the first time that activated Ras can activate ErbB4 receptor in a ligand-independent manner. Expression of constitutively active H-Ras(12V), K-Ras(12V) or N-Ras(13V) in PC12-ErbB4 cells induced ErbB4-receptor phosphorylation, indicating that each of the most abundant Ras isoforms can induce receptor activation. NRG-induced phosphorylation of ErbB4 receptor was blocked by the soluble ErbB4 receptor, which had no effect on the Ras-induced receptor phosphorylation. Moreover, conditioned medium from H-Ras(12V)-transfected PC12-ErbB4 cells had no effect on receptor phosphorylation. It thus indicates that Ras induces ErbB4 phosphorylation in a ligand-independent manner. Each of the Ras effector domain mutants, H-Ras(12V)S35, H-Ras(12V)C40, and H-Ras(12V)G37, which respectively activate Raf1, PI3K, and RalGEF, induced a small but significant receptor phosphorylation. The PI3K inhibitor LY294002 and the MEK inhibitor PD98059 caused a partial inhibition of the Ras-induced ErbB4 receptor phosphorylation. Using a mutant ErbB4 receptor, which lacks kinase activity, we demonstrated that the Ras-mediated ErbB4 phosphorylation depends on the kinase activity of the receptor and facilitates ligand-independent neurite outgrowth in PC12-ErbB4 cells. These experiments demonstrate a novel mechanism controlling ErbB receptor activation. Ras induces ErbB4 receptor phosphorylation in a non-autocrine manner and this activation depends on multiple Ras effector pathways and on ErbB4 kinase activity.     

Inhibition of endoplasmic reticulum stress by neuregulin-1 protects against myocardial ischemia/reperfusion injury

Neuregulin-1 (NRG-1), an endogenously produced polypeptide, is the ligand of cardiomyocyte ErbB receptors, with cardiovascular protective effects. In the present study, we explored whether the cardioprotective effect of NRG-1 against I/R injury is mediated by inhibiting myocardial endoplasmic reticulum (ER) stress. In vitro, NRG-1 directly inhibited the upregulation of ER stress markers such as glucose-regulated protein 78, CCAAT/enhancer binding protein homologous protein and cleaved caspase-12 induced by the ER stress inducers tunicamycin or dithiothreitol in both neonatal and adult ventricular myocytes. Attenuating ErbB signals by an ErbB inhibitor AG1478 or ErbB4 knockdown and preincubation with phosphoinositide 3-kinase inhibitors all reversed the effect of NRG-1 inhibiting ER stress in cultured neonatal rat cardiomyocytes. Concurrently, cardiomyocyte ER stress and apoptosis induced by hypoxia-reoxygenation were decreased by NRG-1 treatment in vitro. Furthermore, in an in vivo rat model of myocardium ischemia/reperfusion (I/R), intravenous NRG-1 administration significantly decreased ER stress and myocardial infarct size induced by I/R. NRG-1 could protect the heart against I/R injury by inhibiting myocardial ER stress, which might be mediated by the phosphoinositide 3-kinase/Akt signaling pathway.     

Binding specificities and affinities of egf domains for ErbB receptors

ErbB receptor activation is a complex process and is dependent upon the type and number of receptors expressed on a given cell. Previous studies with defined combinations of ErbB receptors expressed in mammalian cells have helped elucidate specific biological responses for many of the recognized gene products that serve as ligands for these receptors. However, no study has examined the binding of these ligands in a defined experimental system. To address this issue, the relative binding affinities of the egf domains of eleven ErbB ligands were measured on six ErbB receptor combinations using a soluble receptor-ligand binding format. The ErbB2/4 heterodimer was shown to bind all ligands tested with moderate to very high affinity. In contrast, ErbB3 showed much more restrictive ligand binding specificity and measurable binding was observed only with heregulin, neuregulin2beta, epiregulin and the synthetic heregulin/egf chimera, biregulin. These studies also revealed that ErbB2 preferentially enhances ligand binding to ErbB3 or ErbB4 and to a lesser degree to ErbB1.     

The mucin Muc4 potentiates neuregulin signaling by increasing the cell-surface populations of ErbB2 and ErbB3

Mucins provide a protective barrier for epithelial surfaces, and their overexpression in tumors has been implicated in malignancy. We have previously demonstrated that Muc4, a transmembrane mucin that promotes tumor growth and metastasis, physically interacts with the ErbB2 receptor tyrosine kinase and augments receptor tyrosine phosphorylation in response to the neuregulin-1beta (NRG1beta) growth factor. In the present study we demonstrate that Muc4 expression in A375 human melanoma cells, as well as MCF7 and T47D human breast cancer cells, enhances NRG1beta signaling through the phosphatidylinositol 3-kinase pathway. In examining the mechanism underlying Muc4-potentiated ErbB2 signaling, we found that Muc4 expression markedly augments NRG1beta binding to A375 cells without altering the total quantity of receptors expressed by the cells. Cell-surface protein biotinylation experiments and immunofluorescence studies suggest that Muc4 induces the relocalization of the ErbB2 and ErbB3 receptors from intracellular compartments to the plasma membrane. Moreover, Muc4 interferes with the accumulation of surface receptors within internal compartments following NRG1beta treatment by suppressing the efficiency of receptor internalization. These observations suggest that transmembrane mucins can modulate receptor tyrosine kinase signaling by influencing receptor localization and trafficking and contribute to our understanding of the mechanisms by which mucins contribute to tumor growth and progression.     

Flexible targeting of ErbB dimers that drive tumorigenesis by using genetically engineered T cells

Pharmacological targeting of individual ErbB receptors elicits antitumor activity, but is frequently compromised by resistance leading to therapeutic failure. Here, we describe an immunotherapeutic approach that exploits prevalent and fundamental mechanisms by which aberrant upregulation of the ErbB network drives tumorigenesis. A chimeric antigen receptor named T1E28z was engineered, in which the promiscuous ErbB ligand, T1E, is fused to a CD28 + CD3ζ endodomain. Using a panel of ErbB-engineered 32D hematopoietic cells, we found that human T1E28z⁺ T cells are selectively activated by all ErbB1-based homodimers and heterodimers and by the potently mitogenic ErbB2/3 heterodimer. Owing to this flexible targeting capability, recognition and destruction of several tumor cell lines was achieved by T1E28z⁺ T cells in vitro, comprising a wide diversity of ErbB receptor profiles and tumor origins. Furthermore, compelling antitumor activity was observed in mice bearing established xenografts, characterized either by ErbB1/2 or ErbB2/3 overexpression and representative of insidious or rapidly progressive tumor types. Together, these findings support the clinical development of a broadly applicable immunotherapeutic approach in which the propensity of solid tumors to dysregulate the extended ErbB network is targeted for therapeutic gain.     

Controlled Dimerization of ErbB Receptors Provides Evidence for Differential Signaling by Homo- and Heterodimers

The four members of the ErbB family of receptor tyrosine kinases are involved in a complex array of combinatorial interactions involving homo- and heterodimers. Since most cell types express more than one member of the ErbB family, it is difficult to distinguish the biological activities of different homo- and heterodimers. Here we describe a method for inducing homo- or heterodimerization of ErbB receptors by using synthetic ligands without interference from the endogenous receptors. ErbB receptor chimeras containing synthetic ligand binding domains (FK506-binding protein [FKBP] or FKBP-rapamycin-binding domain [FRB]) were homodimerized with the bivalent FKBP ligand AP1510 and heterodimerized with the bifunctional FKBP-FRB ligand rapamycin. AP1510 treatment induced tyrosine phosphorylation of ErbB1 and ErbB2 homodimers and recruitment of Src homology 2 domain-containing proteins (Shc and Grb2). In addition, ErbB1 and ErbB2 homodimers activated downstream signaling pathways leading to Erk2 and Akt phosphorylation. However, only ErbB1 homodimers were internalized upon AP1510 stimulation, and only ErbB1 homodimers were able to associate with and induce phosphorylation of c-Cbl. Cells expressing AP1510-induced ErbB1 homodimers were able to associate with and induce phosphorylation of c-Cbl. Cells expressing AP1510-induced ErbB1 homodimers were able to form foci; however, cells expressing ErbB2 homodimers displayed a five- to sevenfold higher focus-forming ability. Using rapamycin-inducible heterodimerization we show that c-Cbl is unable to associate with ErbB1 in a ErbB1-ErbB2 heterodimer most likely because ErbB2 is unable to phosphorylate the c-Cbl binding site on ErbB1. Thus, we demonstrate that ErbB1 and ErbB2 homodimers differ in their abilities to transform fibroblasts and provide evidence for differential signaling by ErbB homodimers and heterodimers. These observations also validate the use of synthetic ligands to study the signaling and biological specificity of selected ErbB dimers in any cell type.     

Protein processing and releases of neuregulin-1 are regulated in an activity-dependent manner

Identification of the key molecules that bridge presynaptic neuronal events and long-term modification of the postsynaptic process is an important challenge which will have to be met in order to further our understanding of the mechanisms for learning and memory. This study is focused on neuregulin-1 (NRG-1), a neurotrophic factor, that is known to regulate the development of various tissues and/or the life/death of cells through activation of the ErbB family receptor tyrosine kinases. It was discovered that the soluble form of NRG-1 (sNRG-1) is produced from the transmembrane form of NRG through proteolytic cleavage during electrical stimulation of either cultured cerebellar granule cells (GCs) or pontine nucleus neurons (PNs) that are presynaptic to GCs. sNRG-1 was assayed by measuring the phosphorylation of both the ErbB receptors and cyclic AMP-responsive element-binding protein (CREB), and by means of antibodies to sNRG-1. The cleavage and release of NRG-1 depended on the frequency of electrical stimulation; the peak effect was at 50 Hz in both GCs and PNs. Activation of protein kinase C (PKC) mimicked this effect. The culture apparatus provided along with the mass-electrical stimulation that was employed proved to be a powerful tool for combining neuronal electrical events and chemical events. We conclude from the results that, in mossy fibre (PN axon)-GC synapses, electrical activity controls the proteolytic processing of NRG-1 in a frequency-dependent fashion and involves PKC. Furthermore, cleaved sNRG-1 plays an important functional role in regulating transmission across these synapses.     

Gangliosides influence EGFR/ErbB2 heterodimer stability but they do not modify EGF-dependent ErbB2 phosphorylation

Gangliosides are well-known regulators of cell differentiation through specific interactions with growth factor receptors. Previously, our group provided the first evidence about stable association of ganglioside GM₃ to EGFR/ErbB2 heterodimers in mammary epithelial cells. Goals of the present study were to better define the role of gangliosides in EGFR/ErbB2 heterodimerization and receptor phosphorylation events and to analyze their involvement in mammary cell differentiation. Experiments have been conducted using the ceramide analogue (+/−)-treo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol hydrochloride ([D]-PDMP), which inhibits ceramide glucosyltransferase resulting in the endogenous ganglioside depletion, and the lactogenic hormone mix DIP (dexamethasone, insulin, prolactin), which induces cell differentiation and β-casein mRNA synthesis. In addition, treatments of ganglioside-depleted cells with exogenous GM₃ have been carried out to ascertain the specific involvement of this ganglioside. Results from co-immunoprecipitation and Western blot experiments have shown that the endogenous ganglioside depletion resulted in the disappearance of SDS-stable EGFR/ErbB2 heterodimers and in the appearance of tyrosine-phosphorylated EGFR also in the absence of EGF stimulation; exogenous GM₃ added in combination with [D]-PDMP reversed both these effects. In contrast, the tyrosine phosphorylation of ErbB2 in ganglioside-depleted cells occurred only after EGF stimulation. Moreover, when ganglioside-depleted cells were treated with DIP in absence of EGF, β-casein gene expression appeared strongly down-regulated, and β-casein mRNA levels were partially restored by exogenous GM₃ treatment. Altogether, although the involvement of other ganglioside species cannot be excluded, these findings sustain the ganglioside GM₃ as an essential molecule for EGFR/ErbB2 heterodimer stability and important regulator of EGFR tyrosine phosphorylation, but it is not crucial for tyrosine phosphorylation of the heterodimerization partner ErbB2. Moreover, modulation of EGFR phosphorylation may explain how gangliosides contribute to regulate the lactogenic hormone-induced mammary cell differentiation.     

Diversification of Neu differentiation factor and epidermal growth factor signaling by combinatorial receptor interactions.

The ErbB family includes two receptors, ErbB-1 and ErbB-3, that respectively bind to epidermal growth factor and Neu differentiation factor, and an orphan receptor, ErbB-2. Unlike ErbB-1 and ErbB-2, the intrinsic tyrosine kinase of ErbB-3 is catalytically impaired. By using interleukin-3-dependent cells that ectopically express the three ErbB proteins or their combinations, we found that ErbB-3 is devoid of any biological activity but both ErbB-1 and ErbB-2 can reconstitute its extremely potent mitogenic activity. Transactivation of ErbB-3 correlates with heterodimer formation and is reflected in receptor phosphorylation and the transregulation of ligand affinity. Inter-receptor interactions enable graded proliferative and survival signals: heterodimers are more potent than homodimers, and ErbB-3-containing complexes, especially the ErbB-2/ErbB-3 heterodimer, are more active than ErbB-1 complexes. Nevertheless, ErbB-1 signaling displays dominance over ErbB-3 when the two receptors are coexpressed. Although all receptor combinations activate the mitogen-activated protein kinases ERK and c-Jun kinase, they differ in their rate of endocytosis and in coupling to intervening signaling proteins. It is conceivable that combinatorial receptor interactions diversify signal transduction and confer double regulation, in cis and in trans, of the superior mitogenic activity of the kinase-defective ErbB-3.     

Activation of ErbB2 receptor tyrosine kinase supports invasion of endothelial cells by Neisseria meningitidis

ErbB2 is a receptor tyrosine kinase belonging to the family of epidermal growth factor (EGF) receptors which is generally involved in cell differentiation, proliferation, and tumor growth, and activated by heterodimerization with the other members of the family. We show here that type IV pilus-mediated adhesion of Neisseria meningitidis onto endothelial cells induces tyrosyl phosphorylation and massive recruitment of ErbB2 underneath the bacterial colonies. However, neither the phosphorylation status nor the cellular localization of the EGF receptors, ErbB3 or ErbB4, were affected in infected cells. ErbB2 phosphorylation induced by N. meningitidis provides docking sites for the kinase src and leads to its subsequent activation. Specific inhibition of either ErbB2 and/or src activity reduces bacterial internalization into endothelial cells without affecting bacteria-induced actin cytoskeleton reorganization or ErbB2 recruitment. Moreover, inhibition of both actin polymerization and the ErbB2/src pathway totally prevents bacterial entry. Altogether, our results provide new insight into ErbB2 function by bringing evidence of a bacteria-induced ErbB2 clustering leading to src kinase phosphorylation and activation. This pathway, in cooperation with the bacteria-induced reorganization of the actin cytoskeleton, is required for the efficient internalization of N. meningitidis into endothelial cells, an essential process enabling this pathogen to cross host cell barriers.