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.     

Hsp90 restrains ErbB-2/HER2 signalling by limiting heterodimer formation

ErbB-2/HER2 is an oncogenic tyrosine kinase that regulates a signalling network by forming ligand-induced heterodimers with several growth factor receptors of the ErbB family. Hsp90 and co-chaperones regulate degradation of ErbB-2 but not other ErbB members. Here, we report that the role of Hsp90 in modulating the ErbB network extends beyond regulation of protein stability. The capacity of ErbB-2 to recruit ligand-bound receptors into active heterodimers is limited by Hsp90, which is dissociated from ErbB-2 following ligand-induced heterodimerization. We show that Hsp90 binds a specific loop within the kinase domain of ErbB-2, thereby restraining heterodimer formation and catalytic function. These results define a role for Hsp90 as a molecular switch regulating the ErbB signalling network by limiting formation of ErbB-2-centred receptor complexes.     

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.     

Dynamic equilibrium between multiple active and inactive conformations explains regulation and oncogenic mutations in ErbB receptors

The ErbB growth factor receptor family members are key players in vital physiological and pathological processes. Like other receptor tyrosine kinases, the ErbBs are bi-topic membrane proteins, whose extracellular and intracellular domains are connected by single transmembrane span. In recent years the crystal structures of the extracellular and intracellular domains of some ErbBs have been determined. We integrated the available structural information with phylogenetic, biochemical, biophysical, genetic, and computational data into a suggested model for the regulation and activation of these receptors. According to the model, regulation is maintained by a dynamic equilibrium between monomeric and dimeric states in various conformations. Along this dynamic equilibrium, variations in the points of interactions within the dimers alter the activation state and ligand-binding affinities. The active state was recently shown to be associated with an asymmetric dimer of the kinase domains. That finding enabled us to elucidate, in molecular terms, the directionality observed in the activation process of ErbB heterodimers; it can explain, for example, the preferential activation of ErbB2 by ErbB1 over activation of ErbB1 by ErbB2. Sequence alterations that reverse this directionality lead to aberrant signaling and cancer. Our model also offers molecular interpretations of the effects of various oncogenic alterations that interfere with the regulatory mechanism.     

ErbB2 is necessary for induction of carcinoma cell invasion by ErbB family receptor tyrosine kinases

The epidermal growth factor (EGF) family of tyrosine kinase receptors (ErbB1, -2, -3, and -4) and their ligands are involved in cell differentiation, proliferation, migration, and carcinogenesis. However, it has proven difficult to link a given ErbB receptor to a specific biological process since most cells express multiple ErbB members that heterodimerize, leading to receptor cross-activation. In this study, we utilize carcinoma cells depleted of ErbB2, but not other ErbB receptor members, to specifically examine the role of ErbB2 in carcinoma cell migration and invasion. Cells stimulated with EGF-related peptides show increased invasion of the extracellular matrix, whereas cells devoid of functional ErbB2 receptors do not. ErbB2 facilitates cell invasion through extracellular regulated kinase (ERK) activation and coupling of the adaptor proteins, p130CAS and c-CrkII, which regulate the actin-myosin cytoskeleton of migratory cells. Overexpression of ErbB2 in cells devoid of other ErbB receptor members is sufficient to promote ERK activation and CAS/Crk coupling, leading to cell migration. Thus, ErbB2 serves as a critical component that couples ErbB receptor tyrosine kinases to the migration/invasion machinery of carcinoma cells.     

Molecular modeling of nearly full-length ErbB2 receptor

Members of the epidermal growth factor receptor family play important roles in various cellular processes, both in physiological and in pathological conditions. Dimerization and autophosphorylation of these receptor tyrosine kinases are key events of signal transduction. Details of the molecular events of the signaling are not entirely known. To facilitate the understanding of receptor structure and function at the molecular level, a molecular model was built for the nearly full-length ErbB2 dimer. Modeling was based on the x-ray or nuclear-magnetic resonance structures of extracellular, transmembrane, and intracellular domains. The extracellular domain was positioned above the cell membrane based on the distance determined from experimentally measured fluorescence resonance energy transfer. Favorable dimerization interactions are predicted for the extracellular, transmembrane, and protein kinase domains in the model of a nearly full-length dimer of ErbB2, which may act in a coordinated fashion in ErbB2 homodimerization, and also in heterodimers of ErbB2 with other members of the ErbB family.     

ErbB receptors and their ligands in the breast

ErbB receptor tyrosine kinases are membrane-bound receptors that possess intrinsic, ligand-activated, tyrosine kinase activity. Binding of growth factors to these receptors induces the formation of ErbB homo- and heterodimers and initiates a signalling cascade that traverses the cytoplasm to communicate with the nucleus and the cytoskeleton. The effect of this cascade is the regulation of cellular proliferation, differentiation, apoptosis, migration and adhesion. Although ErbB signalling is important for normal growth and development in the breast, a dysregulation of ErbB activity can lead to tumourigenesis. This review will focus on the role of ErbB signalling in both normal mammary gland development and breast cancer, with an emphasis on the mechanisms behind receptor activation and the therapeutic agents designed to inhibit ErbB activity.     

Heregulin activation of ErbB2/ErbB3 signaling potentiates the integrity of airway epithelial barrier

Background

Members of the ErbB family of the receptor protein tyrosine kinase superfamily mediate heregulin (HRG)-induced cell responses. Here we investigated HRG activation of ErbB receptors, and the role of this activation in the development of the permeability barrier in airway epithelial cells (AECs).

Methods

Two airway epithelial-like cell lines, Calu-3 and 16HBE were exposed to HRG or no stimulus and were evaluated with respect to their paracellular permeability as determined by transepithelial electric resistance (TER) and fluorescein isothiocyanate (FITC)-dextran flux. Tight junctions (TJs) were assessed by immunocytochemical localization of occludin and zonula occludens-1.

Results

HRG promoted the development of the permeability barrier and TJ formation by monolayers of Calu-3 and 16HBE cells. Calu-3 cells expressed ErbB1, ErbB2, and ErbB3, but not ErbB4, on their surface. ErbB3 knockdown by small interference RNA (siRNA) blunted the effects of HRG on the permeability barrier. ErbB3 is known as a kinase-dead receptor and relies on other members of the family for its phosphorylation. To identify its heterodimerization partner, we knocked down the expression of other ErbB family receptors. We found that HRG's effect on the permeability barrier could be significantly attenuated by transfecting cells with ErbB2 siRNA but not with EGFR siRNA.

Conclusion

These results indicate that HRG activation of ErbB2/ErbB3 heterodimers is essential for regulation of the permeability barrier in AECs.     

Mechanism of activation and inhibition of the HER4/ErbB4 kinase

HER4/ErbB4 is a ubiquitously expressed member of the EGF/ErbB family of receptor tyrosine kinases that is essential for normal development of the heart, nervous system, and mammary gland. We report here crystal structures of the ErbB4 kinase domain in active and lapatinib-inhibited forms. Active ErbB4 kinase adopts an asymmetric dimer conformation essentially identical to that observed to be important for activation of the EGF receptor/ErbB1 kinase. Mutagenesis studies of intact ErbB4 in Ba/F3 cells confirm the importance of this asymmetric dimer for activation of intact ErbB4. Lapatinib binds to an inactive form of the ErbB4 kinase in a mode equivalent to its interaction with the EGF receptor. All ErbB4 residues contacted by lapatinib are conserved in the EGF receptor and HER2/ErbB2, which lapatinib also targets. These results demonstrate that key elements of kinase activation and inhibition are conserved among ErbB family members.     

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.     

Structure-Function Relationships of ErbB RTKs in the Plasma Membrane of Living Cells

We review the states of the ErbB family of receptor tyrosine kinases (RTKs), primarily the EGF receptor (EGFR, ErbB1, HER1) and the orphan receptor ErbB2 as they exist in living mammalian cells, focusing on four main aspects: (1) aggregation state and distribution in the plasma membrane; (2) conformational features of the receptors situated in the plasma membrane, compared to the crystallographic structures of the isolated extracellular domains; (3) coupling of receptor disposition on filopodia with the transduction of signaling ligand gradients; and (4) ligand-independent receptor activation by application of a magnetic field.This review deals exclusively with the disposition and function of the human ErbB (HER) family of receptor tyrosine kinases (RTKs) in the plasma membrane of living cells. We have divided the material into four main topics: (1) distribution and aggregation state of ErbB family members; (2) the 3D structure of the ErbB1 and ErbB2 receptors; (3) the role of receptors located on extensions (filopodia) of the cell body; and (4) the phenomenon and implications of nonligand-dependent activation of the receptors.     

The ErbB kinase domain: structural perspectives into kinase activation and inhibition

Epidermal growth factor receptor (EGFR) and its family members, ErbB2, ErbB3 and ErbB4, are receptor tyrosine kinases which send signals into the cell to regulate many critical processes including development, tissue homeostasis, and tumorigenesis. Central to the signaling of these receptors is their intracellular kinase domain, which is activated by ligand-induced dimerization of the receptor and phosphorylates several tyrosine residues in the C-terminal tail. The phosphorylated tail then recruits other signaling molecules and relays the signal to downstream pathways. A model of the autoinhibition, activation and feedback inhibition mechanisms for the ErbB kinase domain has emerged from a number of recent structural studies. Meanwhile, recent clinical studies have revealed the relationship between specific ErbB kinase mutations and the responsiveness to kinase inhibitor drugs. We will review these regulation mechanisms of the ErbB kinase domain, and discuss the binding specificity of kinase inhibitors and the effects of kinase domain mutations found in cancer patients from a structural perspective.     

Kinase Activator-Receiver Preference in ErbB Heterodimers Is Determined by Intracellular Regions and Is Not Coupled to Extracellular Asymmetry

The EGF receptor (EGFR) family comprises four homologs in humans collectively known as the ErbB or HER proteins. ErbB proteins are receptor tyrosine kinases that become activated when ligands bind to their extracellular regions and promote formation of specific homo- and heterodimers with enhanced tyrosine kinase activity. An essential feature of ErbB activation is formation of an asymmetric kinase dimer in which the C-terminal lobe of one kinase serves as the activator or donor kinase by binding the N-terminal lobe of a receiver or acceptor kinase and stabilizing its active conformation. ErbB extracellular regions are also thought to form active asymmetric dimers in which only one subunit binds ligand. The observation that the unliganded ErbB2 kinase preferentially serves as the activator kinase when paired with EGFR/ErbB1 implied that extracellular asymmetry in ErbB proteins might be coupled to intracellular asymmetry with unliganded partners favoring the activator kinase position. Using cell-based stimulation assays and chimeric ErbB proteins, we show that extracellular asymmetry is not coupled to intracellular asymmetry and that ErbB intracellular regions are sufficient to determine relative kinase activator-receiver orientation. We further show a hierarchy of activator-receiver preferences among ErbB proteins, with EGFR/ErbB1 being the strongest receiver, followed by ErbB2 and then ErbB4, and that cis-phosphorylation of EGFR and ErbB2 appears to be negligible. This hierarchy shapes the nature of signaling responses to different ligands in cells expressing multiple ErbB proteins.     

Distinct roles for Src tyrosine kinase in beta2-adrenergic receptor signaling to MAPK and in receptor internalization

G protein-coupled receptors form the largest family of membrane receptors and transmit diverse ligand signals to modulate various cellular responses. After activation by their ligands, some of these G protein-coupled receptors are desensitized, internalized (endocytosed), and down-regulated (degraded). In HEK 293 cells, the G(s)-coupled beta2-adrenergic receptor was postulated to initiate a second wave of signaling, such as the activation of the mitogen-activated protein kinase (MAPK) pathway after the receptor is internalized. The tyrosine kinase c-Src plays a critical role in these events. Here we used mouse embryonic fibroblast (MEF) cells deficient in Src family tyrosine kinases to examine the role of Src in beta2-adrenergic receptor signaling to the MAPK pathway and in receptor internalization. We found that in Src-deficient cells the beta2-adrenergic receptor could activate the MAPK pathway. However, the internalization of beta2-adrenergic receptors was blocked in Src-deficient MEF cells. Furthermore, we observed that in MEF cells deficient in beta-arrestin 2 the internalization of the beta2-adrenergic receptor was impaired, whereas the activation of the MAPK pathway by the beta2-adrenergic receptor was normal. Our data demonstrate that although Src and beta-arrestin 2 play essential roles in beta2-adrenergic receptor internalization, they are not required for the activation of the MAPK pathway by the beta2-adrenergic receptor. In other words, our finding suggests that receptor internalization is not required for beta2-adrenergic receptor signaling to the MAPK pathway in MEF cells.     

Agonist-dependent recruitment of phosphoinositide 3-kinase to the membrane by beta-adrenergic receptor kinase 1. A role in receptor sequestration

Agonist-dependent desensitization of the beta-adrenergic receptor requires translocation and activation of the beta-adrenergic receptor kinase1 by liberated Gbetagamma subunits. Subsequent internalization of agonist-occupied receptors occurs as a result of the binding of beta-arrestin to the phosphorylated receptor followed by interaction with the AP2 adaptor and clathrin proteins. Receptor internalization is known to require D-3 phosphoinositides that are generated by the action of phosphoinositide 3-kinase. Phosphoinositide 3-kinases form a family of lipid kinases that couple signals via receptor tyrosine kinases and G-protein-coupled receptors. The molecular mechanism by which phosphoinositide 3-kinase acts to promote beta-adrenergic receptor internalization is not well understood. In the present investigation we demonstrate a novel finding that beta-adrenergic receptor kinase 1 and phosphoinositide 3-kinase form a cytosolic complex, which leads to beta-adrenergic receptor kinase 1-mediated translocation of phosphoinositide 3-kinase to the membrane in an agonist-dependent manner. Furthermore, agonist-induced translocation of phosphoinositide 3-kinase results in rapid interaction with the receptor, which is of functional importance, since inhibition of phosphoinositide 3-kinase activity attenuates beta-adrenergic receptor sequestration. Therefore, agonist-dependent recruitment of phosphoinositide 3-kinase to the membrane is an important step in the process of receptor sequestration and links phosphoinositide 3-kinase to G-protein-coupled receptor activation and sequestration.     

A hierarchical network of interreceptor interactions determines signal transduction by Neu differentiation factor/neuregulin and epidermal growth factor.

The ErbB family includes four homologous transmembrane tyrosine kinases. Whereas ErbB-1 binds to the epidermal growth factor (EGF), both ErbB-3 and ErbB-4 bind to the Neu differentiation factors (NDFs, or neuregulins), and ErbB-2, the most oncogenic family member, is an orphan receptor whose function is still unknown. Because previous lines of evidence indicated the existence of interreceptor interactions, we used ectopic expression of individual ErbB proteins and their combinations to analyze the details of receptor cross talks. We show that 8 of 10 possible homo-and heterodimeric complexes of ErbB proteins can be hierarchically induced by ligand binding. Although ErbB-2 binds neither ligand, even in a heterodimeric receptor complex, it is the preferred heterodimer partner of the three other members, and it favors interaction with ErbB-3. Selective receptor overexpression in human tumor cells appears to bias the hierarchical relationships. The ordered network is reflected in receptor transphosphorylation, ErbB-2-mediated enhancement of ligand affinities, and remarkable potentiation of mitogenesis by a coexpressed ErbB-2. The observed superior ability of ErbB-2 to form heterodimers, in conjunction with its uniquely high basal tyrosine kinase activity, may explain why ErbB-2 overexpression is associated with poor prognosis.     

ErbB and HB-EGF signaling in heart development and function

The epidermal growth factor (EGF)-ErbB signaling network is composed of multiple ligands of the EGF family and four tyrosine kinase receptors of the ErbB family. In higher vertebrates, these four receptors bind a multitude of ligands. Ligand binding induces the formation of various homo- and heterodimers of ErbB, potentially providing for a high degree of signal diversity. ErbB receptors and their ligands are expressed in a variety of tissues throughout development. Recent advances in gene targeting strategies in mice have revealed that the EGF-ErbB signaling network has fundamental roles in development, proliferation, differentiation, and homeostasis in mammals. The heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that binds to and activates the EGF receptor (EGFR/ErbB1) and ErbB4. Recent studies using several mutant mice lacking HB-EGF expression have revealed that HB-EGF has a critical role in normal heart function and in normal cardiac valve formation in conjunction with ErbB receptors. HB-EGF signaling through ErbB2 is essential for the maintenance of homeostasis in the adult heart, whereas HB-EGF signaling through EGFR is required during cardiac valve development. In this review, we introduce and discuss the role of ErbB receptors in heart function and development, focusing on the physiological function of HB-EGF in these processes.     

Differential association of phosphatidylinositol-5-phosphate 4-kinase with the EGF/ErbB family of receptors.

Phosphatidylinositol-5-phosphate 4-kinase (PIP4K) is required for the production of phosphoinositol-4,5-hisphosphate (PIP2), which has been closely associated with growth factor signalling. Here we have tested the possibility that phosphoinositide kinases may be take part in signal transduction through interactions with the epidermal growth factor (EGF) receptor and the ErbB family of tyrosine kinase receptors. Interactions of the Type IIbeta isoform of PIP4K were observed with the EGF receptor family members in a number of diverse cell lines, including A431, PC12 and MCF7 cells but not with the N6F TrkA receptor. Co-immunoprecipitation experiments indicate that PIP4K interacts with not only the EGF receptor, but also selectively with members of the ErbB tyrosine kinase family. These results demonstrate another enzyme substrate for EGF receptors that facilitates the production of phosphoinositides at the cell membrane.     

Spatial Structure of the Transmembrane Domain Heterodimer of ErbB1 and ErbB2 Receptor Tyrosine Kinases

Growth factor receptor tyrosine kinases of the ErbB family play a significant role in vital cellular processes and various cancers. During signal transduction across plasma membrane, ErbB receptors are involved in lateral homodimerization and heterodimerization with proper assembly of their extracellular single-span transmembrane (TM) and cytoplasmic domains. The ErbB1/ErbB2 heterodimer appears to be the strongest and most potent inducer of cellular transformation and mitogenic signaling compared to other ErbB homodimers and heterodimers. Spatial structure of the heterodimeric complex formed by TM domains of ErbB1 and ErbB2 receptors embedded into lipid bicelles was obtained by solution NMR. The ErbB1 and ErbB2 TM domains associate in a right-handed α-helical bundle through their N-terminal double GG4-like motif T⁶⁴⁸G⁶⁴⁹X₂G⁶⁵²A⁶⁵³ and glycine zipper motif T⁶⁵²X₃S⁶⁵⁶X₃G⁶⁶⁰, respectively. The described heterodimer conformation is believed to support the juxtamembrane and kinase domain configuration corresponding to the receptor active state. The capability for multiple polar interactions, along with hydrogen bonding between TM segments, correlates with the observed highest affinity of the ErbB1/ErbB2 heterodimer, implying an important contribution of the TM helix-helix interaction to signal transduction.