Ligand discrimination in signaling through an ErbB4 receptor homodimer

The epidermal growth factor (EGF)-like family of growth factors elicits cellular responses by stimulating the dimerization, autophosphorylation, and tyrosine kinase activities of the ErbB family of receptor tyrosine kinases. Although several different EGF-like ligands are capable of binding to a single ErbB family member, it is generally thought that the biological and biochemical responses of a single receptor dimer to different ligands are indistinguishable. To test whether an ErbB receptor dimer is capable of discriminating among ligands we have examined the effect of four EGF-like growth factors on signaling through the ErbB4 receptor homodimer in CEM/HER4 cells, a transfected human T cell line ectopically expressing ErbB4 in an ErbB-null background. Despite stimulating similar levels of gross receptor tyrosine phosphorylation, the EGF-like growth factors betacellulin, neuregulin-1beta, neuregulin-2beta, and neuregulin-3 exhibited different biological potencies in a cellular growth assay. Moreover, the different ligands induced different patterns of recruitment of intracellular signaling proteins to the activated receptor and induced differential usage of intracellular kinase signaling cascades. Finally, two-dimensional phosphopeptide mapping of ligand-stimulated ErbB4 revealed that the different growth factors induce different patterns of receptor tyrosine phosphorylation. These results indicate that ErbB4 activation by growth factors is not generic and suggest that individual ErbB receptors can discriminate between different EGF-like ligands within the context of a single receptor dimer. More generally, our observations significantly modify our understanding of signaling through receptor tyrosine kinases and point to a number of possible models for ligand-mediated signal diversification.     

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.     

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.     

Differential activation of ErbB receptors in the rat olfactory mucosa by transforming growth factor-α and epidermal growth factor in vivo

Transforming growth factor-α (TGF-α) and epidermal growth factor (EGF) are members of the EGF family of growth factors. They have a common receptor, the EGF receptor. This belongs to the tyrosine kinase group of receptors called the ErbB receptor family. Other members are ErbB-2, ErbB-3, and ErbB-4. Binding of either ligand to the receptor elicits an increase in tyrosine kinase activity, resulting in the autophosphorylation of the receptor followed by a phosphorylation cascade of other tyrosine kinase substrates including mitogen-activated protein kinase (MAPK). TGF-α and EGF have been shown to stimulate cell division in the olfactory epithelium in vitro and may regulate cell division in vivo. To investigate whether exogenous TGF-α or EGF has a functional effect on the olfactory mucosa in vivo, 12.5–50 μg of each growth factor was administered to rats via the carotid artery. After 2 min, olfactory mucosa and liver samples were collected, homogenized, and immunoprecipitated with antibodies to the ErbB receptors. The immunoprecipitates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western immunoblotting. Using phosphotyrosine antibody, the receptors were probed for phosphorylation. Activation of MAPK was also investigated using MAPK antibody. Exogenous TGF-α activated EGFR, ErbB-2 and MAPK, whereas EGF activated only the EGFR. TGF-α was a more potent activator of EGFR than EGF. Neither ligand had an effect on ErbB-3 and ErbB-4 receptors. These effects were absent in the control animals which received the same solution without the growth factor. These results are consistent with the notion that binding of TGF-α to EGFR may play a role in olfactory cell division in vivo. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 199–210, 1998     

Computational modeling reveals molecular details of epidermal growth factor binding

Background  

The ErbB family of receptors are dysregulated in a number of cancers, and the signaling pathway of this receptor family is a critical target for several anti-cancer drugs. Therefore a detailed understanding of the mechanisms of receptor activation is critical. However, despite a plethora of biochemical studies and recent single particle tracking experiments, the early molecular mechanisms involving epidermal growth factor (EGF) binding and EGF receptor (EGFR) dimerization are not as well understood. Herein, we describe a spatially distributed Monte Carlo based simulation framework to enable the simulation of in vivo receptor diffusion and dimerization.     

Negative constraints underlie the ErbB specificity of epidermal growth factor-like ligands

Epidermal growth factor (EGF)-like growth factors bind their ErbB receptors in a highly selective manner, but the molecular basis for this specificity is poorly understood. We have previously shown that certain residues in human EGF (Ser(2)-Asp(3)) and TGFalpha (Glu(26)) are not essential for their binding to ErbB1 but prevent binding to ErbB3 and ErbB4. In the present study, we have used a phage display approach to affinity-optimize the C-terminal linear region of EGF-like growth factors for binding to each ErbB receptor and thereby shown that Arg(45) in EGF impairs binding to both ErbB3 and ErbB4. By omitting all these so-called negative constraints from EGF, we designed a ligand designated panerbin that binds ErbB1, ErbB3, and ErbB4 with similarly high affinity as their wild-type ligands. Homology models, based on the known crystal structure of TGFalpha-bound ErbB1, showed that panerbin is able to bind ErbB1, ErbB3, and ErbB4 in a highly similar manner with respect to position and number of interaction sites. Upon in silico introduction of the experimentally known negative constraints into panerbin, we found that Arg(45) induced local charge repulsion and Glu(26) induced steric hindrance in a receptor-specific manner, whereas Ser(2)-Asp(3) impaired binding due to a disordered conformation. Furthermore, radiolabeled panerbin was used to quantify the level of all three receptors on human breast cancer cells in a single radioreceptor assay. It is concluded that the ErbB specificity of EGF-like growth factors primarily results from the presence of a limited number of residues that impair the unintended interaction with other ErbB receptors.     

Cytohesins are cytoplasmic ErbB receptor activators

Signaling by ErbB receptors requires the activation of their cytoplasmic kinase domains, which is initiated by ligand binding to the receptor ectodomains. Cytoplasmic factors contributing to the activation are unknown. Here we identify members of the cytohesin protein family as such factors. Cytohesin inhibition decreased ErbB receptor autophosphorylation and signaling, whereas cytohesin overexpression stimulated receptor activation. Monitoring epidermal growth factor receptor (EGFR) conformation by anisotropy microscopy together with cell-free reconstitution of cytohesin-dependent receptor autophosphorylation indicate that cytohesins facilitate conformational rearrangements in the intracellular domains of dimerized receptors. Consistent with cytohesins playing a prominent role in ErbB receptor signaling, we found that cytohesin overexpression correlated with EGF signaling pathway activation in human lung adenocarcinomas. Chemical inhibition of cytohesins resulted in reduced proliferation of EGFR-dependent lung cancer cells in?vitro and in?vivo. Our results establish cytohesins as cytoplasmic conformational activators of ErbB receptors that are of pathophysiological relevance.     

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.     

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.     

Epidermal growth factor-mediated proliferation and sodium transport in normal and PKD epithelial cells

Members of the epidermal growth factor (EGF) family bind to ErbB (EGFR) family receptors which play an important role in the regulation of various fundamental cell processes including cell proliferation and differentiation. The normal rodent kidney has been shown to express at least three members of the ErbB receptor family and is a major site of EGF ligand synthesis. Polycystic kidney disease (PKD) is a group of diseases caused by mutations in single genes and is characterized by enlarged kidneys due to the formation of multiple cysts in both kidneys. Tubule cells proliferate, causing segmental dilation, in association with the abnormal deposition of several proteins. One of the first abnormalities described in cell biological studies of PKD pathogenesis was the abnormal mislocalization of the EGFR in cyst lining epithelial cells. The kidney collecting duct (CD) is predominantly an absorptive epithelium where electrogenic Na⁺ entry is mediated by the epithelial Na⁺ channel (ENaC). ENaC-mediated sodium absorption represents an important ion transport pathway in the CD that might be involved in the development of PKD. A role for EGF in the regulation of ENaC-mediated sodium absorption has been proposed. However, several investigations have reported contradictory results indicating opposite effects of EGF and its related factors on ENaC activity and sodium transport. Recent advances in understanding how proteins in the EGF family regulate the proliferation and sodium transport in normal and PKD epithelial cells are discussed here. This article is part of a Special Issue entitled: Polycystic Kidney Disease.     

Trafficking of the ErbB receptors and its influence on signaling

Although members of the ErbB receptor family are found predominantly at the cell surface, these receptors undergo constant cycling between the plasma membrane and the endosomal compartment. In the absence of an activating ligand, these receptors are slowly internalized (t(1/2) approximately 30 min) but are quickly recycled. The constitutive degradation rate of the epidermal growth factor (EGF) receptor (EGFR) is slower than other ErbB family members and only the EGFR appears to alter its trafficking pattern in response to ligand binding. This altered pattern is characterized by accelerated internalization and enhanced lysosomal targeting. Ligand-regulated trafficking of the EGFR is mediated by a series of motifs distributed through the cytoplasmic domain of the receptor that are exposed by a combination of activation-mediated conformation changes and the binding of proteins such as Grb2. As a consequence of induced internalization, most EGFR signaling occurs within endosomes whereas signaling by the other members of the ErbB family appear to be generated predominantly from the cell surface. Overexpression of ErbB family members can disrupt normal receptor trafficking by driving heterodimerization of receptors with disparate trafficking patterns. Because different ErbB receptor substrates are localized in different cellular compartments, disrupted trafficking could be an important factor in the altered signaling patterns observed as a consequence of receptor overexpression.     

The leucine-rich repeat protein LRIG1 is a negative regulator of ErbB family receptor tyrosine kinases

The molecular mechanisms by which mammalian receptor tyrosine kinases are negatively regulated remain largely unexplored. Previous genetic and biochemical studies indicate that Kekkon-1, a transmembrane protein containing leucine-rich repeats and an immunoglobulin-like domain in its extracellular region, acts as a feedback negative regulator of epidermal growth factor (EGF) receptor signaling in Drosophila melanogaster development. Here we tested whether the related human LRIG1 (also called Lig-1) protein can act as a negative regulator of EGF receptor and its relatives, ErbB2, ErbB3, and ErbB4. We observed that in co-transfected 293T cells, LRIG1 forms a complex with each of the ErbB receptors independent of growth factor binding. We further observed that co-expression of LRIG1 with EGF receptor suppresses cellular receptor levels, shortens receptor half-life, and enhances ligand-stimulated receptor ubiquitination. Finally, we observed that co-expression of LRIG1 suppresses EGF-stimulated transformation of NIH3T3 fibroblasts and that the inducible expression of LRIG1 in PC3 prostate tumor cells suppresses EGF- and neuregulin-1-stimulated cell cycle progression. Our observations indicate that LRIG1 is a negative regulator of the ErbB family of receptor tyrosine kinases and suggest that LRIG1-mediated receptor ubiquitination and degradation may contribute to the suppression of ErbB receptor function.     

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.     

Laminin-1 and epidermal growth factor family members co-stimulate fetal pancreas cell proliferation and colony formation

The epidermal growth factor (EGF) family is implicated in the development and function of multiple cells and organs, including the pancreas. We used a serum-free, low-cell density culture system to investigate the effect of EGFs on fetal pancreas cells. By RT-PCR, the EGF receptors ErbB 1-3 were detected in the developing mouse pancreas between embryonic day (E) 13.5 and E17.5, whereas ErbB4 was not detected until E17.5. The presence but not absence of the basement membrane glycoprotein laminin-1, betacellulin, and to a lesser extent EGF, transforming growth factor alpha, heparin binding EGF, and epiregulin induced E15.5 pancreatic cells to proliferate and form cystoid and solid colonies. These results demonstrate that laminin-1 and EGF signaling pathways interact to promote pancreas development.     

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.     

Human betacellulin structure modeled from other members of EGF family

We have modeled betacellulin (BTC) to gain insight into the structural elements that can explain its properties. The epidermal growth factor (EGF) signal transduction pathway, a significant mediator of several cell functions, is based on four closely related tyrosine kinase receptors. The ErbB receptors are transmembrane glycoproteins and signal transduction is initiated by ligand binding that induces receptor homo- or heterodimerization to form a complex containing two molecules of ligand and two molecules of receptor. The EGF family of ligands can be divided into three groups based on their ability to bind and activate distinct ErbB receptor homo- and heterodimers. Each member of the group formed by BTC, heparin binding EGF (HB-EGF) and epiregulin (EP) can interact with both the EGF receptor (EGFR) and heregulin receptors (ErbB-3 and ErbB-4), and are hence called bispecific ligands. BTC and EP also present the distinctive feature that they activate all possible heterodimeric ErbB receptors. BTC has been modeled with the program MODELLER, using human EGF, human transforming growth factor alpha (hTGF), human HB-EGF and human heregulin one alpha (hHRG-1) as templates. The structure of the model as well as that of the templates were optimized and a simulation of 100 ps was run for all. The main structural properties of the model and the templates were compared and in conclusion the hBTC conformation was closely similar to that of hTGF. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00894-002-0072-2.Electronic Supplementary Material available.     

ErbB-1 and ErbB-2 Acquire Distinct Signaling Properties Dependent upon Their Dimerization Partner

The different epidermal growth factor (EGF)-related peptides elicit a diverse array of biological responses as the result of their ability to activate distinct subsets of ErbB receptor dimers, leading to the recruitment of different intracellular signaling networks. To specifically examine dimerization-dependent modulation of receptor signaling, we constructed NIH 3T3 cell lines expressing ErbB-1 and ErbB-2 singly and in pairwise combinations with each other ErbB family member. This model system allowed the comparison of EGF-activated ErbB-1 with ErbB-1 activated by Neu differentiation factor (NDF)-induced heterodimerization with ErbB-4. In both cases, ErbB-1 coupled to the adaptor protein Shc, but only when activated by EGF was it able to interact with Grb2. Compared to the rapid internalization of EGF-activated ErbB-1, NDF-activated ErbB-1 showed delayed internalization characteristics. Furthermore, the p85 subunit of phosphatidylinositol kinase (PI3-K) associated with EGF-activated ErbB-1 in a biphasic manner, whereas association with ErbB-1 transactivated by ErbB-4 was monophasic. The signaling properties of ErbB-2 following heterodimerization with the other ErbB receptors or homodimerization induced by point mutation or monoclonal antibody treatment were also analyzed. ErbB-2 binding to peptides containing the Src homology 2 domain of Grb2 or p85 and the phosphotyrosine binding domain of Shc varied according to the mode of receptor activation. Finally, tryptic phosphopeptide mapping of both ErbB-1 and ErbB-2 revealed that receptor phosphorylation is dependent on the dimerization partner. Differential receptor phosphorylation may, therefore, be the basis for the differences in the signaling properties observed.     

Caveolin-1 phosphorylation in human squamous and epidermoid carcinoma cells: dependence on ErbB1 expression and Src activation

Previous studies have shown that EGF can induce the tyrosine phosphorylation of caveolin-1 in murine fibroblasts following ErbB1 (EGF receptor) mutation or overexpression, but the cell signaling events linking EGF action with caveolin phosphorylation are not fully established. In this regard, we examined multiple human carcinoma cell lines that express various ErbB family members, including A431 epidermoid carcinoma cells and several squamous carcinoma cell lines. In all cases, EGF treatment induced the tyrosine phosphorylation of caveolin-1 in a time- and EGF dose-dependent manner, and immunoblotting analysis revealed that this phosphorylation occurred at tyrosine-14. The EGF-dependent phosphorylation of caveolin-1 was observed at low temperatures (4 degrees C) and was enhanced by caveolae-disrupting agents (cyclodextrin), suggesting that this EGF-dependent system is in a low temperature-stable arrangement that allows for their interaction under conditions where mobility in the membrane is altered. To further assess the events linking EGF action with caveolin phosphorylation, we evaluated the ligand specificity of these responses and their dependence on known effectors of EGF receptor function. We observed that EGF and HB-EGF, but not heregulin, promoted caveolin-1 phosphorylation in A431 cells, suggesting that these responses are linked to EGF receptor activation and not solely occurring via the activation of other endogenous ErbB family members. In addition, the EGF-induced phosphorylation of caveolin-1 in A431 cells was blocked by the Src kinase antagonists PP1 and PP2, but not by the MEK inhibitor PD98059, the phosphoinositide 3-kinase inhibitors LY294002 and wortmannin, or cytoskeleton-disrupting agents, such as cytochalasin D, colchicine, and nocadazole. Altogether, these data indicate that multiple human carcinoma cells exhibit an EGF receptor-dependent tyrosine phosphorylation of caveolin-1 and that this process is sensitive to Src family kinase inhibitors. These observations support a role for caveolin tyrosine phosphorylation in the profile of cellular responses by which Src potentiates cancer progression following EGF receptor overexpression.     

ErbB-2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling.

We have analyzed ErbB receptor interplay induced by the epidermal growth factor (EGF)-related peptides in cell lines naturally expressing the four ErbB receptors. Down-regulation of cell surface ErbB-1 or ErbB-2 by intracellular expression of specific antibodies has allowed us to delineate the role of these receptors during signaling elicited by: EGF and heparin binding EGF (HB-EGF), ligands of ErbB-1; betacellulin (BTC), a ligand of ErbB-1 and ErbB-4; and neu differentiation factor (NDF), a ligand of ErbB-3 and ErbB-4. Ligand-induced ErbB receptor heterodimerization follows a strict hierarchy and ErbB-2 is the preferred heterodimerization partner of all ErbB proteins. NDF-activated ErbB-3 or ErbB-4 heterodimerize with ErbB-1 only when no ErbB-2 is available. If all ErbB receptors are present, NDF receptors preferentially dimerize with ErbB-2. Furthermore, EGF- and BTC-induced activation of ErbB-3 is impaired in the absence of ErbB-2, suggesting that ErbB-2 has a role in the lateral transmission of signals between other ErbB receptors. Finally, ErbB-1 activated by all EGF-related peptides (EGF, HB-EGF, BTC and NDF) couples to SHC, whereas only ErbB-1 activated by its own ligands associates with and phosphorylates Cbl. These results provide the first biochemical evidence that a given ErbB receptor has distinct signaling properties depending on its dimerization.