Left-Handed Dimer of EphA2 Transmembrane Domain: Helix Packing Diversity among Receptor Tyrosine Kinases

The Eph receptor tyrosine kinases and their membrane-bound ephrin ligands control a diverse array of cell-cell interactions in the developing and adult organisms. During signal transduction across plasma membrane, Eph receptors, like other receptor tyrosine kinases, are involved in lateral dimerization and subsequent oligomerization presumably with proper assembly of their single-span transmembrane domains. Spatial structure of dimeric transmembrane domain of EphA2 receptor embedded into lipid bicelle was obtained by solution NMR, showing a left-handed parallel packing of the transmembrane helices (535-559)₂. The helices interact through the extended heptad repeat motif L⁵³⁵X₃G⁵³⁹X₂A⁵⁴²X₃V⁵⁴⁶X₂L⁵⁴⁹ assisted by intermolecular stacking interactions of aromatic rings of (FF⁵⁵⁷)₂, whereas the characteristic tandem GG4-like motif A⁵³⁶X₃G⁵⁴⁰X₃G⁵⁴⁴ is not used, enabling another mode of helix-helix association. Importantly, a similar motif AX₃GX₃G as was found is responsible for right-handed dimerization of transmembrane domain of the EphA1 receptor. These findings serve as an instructive example of the diversity of transmembrane domain formation within the same family of protein kinases and seem to favor the assumption that the so-called rotation-coupled activation mechanism may take place during the Eph receptor signaling. A possible role of membrane lipid rafts in relation to Eph transmembrane domain oligomerization and Eph signal transduction was also discussed.     

Preparation of pro-oncogenic mutant forms V659E and V659Q of the transmembrane domain of receptor protein kinase ErbB2 for structural studies

Receptor tyrosine kinases (RTKs) play an important role in intercellular signal transduction through the plasma membrane. RTKs are integral membrane proteins activated upon lateral homo- or heterodimerization involving their transmembrane domain. The polymorphism and mutations in RTK transmembrane (TM) domains are directly associated with a number of human diseases. The family of epidermal growth factor receptors, ErbB, is an important class of RTKs participating in human cell growth, development, and differentiation. In order to investigate the influence of pathogenic mutations in ErbB TM domains on the structural and dynamic properties of these receptors and on specific interactions of their TM domains, we have developed highly effective systems of bacterial expression and purification of recombinant transmembrane fragments ErbB2_641–684 with pro-oncogenic substitution of Val⁶⁵⁹ by Glu or Gln. Transmembrane fragments were obtained in Escherichia coli BL21 (DE3) pLysS as a fusion protein with thioredoxin A. The purification protocol includes immobilized metal ion affinity chromatography (IMAC) and cation-exchange chromatography. The application of the protease Thrombin for hybrid protein hydrolysis considerably reduces financial expenditure as compared to the analogous protocols. The described techniques allow obtaining the milligram quantities of ErbB2 transmembrane fragments and its ¹⁵N-/[¹⁵N, ¹³C]-isotope-labeled derivatives for the analysis of their spatial structure using high-resolution heteronuclear NMR spectroscopy in a membrane-mimicking milieu.     

Spatial structure of the dimeric transmembrane domain of the growth factor receptor ErbB2 presumably corresponding to the receptor active state

Proper lateral dimerization of the transmembrane domains of receptor tyrosine kinases is required for biochemical signal transduction across the plasma membrane. The spatial structure of the dimeric transmembrane domain of the growth factor receptor ErbB2 embedded into lipid bicelles was obtained by solution NMR, followed by molecular dynamics relaxation in an explicit lipid bilayer. ErbB2 transmembrane segments associate in a right-handed alpha-helical bundle through the N-terminal tandem GG4-like motif Thr652-X3-Ser656-X3-Gly660, providing an explanation for the pathogenic power of some oncogenic mutations.     

Energetics of ErbB1 Transmembrane Domain Dimerization in Lipid Bilayers

One of the most extensively studied receptor tyrosine kinases is EGFR/ErbB1. Although our knowledge of the role of the extracellular domains and ligands in ErbB1 activation has increased dramatically based on solved domain structures, the exact mechanism of signal transduction across the membrane remains unknown. The transmembrane domains are expected to play an important role in the dimerization process, but the contribution of ErbB1 TM domain to dimer stability is not known, with published results contradicting one another. We address this controversy by showing that ErbB1 TM domain dimerizes in lipid bilayers and by calculating its contribution to stability as −2.5 kcal/mol. The stability calculations use two different methods based on Förster resonance energy transfer, which give the same result. The ErbB1 TM domain contribution to stability exceeds the change in receptor tyrosine kinases dimerization propensities that can convert normal signaling processes into pathogenic processes, and is thus likely important for biological function.     

Insight into the recognition patterns of the ErbB receptor family transmembrane domains: heterodimerization models through molecular dynamics search

ErbB receptors undergo a complex interaction network defining hierarchical and competition relationships. Dimerization is driven entirely by receptor-receptor interactions and the transmembrane domains play a role in modulating the specificity and the selection of the partners during signal transduction. To shed light on the role of the GxxxG-like dimerization motifs in the formation of ErbB transmembrane heterodimers, we propose structural models resulting from conformational search method combined with molecular dynamics simulations. Left-handed structures of the transmembrane heterodimers are found preponderant over right-handed structures. All heterotypic heterodimers undergo two modes of association either via the N-terminal motif or the C-terminal motif. The transmembrane domain of ErbB3 impairs this C-terminal motif but also associates with the other partners owing to the presence of Gly residues. The two dimerization modes involve different orientations of the two helices. Thus, a molecular-switch model allowing the transition between the two dimerizing states may apply to the heterodimers and could help interpret receptor competition for the formation of homodimers and heterodimers. The comparison between experimental and theoretical results on the dimerization hierarchy of the transmembrane domains is not straightforward. However, we demonstrate that the intrinsic properties of the transmembrane sequences are an important component in heterodimer formation and that the ErbB2 and ErbB3 transmembrane domains have a strong power for heterodimerization as observed experimentally.     

The single transmembrane domains of ErbB receptors self-associate in cell membranes.

Members of the epidermal growth factor receptor, or ErbB, family of receptor tyrosine kinases have a single transmembrane (TM) alpha-helix that is usually assumed to play a passive role in ligand-induced dimerization and activation of the receptor. However, recent studies with the epidermal growth factor receptor (ErbB1) and the erythropoietin receptor have indicated that interactions between TM alpha-helices do contribute to stabilization of ligand-independent and/or ligand-induced receptor dimers. In addition, not all of the expected ErbB receptor ligand-induced dimerization events can be recapitulated using isolated extracellular domains, suggesting that other regions of the receptor, such as the TM domain, may contribute to dimerization in vivo. Using an approach for analyzing TM domain interactions in Escherichia coli cell membranes, named TOXCAT, we find that the TM domains of ErbB receptors self-associate strongly in the absence of their extracellular domains, with the rank order ErbB4-TM > ErbB1-TM equivalent to ErbB2-TM > ErbB3-TM. A limited mutational analysis suggests that dimerization of these TM domains involves one or more GXXXG motifs, which occur frequently in the TM domains of receptor tyrosine kinases and are critical for stabilizing the glycophorin A TM domain dimer. We also analyzed the effect of the valine to glutamic acid mutation in ErbB2 that constitutively activates this receptor. Contrary to our expectations, this mutation reduced rather than increased ErbB2-TM dimerization. Our findings suggest a role for TM domain interactions in ErbB receptor function, possibly in stabilizing inactive ligand-independent receptor dimers that have been observed by several groups.     

Two motifs within a transmembrane domain, one for homodimerization and the other for heterodimerization

Protein assembly is a critical process involved in a wide range of cellular events and occurs through extracellular and/or transmembrane domains (TMs). Previous studies demonstrated that a GXXXG motif is crucial for homodimer formation. Here we selected the TMs of ErbB1 and ErbB2 as a model since these receptors function both as homodimers and as heterodimers. Both TMs contain two GXXXG-like motifs located at the C and N termini. The C-terminal motifs were implicated previously in homodimer formation, but the role of the N-terminal motifs was not clear. We used the ToxR system and expressed the TMs of both ErbB1 and ErbB2 containing only the N-terminal GXXXG motifs. The data revealed that the ErbB2 but not the ErbB1 construct formed homodimers. Importantly, a synthetic ErbB1 TM peptide was able to form a heterodimer with ErbB2, by displacing the ErbB2 TM homodimer. The specificity of the interaction was demonstrated by using three controls: (i) Two single mutations within the GXXXG-like motif of the ErbB1 peptide reduced or preserved its activity, in agreement with similar mutations in glycophorin A. (ii) A TM peptide of the bacterial Tar receptor did not assemble with the ErbB2 construct. (iii) The ErbB1 peptide had no effect on the dimerization of a construct containing the TM-1 domain of the Tar receptor. Fluorescence microscopy demonstrated that all the peptides localized on the membrane. Furthermore, incubation with the peptides had no effect on bacterial growth and protein expression levels. Our results suggest that the N-terminal GXXXG-like motif of the ErbB1 TM plays a role in heterodimerization with the ErbB2 transmembrane domain. To our knowledge, this is the first demonstration of a transmembrane domain with two distinct recognition motifs, one for homodimerization and the other for heterodimerization.     

Structural and thermodynamic insight into the process of “weak” dimerization of the ErbB4 transmembrane domain by solution NMR

Specific helix–helix interactions between the single-span transmembrane domains of receptor tyrosine kinases are believed to be important for their lateral dimerization and signal transduction. Establishing structure–function relationships requires precise structural-dynamic information about this class of biologically significant bitopic membrane proteins. ErbB4 is a ubiquitously expressed member of the HER/ErbB family of growth factor receptor tyrosine kinases that is essential for the normal development of various adult and fetal human tissues and plays a role in the pathobiology of the organism. The dimerization of the ErbB4 transmembrane domain in membrane-mimicking lipid bicelles was investigated by solution NMR. In a bicellar DMPC/DHPC environment, the ErbB4 membrane-spanning α-helices (651–678)₂ form a right-handed parallel dimer through the N-terminal double GG4-like motif A⁶⁵⁵GxxGG⁶⁶⁰ in a fashion that is believed to permit proper kinase domain activation. During helix association, the dimer subunits undergo a structural adjustment (slight bending) with the formation of a network of inter-monomeric polar contacts. The quantitative analysis of the observed monomer–dimer equilibrium provides insights into the kinetics and thermodynamics of the folding process of the helical transmembrane domain in the model environment that may be directly relevant to the process that occurs in biological membranes. The lipid bicelles occupied by a single ErbB4 transmembrane domain behave as a true (“ideal”) solvent for the peptide, while multiply occupied bicelles are more similar to the ordered lipid microdomains of cellular membranes and appear to provide substantial entropic enhancement of the weak helix–helix interactions, which may be critical for membrane protein activity.     

Transmembrane peptides as inhibitors of ErbB receptor signaling

Receptor tyrosine kinases have a single transmembrane (TM) segment that is usually assumed to play a passive role in ligand-induced dimerization and activation of the receptor. However, mutations within some of these receptors, and recent studies with the epidermal growth factor (EGF) and ErbB2 receptors have indicated that interactions between TM domains do contribute to stabilization of ligand-independent and/or ligand-induced receptor dimerization and activation. One consequence of the importance of these interactions is that short hydrophobic peptides corresponding to these domains should act as specific inhibitors. To test this hypothesis, we constructed expression vectors encoding short fusion peptides encompassing native or mutated TM domains of the EGF, ErbB2, and insulin receptors. In human cell lines overexpressing the wild-type EGF receptor or ErbB2, we observed that the peptides are expressed at the cell surface and that they inhibit specifically the autophosphorylation and signaling pathway of their cognate receptor. Identical results were obtained with peptides chemically synthesized. Mechanism of action involves inhibition of dimerization of the receptors as shown by the lack of effects of mutant nondimerizing sequences, completed by density centrifugation and covalent cross-linking experiments. Our findings stress the role of TM domain interactions in ErbB receptor function, and possibly for other single-spanning membrane proteins.     

N-glycan of ErbB family plays a crucial role in dimer formation and tumor promotion

More and more evidence indicates that N-glycan regulates signal transduction by modulating receptor functions. Previous studies suggested that glycosylation of EGFR is involved in dimerization and endocytosis. We further determined the role of N-glycosylation of ErbB family. A series of human ErbB3 mutants that lack each of the 10 N-glycosylation sites were prepared and transfected to Flp-In-CHO cells for stable expression. A crosslinking study showed that Asn 418 to Gln mutant (N418Q) of ErbB3 underwent autodimerization without its ligand, heregulin, and the heterodimer formation with ErbB2 was also increased. The N418Q mutant of ErbB3 co-expressed with ErbB2 promoted downstream signaling, anchorage-independent cell growth and the tumor growth in athymic mice. These findings suggest that the specific N-glycan in domain III of ErbB family plays an essential role in regulating receptor dimerization and transforming activity. We assume that the N-glycans affect the conformation of ErbB family, which is crucial for their activity. Together with findings from other laboratories, it is suggested that N-glycosylation controls ErbB signaling by various mechanisms.     

Coupled Stochastic Spatial and Non-Spatial Simulations of ErbB1 Signaling Pathways Demonstrate the Importance of Spatial Organization in Signal Transduction

Background

The ErbB family of receptors activates intracellular signaling pathways that control cellular proliferation, growth, differentiation and apoptosis. Given these central roles, it is not surprising that overexpression of the ErbB receptors is often associated with carcinogenesis. Therefore, extensive laboratory studies have been devoted to understanding the signaling events associated with ErbB activation.

Methodology/Principal Findings

Systems biology has contributed significantly to our current understanding of ErbB signaling networks. However, although computational models have grown in complexity over the years, little work has been done to consider the spatial-temporal dynamics of receptor interactions and to evaluate how spatial organization of membrane receptors influences signaling transduction. Herein, we explore the impact of spatial organization of the epidermal growth factor receptor (ErbB1/EGFR) on the initiation of downstream signaling. We describe the development of an algorithm that couples a spatial stochastic model of membrane receptors with a nonspatial stochastic model of the reactions and interactions in the cytosol. This novel algorithm provides a computationally efficient method to evaluate the effects of spatial heterogeneity on the coupling of receptors to cytosolic signaling partners.

Conclusions/Significance

Mathematical models of signal transduction rarely consider the contributions of spatial organization due to high computational costs. A hybrid stochastic approach simplifies analyses of the spatio-temporal aspects of cell signaling and, as an example, demonstrates that receptor clustering contributes significantly to the efficiency of signal propagation from ligand-engaged growth factor receptors.     

Monitoring the size and lateral dynamics of ErbB1 enriched membrane domains through live cell plasmon coupling microscopy

To illuminate the role of the spatial organization of the epidermal growth factor receptor (ErbB1) in signal transduction quantitative information about the receptor topography on the cell surface, ideally on living cells and in real time, are required. We demonstrate that plasmon coupling microscopy (PCM) enables to detect, size, and track individual membrane domains enriched in ErbB1 with high temporal resolution. We used a dendrimer enhanced labeling strategy to label ErbB1 receptors on epidermoid carcinoma cells (A431) with 60 nm Au nanoparticle (NP) immunolabels under physiological conditions at 37°C. The statistical analysis of the spatial NP distribution on the cell surface in the scanning electron microscope (SEM) confirmed a clustering of the NP labels consistent with a heterogeneous distribution of ErbB1 in the plasma membrane. Spectral shifts in the scattering response of clustered NPs facilitated the detection and sizing of individual NP clusters on living cells in solution in an optical microscope. We tracked the lateral diffusion of individual clusters at a frame rate of 200 frames/s while simultaneously monitoring the configurational dynamics of the clusters. Structural information about the NP clusters in their membrane confinements were obtained through analysis of the electromagnetic coupling of the co-confined NP labels through polarization resolved PCM. Our studies show that the ErbB1 receptor is enriched in membrane domains with typical diameters in the range between 60–250 nm. These membrane domains exhibit a slow lateral diffusion with a diffusion coefficient of  = |0.0054±0.0064| µm²/s, which is almost an order of magnitude slower than the mean diffusion coefficient of individual NP tagged ErbB1 receptors under identical conditions.     

Point Mutations in Dimerization Motifs of the Transmembrane Domain Stabilize Active or Inactive State of the EphA2 Receptor Tyrosine Kinase

The EphA2 receptor tyrosine kinase plays a central role in the regulation of cell adhesion and guidance in many human tissues. The activation of EphA2 occurs after proper dimerization/oligomerization in the plasma membrane, which occurs with the participation of extracellular and cytoplasmic domains. Our study revealed that the isolated transmembrane domain (TMD) of EphA2 embedded into the lipid bicelle dimerized via the heptad repeat motif L⁵³⁵X₃G⁵³⁹X₂A⁵⁴²X₃V⁵⁴⁶X₂L⁵⁴⁹ rather than through the alternative glycine zipper motif A⁵³⁶X₃G⁵⁴⁰X₃G⁵⁴⁴ (typical for TMD dimerization in many proteins). To evaluate the significance of TMD interactions for full-length EphA2, we substituted key residues in the heptad repeat motif (HR variant: G539I, A542I, G553I) or in the glycine zipper motif (GZ variant: G540I, G544I) and expressed YFP-tagged EphA2 (WT, HR, and GZ variants) in HEK293T cells. Confocal microscopy revealed a similar distribution of all EphA2-YFP variants in cells. The expression of EphA2-YFP variants and their kinase activity (phosphorylation of Tyr⁵⁸⁸ and/or Tyr⁵⁹⁴) and ephrin-A3 binding were analyzed with flow cytometry on a single cell basis. Activation of any EphA2 variant is found to occur even without ephrin stimulation when the EphA2 content in cells is sufficiently high. Ephrin-A3 binding is not affected in mutant variants. Mutations in the TMD have a significant effect on EphA2 activity. Both ligand-dependent and ligand-independent activities are enhanced for the HR variant and reduced for the GZ variant compared with the WT. These findings allow us to suggest TMD dimerization switching between the heptad repeat and glycine zipper motifs, corresponding to inactive and active receptor states, respectively, as a mechanism underlying EphA2 signal transduction.     

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.     

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.     

Folding Topology of a Bimolecular DNA Quadruplex Containing a Stable Mini-hairpin Motif within the Diagonal Loop

We describe the NMR structural characterisation of a bimolecular anti-parallel DNA quadruplex d(G₃ACGTAGTG₃)₂ containing an autonomously stable mini-hairpin motif inserted within the diagonal loop. A folding topology is identified that is different from that observed for the analogous d(G₃T₄G₃)₂ dimer with the two structures differing in the relative orientation of the diagonal loops. This appears to reflect specific base stacking interactions at the quadruplex-duplex interface that are not present in the structure with the T₄-loop sequence. A truncated version of the bimolecular quadruplex d(G₂ACGTAGTG₂)₂, with only two core G-tetrads, is less stable and forms a heterogeneous mixture of three 2-fold symmetric quadruplexes with different loop arrangements. We demonstrate that the nature of the loop sequence, its ability to form autonomously stable structure, the relative stabilities of the hairpin loop and core quadruplex, and the ability to form favourable stacking interactions between these two motifs are important factors in controlling DNA G-quadruplex topology.     

A Novel Assay for Assessing Juxtamembrane and Transmembrane Domain Interactions Important for Receptor Heterodimerization

Understanding the basis of specificity in receptor homodimerization versus heterodimerization is essential in determining the role receptor plays in signal transduction. Specificity in each of the interfaces formed during signal transduction involves cooperative interactions between receptor extracellular, transmembrane (TM), and cytoplasmic domains. While methods exist for studying receptor heterodimerization in cell membranes, they are limited to either TM domains expressed in an inverted orientation or capture only heterodimerization in a single assay. To address this limitation, we have developed an assay (DN-AraTM) that enables simultaneous measurement of homodimerization and heterodimerization of type I receptor domains in their native orientation, including both soluble and TM domains. Using integrin α_IIb and RAGE (receptor for advanced glycation end products) as model type I receptor systems, we demonstrate both specificity and sensitivity of our approach, which will provide a novel tool to identify specific domain interactions that are important in regulating signal transduction.     

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.     

Nrg1/ErbB signaling networks in Schwann cell development and myelination

Neuregulin-1 (Nrg1) provides a key axonal signal that regulates Schwann cell proliferation, migration and myelination through binding to ErbB2/3 receptors. The analysis of a number of genetic models has unmasked fundamental mechanisms underlying the specificity of the Nrg1/ErbB signaling axis. Differential expression of Nrg1 isoforms, Nrg1 processing, and ErbB receptor localization and trafficking represent important regulatory themes in the control of Nrg1/ErbB function. Nrg1 binding to ErbB2/3 receptors results in the activation of intracellular signal transduction pathways that initiate changes in Schwann cell behavior. Here, we review data that has defined the role of key Nrg1/ErbB signaling components like Shp2, ERK1/2, FAK, Rac1/Cdc42 and calcineurin in development of the Schwann cell lineage in vivo. Many of these regulators receive converging signals from other cues that are provided by Notch, integrin or G-protein coupled receptors. Signaling by multiple extracellular factors may act as key modifiers and allow Schwann cells at different developmental stages to respond in distinct manners to the Nrg1/ErbB signal.     

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.