An Immunological Approach Reveals Biological Differences between the Two NDF/Heregulin Receptors, ErbB-3 and ErbB-4

Abstract

The group of subtype I transmembrane tyrosine kinases includes the epidermal growth factor (EGF) receptor (ErbB-1), an orphan receptor (ErbB-2), and two receptors for the Neu differentiation factor (NDF/ heregulin), namely: ErbB-3 and ErbB-4. Here we addressed the distinct functions of the two NDF receptors by using an immunological approach. Two sets of monoclonal antibodies (mAbs) to ErbB-3 and ErbB-4 were generated through immunization with recombinant ectodomains of the corresponding receptors that were fused to immunoglobulin. We found that the shared ligand binds to highly immunogenic, but immunologically distinct sites of ErbB-3 and ErbB-4. NDF receptors differed also in their kinase activities; whereas the catalytic activity of ErbB-4 was activable by mAbs, ErbB-3 underwent no activation by mAbs in living cells. Likewise, down-regulation of ErbB-4, but not ErbB-3, was induced by certain mAbs. By using the generated mAbs, we found that the major NDF receptor on mammary epithelial cells is a heterodimer of ErbB-3 with ErbB-2, whereas an ErbB-1/ErbB-2 heterodimer, or an ErbB-1 homodimer, is the predominant species that binds EGF. Consistent with ErbB-2 being a shared receptor subunit, its tyrosine phosphorylation was increased by both heterologous ligands and it mediated a trans-inhibitory effect of NDF on EGF binding. Last, we show that the effect of NDF on differentiation of breast tumor cells can be mimicked by anti-ErbB-4 antibodies, but not by mAbs to ErbB-3. Nevertheless, an ErbB-3-specific mAb partially inhibited the effect of NDF on cellular differentiation. These results suggest that homodimers of ErbB-4 are biologically active, but heterodimerization of the kinase-defective ErbB-3, probably with ErbB-2, is essential for transmission of NDF signals through ErbB-3.Signals for growth and differentiation are mediated by binding of soluble growth factors to transmembrane receptors, that carry an intrinsic tyrosine kinase activity (1). The group of subtype I receptor tyrosine kinases includes four members that are characterized by ectodomains with two cysteine-rich sequences. Despite extensive structural homology, these receptors differ in their ligand specificities. Thus, ErbB-1 (also called HER-1) binds several distinct ligands whose prototype is the epidermal growth factor (EGF), 1 whereas ErbB-3 and ErbB-4 are the respective low and high affinity receptors for more than dozen isoforms of the Neu differentiation factor (NDF/heregulin) (2-4). The fourth member of the family, ErbB-2/Neu remains an orphan receptor because no fully characterized ligand of this receptor has been reported (5). Besides the interest in ErbB proteins as mediators of signal transduction, these receptors attracted attention due to their involvement in cancer development (6). Both ErbB-1 and ErbB-2 are oncogenic when overexpressed in murine fibroblasts (7,8), and their overexpression in human adenocarcinomas is associated with poor prognosis (9, 10). Likewise, ErbB-3 is overexpressed in some adenocarcinomas, but its prognostic s...

The role of transforming growth factor alpha production and ErbB-2 overexpression in induction of tumorigenicity of lung epithelial cells

“…A very complex series of ligand-receptor interactions regulating the biological function of ErbB family receptors is emerging (Riese et al, 1995;Chen et al, 1996;Karunagaran et al, 1996). It is becoming clear that there exist ligand-dependent hierarchies of heterodimer formation among ErbB receptors in response to specific ligands that continue to be discovered (Carraway et al, 1997;Chang et al, 1997).…”

Regulation of Cyclooxygenase-2 pathway by HER2 receptor

“…In order to understand the possible nature of the cellular-derived factor(s) that may regulate the observed constitutive activation of HER2/HER3 receptors, we next explored the possibility of endogenously secreted HRG or HRG-like ligand(s) on the formation of HER2/HER3 dimers in LS174T cells, using an anti-HER3 mAb against the HRG binding site on HER3 receptor (Chen et al, 1996). As illustrated in Figure 3B, pretreatment of cells with anti-HER3 mAb resulted in a signi®cant inhibition of heterodimerization between HER3 and HER2 and this was also true when cells were stimulated with NDF.…”

Differential expression of NDF/neuregulin receptors ErbB-3 and ErbB-4 and involvement in inhibition of neuronal differentiation

“…Separate cultures were incubated for 4 days with no added factor (CONTROL), or with one of the following proteins, as indicated: soluble forms of ErbB-4 or ErbB-2, in the form of immunoglobulin fusion proteins (denoted IgB-4 and IgB-2, respectively), each at a concentration of 4 mg/ml, or NDFb1 14 ± 246 (100 ng/ml Figure 5b, and data not shown). To examine the possibility that endogenous NDF/neuregulin was masking the e ect of the exogenously added ligand, we attempted to block the neuron-and astroglia-derived factor by using a soluble form of ErbB-4, that we have previously characterized as an e ective antagonist of NDF (Dong et al, 1995;Chen et al, 1996). This recombinant protein, denoted IgB-4, is a fusion between the extracellular ligand binding portion of ErbB-4 and the Fc portion of immunoglobulin G 1 .…”