Barry J. Ratzkin

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

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

ErbB-2 is a common auxiliary subunit of NDF and EGF receptors: implications for breast cancer.

Overexpression of the erbB‐2 gene contributes to aggressive behavior of various human adenocarcinomas, including breast cancer, through an unknown molecular mechanism. The erbB‐2‐encoded protein is a member of the ErbB family of growth factor receptors, but no direct ligand of ErbB‐2 has been reported. We show that in various cells ErbB‐2 can form heterodimers with both EGF receptor (ErbB‐1) and NDF receptors (ErbB‐3 and ErbB‐4), suggesting that it may affect the action of heterologous ligands without the involvement of a direct ErbB‐2 ligand. This possibility was addressed in breast cancer cells through either overexpression of ErbB‐2 or by blocking its delivery to the cell surface by means of an endoplasmic reticulum‐trapped antibody. We report that ErbB‐2 overexpression enhanced binding affinities to both EGF and NDF, through deceleration of ligand dissociation rates. Likewise, removal of ErbB‐2 from the cell surface almost completely abolished ligand binding by accelerating dissociation of both growth factors. The kinetic effects resulted in enhancement and prolongation of the stimulation of two major cytoplasmic signaling pathways, namely: MAP kinase (ERK) and c‐Jun kinase (SAPK), by either ligand. Our results imply that ErbB‐2 is a pan‐ErbB subunit of the high affinity heterodimeric receptors for NDF and EGF. Therefore, the oncogenic action of ErbB‐2 in human cancers may be due to its ability to potentiate in trans growth factor signaling.

Molecular characterization of ALK, a receptor tyrosine kinase expressed specifically in the nervous system

The 2;5 chromosomal translocation is frequently associated with anaplastic large cell lymphomas (ALCLs). The translocation creates a fusion gene consisting of the alk (anaplastic lymphoma kinase) gene and the nucelophosmin (npm) gene: the 3′ half of alk derived from chromosome 2 is fused to the 5′ portion of npm from chromosome 5. A recent study shows that the product of the npm-alk fusion gene is oncogenic. To help understand how the npm-alk oncogene transform cells, it is important to investigate the normal biological function of the alk gene product, ALK. Here, we show molecular cloning of cDNAs for both the human and mouse ALK proteins. The deduced amino acid sequences reveal that ALK is a novel receptor protein-tyrosine kinase having a putative transmembrane domain and an extracellular domain. These sequences are absent in the product of the transforming npm-alk gene. ALK shows the greatest sequence similarity to LTK (leukocyte tyrosine kinase) whose biological function is presently unknown. RNA blot hybridization analysis of various tissues reveals that the alk mRNA is dominantly detected in the brain and spinal cord. Immunoblotting with anti-ALK antibody shows that ALK is highly expressed in the neonatal brain. Furthermore, RNA in situ hybridization analysis shows that the alk mRNA is dominantly expressed in neurons in specific regions of the nervous system such as the thalamus, mid-brain, olfactory bulb, and ganglia of embryonic and neonatal mice. These data suggest that ALK plays an important role(s) in the development of the brain and exerts its effects on specific neurons in the nervous system.

Bivalence of EGF-like ligands drives the ErbB signaling network

Signaling by epidermal growth factor (EGF)‐like ligands is mediated by an interactive network of four ErbB receptor tyrosine kinases, whose mechanism of ligand‐induced dimerization is unknown. We contrasted two existing models: a conformation‐driven activation of a receptor‐intrinsic dimerization site and a ligand bivalence model. Analysis of a Neu differentiation factor (NDF)‐induced heterodimer between ErbB‐3 and ErbB‐2 favors a bivalence model; the ligand simultaneously binds both ErbB‐3 and ErbB‐2, but, due to low‐affinity of the second binding event, ligand bivalence drives dimerization only when the receptors are membrane anchored. Results obtained with a chimera and isoforms of NDF/neuregulin predict that each terminus of the ligand molecule contains a distinct binding site. The C‐terminal low‐affinity site has broad specificity, but it prefers interaction with ErbB‐2, an oncogenic protein acting as a promiscuous low‐affinity subunit of the three primary receptors. Thus, ligand bivalence enables signal diversification through selective recruitment of homo‐ and heterodimers of ErbB receptors, and it may explain oncogenicity of erbB‐2/HER2.

Neu Differentiation Factor/Neuregulin Isoforms Activate Distinct Receptor Combinations

The multiple isoforms of Neu differentiation factor (NDF/neuregulin) induce a pleiotropic cellular response that is isoform-specific and cell type-dependent. The molecular basis of this heterogeneity was addressed by comparing the two major groups of isoforms, α and β. Both groups bind to the catalytically impaired receptor tyrosine kinase ErbB-3, whose mitogenic stimulation by NDF requires transactivation by other ErbB proteins, either ErbB-1 or ErbB-2. By expressing each pair of receptors in interleukin 3-dependent myeloid cells, we found that both isoforms induced mitogenic signals in cells co-expressing the combination of ErbB-3 with ErbB-2. However, only the β isoform stimulated cells that expressed both ErbB-3 and ErbB-1, and neither isoform was active on cells expressing ErbB-3 alone. Both isoforms bind to all ErbB-3-expressing cells, albeit with different affinities, but the co-stimulatory mitogenic effect is correlated with the ability of each auxiliary receptor to transphosphorylate ErbB-3. These results imply that NDF isoforms differ in their ability to induce receptor heterodimers; whereas both types of isoforms signal through ErbB-3/ErbB-2 heterodimers, only β isoforms are able to stabilize ErbB-3/ErbB-1 heterodimers.

ErbB tyrosine kinases and the two neuregulin families constitute a ligand-receptor network.

ABSTRACT The recently isolated second family of neuregulins, NRG2, shares its primary receptors, ErbB-3 and ErbB-4, and induction of mammary cell differentiation with NRG1 isoforms, suggesting functional redundancy of the two growth factor families. To address this possibility, we analyzed receptor specificity of NRGs by using an engineered cellular system. The activity of isoform-specific but partly overlapping patterns of specificities that collectively activate all eight ligand-stimulatable ErbB dimers was revealed. Specifically, NRG2-β, like NRG1-α, emerges as a narrow-specificity ligand, whereas NRG2-α is a pan-ErbB ligand that binds with different affinities to all receptor combinations, including those containing ErbB-1, but excluding homodimers of ErbB-2. The latter protein, however, displayed cooperativity with the direct NRG receptors. Apparently, signaling by all NRGs is funneled through the mitogen-activated protein kinase (MAPK). However, the duration and potency of MAPK activation depend on the identity of the stimulatory ligand-receptor ternary complex. We conclude that the NRG-ErbB network represents a complex and nonredundant machinery developed for fine-tuning of signal transduction.

Binding of Neu Differentiation Factor with the Extracellular Domain of Her2 and Her3

The interaction of neu differentiation factor (NDF) with the extracellular domains of Her2 (sHer2) and Her3 (sHer3) have been studied using native gels, light scattering, and sedimentation equilibrium. The full-length NDFβ2 was shown to bind sHer3 with a dissociation constant of 26 ± 9 nM, while it showed a 1000-fold weaker binding to sHer2. Taken together, these results demonstrate that NDF is a high affinity ligand for Her3, but not for Her2. No increase in affinity of the NDFβ2 for sHer3 was observed upon addition of sHer2 to the NDFβ2-sHer3 mixture. Binding of NDFβ2 to sHer3 did not induce receptor dimerization or oligomerization, the stoichiometry being one sHer3 per one NDF molecule. This finding suggests that transmembrane and/or intracellular domains of receptor family members or perhaps additional unidentified components may be involved in NDF induced dimerization and autophosphorylation, or alternatively, that dimerization is not the mechanism for Her3 autophosphorylation and signal transduction.

Fractionation of polyclonal antibodies to fragments of a neuroreceptor using three increasingly chaotropic solvents

We have developed specific antibodies against fragments of anaplastic lymphoma kinase (ALK) in order to develop tools for characterizing the expression and biological function of this orphan receptor. The first fragment consisted of residues 280 to 480 of the murine extracellular domain, was expressed in Escherichia coli (E. coli), purified in the presence of urea from the pellet of mechanically lysed cells and injected into rabbits as an unfolded protein in urea. The second fragment consisted of residues 1519 to 1619 of the murine sequence, corresponding to the C-terminal side of the kinase domain. It was expressed in E. coli as a soluble glutathione-S-transferase fusion protein, purified from the supernatant of broken cells and injected into rabbits as a folded protein. Both antisera were purified using antigen affinity chromatography, with the polyclonal antibodies eluted stepwise using three different buffers, 0.1 M glycine, pH 2.9, followed by 7 M urea, pH 4, followed by 6 M guanidine-HCl (GdnHCl), pH 4. Antisera prepared against either antigen contained antibodies that eluted in each of the three pools, indicating that solvents more chaotropic than acid were required to elute antibody populations that were tightly bound to the antigen column. All three antibody pools were reactive towards their respective antigens upon Western blot analysis. Purified polyclonal antibodies (pAbs) to both fragments also recognized the full-length protein expressed in Chinese hamster ovary cells. In every case, the pAbs eluting in GdnHCl were the most sensitive for detecting full-length ALK.

NDF INDUCES EXPRESSION OF A NOVEL 46 KD PROTEIN IN ESTROGEN RECEPTOR POSITIVE BREAST CANCER CELLS

Most human breast tumors start as estrogen‐dependent, but during the course of the disease become refractory to hormone therapy. The transition of breast tumors from estrogen dependent to independent behavior may be regulated by autocrine and/or paracrine growth factor(s) that are independent of the estrogen receptor (ER). We have investigated the role(s) of NDF (neu‐differentiation factor) in the biology of estrogen positive breast cancer cells by using MCF‐7 cells as a model system. Treatment of MCF‐7 cells with human recombinant NDF‐β2 (NDF) inhibited the ER expression by 70% and this was associated with growth stimulation in an estrogen‐independent manner. To explore the mechanism(s) of action of NDF in MCF‐7 cells, we examined the expression of NDF‐inducible gene products. We report here that NDF stimulated the levels of expression of a 46 kD protein (p46) (in addition to few minor proteins) in ER positive breast cancer cells including MCF‐7, T‐47‐D, and ZR‐75‐R cells but not in ER negative breast cancer cells including MDA‐231, SK‐BP‐3, and MDA‐468 cells. This effect of NDF was due to induction in the rate of synthesis of new p46. The observed NDF‐mediated induction of p46 expression was specific as there was no such effect by epidermal growth factor or 17‐β‐estradiol, and inclusion of actinomycin D partially inhibited the p46 induction elicited by NDF. NDF‐inducible stimulation of p46 expression was an early event (2–6 h) which preceded the period of down‐regulation of ER expression by NDF. These results support the existence of NDF‐responsive specific cellular pathway(s) that may regulate ER, and these interactions could play a role(s) in hormone‐independence of ER positive breast cancer cells.