Neuregulin-1 only induces trans-phosphorylation between ErbB receptor heterodimer partners

ErbB2, ErbB3 and ErbB4 are members of the Epidermal Growth Factor Receptor (EGFR) sub-family of Receptor Tyrosine Kinases (RTKs). Neuregulin-1 (NRG-1) is a ligand of ErbB3 and ErbB4 receptors. NRG-1-induced ErbB2/ErbB3 or ErbB2/ErbB4 heterodimerization, followed by receptor phosphorylation, plays multiple biological roles. To precisely determine the phosphorylation status of each ErbB receptor in ErbB2/ErbB3 and ErbB2/ErbB4 heterodimers, an immunoprecipitation-recapture of the ErbB receptors was performed to exclude any co-immunoprecipitated heterodimer partners from cells with co-expression of ErbB2/ErbB3, ErbB2/ErbB4, or ErbB2/ErbB4D843N, a kinase-inactive ErbB4 mutant, in which the aspartic acid at 843 (D843) was replaced by an asparagine (N). Here, we provide direct biochemical evidence that ErbB2 was only trans-phosphorylated by ErbB4, but not by ErbB3 or ErbB4D843N. By contrast, ErbB3, ErbB4 and ErbB4D843N were trans-phosphorylated by ErbB2 in the co-transfected cells. Therefore, we conclude that trans-phosphorylation, but not cis-phosphorylation occurred between ErbB2/ErbB3 and ErbB2/ErbB4 heterodimer partners by NRG-1 stimulation.     

ErbB2 and ErbB3 do not quantitatively modulate ligand-induced ErbB4 tyrosine phosphorylation

The ErbB family of receptor tyrosine kinases consists of four members: the epidermal growth factor receptor (EGFR/ErbB1), ErbB2/HER2/Neu, ErbB3/HER3, and ErbB4/HER4. ErbB2 is an "orphan" for which there is no naturally occurring, soluble ligand. ErbB3 lacks tyrosine kinase activity. Thus, we hypothesized that ErbB2 enhances ligand-induced ErbB family receptor signalling through mass action. In contrast, we hypothesized that ErbB3 reduces ligand-induced ErbB family receptor signalling by forming receptor heterodimers that cannot undergo bidirectional cross-phosphorylation. We tested these hypotheses using three cell lines that express equal levels of ErbB4. One expresses ErbB4 alone, the second expresses ErbB2 and ErbB4, and the third expresses ErbB3 and ErbB4. We treated the cells with the ErbB4 ligands betacellulin (BTC) and neuregulin1beta (NRG1 beta) and assayed ErbB4 tyrosine phosphorylation. ErbB2 and ErbB3 do not affect the amount of ligand-induced ErbB4 tyrosine phosphorylation. We will discuss these findings within the context of a model for ErbB receptor signalling.     

Enhanced drug resistance in cells coexpressing ErbB2 with EGF receptor or ErbB3

Overexpression of ErbB2 has been found in approximately 25-30% of human breast cancers and has been shown to render the cancer cells more resistant to chemotherapy. However, it is not clear whether ErbB2 overexpression renders the cells more resistant to specific anti-cancer drugs or renders the cells more resistant to a broad range of anti-cancer drugs. It is not clear how the function of ErbB2 in drug resistance is related to expression and activation of the other ErbB receptors. In this communication, we showed that several breast cancer cell lines including BT20, BT474, MCF-7, MDA-MB-453, and SKBR-3 cells had a similar pattern of resistance to a broad range of anti-cancer drugs including 5-Fluorouracil, Cytoxan, Doxorubincin, Taxol, and Vinorelbin, suggesting a mechanism of multidrug resistance. High expression of P-glycoprotein and the ErbB receptors contribute to drug resistance of these breast cancer cells; however, overexpression of ErbB2 alone is not a major factor in determining drug resistance. To further determine the role of the ErbB receptors in drug resistance, we selected various NIH 3T3 cell lines that specifically expressed EGF receptor (EGFR), ErbB2, ErbB3, EGFR/ErbB2, EGFR/ErbB3, or ErbB2/ErbB3. A cytotoxicity assay showed that expression of ErbB2 alone did not significantly enhance drug resistance, whereas coexpression of either EGFR or ErbB3 with ErbB2 significantly enhanced drug resistance. Moreover, ErbB2 was highly phosphorylated in NIH 3T3 cells that coexpress ErbB2 with either EGFR or ErbB3, but not in NIH 3T3 cells that express ErbB2 alone. Together, our results suggest that coexpression of EGFR or ErbB3 with ErbB2 induces high phosphorylation of ErbB2 and renders the cells more resistant to various anti-cancer drugs.     

Identification of prolidase as a high affinity ligand of the ErbB2 receptor and its regulation of ErbB2 signaling and cell growth

ErbB2, an important membrane-bound receptor tyrosine kinase, was discovered nearly 30 years ago, but a natural ligand has never been found previously. ErbB2 is also an important oncogene and anticancer target, and its overexpression in cancer is associated with poor disease prognosis. Here, we report that human prolidase (PEPD) is a high affinity ligand of ErbB2 and binds as a homodimer to subdomain 3 in the extracellular domain of this receptor. In ErbB2-overexpressing cells, both ErbB2 monomers and activated dimers exist. PEPD bound to ErbB2 monomers relatively slowly but caused ErbB2 dimerization, ErbB2 phosphorylation and downstream signaling. In contrast, PEPD bound rapidly to ErbB2 homodimers and rapidly silenced ErbB2 dimer-Src signaling, a key oncogenic pathway of ErbB2, by disrupting the association of Src with ErbB2. PEPD also caused pronounced ErbB2 depletion, resulting from ErbB2 internalization and degradation. Moreover, PEPD strongly inhibited the DNA synthesis, anchorage-independent growth and invasion and migration of cells that overexpressed ErbB2 but had no effect on cells without ErbB2 overexpression. Cells became sensitized to PEPD upon achieving stable ErbB2 overexpression. Thus, the impact of PEPD on ErbB2 is predominantly inhibitory, and PEPD targets cells addicted to ErbB2. PEPD is also a dipeptidase, but its enzymatic function is not involved in ErbB2 modulation. These findings revise our understanding of ErbB2 and PEPD and may be especially important for combating ErbB2-positive cancers.     

Sensitivity of mature Erbb2 to geldanamycin is conferred by its kinase domain and is mediated by the chaperone protein Hsp90

ErbB receptors are a family of ligand-activated tyrosine kinases that play a central role in proliferation, differentiation, and oncogenesis. ErbB2 is overexpressed in >25% of breast and ovarian cancers and is correlated with poor prognosis. Although ErbB2 and ErbB1 are highly homologous, they respond quite differently to geldanamycin (GA), an antibiotic that is a specific inhibitor of the chaperone protein Hsp90. Thus, although both mature and nascent ErbB2 proteins are down-regulated by GA, only nascent ErbB1 is sensitive to the drug. To reveal the underlying mechanism behind these divergent responses, we made a chimeric receptor (ErbB1/2) composed of the extracellular and transmembrane domains of ErbB1 and the intracellular domain of ErbB2. The ErbB1/2 protein is functional since its kinase activity was stimulated by epidermal growth factor. The sensitivity of ErbB1/2 to GA was similar to that of ErbB2 and unlike that of ErbB1, indicating that the intracellular domain of the chimera confers GA sensitivity. This finding also suggests that the GA sensitivity of mature ErbB2 depends on cytosolic Hsp90, rather than Grp94, a homolog of Hsp90 that is restricted to the lumen of the endoplasmic reticulum, although both chaperones bind to and are inhibited by GA. Lack of Grp94 involvement in mediating ErbB2 sensitivity to GA is further suggested by the fact that a GA derivative with low affinity for Grp94 efficiently depleted ErbB2 protein in treated cells. To localize the specific region of ErbB2 that confers GA sensitivity, we made truncated receptors with progressive deletions of the cytoplasmic domain and tested the GA sensitivity of these molecules. We found that ErbB2 constructs containing an intact kinase domain retained GA sensitivity, whereas those lacking the kinase domain (ErbB2/DK) lost responsiveness to GA completely. Hsp90 co-immunoprecipitated with all ErbB2 constructs that were sensitive to GA, but not with ErbB2/DK or ErbB1. Both tyrosine-phosphorylated and non-phosphorylated ErbB2 proteins were similarly sensitive to GA, as was a kinase-dead ErbB2 mutant. These data suggest that Hsp90 uniquely stabilizes ErbB2 via interaction with its kinase domain and that GA stimulates ErbB2 degradation secondary to disruption of ErbB2/Hsp90 association.     

ErbB3和ErbB4特异性配体筛选系统的建立及neuregulin-1突变体的筛选

Neuregulin-1(NRG1,纽兰格林)通过活化ErbB2/ErbB4二聚体具有治疗心衰的作用,目前已完成临床二期。为避免作为心衰治疗药物时同时激活ErbB3并产生副作用,因此用NRG1变异体的方法寻找能对ErbB4专一性激活的配体。文章构建了带有不同筛选标记的ErbB2、ErbB3、ErbB4细胞表达质粒,将ErbB2/ErbB3、ErbB2/ErbB4质粒共转染至CHO细胞,建立了ErbB2/ErbB3特异性表达和ErbB2/ErbB4特异性表达的细胞株。通过与新生大鼠原代心肌细胞比较,证明ErbB2/ErbB4细胞株信号传导功能与心肌细胞相似,NRG1可以激活下游的AKT信号途径、PI3K信号途径,并表现出良好的剂量效应。因此可以通过检测与心肌功能密切相关的下游信号AKT磷酸化水平快速筛选抗心衰药物,并通过与ErbB2/ErbB3信号激活水平比较鉴定其对心肌细胞的特异性。文章还构建了31个不同的NRG1突变体并在大肠杆菌中成功的表达和纯化。将这些突变体用于刺激两个细胞株,通过检测AKT磷酸化水平,发现这些突变体对ErbB2/ErbB3与ErbB2/ErbB4受体的激活能力不同。进一步检测其中5个ErbB2/ErbB4激活特异性发生改变的突变体与两对受体的亲和力,发现这些突变体和ErbB2/ErbB4与ErbB2/ErbB3受体亲和力的变化有一致性。最终筛选到了4个可以更特异性激活ErbB2/ErbB4受体的突变体作为更有效治疗心衰的候选药物。     

Changes in ErbB2 (her-2/neu), ErbB3, and ErbB4 during growth, differentiation, and apoptosis of normal rat mammary epithelial cells.

Studies were undertaken to examine the natural role of ErbB2, ErbB3, and ErbB4 during the development of normal rat mammary epithelial cells (MECs) in vivo and in vitro. Immunohistochemical analysis demonstrated that mammary gland terminal end buds expressed abundant ErbB2 and ErbB4 but limited ErbB3 in pubescent rats, whereas luminal epithelial cells in nulliparous rats expressed ErbB2, ErbB3, and/or ErbB4. During pregnancy, ductal epithelial cells and stromal cells expressed abundant ErbB3 but limited ErbB2. Although ErbB2 and ErbB3 were downregulated throughout lactation, both receptors were re-expressed during involution. In contrast, ErbB4 was downregulated throughout pregnancy, lactation, and involution. Immunoblotting and immunoprecipitation studies confirmed the developmental expression of ErbB2 and ErbB3 in the mammary gland and the co-localization of distinct ErbB receptors in the mammary gland of nulliparous rats. In agreement with our in vivo findings, primary culture studies demonstrated that ErbB2 and ErbB3 were expressed in functionally immature, terminally differentiated and apoptotic MECs, and downregulated in functionally differentiated MECs. ErbB receptor signaling was required for epithelial cell growth, functional differentiation, and morphogenesis of immature MECs, and the survival of terminally differentiated MECs. Finally, ErbB4 expression did not interfere with functional differentiation and apoptosis of normal MECs.     

The crystal structure of a truncated ErbB2 ectodomain reveals an active conformation,poised to interact with other ErbB receptors

ErbB2 does not bind ligand, yet appears to be the major signaling partner for other ErbB receptors by forming heteromeric complexes with ErbB1, ErbB3, or ErbB4. The crystal structure of residues 1-509 of ErbB2 at 2.5 A resolution reveals an activated conformation similar to that of the EGFR when complexed with ligand and very different from that seen in the unactivated forms of ErbB3 or EGFR. The structure explains the inability of ErbB2 to bind known ligands and suggests why ErbB2 fails to form homodimers. Together, the data suggest a model in which ErbB2 is already in the activated conformation and ready to interact with other ligand-activated ErbB receptors.     

Curcumin-induced degradation of ErbB2: A role for the E3 ubiquitin ligase CHIP and the Michael reaction acceptor activity of curcumin

We investigated the molecular mechanism underlying curcumin depletion of ErbB2 protein. Curcumin induced ErbB2 ubiquitination but pretreatment with proteasome inhibitors neither prevented curcumin depletion of ErbB2 protein nor further accumulated ubiquitinated ErbB2. Curcumin increased association of endogenous and ectopically expressed CHIP, a chaperone-dependent ubiquitin ligase, with ErbB2. In COS7 cells cotransfected with ErbB2 and various CHIP plasmids followed by curcumin treatment, CHIP-H260Q (a mutant lacking ubiquitin ligase activity) promoted less curcumin-induced ErbB2 ubiquitination than did wild type CHIP, and CHIP-K30A (a mutant incapable of binding Hsp90 and Hsp70) neither associated with ErbB2 nor promoted its ubiquitination. ErbB2 mutants lacking the kinase domain failed to associate with CHIP and were completely resistant to ubiquitination and depletion induced by curcumin. Finally, curcumin's Michael reaction acceptor functionality was required for both covalent association of curcumin with ErbB2 and curcumin-mediated ErbB2 depletion. These data suggest (1) that CHIP-dependent ErbB2 ubiquitination is implicated in curcumin-stimulated ErbB2 depletion, and (2) that covalent modification of ErbB2 by curcumin is the proximal signal which initiates this process.     

Constitutively active ErbB4 and ErbB2 mutants exhibit distinct biological activities.

ErbB4 is a member of the epidermal growth factor receptor(EGFR) family of tyrosine kinases, which includes EGFR/ErbB1, ErbB2/HER2/Neu, and ErbB3/HER3. These receptors play important roles both in normal development and in neoplasia. For example, deregulated signaling by ErbB1 and ErbB2 is observed in many human malignancies. In contrast, the roles that ErbB4 plays in tumorigenesis and normal biological processes have not been clearly defined. To identify the biological responses that are coupled to ErbB4, we have constructed three constitutively active ErbB4 mutants. Unlike a constitutively active ErbB2 mutant, the ErbB4 mutants are not coupled to increased cell proliferation, loss of contact inhibition, or anchorage independence in a rodent fibroblast cell line. This suggests that ErbB2 and ErbB4 may play distinct roles in tumorigenesis in vivo.     

Differential cytoskeletal association of ErbB1 and ErbB2 during keratinocyte differentiation

ErbB1 and ErbB2 display differential subcellular localization in human skin and cultured keratinocytes. To determine whether ErbB1 and ErbB2 also differ in cytoskeletal binding properties, normal human keratinocytes grown under conditions favoring a basal or differentiated phenotype were repeatedly extracted in a non-ionic detergent buffer. In basaloid keratinocytes, cytoskeletal association of ErbB1 and ErbB2 was limited. ErbB1 ( approximately 5%) was tightly associated with the cytoskeleton, compared to <1% of ErbB2 (p=0.004). After EGF stimulation, activated ErbB1 and ERK associated with the cytoskeleton to a greater extent than did total ErbB1 and total ERK. Association of ErbB2 increased markedly in differentiated keratinocytes, whereas association of ErbB1 was similar in basaloid and differentiated cells. Cytoskeletal association of ErbB2 correlated with plasma membrane localization. These results suggest that ErbB1 and ErbB2 employ different mechanisms of plasma membrane targeting during keratinocyte differentiation, and that cytoskeletal association may facilitate the coupling of activated ErbB1 and ERK.     

Mutational analysis of ErbB2 intracellular localization

In the present study, the sub-cellular localization of ErbB2 and its mutants expressed as GFP-tagged proteins in MCF-7 cells or endogenous ErbB2 in SKBR3 cells was examined. The data presented here demonstrate that the full-length ErbB2 was localized at the cytoplasmic membrane and ErbB2 ICD localized in the nucleus predominantly. The sequence of ErbB2 ICD contains the information supporting its nuclear translocation and cytoplasmic retention. A region (residues 721–970) harboring an arginine triplet is essential for the cytoplasmic trafficking of ErbB2. The results indicate that differential sub-cellular localization of ErbB2 ICD and the full-length ErbB2 is dependent on their structural determinants. The present results give initial clues for further analysis of the mechanism of ErbB2 intracellular localization.     

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.     

Identification of the domain in ErbB2 that restricts ligand-induced degradation

Ligand-induced receptor degradation is an important process for down-regulation of plasma membrane receptors. While epidermal growth factor receptor (EGFR) is rapidly internalised and degraded upon ligand stimulation, ErbB2, the closest member to EGFR in ErbB receptor family, is resistant in ligand-induced degradation. To understand the molecular mechanisms underlying the impairment in ligand-induced degradation of ErbB2, we attempted to determine structural factor in ErbB2 that restricts the degradation. By analysis of ligand-induced degradation of EGFR/ErbB2 chimeras, we have identified a region between amino acid residues F1030 and L1075 in ErbB2 as the domain that restricts the ligand-induced degradation. We designated this domain as the Blocking ErbB2 Degradation or the BED domain. Replacement of the BED domain in an EGFR/ErbB2 chimera with the corresponding region of EGFR changed this chimera from a non-degradable to a degradable receptor, indicating that the BED domain is the factor restricting the ligand-induced degradation of ErbB2. In addition, we found that a non-degradable EGFR/ErbB2 chimera was not defective in tyrosine phosphorylation, ubiquitination and interaction with c-Cbl, rather, was defective in ligand-induced internalisation, suggesting that the endocytosis defect is the cause restricting the degradation of ErbB2, and that c-Cbl-catalysed mono-ubiquitination is not involved in the impairment in ligand-induced degradation of ErbB2.     

Induction or suppression of expression of cytochrome C oxidase subunit II by heregulin beta 1 in human mammary epithelial cells is dependent on the levels of ErbB2 expression

The ErbB family of receptor kinases is composed of four members: epidermal growth factor receptor (EGFR/ErbB1), ErbB2/neu, ErbB3, and ErbB4. Amplification of the ErbB2/neu is found in about 30% of breast cancer patients and is associated with a poor prognosis. Heregulin (HRG) activates the ErbB2 via induction of heterodimerization with ErbB3 and ErbB4 receptors. With suppression subtractive hybridization, we demonstrated that the expression of cytochrome c oxidase subunit II (COXII) is HRG-responsive. Two nontransformed human mammary epithelial cell lines, the HB2 and the HB2(ErbB2) (the HB2 engineered to overexpress ErbB2), displayed an opposite response to HRG-mediated regulation. HRG upregulated mRNA expression of COXII in the HB2 cells, but suppressed COXII expression in the HB2(ErbB2) cells. A human breast cancer cell line (T47D), which expresses ErbB2 at a level similar to that of the HB2 cells, also responded to HRG by increasing COXII mRNA levels. Therefore, HRG regulation of COXII expression depends on the levels of ErbB2 expression. Furthermore, the expression of COXII was inversely correlated to the levels of ErbB2, i.e., the cells overexpressing ErbB2 exhibited lower COXII levels. HRG-evoked signal transduction differed between the cells with normal ErbB expression and the cells overexpressing ErbB2. The activation of both ERK and PI3-K was essential for HRG regulation of COXII, i.e., blockage of either pathway eliminated HRG-mediated alteration. This is the first report demonstrating that the expression of mitochondria-encoded COXII is HRG-responsive. The levels of ErbB2 expression are decisive for the diverse biological activities of HRG.     

Expression,localization, and regulation of the neuregulin receptor ErbB3 in mouse heart

Neuregulins (NRG) belong to the EGF family of growth factors, which are ligands of the ErbB receptors. Their expression in the adult heart is essential, especially when the heart is submitted to cardiotoxic stress such as that produced by anthracyclines. It is considered that ErbB4 is the only NRG receptor expressed by the adult heart. Upon binding, ErbB4 may dimerize with ErbB2 to generate signals inside cells. However, here we show the presence of ErbB3 in the mouse heart from birth to adulthood by Western blotting and real‐time RT‐PCR. The expression level of ErbB3 mRNA was lower than that of ErbB2 or ErbB4, but was more stable throughout postnatal development. In isolated heart myocytes, ErbB3 localized to the Z‐lines similarly to ErbB1. Perfusion of isolated hearts with NRG‐1β induced phosphorylation of ErbB3, as well as ErbB2 and ErbB4. In adult mice, both ErbB2 and ErbB3, but not ErbB1 or ErbB4, were rapidly down‐regulated upon the induction of heart hypertrophy. In conclusion, our results demonstrate that ErbB3, in addition to ErbB4, is a receptor for neuregulin‐1β in the adult mouse heart. J. Cell. Physiol. 226: 450–455, 2011. © 2010 Wiley‐Liss, Inc.     

ErbB2 and ErbB4 Cbl binding sites can functionally replace the ErbB1 Cbl binding site

Poor downregulation of ErbB receptors is associated with enhanced downstream signaling and tumorigenesis. It has been suggested that poor downregulation of ErbB-2, -3 and -4 receptors when compared to ErbB1 is due to decreased recruitment of Cbl E3 ligase proteins. However, a highly conserved Cbl binding site is not only present in ErbB1/EGFR (FLQRpY¹⁰⁴⁵SSDP), but also in ErbB2 (PLQRpY¹⁰⁹¹SEDP) and ErbB4 (STQRpY¹¹⁰³SADP). We therefore replaced the ErbB1 Cbl binding site by that of ErbB2 and ErbB4. Whereas retrovirally infected NIH3T3 cells containing the EGFR Y1045F mutation showed dramatically impaired Cbl recruitment, EGFR ubiquitination and delayed EGFR degradation, replacement of the EGFR Cbl binding site by that of ErbB2 or ErbB4 did not affect Cbl recruitment, receptor-ubiquitination, -degradation, -downregulation or ligand degradation. We conclude that poor downregulation of ErbB2 and ErbB4 receptors is not due to sequence variations in the Cbl binding site of these receptors.     

Muc4 is required for activation of ErbB2 in signet ring carcinoma cell lines

Signet-ring cell carcinoma is one of the most malignant tumors, classified histologically as a poorly differentiated adenocarcinoma. The ErbB2/ErbB3 complex is often constitutively activated, which suggests that the ErbB2/ErbB3 signaling pathway may be important for malignancy of this tumor. However, the mechanism underlying this activation has not been understood. Here, we show that ErbB2 and Muc4 bind in signet ring carcinoma cells, which was not seen in highly differentiated adenocarcinoma cell lines. ErbB3 was suggested to be a substrate of ErbB2 because knockdown of ErbB2 resulted in less phosphorylation of ErbB3. Inhibition of expression of Muc4 at the cell surface by the treatment of the cells with benzyl-GalNac, an inhibitor of mucin secretion, blocked phosphorylation of ErbB3, suggesting that activity of ErbB2 depends on the expression of Muc4. These results supply the biochemical backgrounds in recent studies suggesting the contribution of Muc4 in the tumorigenesis.