Tyrosine kinases, which are important regulators of intracellular signal-transduction pathways, have mutated forms that are often associated with oncogenesis and are attractive targets for therapeutic intervention. Recently, systematic mutational analyses of tyrosine kinases revealed that a minimum of 30% of colorectal cancer contain at least one mutation in the tyrosine kinases. To further explore these mutations, we examined all reported mutations of NTRK3, FES, KDR, EPHA3, NTRK2, JAK1, PDGFRA, EPHA7, EPHA8, ERBB4, FGFR1, MLK4 and GUCY2F genes in the 24 colorectal cancer cell lines. Unexpectedly, among 24 colorectal cancer cell lines, only two cell lines (LoVo and CaR1) harbored mutation C1408T (R470C) in MLK4 gene. The mutation rate was extremely low compared to that previously reported. Therefore, we analyzed mutations in 46 colorectal cancer samples resected from the same number of Japanese patients. Surprisingly, none of the 46 samples contained any of the mutations reported. Based on our study, we advise that a more comprehensive tyrosine kinase gene mutation assay is necessary in the future.

The ErbB tyrosine kinases (epidermal growth factor receptor (EGFR), ErbB2/HER2, ErbB3, and ErbB4) are cell surface growth factor receptors widely expressed in many developing mammalian tissues, including in the intestinal tract. Signaling elicited by these receptors promotes epithelial cell growth and survival, and ErbB ligands have been proposed as therapeutic agents for intestinal diseases of pediatric populations, including inflammatory bowel disease (IBD), necrotizing enterocolitis (NEC), and inflammation associated with total parenteral nutrition (TPN). Furthermore, emerging evidence points to reduced ErbB ligand expression and thus reduced ErbB activity in IBD, NEC, and TPN models. This review will discuss the current understanding of the role of ErbB receptors in the pathogenesis and potential treatment of pediatric intestinal inflammation, with focus on the altered signaling in disease and the molecular mechanisms by which exogenous ligands are protective.

Alzheimer's disease (AD) is characterized by the deposition of beta-amyloid protein (Aβ) and extensive neuronal cell death. Apoptosis plays a crucial role in loss of neurons in AD. Neuregulin1 (NRG1) has been found to protect neurons from oxygen glucose deprivation induced apoptosis and hypoxia ischemia induced apoptosis. However, the relationship between NRG1 and apoptosis related protein expression in AD and its mechanism remain uncertain. The present study explores the effects of NRG1 on Aβ-induced apoptosis in AD. In this study, extracellular domain of NRG1beta1 (NRG1β1-ECD) promoted the expression of p-ErbB4 receptor, p-Akt and increased the level of Bcl-2 both in APP/PS1 transgenic mice and in vitro. In primary culture of neurons, the level of Bcl-2 protein decreased significantly after Aβ treatment. These changes were inhibited by pretreatment of neurons with NRG1β1-ECD. A specific inhibitor of PI3-kinase/Akt pathway, wortmannin, significantly abrogated the effects of NRG1β1-ECD on p-Akt and Bcl-2 levels. Furthermore, the expression of PI3-kinase/Akt by NRG1β1-ECD was ErbB4-dependent. Our data demonstrated that NRG1β1-ECD might serve as an obvious neuroprotection in AD, and the possible protective mechanism occurs most likely via ErbB4-dependent activation of PI3-kinase/Akt pathway.

The neuregulin 1 (NRG1)-ErbB4 signaling pathway has been implicated in the pathophysiology of schizophrenia. Recent studies suggest that this pathway may interact with the N-methyl-d-aspartate receptor (NMDAR) via the postsynaptic scaffold protein PSD-95. This interaction is of particular interest given the leading role of the NMDAR hypofunction in schizophrenia. The present study investigated the short- and long-term effects of chronic NMDAR blockade on the functional interaction between the two systems in rat prefrontal cortex and hippocampus using immunoprecipitation. Adult male Wistar rats were treated intraperitoneally with MK-801 (0.25 mg/kg) or saline for 28 days. Twenty-four hours after the last injection, the associations of ErbB4 with PSD-95 and NMDAR were enhanced in the prefrontal cortex, whereas only phosphorylated-ErbB4 relative to ErbB4 was increased in the hippocampus. These effects, however, were not detectable 12 days after the last MK-801 treatment, indicating the reversible nature of these changes. We also investigated the effects of chronic MK-801 treatment on locomotion, prepulse inhibition, recognition memory, and spatial working memory. The results showed that this treatment led to decreased locomotor activity, reduced exploration in the center arena, and elevated startle magnitudes, indicating an anxiety-like phenotype. Taken together, our findings suggest that the NRG1-ErbB4 signaling could be modulated by repeated NMDAR blockade, and provide further evidence for the cross-talk between the two signaling pathways.

Nestin-expressing neural progenitor cells (NPCs) have been isolated from hippocampus of brains and propagated with epidermal growth factor and basic fibroblast growth factor (bFGF). However, the underlying signaling mechanisms regulating NPC proliferation remain elusive. Here we showed that neuregulinbeta1 (NRG), like bFGF, effectively promoted the proliferation of hippocampus-derived NPCs and maintained the progenitor states of NPCs. Activation of protein kinase C (PKC), a downstream effector of phospholipase C (PLC), with 12-O-tetradecanoylphorbol-13-acetate (TPA) mimicked the NRG-induced proliferation of NPCs. The synergic effect of TPA plus NRG on neurosphere growth further prompted us to find that NRG induced NPC propagation through PLC/PKC signaling pathway. ErbB4, an important functional receptor of NRG, had an interaction with PLCgamma1 protein. In addition, inactivation of PLC pathway led to severe proliferative suppression of NPCs. Our study suggests that activation of PLC/PKC pathway plays an essential role in the NRG-induced proliferation of hippocampus-derived NPCs.

Integrin alpha(6)beta(4) is abundantly expressed in normal epithelial cells and forms hemidesmosomes, one of cell-extracellular matrix junctions. In many types of cancer cells, integrin alpha(6)beta(4) is up-regulated, laminin, an integrin alpha(6)beta(4)-binding extracellular matrix protein, is cleaved, and hemidesmosomes are disrupted, eventually causing an enhancement of cancer cell movement and a facilitation of their invasion. It was previously shown that integrin alpha(6)beta(4) interacts with ErbB1 and ErbB2 and enhances cell proliferation and motility. Here we show that integrin alpha(6)beta(4) interacts with ErbB3 but not with ErbB1, ErbB2 or ErbB4, and enhances the heregulin-induced, ErbB3/ErbB2 heterodimer-mediated DNA synthesis, but not cell motility, in A549 cells.

A constitutively active epidermal growth factor receptor (EGFR) mutant, EGFR variant III (EGFRvIII), has been detected at high frequencies in certain human cancers. This study evaluated transactivation and trafficking of erbB family members as a result of constitutive EGFR activity in a cancer cell line. Expression of EGFRvIII modulated erbB family members through different mechanisms; the erbB3 mRNA level was reduced, whereas wild-type EGFR (wtEGFR) and erbB2 protein levels were diminished, with no change in their mRNA levels, and there was no change in the erbB4 expression level. Both EGFR and erbB2 were internalized as a result of EGFRvIII's activity and redistributed to the cell surface upon addition of AG1478, an inhibitor of wtEGFR/EGFRvIII catalytic activity. Acute activation of EGFRvIII by removing AG1478 from cells increased phosphorylation of both wtEGFR and erbB2 and caused differential trafficking of EGFRvIII's activation partners; wtEGFR was directed primarily to lysosomal compartments and partially to recycling compartments, whereas erbB2 was directed primarily to recycling compartments and partially to lysosomal compartments. Our data demonstrate that the constitutive activity of EGFRvIII is sufficient to trigger endocytosis and trafficking of wtEGFR and erbB2, which may play a role in activating signaling pathways that are triggered during receptor endocytosis.

Epidermal growth factor (EGF) belongs to a family of growth factor ligands and receptors. At present, five ligands have been recognized which as EGF exert their effects via binding to the same EGF receptor. The family has three other receptors erbB2, erbB3, and erbB4, which have their own ligands (the heregulins). The system is ubiquitously distributed in mammals, and has important roles in normal development, and in regenerative and neoplastic growth. Mouse and human EGF were discovered in 1962 and 1975 by Stanley Cohen and Harry Gregory, respectively, due to EGFs potent systemic effects. EGF accelerated eyelid opening in newborn mice and inhibited gastric acid secretion in humans. Already in the late thirties, a factor in human urine was recognized which prevented or accelerated healing of experimental damage in the gastrointestinal tract. This factor appeared to be EGF. Around 1980, an effect of commercial interest was described-EGF caused shedding of the fleece in sheep. In line with the original observations, several studies have examined effects of EGF on developmental processes. Amongst other effects, EGF accelerates lung and intestinal maturation before birth and in newborn mammals. Due to the possible use of EGF in the wool industry, it was mandatory to know more about EGF. Amongst other effects in mature sheep and other animals are haemodynamic changes, changes in electrolyte homeostasis, and endocrinological changes. In relation to experimental damage, the therapeutic potential of systemic EGF has been demonstrated in all parts of the gastrointestinal tract, in the kidneys, in the liver and in the trachea. EGF has even been tried in humans in gastric ulcer healing and in necrotising enterocolitis. Studies on prolonged treatment with EGF have first recently appeared. We described effects of 4-5 weeks of treatment in Goettingen minipigs and in rats, and two other groups described effects in monkeys and in rats. In summary, species differences were observed. The species of higher order were most sensitive to treatment with EGF. EGF did not consistently change the total body weight despite EGF consistently reduced circulating levels of insulin-like growth factor I (IGF-I) in Goettingen minipigs as well as in rats. Low circulating levels of IGF-I are usually associated with retarded growth. This review mostly focuses on the organs which appeared to be most sensitive to EGF, the urinary and gastrointestinal tracts including the liver and the pancreas. The histopathological changes consisted mainly of epithelial proliferations in the gastrointestinal, urinary and respiratory tracts. These findings match the knowledge obtained from animals overexpressing the EGF agonist, transforming growth factor alpha (TGF alpha), and the mice with a knock out of the gene encoding for the EGF receptor. EGF receptor hyperstimulation (TGF alpha overexpression) in the context of the whole animal leads to epithelial proliferations whereas hypostimulation (EGF receptor knock out) leads to epithelial immaturities. In the minipigs, the epithelia of the oesophagus, ducts of the pancreas, and the urothelium were hyperplastic, the latter two epithelia with accumulation of glycoconjugates. In the rats, the epithelial hyperplasias in these tissues and in the small and large intestines were without glycoconjugate accumulations. In rats, the mucosal proliferations in the intestines resulted in increased mucosal surface area. Mesenchymal growth effects were also noted. In the ureters of the minipigs, smooth muscle cell hyperplasia and hypertrophia were found. The heart of the minipigs was also enlarged, an interesting finding regarding interactions between the different parts of the EGF system, as knock out mice of the receptors erbB2 and erbB4 die due to maldevelopments in the heart. Measurements in blood and serum also revealed consistent changes. (ABSTRACT TRUNCATED)

We previously developed and validated a strategy for stimulating heart regeneration by administration of recombinant neuregulin (rNRG1), a growth factor, in mice. rNRG1 stimulated proliferation of heart muscle cells, cardiomyocytes, and was most effective when administration began during the neonatal period. Our results suggested the use of rNRG1 to treat pediatric patients with heart failure. However, administration in this age group may stimulate growth outside of the heart.

NRG1 and ErbB receptor expression was determined by RT-PCR. rNRG1 concentrations in serum were quantified by ELISA. Mice that received protocols of recombinant neuregulin1-β1 administration (rNRG1, 100 ng/g body weight, daily subcutaneous injection for the first month of life), previously shown to induce cardiac regeneration, were examined at pre-determined intervals. Somatic growth was quantified by weighing. Organ growth was quantified by MRI and by weighing. Neoplastic growth was examined by MRI, visual inspection, and histopathological analyses. Phospho-ERK1/2 and S6 kinase were analyzed with Western blot and ELISA, respectively.

Lung, spleen, liver, kidney, brain, and breast gland exhibited variable expression of the NRG1 receptors ErbB2, ErbB3, ErbB4, and NRG1. Body weight and tibia length were not altered in mice receiving rNRG1. MRI showed that administration of rNRG1 did not alter the volume of the lungs, liver, kidneys, brain, or spinal cord. Administration of rNRG1 did not alter the weight of the lungs, spleen, liver, kidneys, or brain. MRI, visual inspection, and histopathological analyses showed no neoplastic growth. Follow-up for 6 months showed no alteration of somatic or organ growth. rNRG1 treatment increased the levels of phospho-ERK1/2, but not phospho-S6 kinase.

Administration protocols of rNRG1 for stimulating cardiac regeneration in mice during the first month of life did not induce unwanted growth effects. Further studies may be required to determine whether this is the case in a corresponding human population.

Non-small cell lung cancer (NSCLC) is a common cancer with a poor prognosis. The aim of this study was to screen Finnish NSCLC tumor samples for common cancer-related mutations by targeted next generation sequencing and to determine their concurrences and associations with clinical features.

Sequencing libraries were prepared from DNA isolated from formalin-fixed, paraffin-embedded tumor material of 425 patients using the AmpliSeq Colon and Lung panel covering mutational hot spot regions of 22 cancer genes. Sequencing was performed with the Ion Torrent Personal Genome Machine (PGM).

Data analysis of the hot spot mutations revealed mutations in 77% of the patients, with 7% having 3 or more mutations reported in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. Two of the most frequently mutated genes were TP53 (46%) and KRAS (25%). KRAS codon 12 mutations were the most recurrently occurring mutations. EGFR mutations were significantly associated with adenocarcinoma, female gender and never/light-smoking history; CTNNB1 mutations with light ex-smokers, PIK3CA and TP53 mutations with squamous cell carcinoma, and KRAS with adenocarcinoma. TP53 mutations were most prevalent in current smokers and ERBB2, ERBB4, PIK3CA, NRAS, NOTCH1, FBWX7, PTEN and STK11 mutations occurred exclusively in a group of ever-smokers, however the association was not statistically significant. No mutation was found that associated with asbestos exposure.

Finnish NSCLC patients have a similar mutation profile as other Western patients, however with a higher frequency of BRAF mutations but a lower frequency of STK11 and ERBB2 mutations. Moreover, TP53 mutations occurred frequently with other gene mutations, most commonly with KRAS, MET, EGFR and PIK3CA mutations.

BACKGROUND

The ErbB family consists of four proteins including (EGFR)/ErbB1, ErbB2, ErbB3, and ErbB4, and plays a crucial role in the promotion of multiple tumorigenic processes. In addition to the traditional pathways of EGFR signaling, EGFR translocates to the nucleus and acts as a transcription factor in the proliferation of cancer cells. Heregulin is known as both an ErbB3 and an ErbB4 ligand. This study aimed to investigate the expression of heregulin and its relevant EGFR family members as well as their phosphorylated forms in human colorectal cancer (CRC) tissues and to determine the relationship between their expression and clinicopathological factors including patient prognosis.

METHODS

We analyzed the effects of exogenous heregulin on ErbB2, ErbB3 and ErbB4 phosphorylation in Caco-2, DLD-1, and HCT 116 colon cancer cell lines by western blot analysis. We examined 155 surgical resections from colorectomy patients. Cellular localization of ErbB1-4, their phosphorylated forms and heregulin protein was analyzed in CRC surgical resections by immunohistochemical analysis. Immunohistochemical results were compared with clinicopathological factors and patient prognosis.

RESULTS

Phosphorylated ErbB2 (pErbB2) and phosphorylated ErbB3 (pErbB3) were detected in both nuclear and cytosolic fractions of Caco-2 and DLD-1 cells stimulated by exogenous heregulin. Whereas, phosphorylated ErbB4 (pErbB4) was detected only in cytosolic fractions of HCT 116 cells stimulated by exogenous heregulin. Phosphorylated EGFR (pEGFR) immunoreactivity was observed in the cytoplasm and nuclei of cancer cells, whereas the pattern of EGFR staining was membranous and cytoplasmic. Subcellular localization of pErbB2, cytoplasmic, membranous, or nuclear, varied among cases. pErbB3 immunoreactivity was exclusively observed in the nuclei of cancer cells. pErbB4 immunoreactivity was observed in the cell membrane of cancer cells. Statistically, heregulin immunoreactivity correlated with pErbB2 and pErbB4 expression. In multivariate analysis for disease free survival, lymph node status, pErbB3 and pErbB4 expression retained independent prognostic significance. In multivariate analysis for overall survival, lymph node status, pEGFR and pErbB4 retained independent prognostic significance.

CONCLUSIONS

ErbB2 and ErbB3 phosphorylated by heregulin localized in the nucleus of CRC cells. Phosphorylated ErbB1-4 and heregulin contribute to poorer patient prognosis in CRC. This heregulin-ErbB family member autocrine loop may be a candidate for targeted treatment of CRC.

Malignant melanoma is a life-threatening skin cancer increasingly diagnosed in the western world. In advanced disease the prognosis is grave. Growth and metastasis formation in melanomas are regulated by a network of cytokines, cytokine-receptors, and adhesion molecules. However, little is known about surface antigens and target expression profiles in human melanomas. We examined the cell surface antigen profile of human skin melanoma cells by multicolor flow cytometry, and compared their phenotype with 4 melanoma cell lines (A375, 607B, Mel-Juso, SK-Mel28). Melanoma cells were defined as CD45-/CD31- cells co-expressing one or more melanoma-related antigens (CD63, CD146, CD166). In most patients, melanoma cells exhibited ErbB3/Her3, CD44/Pgp-1, ICAM-1/CD54 and IGF-1-R/CD221, but did not express CD20, ErbB2/Her2, KIT/CD117, AC133/CD133 or MDR-1/CD243. Melanoma cell lines were found to display a similar phenotype. In most patients, a distinct subpopulation of melanoma cells (4-40%) expressed the erythropoietin receptor (EPO-R) and ErbB4 together with PD-1 and NGF-R/CD271. Both the EPO-R+ and EPO-R- subpopulations produced melanoma lesions in NOD/SCID IL-2Rgamma(null) (NSG) mice in first and secondary recipients. Normal skin melanocytes did not express ErbB4 or EPO-R, but expressed a functional KIT receptor (CD117) as well as NGF-R, ErbB3/Her3, IGF-1-R and CD44. In conclusion, melanoma cells display a unique composition of surface target antigens and cytokine receptors. Malignant transformation of melanomas is accompanied by loss of KIT and acquisition of EPO-R and ErbB4, both of which are co-expressed with NGF-R and PD-1 in distinct subfractions of melanoma cells. However, expression of EPO-R/ErbB4/PD-1 is not indicative of a selective melanoma-initiating potential.

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.

ErbB receptor tyrosine kinases are important in maintaining the long-term structural integrity of the heart and in the induction of hypertrophy. In addition, in vivo activation of ErbB1 by epidermal growth factor (EGF) protects the heart against acute stress-induced damage. We examined here whether the ErbB sytem acutely protects the isolated heart in which stress was induced in vitro by ischemia combined with epinephrine infusion (EPI). In perfused mouse hearts, EGF induced Tyr-phosphorylation of ErbB1 but not ErbB2. Neuregulin-1beta (NRG-1beta) induced Tyr-phosphorylation of both ErbB4 and ErbB2. We also found differences in the signaling cascades activated by each growth factor. To stress the perfused mouse heart, we combined EPI with low-flow ischemia. This resulted in (i) loss of left ventricle contraction force ( + dP/dt(max)) and developed pressure (LVDP) after a short period of hypercontractility, (ii) enhanced anaerobic metabolism (lactate production), and (iii) myocyte injury (lactate dehydrogenase (LDH) release). EGF and NRG-1beta had different effects on stressed-heart contractility. EGF reduced to a half the loss of both + dP/dt(max) and LVDP. In contrast, NRG-1beta exacerbated the hypercontractility soon after reperfusion. This is coincident with a transient increase in coronary flow after reperfusion. In spite of these differences in contraction, both EGF and NRG-1beta induced similar early protection as shown by the reduction of LDH release. Our results show that the ErbB system protects the perfused heart against damage induced by acute stress. They reinforce the relevance of ErbB receptors and ligands in cardiac physiology.

There is a relatively high genetic heritability of schizophrenia as shown by family, twin and adoption studies. A large number of hypotheses on the causes of schizophrenia occurred over time. In this review we focus on genetic findings related to potential alterations of intracellular Ca-homeostasis in association with schizophrenia. First, we provide evidence for the NMDA/glutamatergic theory of schizophrenia including calcium processes. We mainly focus on genes including: DAO (D-amino acid oxidase), DAOA (D-amino acid oxidase activator), DTNBP1 (Dysbindin 1, dystrobrevin-binding protein 1), NRG1 (Neuregulin 1), ERBB4 (v-erb-a erythroblastic leukemia viral oncogene homolog 4, avian), NOS1 (nitric oxide synthase 1, neuronal) and NRGN (Neurogranin). Furthermore, a gene coding for a calcium channel subunit (CACNA1C: calcium channel, voltage-dependent, L type, alpha 1C subunit) is discussed in the light of schizophrenia whereas genetic findings related to alterations in the intracellular Ca-homeostasis associated specifically with dopaminergic and serotonergic neurotransmission in schizophrenia are not herein closer reviewed. Taken together there is converging evidence for the contribution of genes potentially related to alterations in intracellular Ca-homeostasis to the risk of schizophrenia. Replications and functional studies will hopefully provide further insight into these genetic variants and the underlying processes.

Schizophrenia is a severe psychiatric disorder with lifetime prevalence of ~1% worldwide. A genotyping study was conducted using a custom panel of Illumina 1536 SNPs in 840 schizophrenia cases and 876 controls (351 patients and 385 controls from North India; and 436 patients, 401 controls and 143 familial samples with 53 probands containing 37 complete and 16 incomplete trios from South India). Meta-analysis of this population of Indo-European and Dravidian ancestry identified three strongly associated variants with schizophrenia: STT3A (rs548181, p=1.47×10(-5)), NRG1 (rs17603876, p=8.66×10(-5)) and GRM7 (rs3864075, p=4.06×10(-3)). Finally, a meta-analysis was conducted comparing our data with data from the Schizophrenia Psychiatric Genome-Wide Association Study Consortium (PGC-SCZ) that supported rs548181 (p=1.39×10(-7)). In addition, combined analysis of sporadic case-control association and a transmission disequilibrium test in familial samples from South Indian population identified three associations: rs1062613 (p=3.12×10(-3)), a functional promoter variant of HTR3A; rs6710782 (p=3.50×10(-3)), an intronic variant of ERBB4; and rs891903 (p=1.05×10(-2)), an intronic variant of EBF1. The results support the risk variants observed in the earlier published work and suggest a potential role of neurodevelopmental genes in the schizophrenia pathogenesis.

Receptor tyrosine kinases (RTKs) in the ErbB family (EGFR, ErbB2, ErbB3, and ErbB4) are implicated in a variety of human malignancies. Accordingly, determination of both expression and activation (dimerization/heterodimerization and phosphorylation) of ErbB proteins is critical in defining their functional role in cancer. Efficient and comprehensive methods to study molecular functions of ErbB family of RTKs are needed not only for improvements in diagnostics but also for early screening of targeted drugs (eg, small molecule inhibitors and therapeutic antibodies). We report development of 3 multiplex microbead immunoassays for simultaneous detection of expression, protein-protein interactions, and phosphorylation of these RTKs. These novel multiplex immunoassays were used to study ErbB RTKs under different cell activation conditions in 2 breast cancer cell lines (MDA-MB-453 and MDA-MB-468) and an epidermoid cancer cell line (A431). The results were confirmed by immunoprecipitation/western blot. Importantly, the multiplex immunoassay facilitated time-course studies in these cell lines after cell activation with EGF and neuregulin, revealing the kinetics of phosphorylation of the ErbB family RTKs. This study demonstrates the utility of the Luminex(R) multiplex system as an efficient and comprehensive approach to study different aspects of molecular roles of these RTKs. Importantly, the study provides proof-of-concept for the utility of the multiplex microbead immunoassay approach for potential use in efficient, robust, and rapid screening of drugs, particularly those targeting functional aspects of these potent signaling molecules. In addition, the assays described here may be useful for cancer diagnostics and monitoring efficacy of therapy targeting the ErbB family of RTKs.

Important biological events associated with plasma membranes, such as signal transduction, cell adhesion, and protein trafficking, are mediated through the membrane microdomains. We have developed a novel method termed enzyme-mediated activation of radical sources (EMARS) to identify coclustering molecules on the cell surface under living conditions, which features a radical formation from an aryl azide reagent by horseradish peroxidase (HRP). For identification of molecules labeled by the EMARS reaction, antibody array system and mass spectrometry-based proteomics approaches are available. Spatio- temporally-regulated interaction between b1 integrin and ErbB4 involved in fibronectin-dependent cell migration and therapeutic antibody-stimulated interaction between FGFR3 and CD20 were discovered using the EMARS method.

Molecular cloning of the partial cDNA coding sequences of the four erbB receptors and the epidermal growth factor (EGF)-like ligands EGF, transforming growth factor alpha (TGF), and heparin-binding EGF (HB-EGF) has provided the basis for a comprehensive analysis of the spatiotemporal expression pattern of the EGF receptor/ligand system during the peri-implantation period in the rabbit. Employing nonradioactive in situ hybridization and immunolocalization, we observed differential expression of erbB1-erbB3 within the trophectoderm of the blastocyst. ErbB1 was strongly expressed in the cytotrophoblast but was downregulated upon syncytium formation. ErbB3 was a product of both the cyto- and syncytiotrophoblast. Despite the expression of erbB2 mRNA, the trophectoderm was devoid of immunoreactive ErbB2. ErbB4 gene activity was exclusively detected in the trophoblast at midpregnancy. The luminal and glandular epithelium and stroma of the nonpregnant, pseudopregnant, and pregnant rabbit uterus at Day 6 of gestation also expressed ErbB1-ErbB3. In the peri-implantation period, gene activities of erbB1-erbB3 were upregulated upon decidualization. At the site of implantation, uterine luminal epithelial cells apposing the preimplantation blastocyst displayed a distinct membrane immunolocalization of ErbB2, identifying the uterine epithelium as target for EGF, TGFalpha, and HB-EGF derived from both the embryonic trophectoderm and the uterine epithelium. In the luminal epithelium at the antimesometrial uterine site, HB-EGF gene activity was upregulated at the time of blastocyst attachment, but this upregulation was not reflected in an increase in immunoreactive HB-EGF. The detection of tyrosine phosphorylated ErbB2 in the rabbit placenta indicated the presence of a functional ErbB/EGF-like system in the pregnant rabbit uterus. This study provides strong evidence for a role of the ErbB/EGF-like system in embryo/maternal interactions during the peri-implantation period in the rabbit.

The Neuregulins (NRGs) are members of the epidermal growth factor (EGF) family of growth factors. EGF family members regulate the signaling of ErbB family receptor tyrosine kinases, including the epidermal growth factor receptor (EGFR/ErbB1), ErbB2/HER2/Neu, ErbB3/HER3 and ErbB4/HER4. We have previously demonstrated that the EGF family hormone NRG2beta is a potent ErbB4 agonist, whereas NRG2alpha is a weak ErbB4 agonist. We have also previously demonstrated that Phe45 of NRG2beta regulates the potency of NRG2beta. Here, we address the hypotheses that Phe45 regulates the potency of NRG2beta by regulating the affinity of NRG2beta for ErbB4. We demonstrate that Phe45 of NRG2beta indeed regulates the affinity of NRG2beta for ErbB4. Furthermore, a hydrophobic or uncharged amino acid side chain at residue 45 contributes to NRG2beta binding to ErbB4. These data indicate that Phe45 of NRG2beta may regulate the affinity of NRG2beta for ErbB4 by interacting with hydrophobic amino acids in ErbB4.

A rapid, sensitive, and high-throughput assay has been developed to quantify ligand-induced receptor tyrosine kinase activation in terms of receptor phosphorylation. The assay, termed a kinase receptor activation enzyme-linked immunosorbant assay (KIRA-ELISA), consists of two separate microtiter plates, one for cell culture, ligand stimulation, and cell lysis/receptor solubilization and the other plate for receptor capture and phosphotyrosine ELISA. The assay was developed for analysis of heregulin-induced ErbB2 activation and utilizes the stimulation of intact receptor on the adherent breast carcinoma cell line, MCF-7. Membrane proteins are solubilized via Triton X-100 lysis and the receptor is captured in ELISA wells coated with ErbB2-specific antibodies with no cross-reaction to ErbB3 or ErbB4. The degree of receptor phosphorylation is then quantified by antiphosphotyrosine ELISA. A reproducible standard curve is generated with a EC(50) of approximately 360 pM for heregulin beta 1(177-244) (HRG beta 1(177-244). When identical samples of HRG beta 1(177-244) are analyzed by both the KIRA-ELISA and quantitative antiphosphotyrosine Western blot analysis, the results correlate very closely with one another. The assay described in this report is able to specifically quantify tyrosine phosphorylation of ErbB2 that results from the interaction of HRG with ErbB3 and/or ErbB4.

Extra-virgin olive oil (EVOO) has been hypothesized to have chemopreventive effects on breast cancer, unlike high corn oil (HCO) diets that stimulate it. We have investigated mechanisms of these differential modulatory actions on experimental mammary cancer. In 7,12-dimethylbenz(a)anthracene adenocarcinomas of rats fed a high EVOO, HCO and control diets (n = 20 for each group), we have analyzed the expression and activity of ErbB receptors, p21Ras and its extracellular signal-regulated kinase (ERK) 1/2, Akt and RalA/B effectors by immunoblotting analyses. We explored the Ha-ras1 mutation status by Southern blot, mismatch amplification mutation assay and sequencing, and the 3-hydroxy-3-methylglutaryl-coenzyme A reductase and squalene synthase messenger RNA expression by real-time polymerase chain reaction. We analyzed the tumor mitotic index, proliferating cell nuclear antigen (PCNA) levels, and apoptosis through Caspase-3 analysis and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assays. Finally, we measured the 8-oxo-2'-deoxyguanosine levels. Non-parametrical statistics were used. The EVOO diet decreased Ras activation, downregulated the Ras/phosphatidyl inositol 3-kinase/Akt pathway and upregulated the Raf/Erk pathway, compared with the control. In contrast, the HCO diet did not modify Ras activity but rather enhanced the Raf/Erk pathway. The EVOO diet decreased the cleaved ErbB4 levels, compared with the HCO diet, increased apoptosis and diminished the mono-ubiquitylated PCNA levels, which is related to DNA damage. Tumors from rats fed the EVOO diet displayed a more benign phenotype, whereas those from rats fed the HCO diet were biologically more aggressive. In conclusion, high EVOO and corn oil diets exert their modulatory effects on breast cancer through a different combination of Ras signaling pathways, a different proliferation-apoptosis balance and probably distinct levels of DNA damage.

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.

ERBB receptors have an important function in mammalian development and normal physiology, but overexpression and poor downregulation of ERBB receptors have been associated with malignant growth. Ligand-induced ERBB receptor signaling is terminated by clathrin-dependent receptor endocytosis, followed by incorporation of activated receptor complexes into multi-vesicular bodies and subsequent degradation in lysosomes. In the case of ERBB1, also known as the EGF receptor, it has been shown that ubiquitination serves as a signal to facilitate internalization and subsequent endosomal sorting, but little is known about the role of ubiquitination of other ERBB receptors. In the present study we investigated the regulation of ubiquitination and deubiquitination of the ERBB4 CYT-1 and CYT-2 isoforms in the context of chimeric EGFR-ERBB4 receptors. We demonstrate that EGFR-ERBB4 CYT-2 chimera shows decreased ligand-induced downregulation and EGF-degradation, as well as enhanced EGF recycling, when compared to EGFR-ERBB4 CYT-1. Moreover we show that the mutation Y1103F in the binding site for Cbl which is present in both CYT-1 and CYT-2, does not influence ERBB4 endosomal trafficking. We further demonstrate that total ligand-induced ubiquitination of CYT-1 is higher than that of CYT-2, whereby CYT-1 ubiquitination is mainly dependent on the PPXY(1056) Itch binding site for the E3-ligase Itch which is only present in CYT-1, while that of CYT-2 is dependent on the Y1103 Cbl binding site. The E3-ligase c-Cbl is more efficiently phosphorylated upon EGF stimulation of the CYT-2 than the CYT-1 isoform. Moreover our data show that the pY1103 Cbl binding site is required for K48-polyubiquitination of both CYT-1 and CYT-2, whereas the PPXY(1056) Itch binding site is required for K63-polyubiquitination of CYT-1. We further demonstrate that EGF stimulation of EGFR-ERBB4 CYT-1 and CYT-2 does not result in efficient binding to and tyrosine phosphorylation of the ESCRT-0 subunit Hrs. Finally, even though CYT-1 shows ligand-induced K63-polyubiquitination, it is not subjected to deubiquitination by the K63 polyubiquitin-specific AMSH deubiquitinating enzyme, while CYT-1 is slightly deubiquitinated by USP8. We conclude that Cbl and Itch binding sites in ERBB4 CYT-1 and CYT-2 mediate K48- and K63-polyubiquitination, respectively.