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Neuregulin interaction with receptors

Two members of the epidermal growth factor receptor family of receptor/tyrosine kinases, p180erbB3 and p180erbB4, serve as receptors for the Heregulin ligand (a homolog of Vein). While HRG appears to be capable of stimulating the autophosphorylation activity of p180erbB4, the co-expression of p185erbB2/neu with p180erbB3 is necessary for the HRG-stimulated tyrosine phosphorylation of both of these receptors. On the basis of the sequences surrounding their putative tyrosine phosphorylation sites, it is predicted that the different HRG-responsive receptors couple to different intracellular SH2 domain-containing proteins. Hence, the different receptors may mediate different cellular responses to the HRG ligand. HRG beta 1 is mitogenic for erbB3-transfected DHFR/G8 cells, an NIH3T3 mouse fibroblast derivative that over-expresses p185erbB2/neu. HRG was not mitogenic for parental DHFR/G8 cells that do not express the ErbB3 protein. Phosphatidylinositol (PI) 3-kinase, an enzyme believed to be important in cellular growth regulation by growth factors and oncogenes, is predicted to couple to tyrosine-phosphorylated ErbB3. HRG stimulates the association of PI 3-kinase with both p185erbB2/neu and ErbB3 in transfected DHFR/G8 cells, but not in the parental cell line. It is concluded that the ErbB3 protein is capable of mediating a proliferative response of fibroblasts to HRG, and that the activation of PI 3-kinase is an integral part of the growth signaling mechanism (Carraway, 1995).

Deregulated signaling by the four members of the epidermal growth factor receptor tyrosine kinase family (erbB family) is implicated in the genesis or progression of human cancers. However, efforts to analyze signaling by these receptors have been hampered by the diversity of ligands and extensive interreceptor cross talk. The four human erbB family receptors were expressed, singly and in pairwise combinations, in a pro-B-lymphocyte cell line (Ba/F3). The range of interactions activated by the epidermal growth factor homology domain of the agonist neuregulin beta was investigated. The results provide the first comprehensive analysis of the response of this receptor family to a single peptide agonist. This peptide induces complex patterns of receptor tyrosine phosphorylation and regulation of Ba/F3 cell survival and proliferation. These data demonstrate the existence of several previously undocumented receptor interactions driven by neuregulin (Riese, 1995).

The multiple isoforms of Neu differentiation factor (NDF/neuregulin), a homolog of Drosophila Vein, 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, alpha and beta. 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, it was found that both isoforms induced mitogenic signals in cells co-expressing the combination of ErbB-3 with ErbB-2. However, only the beta isoform stimulate cells that expressed both ErbB-3 and ErbB-1, and neither isoform is 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 beta isoforms are able to stabilize ErbB-3/ErbB-1 heterodimers (Pinkas-Kramarski, 1996a).

The ErbB family includes three receptors: the first two, ErbB-1 and ErbB-3 bind (respectively) to epidermal growth factor and Neu differentiation factor; the third is 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, it was found that ErbB-3 is devoid of any biological activity but either ErbB-1 or 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 (Pinkas-Kramarski, 1996).

The neuregulins comprise a subfamily of epidermal growth factor (EGF)-like growth factors that elicit diverse cellular responses by activating members of the ErbB family of receptor tyrosine kinases. Although neuregulin-1 and neuregulin-2 are both binding ligands for the ErbB3 and ErbB4 receptors, they exhibit distinct biological activities depending on cellular context. In MDA-MB-468 human mammary tumor cells, neuregulin-2beta (NRG2beta) inhibits cell growth, whereas neuregulin-1beta (NRG1beta) does not. In these cells, NRG2beta appears to preferentially act through the EGF receptor, stimulating receptor tyrosine phosphorylation and the recruitment of phospholipase C-gamma, Cbl, SHP2, and Shc to that receptor. NRG1beta preferentially acts through ErbB3 in these cells by stimulating the tyrosine phosphorylation and recruitment of phosphatidylinositol 3-kinase and Shc to that receptor. In MDA-MB-453 cells, both NRG1beta and NRG2beta stimulate the tyrosine phosphorylation of the ErbB2 and ErbB3 receptors to similar extents, but only NRG1beta potently stimulates morphological changes consistent with their differentiation. The profiles of SH2 domain-containing proteins that are efficiently recruited to activated receptors differ for the two factors. These observations indicate that despite their overlapping receptor specificity, the neuregulins exhibit distinct biological and biochemical properties. Since both of these cell lines express only two of the known ErbB receptors, these results imply that EGF-like ligands might elicit differential signaling within the context of a single receptor heterodimer (Crovello, 1998).

Many different growth factor ligands, including epidermal growth factor (EGF) and the neuregulins (NRGs), regulate members of the erbB/HER family of receptor tyrosine kinases. These growth factors induce erbB receptor oligomerization, and their biological specificity is thought to be defined by the combination of homo- and hetero-oligomers that they stabilize upon binding. One model proposed for ligand-induced erbB receptor hetero-oligomerization involves simple heterodimerization; another suggests that higher order hetero-oligomers are 'nucleated' by ligand-induced homodimers. To distinguish between these possibilities, the abilities of EGF and NRG1-beta1 to induce homo- and hetero-oligomerization of purified erbB receptor extracellular domains was determined. EGF and NRG1-beta1 induce efficient homo-oligomerization of the erbB1 and erbB4 extracellular domains, respectively. In contrast, ligand-induced erbB receptor extracellular domain hetero-oligomers do not form (except for s-erbB2-s-erbB4 hetero-oligomers). These findings argue that erbB receptor extracellular domains do not recapitulate most heteromeric interactions of the erbB receptors, yet reproduce their ligand-induced homo-oligomerization properties very well. This suggests that mechanisms for homo- and hetero-oligomerization of erbB receptors are different, and contradicts the simple heterodimerization hypothesis prevailing in the literature (Ferguson, 2000).

Neuregulin expression patterns and alternative splicing

The distribution of neuregulin transcripts in rat brains was studied by both RNA blotting and in situ hybridization. Multiple neuregulin transcripts are expressed in neurons of the basal forebrain, the hippocampus, the diencephalon, the cerebellum, the brainstem, and the spinal cord. Developmental changes in the distribution of neuregulin transcripts are observed only in the cerebellum and the hippocampus. The intense neuregulin hybridization signals in the motor and sensory nuclei of the brainstem and the spinal motor neurons are suggestive of a functional involvement of neuregulins in motor and sensory systems. The expression of neuregulins in other parts of the brain also indicates that these factors are involved in a variety of central nervous system functions (Chen, 1994).

Sites of neuregulin expression correspond to mesenchymal cells of various parenchymal organs. This implies a function of neuregulin as a mesenchymal factor that acts on epithelia. The mesenchymal expression of neuregulin could provide a molecular basis for the biological phenomenon of mesenchymal-epithelial interactions. These findings also have implications for the molecular mechanism by which amplification of c-neu can affect tumor progression of carcinomas. In addition, neuregulin expression is found in neuronal cells during development. Distinct isoforms of neuregulin are expressed in the brain. Therefore neuregulins have a dual role as a mesenchymal and a neuronal factor (Meyer, 1994).

ARIA, heregulin, neu differentiation factor, and glial growth factor are members of a new family of growth and differentiation factors whose effects have been assayed on Schwann cells, skeletal muscle cells, and mammary tumor cell lines. To gain insight into their roles in the CNS, the expression of ARIA in the rat brain was studied. ARIA mRNA is found in all cholinergic neurons throughout the CNS, including motor neurons and cells of the medial septal nucleus and the nucleus basalis of Meynert. ARIA induces tyrosine phosphorylation of a 185 kDa protein in central and peripheral targets of these cholinergic neurons. ARIA mRNA, however, is not restricted to cholinergic neurons, suggesting that it may also play a role at other types of synapses. Its distribution in germinal layers of the telencephalon and cerebellum suggests that it may also play a role in the proliferation and/or migration of neuronal and glial precursor cells (Corfas, 1995).

Neuregulin (also known as NDF, heregulin, ARIA, GGF or SMDF) induces cell growth and differentiation. Biological effects of neuregulin are mediated by members of the erbB family of tyrosine kinase receptors. Neuregulin-induced cellular responses are mediated by tyrosine kinase receptors of the erbB family: neuregulin binds erbB3 and erbB4 with high affinity, but not the erB2 (Her2) and erbB1 (EGF) receptors. Three major neuregulin isoforms are produced from the gene, which differ substantially in sequence and in overall structure. Type I (neu differentiation factor) possesses an Ig domain, an EGF domain, a hydrophobic sequence, and a unique C-terminal domain; Type II (glial growth factor) posseses an N terminal signal peptide, a kringle-like domain and a C-terminal EGF domain, and Type III (sensory and motor neuron-derived factor, SMDF) possesses a unique N-terminal domain, including a hydrophobic region and a C-terminal EGF domain. Ablation of the neuregulin gene in the mouse has demonstrated multiple and independent functions of this factor in development of both the nervous system and the heart. Targeted mutations that affect different isoforms result in distinct phenotypes, demonstrating that isoforms can take over specific functions in vivo. Type I neuregulin is required for generation of neural crest-derived neurons in cranial ganglia and for trabeculation of the heart ventricle, whereas type III neuregulin plays an important role in the early development of Schwann cells. The complexity of neuregulin function in development is therefore due to independent roles played by distinct isoforms (Meyer, 1997).

Members of the epidermal growth factor family of receptors have long been implicated in the pathogenesis of various tumors, and more recently, apparent roles in the developing heart and nervous system have been described. Numerous ligands that activate these receptors have been isolated. The cloning and initial characterization of a second ligand for the erbB family of receptors is reported. This factor (termed Don-1 [divergent of neuregulin 1]) has structural similarity with the neuregulins. Four splice variants, two each from human and mouse, have been isolated. They are capable of inducing tyrosine phosphorylation of erbB3, erbB4, and erbB2. In contrast to those of neuregulin, high levels of expression of Don-1 are restricted to the cerebellum and dentate gyrus in the adult brain and to fetal tissues (Busfield, 1997).

Regulation of Neuregulin expression

Neuregulins (NRGs) are expressed in spinal cord motor neurons and accumulate at the neuromuscular junction where they may increase the synthesis of postsynaptic acetylcholine receptors and voltage-gated sodium channels. NRG expression is selectively increased in rat ventral spinal cord neurons at approximately the time that nerve-muscle synapses first form. A rapid increase in NRG mRNA and protein expression is induced in vitro in cultured rat spinal motor neurons by brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4, or glial-cell-line-derived neurotrophic factor. Agrin expression is not affected by these factors over the same time course. Brain-derived neurotrophic factor, but not neurotrophin-3, selectively regulate immunoglobulin domain-containing splice variants of NRG, which are likely to be important for binding to the synaptic basal lamina. Regulation of NRG expression in motor neurons by muscle-derived neurotrophic factors may represent one portion of a reciprocal, regulatory loop that promotes neuromuscular synapse development (Loeb, 1997).

Neuregulin signal transduction and targets

The heregulin-beta 1 isoform is rapidly internalized and translocated to the nucleus of SK-BR-3 breast cancer cells as an intact molecule. Heregulin-beta 1 treatment up-regulates expression of c-myc mRNA and protein, which is also observed to undergo its own translocation from the cytosol to the nucleus. c-myc thus appears to be a cellular target gene of HRGbeta 1, and its induction may be related to the nuclear translocation of heregulin (Li, 1996).

Two different signaling pathways mediate the localization of acetylcholine receptors (AChRs) to synaptic sites in skeletal muscle. The signal for one pathway is agrin, a protein that triggers a redistribution of previously unlocalized cell surface AChRs to synaptic sites. The signal for the other pathway is not known, but this signal stimulates transcription of AChR genes in myofibre nuclei near the synaptic site. Neuregulins, identified originally as a potential ligand for erbB2 (Neu differentiation factor, NDF), stimulate proliferation of Schwann cells (glial growth factor, GGF), increase the rate of AChR synthesis in cultured muscle cells (AChR-inducing activity) and are expressed in motor neurons. These results raise the possibility that neuregulin is the signal that activates AChR genes in synaptic nuclei. Neuregulin activates AChR gene expression in C2 muscle cells. The neuregulin response element in the AChR delta-subunit gene is contained in the same 181 base pairs that confer synapse-specific expression in transgenic mice. Neuregulins are concentrated at synaptic sites. Like the extracellular signal that stimulates synapse-specific expression, neuregulins remain at synaptic sites in the absence of nerve and muscle. C2 muscle cells contain erbB2 and erbB3 messenger RNA but little or no erbB4 mRNA. Neuregulin stimulates tyrosine phosphorylation of erbB2 and erbB3, indicating that neuregulin signalling in skeletal muscle may be mediated by a complex of erbB2 and erbB3 (Jo, 1995).

beta neuregulins (also called NDF, GGF, ARIA, and heregulins) are neuron-derived molecules that are likely to be responsible for Schwann cell precursor survival, proliferation, and maturation, both in vivo and in vitro. Although the receptors to which beta neuregulins bind have been defined, little is known about the transcription factors these important ligands activate. Using antibodies, quantitative imaging methods and Western blotting, it has been shown that beta neuregulin induces a high level of phosphorylation of the transcription factor cyclic AMP response element binding protein (CREB) on Ser-133 in cultured rat Schwann cells and that the phosphorylation is prolonged over several hours. In contrast, neurotrophins, CNTF, FGF-2, EGF, and TGFbeta induce little or no phosphorylation of CREB despite the fact that receptors for these factors are present on Schwann cells. As expected, CREB phosphorylation is detected following cAMP elevation, and it is also induced by elevation of cytoplasmic Ca2+, endothelin 1, and PDGF-BB. The signal is lower than that seen in response to beta neuregulin, and transient, unlike the sustained CREB activation induced by beta neuregulin. These results suggest that the sustained phosphorylation of CREB on Ser-133 may contribute to the broad spectrum of effects that beta neuregulins have on cells of the Schwann cell lineage and that the CREB pathway may be important for transduction of neuregulin signals in Schwann cells (Tabernero, 1998).

The molecular mechanisms by which mammalian receptor tyrosine kinases are negatively regulated remain largely unexplored. Previous genetic and biochemical studies indicate that Kekkon-1, a transmembrane protein containing leucine-rich repeats and an immunoglobulin-like domain in its extracellular region, acts as a feedback negative regulator of epidermal growth factor (EGF) receptor signaling in Drosophila development. Whether the related human LRIG1 (also called Lig-1) protein can act as a negative regulator of EGF receptor and its relatives, ErbB2, ErbB3, and ErbB4, was tested. In co-transfected 293T cells, LRIG1 forms a complex with each of the ErbB receptors independent of growth factor binding. Co-expression of LRIG1 with EGF receptor suppresses cellular receptor levels, shortens receptor half-life, and enhances ligand-stimulated receptor ubiquitination. Finally, it was observed that co-expression of LRIG1 suppresses EGF-stimulated transformation of NIH3T3 fibroblasts and that the inducible expression of LRIG1 in PC3 prostate tumor cells suppresses EGF- and neuregulin-1-stimulated cell cycle progression. These observations indicate that LRIG1 is a negative regulator of the ErbB family of receptor tyrosine kinases and suggest that LRIG1-mediated receptor ubiquitination and degradation may contribute to the suppression of ErbB receptor function (Laederich, 2004).

Neuregulin-1 (Nrg-1) contains an intracellular domain (Nrg-ICD) that translocates into the nucleus, where it may regulate gene expression upon neuronal depolarization. However, the identity of its target promoters and the mechanisms by which it regulates transcription have been elusive. In the mouse cochlea, synaptic activity increases the level of nuclear Nrg-ICD and upregulates postsynaptic density protein-95 (PSD-95), a scaffolding protein that is enriched in post-synaptic structures. Nrg-ICD enhances the transcriptional activity of the PSD-95 promoter by binding to a zinc-finger transcription factor, Eos. The Nrg-ICD-Eos complex induces endogenous PSD-95 expression in vivo through a signaling pathway that is mostly independent of gamma-secretase regulation. This upregulation of PSD-95 expression by the Nrg-ICD-Eos complex provides a molecular basis for activity-dependent synaptic plasticity (Bao, 2004).

Neuregulin (NRG) (also known as ARIA, GGF, and other names) is a heparin sulfate proteoglycan secreted into the neuromuscular junction by innervating motor and sensory neurons. An integral part of synapse formation, NRG-induced changes in gene expression were analyzed over 48 h in primary human myotubes. In addition to increasing the expression of acetylcholine receptors on the myotube surface, NRG treatment results in a transient increase of several members of the early growth response (Egr) family of transcription factors. Three Egrs (Egr1, -2, and -3) are induced within the first hour of NRG treatment, with Egr1 and -3 RNA levels showing the most significant increases of approximately 9- and 16-fold, respectively. Also noted was a corresponding increase in protein levels for both of these transcription factors. Previous literature indicates that Egr3 expression is required for the formation of muscle spindle fibers, sensory organs that are distinct from skeletal muscle contractile fibers. At the molecular level, muscle spindle fibers express a unique subset of myosin heavy chains. Two isoforms of the myosin heavy chain, the slow development and neonatal, were found to be increased in myotube cultures after 48 h of treatment with NRG. Taken together, these results indicate that not only can NRG induce the expression of a transcription factor key to spindle fiber development (Egr3), but that a portion of this developmental process can be replicated in vitro (Jacobson, 2004).

ErbB signaling regulates cell adhesion and movements during Xenopus gastrulation, but the downstream pathways involved have not been elucidated. This study shows that phosphatidylinositol-3 kinase (PI3K) and Erk mitogen-activated protein kinase (MAPK) mediate ErbB signaling to regulate gastrulation. Both PI3K and MAPK function sequentially in mesoderm specification and movements, and ErbB signaling is important only for the late phase activation of these pathways to control cell behaviors. Activation of either PI3K or Erk MAPK rescues gastrulation defects in ErbB4 morphant embryos, and restores convergent extension in the trunk mesoderm as well as coherent cell migration in the head mesoderm. The two signals preferentially regulate different aspects of cell behaviors, with PI3K more efficient in rescuing cell adhesion and spreading and MAPK more effective in stimulating the formation of filopodia. PI3K and MAPK also weakly activate each other, and together they modulate gastrulation movements. These results reveal that PI3K and Erk MAPK, which have previously been considered as mesodermal inducing signals, also act downstream of ErbB signaling to participate in regulation of gastrulation morphogenesis (Nie, 2007).

Table of contents

vein: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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