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Mammalian neuregulin genes

The only known ligands that have both EGF-like and Ig-like domains, and thus considered to be homologs of Drosophila Vein, are the vertebrate neuregulins (also known as Neu differentiation factor), heregulins, ARIA and glial growth factors. The neuregulins were described initially as direct ligands for neu but later shown to bind its relatives erbB3 and erbB4. Signaling by the neuregulins can occur through receptor heterodimers (Carraway, 1994).

A set of new neuregulin-like growth factors, which is called neuregulin-2 (NRG-2), has been identified: members of this set are encoded by their own gene and exhibit a distinct expression pattern in adult brain and developing heart. Like NRG-1, the EGF-like domain of the new ligands binds to both the ErbB3- and ErbB4-receptor tyrosine kinases. However, NRG-2 stimulates different ErbB-receptor tyrosine-phosphorylation profiles from NRG-1. These results indicate that NRG-1 and NRG-2 mediate distinct biological processes by acting at different sites in tissues and eliciting different biochemical responses in cells (Carraway, 1997).

Neuregulins (also called ARIA, GGF, heregulin or NDF) are a group of polypeptide factors that arise from alternative RNA splicing of a single gene. Through their interaction with the ErbB family of receptors (ErbB2, ErbB3 and ErbB4), neuregulins help to regulate cell growth and differentiation in many tissues. The cloning of a second neuregulin-like gene, neuregulin-2, is reported. The encoded product of the neuregulin-2 gene has a motif structure similar to that of neuregulins. An alternative splicing site in the epidermal growth factor(EGF)-like domain gives rise to two isoforms (alpha and beta). Northern blot and in situ hybridization analysis of adult rat tissues indicate that expression of neuregulin-2 is highest in the cerebellum, and the expression pattern is different from that of neuregulins. Recombinant neuregulin-2beta induces the tyrosine-phosphorylation of ErbB2, ErbB3 and ErbB4 in cell lines expressing all of these ErbB-family receptors. However, in cell lines with defined combinations of ErbBs, neuregulin-2beta only activates those with ErbB3 and/or ErbB4, suggesting that signaling by neuregulin-2 is mediated by ErbB3 and/or ErbB4 receptors (Chang, 1997).

Described here is the identification of Neuregulin-3 (NRG3), a novel protein that is structurally related to the neuregulins (NRG1). The NRG1/neuregulins are members of a diverse family of proteins that arise by alternative splicing from a single gene. These proteins play an important role in controlling the growth and differentiation of glial, epithelial, and muscle cells. The biological effects of NRG1 are mediated by receptor tyrosine kinases ErbB2, ErbB3, and ErbB4. However, genetic studies have suggested that the activity of ErbB4 may also be regulated in the central nervous system by a ligand distinct from NRG1. NRG3 is predicted to contain an extracellular domain with an epidermal growth factor (EGF) motif, a transmembrane domain, and a large cytoplasmic domain. The EGF-like domain of NRG3 binds to the extracellular domain of ErbB4 in vitro. Moreover, NRG3 binds to ErbB4 expressed on cells and stimulates tyrosine phosphorylation of this receptor. The expression of NRG3 is highly restricted to the developing and adult nervous system. These data suggest that NRG3 is a novel, neural-enriched ligand for ErbB4 (Zhang, 1997).

Mutation of Neuregulins

Mice with the active domain of NRG-2 deleted were generated in an effort to characterize the biological function of NRG-2 in vivo. In contrast to the NRG-1 knockout animals, NRG-2 knockouts have no apparent heart defects and survive embryogenesis. Mutant mice display early growth retardation and reduced reproductive capacity. No obvious histological differences were observed in the major sites of NRG-2 expression. These results indicate that in vivo NRG-2 activity differs substantially from that of NRG-1 and that it is not essential for normal development in utero (Britto, 2004).

EGF in C. elegans

Information is provided about EGF in C. elegans because a localized source of EGF in the worm serves to restrict Epidermal growth factor receptor signaling, the same function served by Vein in Drosophila. Precursor cells of the vulva of the C. elegans hermaphrodite choose between two vulval cell fates (I and II) and a non-vulval epidermal fate (III) in response to three intercellular signals. An inductive signal produced by the anchor cell induces the vulval precursors to assume the I and II vulval fates. This inductive signal is an EGF-like growth factor encoded by the gene lin-3. An inhibitory signal mediated by lin-15, and which may originate from the surrounding epidermis, prevents the vulval precursors from assuming vulval fates in the absence of the inductive signal. A short range lateral signal, which acts through the gene lin-12, regulates the pattern of I and II fates assumed by the induced vulval precursors. The combined action of the three signals precisely directs the six vulval precursors to adopt a III, III, II, I, II, III and III pattern of fates. The amount of inductive signal produced by the anchor cell appears to determine the number of vulval precursors that assume vulval fates. The three induced vulval precursors most proximal to the anchor cell are proposed to adopt the II, I, II pattern of fates in response to a gradient of the inductive signal and also in response to lateral signaling that inhibits adjacent vulval precursor cells from both assuming the I fate (Hill, 1993).

The epidermal growth factor-like domain of the LIN-3 protein can induce either of two distinct vulval cell fates: a high dose of LIN-3 induces a I fate; a lower dose of LIN-3 induces a II fate. A high dose of LIN-3 can also induce adjacent vulval precursor cells to assume I fates; thus, high levels of LIN-3 can override the lateral signaling that normally inhibits formation of adjacent I fates. It is proposed that the invariant pattern of vulval cell fates is generated by a graded distribution of LIN-3 that promotes different vulval fates according to local concentration and by a lateral signal that reinforces this initial bias (Katz, 1995).

How do temporal and spatial interactions between multiple intercellular and intracellular factors specify the fate of a single cell in Caenorhabditis elegans? P12, which is a ventral cord neuroectoblast, divides postembryonically to generate neurons and a unique epidermal cell. Three classes of proteins are involved in the specification of P12 fate: the LIN-3/LET-23 epidermal growth factor signaling pathway; a Wnt protein LIN-44 and its candidate receptor LIN-17, and a homeotic gene product EGL-5. lin-3 encodes a membrane-spanning protein with a single extracellular EGF domain that is similar in structure to members of the EGF family of growth factors. LIN-3 is an inductive signal sufficient to promote the P12 fate, and the conserved EGF signaling pathway is utilized for P12 fate specification: egl-5, an AbdominalB homolog, is a downstream target of the lin-3/let-23 pathway in specifying P12 fate, and LIN-44 and LIN-17 act synergistically with lin-3 in the specification of the P12 fate. The Wnt pathway may function early in development to regulate the competence of the cells to respond to the LIN-3 inductive signal (Jiang, 1998).

In C. elegans there are twelve ventral cord precursor cells, P1-P12, numbered from anterior to posterior along the body axis. These cells divide postembryonically to generate cells of the ventral nervous system, as well as the vulva. P11/P12 are the most posterior pair of the ventral cord precursors. At hatching, the cells AB.plapappa (left side) and AB.prapappa (right side) are disposed laterally. In hermaphrodites, they start to migrate ventrally several hours after hatching and enter the ventral cord about 8-9 hours after hatching. The left cell migrates to the anterior and becomes P11; the right cell migrates to the posterior and becomes P12. Two hours later they each divide once. The anterior daughters, P11.a and P12.a are neuroblasts that will divide for three more rounds to generate several ventral cord neurons. These neurons are morphologically indistinguishable under Nomarski optics. The posterior daughter of P11, P11.p, does not divide but rather fuses with the large epidermal syncytium hyp7. P12.p divides once more about 1 hour prior to L1 molt to generate two cells:, which becomes a unique epidermal cell (hyp12) and P12.pp, which undergoes cell death. P11.p and can be distinguished by their different nuclear morphologies and positions observed with Nomarski optics. Prior to migration, both P11 and P12 cells are able to express the P12 fate: if only a single cell is present, it will adopt the P12- like fate. Therefore, P12 represents a primary fate, while P11 is a secondary fate (Jiang, 1998).

Mutants of several lin-3/let-23 pathway components have been reported to display defects in P11/P12 cell fate specification. Loss-of-function alleles of let-23 show a loss of the cell with concommitant duplication of P11.p in the hermaphrodite tail. Lineage analysis in males indicates that this defect likely represents a transformation of P12 to P11 fate, since the anterior branch is also affected. Mutations at the lin-15 locus, which encodes negative regulators of the lin-3/let-23 pathway, have the opposite defect: P11 to P12 cell fate transformation. Other components, sem-5 and let-60, which encode a SH2/SH3 domain protein and a RAS protein, respectively, are also involved in P11/P12 cell fate specification (Jiang, 1998).

egl-5 might play a permissive role in P12 fate specification by setting up the competence of the cell to respond to the LIN-3 inductive signal, or egl-5 might be an instructive factor for P12 fate specification. To distinguish between these hypotheses, a test was performed of the effect of overexpression of EGL-5 on P11/P12 cell fate specification. Overexpression of EGL-5 in wild-type animals does lead to a P11 to P12 fate transformation. Could overexpression of EGL-5 suppress the P11/P12 defect of let-23 mutants? If egl-5 is a permissive factor for P12 fate, no rescue of the P11/P12 defect would be expected; whereas if egl-5 is an instructive factor, overexpression of EGL-5 should be able to rescue the P11/P12 defect caused by let-23 mutation, and may additionally result in a P11 to P12 cell fate transformation. Overexpression of EGL-5 suppresses the P11/P12 defect of let-23 mutants, as predicted by the instructive model. Therefore, egl-5 plays an active role in P12 fate specification and acts downstream of let-23. egl-5has been shown to be a downstream target of the lin-3/let-23 pathway for P12 fate specification (Jiang, 1998).

Next the interactions between lin-3 and lin-44 were tested by examining the P11/P12 defect in a strain defective in both genes. The strong synergy observed between lin-3 and lin-44 is consistent with the two signals acting in parallel. To confirm the interaction between lin-3 and lin-44, a test of synergy between lin-3 and lin-17 mutations was performed. lin-17, which encodes a putative seven-transmembrane protein similar to the Drosophila Frizzled protein, has been suggested to be a receptor for the LIN-44 protein. Similar synergistic interactions are found between lin-3 and lin-17 as have been found between lin-3 and lin-44. A synergistic interaction is also found between mutations of let-23, the EGF receptor for LIN-3 signal, and the Wnt signal LIN-44. These data support the hypothesis that both of the two signaling pathways, lin-3 and lin-44, are required for the P12 fate specification. How do these two signaling pathways function in concert to specify P12 fate? The favored model is that both pathways are required for proper P12 fate specification and that they act at different developmental times. Three sources of evidence support this model: (1) LIN-3 overexpression experiments indicate that LIN-3 signal is required in early L1 before P11/P12 enter the ventral cord to induce P12 fate. (2) lin-44 expression is turned on during embryogenesis, much earlier than the time of P11/P12 induction. (3) The unique effect of overexpression of LIN-3EGF during late embryogenesis in lin-44 mutants suggests that LIN-44 function may be important in the early phase of P12 fate specification. It is possible that the Wnt pathway regulates the competence of the cells to respond to the LIN-3 inductive signal. But the Wnt signal alone is not sufficient to promote P12 fate, since overexpression of LIN-44 in wild-type animals has no effect on P11/P12 cell fate specification (Jiang, 1998).

The following is a model for P12 neuroectoblast fate specification: in newly hatched larvae, LIN-44 signal acts via receptor LIN-17 to set up the competence of P11 and P12 cells to respond to the inductive signal and be able to express P12 fate. egl-5 expression is kept off in both P11 and P12 cells. Later, an inductive signal LIN-3 coming from the posterior region activates LET-23 receptor activity in the posterior cell of the P11/P12 pair. Activation of the lin-3/let-23 pathway turns on egl-5 expression, which specifies the posterior cell to take on P12 fate. lin-15 negatively regulates let-23 activity and prevents the anterior cell from becoming P12. Information from the Wnt signaling pathway may be integrated into the lin-3/let-23 EGF signaling pathway either at the level of LIN-3 signal, LET-23 receptor or egl-5 transcription. Thus the temporal and spatial co-ordination and interactions between the Wnt signal, EGF signal and HOM-C transcription factor are important for P12 fate specification (Jiang, 1998).

The epidermal growth factor receptor (EGFR)/ErbB receptor tyrosine kinases regulate several aspects of development, including the development of the mammalian nervous system. ErbB signaling also has physiological effects on neuronal function, with influences on synaptic plasticity and daily cycles of activity. However, little is known about the effectors of EGFR activation in neurons. This study shows that EGF signaling has a nondevelopmental effect on behavior in Caenorhabditis elegans. Ectopic expression of the EGF-like ligand LIN-3 at any stage induces a reversible cessation of feeding and locomotion. These effects are mediated by neuronal EGFR (also called LET-23) and phospholipase C-gamma (PLC-gamma), diacylglycerol-binding proteins, and regulators of synaptic vesicle release. Activation of EGFR within a single neuron, ALA, is sufficient to induce a quiescent state. This pathway modulates the cessation of pharyngeal pumping and locomotion that normally occurs during the lethargus period that precedes larval molting. These results reveal an evolutionarily conserved role for EGF signaling in the regulation of behavioral quiescence (Van Buskirk, 2007).

Proteolytic processing of neuregulins

ARIA, or acetylcholine receptor-inducing activity, is a polypeptide that stimulates the synthesis of acetylcholine receptors in skeletal muscle. The ability of ARIA to induce phosphorylation of its receptor in muscle is blocked by highly charged glycosaminoglycans. ARIA constructs lacking the NH2-terminal portion, containing an immunoglobulin-like domain, are fully active and are not inhibited by glycosaminoglycans. Limited proteolysis of ARIA with subtilisin blocks the glycosaminoglycan interaction by degrading this NH2-terminal portion, but preserves the active, EGF-like domain. ARIA can be released from freshly dissociated cells of the embryonic chick spinal cord and cerebellum by either heparin, high salt or limited proteolysis with subtilisin, suggesting that ARIA is bound to the extracellular matrix through charged interactions. A model is presented for how ARIA may be stored in extracellular matrix at developing synapses and how its release may be mediated by local proteolysis (Loeb, 1995).

How the transmembrane precursor proARIA is processed to ARIA (acetylcholine receptor-inducing activity) has been investigated. Pulse-chase labeling in transfected Chinese hamster ovary (CHO) cells show that proARIA is cleaved to release ARIA into the medium. Cell surface biotin-labeling experiments demonstrate that proARIA is first expressed on the cell surface before being rapidly cleaved to release biotin-labeled ARIA into the medium. While not essential for proteolytic cleavage of proARIA, serum or phorbol-12-myristate-13-acetate (PMA), which activates protein kinase C (PKC), is needed for the efficient release of the processed ARIA. Proteolytic cleavage is blocked by brefeldin A, suggesting that processing occurs distal to Golgi compartments, and by NH4Cl, suggesting a need for intracellular acidic compartments. Serum and PMA also stimulate ARIA release from cultured sensory neurons, suggesting that a similar regulated release mechanism occurs in neurons and may be important in determining where ARIA is released in the developing nervous system (Loeb, 1998).

Neuregulins and the extracellular matrix

Neuregulins are a family of growth and differentiation factors that act through activation of cell-surface erbB receptor tyrosine kinases and have essential functions both during development and on the growth of cancer cells. One alternatively spliced neuregulin-1 form has a distinct heparin-binding immunoglobulin-like domain that enables it to adhere to heparan sulfate proteoglycans at key locations during development and substantially potentiates its activity. The structural specificity needed for neuregulin-1-heparin interactions was examined using a gel mobility shift assay together with an assay that measures the ability of specific oligosaccharides to block erbB receptor phosphorylation in L6 muscle cells. Whereas the N-sulfate group of heparin was most important, the 2-O-sulfate and 6-O-sulfate groups also contributed to neuregulin-1 binding in these two assays. Optimal binding to neuregulin-1 requires eight or more heparin disaccharides; however, as few as two disaccharides are still able to bind neuregulin-1 to a lesser extent. The physiological importance of this specificity was shown both by chemical and siRNA treatment of cultured muscle cells. Pretreatment of muscle cells with chlorate that blocks all sulfation or with an siRNA that selectively blocks N-sulfation significantly reduces erbB receptor activation by neuregulin-1 but has no effect on the activity of neuregulin-1 that lacks the heparin-binding domain. These results suggest that the regulation of glycosaminoglycan sulfation is an important biological mechanism that can modulate both the localization and potentiation of neuregulin-1 signaling (Pankonin, 2005).

Heparin-binding EGF-like growth factor (HB-EGF), a member of the EGF family of growth factors, plays an important role in cardiac valve development by suppressing mesenchymal cell proliferation. This study showed that HB-EGF must interact with heparan sulfate proteoglycans (HSPGs) to properly function in this process. In developing valves, HB-EGF is synthesized in endocardial cells but accumulates in the mesenchyme by interacting with HSPGs. Disrupting the interaction between HB-EGF and HSPGs in an ex vivo model of endocardial cushion explants resulted in increased mesenchymal cell proliferation. Moreover, homozygous knock-in mice (HBδhb/δhb) expressing a mutant HB-EGF that cannot bind to HSPGs developed enlarged cardiac valves with hyperproliferation of mesenchymal cells; this resulted in a phenotype that resembled that of Hbegf-null mice. Interestingly, although Hbegf-null mice had abnormal heart chambers and lung alveoli, HBδhb/δhb mice did not exhibit these defects. These results indicate that interactions with HSPGs are essential for the function of HB-EGF, especially in cardiac valve development, in which HB-EGF suppresses mesenchymal cell proliferation (Iwamoto, 2010).

Table of contents

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

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