Signaling by ERBB4

ERBB4, also known as HER4, belongs to the ERBB family of receptors, which also includes ERBB1 (EGFR/HER1), ERBB2 (HER2/NEU) and ERBB3 (HER3). Similar to EGFR, ERBB4 has an extracellular ligand binding domain, a single transmembrane domain and a cytoplasmic domain which contains an active tyrosine kinase and a C-tail with multiple phosphorylation sites. At least three and possibly four splicing isoforms of ERBB4 exist that differ in their C-tail and/or the extracellular juxtamembrane regions: ERBB4 JM-A CYT1, ERBB4 JM-A CYT2 and ERBB4 JM-B CYT1 (the existence of ERBB4 JM-B CYT2 has not been confirmed). <br><br>ERBB4 becomes activated by binding one of its seven ligands, three of which, HB-EGF, epiregulin EPR and betacellulin BTC, are EGF-like (Elenius et al. 1997, Riese et al. 1998), while four, NRG1, NRG2, NRG3 and NRG4, belong to the related neuregulin family (Tzahar et al. 1994, Carraway et al. 1997, Zhang et al. 1997, Hayes et al. 2007). Upon ligand binding, ERBB4 forms homodimers (Sweeney et al. 2000) or it heterodimerizes with ERBB2 (Li et al. 2007). Dimers of ERBB4 undergo trans-autophosphorylation on tyrosine residues in the C-tail (Cohen et al. 1996, Kaushansky et al. 2008, Hazan et al. 1990, Li et al. 2007), triggering downstream signaling cascades. The pathway Signaling by ERBB4 only shows signaling by ERBB4 homodimers. Signaling by heterodimers of ERBB4 and ERBB2 is shown in the pathway Signaling by ERBB2. Ligand-stimulated ERBB4 is also able to form heterodimers with ligand-stimulated EGFR (Cohen et al. 1996) and ligand-stimulated ERBB3 (Riese et al. 1995). Dimers of ERBB4 with EGFR and dimers of ERBB4 with ERBB3 were demonstrated in mouse cell lines in which human ERBB4 and EGFR or ERBB3 were exogenously expressed. These heterodimers undergo trans-autophosphorylation. The promiscuous heteromerization of ERBBs adds combinatorial diversity to ERBB signaling processes. As ERBB4 binds more ligands than other ERBBs, but has restricted expression, ERBB4 expression channels responses to ERBB ligands. The signaling capabilities of the four receptors have been compared (Schulze et al. 2005).<br><br>As for other receptor tyrosine kinases, ERBB4 signaling effectors are largely dictated through binding of effector proteins to ERBB4 peptides that are phosphorylated upon ligand binding. All splicing isoforms of ERBB4 possess two tyrosine residues in the C-tail that serve as docking sites for SHC1 (Kaushansky et al. 2008, Pinkas-Kramarski et al. 1996, Cohen et al. 1996). Once bound to ERBB4, SHC1 becomes phosphorylated on tyrosine residues by the tyrosine kinase activity of ERBB4, which enables it to recruit the complex of GRB2 and SOS1, resulting in the guanyl-nucleotide exchange on RAS and activation of RAF and MAP kinase cascade (Kainulainen et al. 2000). <br><br>The CYT1 isoforms of ERBB4 also possess a C-tail tyrosine residue that, upon trans-autophosphorylation, serves as a docking site for the p85 alpha subunit of PI3K (Kaushansky et al. 2008, Cohen et al. 1996), leading to assembly of an active PI3K complex that converts PIP2 to PIP3 and activates AKT signaling (Kainulainen et al. 2000). <br><br>Besides signaling as a conventional transmembrane receptor kinase, ERBB4 differs from other ERBBs in that JM-A isoforms signal through efficient release of a soluble intracellular domain. Ligand activated homodimers of ERBB4 JM-A isoforms (ERBB4 JM-A CYT1 and ERBB4 JM-A CYT2) undergo proteolytic cleavage by ADAM17 (TACE) in the juxtamembrane region, resulting in shedding of the extracellular domain and formation of an 80 kDa membrane bound ERBB4 fragment known as ERBB4 m80 (Rio et al. 2000, Cheng et al. 2003). ERBB4 m80 undergoes further proteolytic cleavage, mediated by the gamma-secretase complex, which releases the soluble 80 kDa ERBB4 intracellular domain, known as ERBB4 s80 or E4ICD, into the cytosol (Ni et al. 2001). ERBB4 s80 is able to translocate to the nucleus, promote nuclear translocation of various transcription factors, and act as a transcription co-factor. For example, in mammary cells, ERBB4 binds SH2 transcription factor STAT5A. ERBB4 s80 shuttles STAT5A to the nucleus, and actsa as a STAT5A co-factor in binding to and promoting transcription from the beta-casein (CSN2) promoter, and may be involved in the regulation of other lactation-related genes (Jones et al. 1999, Williams et al. 2004, Muraoka-Cook et al. 2008). ERBB4 s80 binds activated estrogen receptor in the nucleus and acts as a transcriptional co-factor in promoting transcription of some estrogen-regulated genes, including progesterone receptor gene NR3C3 and CXCL12 (SDF1) (Zhu et al. 2006). In neuronal precursors, ERBB4 s80 binds the complex of TAB and NCOR1, helps to move the complex into the nucleus, and is a co-factor of TAB:NCOR1-mediated inhibition of expression of astrocyte differentiation genes GFAP and S100B (Sardi et al. 2006).<br><br>The C-tail of ERBB4 possesses several WW-domain binding motifs (three in CYT1 isoform and two in CYT2 isoform), which enable interaction of ERBB4 with WW-domain containing proteins. ERBB4 s80, through WW-domain binding motifs, interacts with YAP1 transcription factor, a known proto-oncogene, and is a co-regulator of YAP1-mediated transcription in association with TEAD transcription factors (Komuro et al. 2003, Omerovic et al. 2004). Hence, the WW binding motif couples ERBB4 to the major effector arm of the HIPPO signaling pathway. The tumor suppressor WWOX, another WW-domain containing protein, competes with YAP1 in binding to ERBB4 s80 and prevents translocation of ERBB4 s80 to the nucleus (Aqeilan et al. 2005).<br><br>WW-domain binding motifs in the C-tail of ERBB4 play an important role in the downregulation of ERBB4 receptor signaling, enabling the interaction of intact ERBB4, ERBB4 m80 and ERBB4 s80 with NEDD4 family of E3 ubiquitin ligases WWP1 and ITCH. The interaction of WWP1 and ITCH with intact ERBB4 is independent of receptor activation and autophosphorylation. Binding of WWP1 and ITCH ubiquitin ligases leads to ubiquitination of ERBB4 and its cleavage products, and subsequent degradation through both proteasomal and lysosomal routes (Omerovic et al. 2007, Feng et al. 2009). In addition, the s80 cleavage product of ERBB4 JM-A CYT-1 isoform is the target of NEDD4 ubiquitin ligase. NEDD4 binds ERBB4 JM-A CYT-1 s80 (ERBB4jmAcyt1s80) through its PIK3R1 interaction site and mediates ERBB4jmAcyt1s80 ubiquitination, thereby decreasing the amount of ERBB4jmAcyt1s80 that reaches the nucleus (Zeng et al. 2009).<br><br>ERBB4 also binds the E3 ubiquitin ligase MDM2, and inhibitor of p53 (Arasada et al. 2005). Other proteins that bind to ERBB4 intracellular domain have been identified by co-immunoprecipitation and mass spectrometry (Gilmore-Hebert et al., 2010), and include transcriptional co-repressor TRIM28/KAP1, which promotes chromatin compaction. DNA damage signaling through ATM releases TRIM28-associated heterochromatinization. Interactions of ERBB4 with TRIM28 and MDM2 may be important for integration of growth factor responses and DNA damage responses.<br><br>In human breast cancer cell lines, ERBB4 activation enhances anchorage-independent colony formation in soft agar but inhibits cell growth in a monolayer culture. Different ERBB4 ligands induce different gene expression changes in breast cancer cell lines. Some of the genes induced in response to ERBB4 signaling in breast cancer cell lines are RAB2, EPS15R and GATA4. It is not known if these gene are direct transcriptional targets of ERBB4 (Amin et al. 2004).<br><br>Transcriptome and ChIP-seq comparisons of full-length and intracellular domain isoforms in isogenic MCF10A mammary cell background have revealed the diversification of ERBB4 signaling engendered by alternative splicing and cleavage (Wali et al., 2014). ERBB4 broadly affected protease expression, cholesterol biosynthesis, HIF1-alpha signaling, and HIPPO signaling pathways, and other pathways were differentially activated by CYT1 and CYT2 isoforms. For example, CYT1 promoted expression of transcription factors TWIST1 and SNAIL1 that promote epithelial-mesenchymal transition. HIF1-alpha and HIPPO signaling are mediated, respectively, by binding of ERBB4 to HIF1-alpha and to YAP (Paatero et al., 2012, Komuro et al., 2003). ERBB4 increases activity of the transcription factor SREBF2, resulting in increased expression of SREBF2-target genes involved in cholesterol biosynthesis. The mechanism is not known and may involve facilitation of SREBF2 cleavage through ERBB4-mediated PI3K signaling (Haskins et al. 2016).<br><br>In some contexts, ERBB4 promotes growth suppression or apoptosis (Penington et al., 2002). Activation of ERBB4 in breast cancer cell lines leads to JNK dependent increase in BRCA1 mRNA level and mitotic cell cycle delay, but the exact mechanism has not been elucidated (Muraoka Cook et al. 2006). The nature of growth responses may be connected with the spliced isoforms expressed. In comparisons of CYT1 vs CYT2 (full-length and ICD) expression in mammary cells, CYT1 was a weaker growth inducer, associated with attenuated MAPK signaling relative to CYT2 (Wali et al., 2014). ERBB4 s80 is also able to translocate to the mitochondrial matrix, presumably when its nuclear translocation is inhibited. Once in the mitochondrion, the BH3 domain of ERBB4, characteristic of BCL2 family members, may enable it to act as a pro apoptotic factor (Naresh et al. 2006).<br><br>ERBB4 plays important roles in the developing and adult nervous system. Erbb4 deficiency in somatostatin-expressing neurons of the thalamic reticular nucleus alters behaviors dependent on sensory selection (Ahrens et al. 2015). NRG1-activated ERBB4 signaling enhances AMPA receptor responses through PKC-dependent AMPA receptor exocytosis. This results in an increased excitatory input to parvalbumin-expressing inhibitory neurons in the visual cortex and regulates visual cortical plasticity (Sun et al. 2016). NRG1-activated ERBB4 signaling is involved in GABAergic activity in amygdala which mediates fear conditioning (fear memory) (Lu et al. 2014). Conditional Erbb4 deletion from fast-spiking interneurons, chandelier and basket cells of the cerebral cortex leads to synaptic defects associated with increased locomotor activity and abnormal emotional, social and cognitive function that can be linked to some of the schizophrenia features. The level of GAD1 (GAD67) protein is reduced in the cortex of conditional Erbb4 mutants. GAD1 is a GABA synthesizing enzyme. Cortical mRNA levels of GAD67 are consistently decreased in schizophrenia (Del Pino et al. 2014). Erbb4 is expressed in the GABAergic neurons of the bed nucleus stria terminalis, a part of the extended amygdala. Inhibition of NRG1-triggered ERBB4 signaling induces anxiety-like behavior, which depends on GABAergic neurotransmission. NRG1-ERBB4 signaling stimulates presynaptic GABA release, but the exact mechanism is not known (Geng et al. 2016). NRG1 protects cortical interneurons against ischemic brain injury through ERBB4-mediated increase in GABAergic transmission (Guan et al. 2015). NRG2-activated ERBB4 can reduce the duration of GABAergic transmission by binding to GABA receptors at the postsynaptic membrane via their GABRA1 subunit and promoting endocytosis of GABA receptors (Mitchell et al. 2013). NRG1 promotes synchronization of prefrontal cortex interneurons in an ERBB4 dependent manner (Hou et al. 2014). NRG1-ERBB4 signaling protects neurons from the cell death induced by a mutant form of the amyloid precursor protein (APP) (Woo et al. 2012).<br><br>Clinical relevance of ERBB4 has been identified in several contexts. In cancer, putative and validated gain-of-function mutations or gene amplification that may be drivers have been identified at modest frequencies, and may also contribute to resistance to EGFR and ERBB2-targeted therapies. This is noteworthy as ERBB4 kinase activity is inhibited by pan-ERBB tyrosine kinase inhibitors, including lapatinib, which is approved by the US FDA. The reduced prevalence relative to EGFR and ERBB2 in cancer may reflect more restricted expression of ERBB4, or differential signaling, as specific ERBB4 isoforms have been linked to growth inhibition or apoptosis in experimental systems. ERBB2/ERBB4 heterodimers protect cardiomyocytes, so reduced activity of ERBB4 in patients treated with the ERBB2-targeted therapeutic antibody trastuzumab may contribute to the cardiotoxicity of this agent when used in combination with (cardiotoxic) anthracyclines.<br><br>With the importance of ERBB4 in developing and adult nervous system, NRG1 and/or ERBB4 polymorphisms, splicing aberrations and mutations have been linked to nervous system disorders including schizophrenia and amyotrophic lateral sclerosis, although these findings are not yet definitive.

ERBB4，亦称HER4，隶属于ERBB受体家族，该家族还包括ERBB1（EGFR/HER1）、ERBB2（HER2/NEU）和ERBB3（HER3）。与EGFR相似，ERBB4具有细胞外配体结合域、单个跨膜域以及含有活性酪氨酸激酶和多个磷酸化位点的C端。至少存在三种，可能四种剪接异构体，ERBB4在C端和/或细胞外邻膜区域有所不同：ERBB4 JM-A CYT1、ERBB4 JM-A CYT2和ERBB4 JM-B CYT1（ERBB4 JM-B CYT2的存在尚未得到证实）。<br><br>ERBB4通过与其七个配体之一结合而被激活，其中三个，即HB-EGF、epiregulin EPR和betacellulin BTC，属于EGF样（Elenius等，1997，Riese等，1998），而另外四个，即NRG1、NRG2、NRG3和NRG4，属于相关的神经调节素家族（Tzahar等，1994，Carraway等，1997，Zhang等，1997，Hayes等，2007）。在配体结合后，ERBB4形成同源二聚体（Sweeney等，2000）或与ERBB2异源二聚化（Li等，2007）。ERBB4的二聚体在C端酪氨酸残基上发生转自磷酸化（Cohen等，1996，Kaushansky等，2008，Hazan等，1990，Li等，2007），从而触发下游信号级联反应。ERBB4同源二聚体信号通路仅显示ERBB4同源二聚体的信号。ERBB4和ERBB2异源二聚体的信号在ERBB2信号通路中展示。配体激活的ERBB4也能与配体激活的EGFR（Cohen等，1996）和配体激活的ERBB3（Riese等，1995）形成异源二聚体。在将人ERBB4、EGFR或ERBB3外源性表达的鼠细胞系中，已经证明了ERBB4与EGFR和ERBB4与ERBB3的二聚体。这些异源二聚体经历转自磷酸化。ERBBs的杂合异源二聚化增加了ERBB信号过程的组合多样性。由于ERBB4比其他ERBB结合更多的配体，但表达受限，因此ERBB4的表达渠道响应ERBB配体。四种受体的信号能力已进行比较（Schulze等，2005）。<br><br>对于其他受体酪氨酸激酶，ERBB4信号效应器主要通过效应蛋白与ERBB4配体结合而受到调控。所有剪接异构体的ERBB4都拥有C端两个酪氨酸残基，这些残基作为SHC1的结合位点。一旦结合到ERBB4，SHC1就会在酪氨酸残基上被ERBB4的酪氨酸激酶活性磷酸化，从而使其能够募集GRB2和SOS1的复合物，导致RAS的鸟苷酸交换和RAF和MAP激酶级联反应的激活（Kainulainen等，2000）。<br><br>ERBB4的CYT1异构体也拥有C端酪氨酸残基，在转自磷酸化后，该残基作为PI3K p85α亚基的结合位点。这一结合导致活性PI3K复合物的组装，将PIP2转化为PIP3并激活AKT信号（Kainulainen等，2000）。<br><br>除了作为传统跨膜受体激酶进行信号外，ERBB4与其他ERBB的不同之处在于JM-A异构体通过有效释放可溶性细胞内域进行信号。ERBB4 JM-A异构体（ERBB4 JM-A CYT1和ERBB4 JM-A CYT2）的配体激活的同源二聚体在邻膜区域被ADAM17（TACE）蛋白水解酶裂解，导致细胞外域的脱落和80 kDa膜结合的ERBB4片段（ERBB4 m80）的形成（Rio等，2000，Cheng等，2003）。ERBB4 m80进一步被γ-分泌酶复合物介导的蛋白水解酶裂解，释放可溶性80 kDa的ERBB4细胞内域，称为ERBB4 s80或E4ICD，进入细胞质（Ni等，2001）。ERBB4 s80能够转移到细胞核中，促进各种转录因子的核转位，并作为转录共因子。例如，在乳腺细胞中，ERBB4与SH2转录因子STAT5A结合。ERBB4 s80将STAT5A转运到细胞核中，并在结合和促进β-酪蛋白（CSN2）启动子的转录中作为STAT5A的共因子，并可能参与其他泌乳相关基因的调节（Jones等，1999，Williams等，2004，Muraoka-Cook等，2008）。ERBB4 s80与细胞核中的激活的雌激素受体结合，并在促进某些雌激素调节基因的转录中作为转录共因子，包括孕酮受体基因NR3C3和CXCL12（SDF1）（Zhu等，2006）。在神经元前体细胞中，ERBB4 s80与TAB和NCOR1的复合物结合，帮助复合物进入细胞核，并作为TAB:NCOR1介导的胶质细胞分化基因GFAP和S100B表达抑制的共因子（Sardi等，2006）。<br><br>ERBB4的C端具有多个WW结构域结合基序（CYT1异构体中有三个，CYT2异构体中有两个），这使ERBB4能够与包含WW结构域的蛋白质相互作用。ERBB4 s80通过WW结构域结合基序与YAP1转录因子相互作用，YAP1是一种已知的原癌基因，并与TEAD转录因子相关联，是YAP1介导的转录的共调节因子（Komuro等，2003，Omerovic等，2004）。因此，WW结合基序将ERBB4耦合到HIPPO信号通路的主要效应臂。肿瘤抑制因子WWOX，另一种包含WW结构域的蛋白质，与YAP1竞争结合ERBB4 s80，防止ERBB4 s80转移到细胞核（Aqeilan等，2005）。<br><br>ERBB4的C端WW结构域结合基序在ERBB4受体信号下调中发挥着重要作用，使得完整的ERBB4、ERBB4 m80和ERBB4 s80能够与NEDD4家族的E3泛素连接酶WWP1和ITCH相互作用。WWP1和ITCH与完整ERBB4的相互作用不受受体激活和自磷酸化的影响。WWP1和ITCH泛素连接酶的结合导致ERBB4及其裂解产物的泛素化，并通过蛋白酶体和溶酶体途径进行降解（Omerovic等，2007，Feng等，2009）。此外，ERBB4 JM-A CYT-1异构体的s80裂解产物是NEDD4泛素连接酶的目标。NEDD4通过其PIK3R1相互作用位点结合ERBB4 JM-A CYT-1 s80（ERBB4jmAcyt1s80），介导ERBB4jmAcyt1s80的泛素化，从而减少到达细胞核的ERBB4jmAcyt1s80的数量（Zeng等，2009）。<br><br>ERBB4还结合E3泛素连接酶MDM2和p53抑制剂。通过共免疫沉淀和质谱分析已经鉴定出与ERBB4细胞内域结合的其他蛋白质（Gilmore-Hebert等，2010），包括转录共抑制因子TRIM28/KAP1，它促进染色质紧缩。通过ATM介导的DNA损伤信号释放TRIM28相关的异染色质化。ERBB4与TRIM28和MDM2的相互作用可能对于整合生长因子反应和DNA损伤反应很重要。<br><br>在人乳腺癌细胞系中，ERBB4的激活增强了在软琼脂中的锚定独立性集落形成，但在单层培养中抑制了细胞生长。不同的ERBB4配体在乳腺癌细胞系中诱导不同的基因表达变化。一些在乳腺癌细胞系中对ERBB4信号反应诱导的基因是RAB2、EPS15R和GATA4。这些基因是否是ERBB4的直接转录靶点尚不清楚（Amin等，2004）。<br><br>在等基因MCF10A乳腺细胞背景中，全长和细胞内域异构体的转录组学和ChIP-seq比较揭示了由选择性剪接和裂解引起的ERBB4信号多样化（Wali等，2014）。ERBB4广泛影响蛋白酶表达、胆固醇生物合成、HIF1-alpha信号和HIPPO信号通路，CYT1和CYT2异构体通过不同的信号通路激活其他通路。例如，CYT1促进转录因子TWIST1和SNAIL1的表达，这些转录因子促进上皮间质转化。HIF1-alpha和HIPPO信号分别通过ERBB4与HIF1-alpha和YAP的结合介导（Paatero等，2012，Komuro等，2003）。ERBB4增加转录因子SREBF2的活性，导致SREBF2靶基因（涉及胆固醇生物合成）的表达增加。该机制尚不清楚，可能涉及通过ERBB4介导的PI3K信号促进SREBF2的裂解（Haskins等，2016）。<br><br>在某些情况下，ERBB4促进生长抑制或细胞凋亡（Penington等，2002）。在乳腺癌细胞系中激活ERBB4会导致JNK依赖性BRCA1 mRNA水平的增加和有丝分裂细胞周期的延迟，但其确切机制尚未阐明（Muraoka Cook等，2006）。生长反应的性质可能与表达的剪接异构体有关。在比较乳腺细胞中CYT1与CYT2（全长和ICD）表达时，CYT1是一种较弱的生长诱导剂，与相对于CYT2的减弱的MAPK信号相关（Wali等，2014）。ERBB4 s80也能够转移到线粒体基质中，这可能是当其核转位被抑制时。一旦进入线粒体，ERBB4的BH3结构域（BCL2家族成员的特征）可能使其能够作为促凋亡因子发挥作用（Naresh等，2006）。<br><br>ERBB4在发育和成年神经系统中的作用至关重要。NRG1和/或ERBB4的多态性、剪接异常和突变与包括精神分裂症和肌萎缩侧索硬化症在内的神经系统疾病相关，尽管这些发现尚未得到最终证实。