The structural basis of biased signaling through HER4 receptor complexes

人表皮生长因子受体（HER）酪氨酸激酶，以结构相关蛋白EGFR、HER2、HER3和HER4为代表，是细胞稳态的守门人。它们通过连接细胞外生长因子结合和细胞内化学事件来控制细胞的生长、存活和运动。尽管HER4在心脏和神经组织的发育和功能中起着至关重要的作用，但它是最不为人所知的家族成员。在心脏中，HER4与HER2形成复合物，指导胚胎心脏发育和成人心脏功能。HER4和HER4/HER2信号的错误调控与心脏病和神经系统疾病如精神分裂症和阿尔茨海默病密切相关。虽然HER2不与配体结合，但HER4可以被神经调节蛋白生长因子家族（NRG1-4）的所有四种成员激活，并且具有区分它们彼此的显著能力，从而将NRG类型不同的信息传递到细胞中。这种偏向性激动作用导致配体特异性功能对细胞的影响，并决定细胞是否增殖、死亡或分化。然而，我们目前还不了解不同配体结合到HER4细胞外结构域影响其细胞内信号传导结构域-激酶的机制。这是因为没有细胞外结构域和激酶结构域同时存在的高分辨率结构。在本提案中，我的目标是通过获得与不同神经调节因子生长因子和HER2复合物中接近全长的HER4受体的第一个高分辨率结构来填补这一空白。为此，我将使用冷冻电镜和我们最近获得的纯化HER4和HER2的能力，以进行大量的结构研究。在我们的初步工作中，我们表明我们能够形成nrg1结合的HER4和HER2/HER4异质复合物。通过负染色和低温电镜对这些受体的初步表征已经揭示了在已发表的分离受体结构域的晶体结构中未观察到的受体特征。这些结果证明了我们的方法的可行性及其在理解受体激活机制和不同配体微调方面的应用。我们将通过诱变在细胞增殖试验中验证我们的结构-功能发现。我们的研究对于揭示配体诱导的HER4受体信号传导的基本原理以及心血管和神经疾病的靶向治疗设计将是非常宝贵的。

The Human Epidermal growth factor Receptor (HER) tyrosine kinases, represented by the structurally-related proteins EGFR, HER2, HER3 and HER4, are gatekeepers of cellular homeostasis. They control cell growth, survival, and motility by linking extracellular growth factor binding to intracellular chemical events. The least characterized family member is HER4, despite its crucial role in the development and function of heart and neuronal tissues. In the heart, HER4 forms complexes with HER2 to guide heart development in the embryo and function in the adult. Missregulation of HER4 and HER4/HER2 signaling are strongly correlated with heart disease and neurological disorders such as schizophrenia and Alzheimer’s Disease. While HER2 does not bind to ligands, HER4 can be activated by all four members of the Neuregulin family of growth factors (NRG1-4), and has a remarkable ability to distinguish them from one another to relay NRG type-distinct messages into the cell. This biased agonism leads to ligand-specific functional consequences for the cell and determines whether a cell proliferates, dies, or differentiates. However, we do not currently understand the mechanism by which binding of different ligands to the extracellular domains of HER4 influences its intracellular signaling domains - kinases. This is because there are no high-resolution structures in which both extracellular domains and kinase domains are present simultaneously. In this proposal, I aim to fill this gap by obtaining the first high-resolution structures of the near full-length HER4 receptor in complex with different Neuregulin growth factors, and with HER2. To this end, I will use cryo-EM and our recently gained ability to purify HER4 and HER2 in robust quantities permitting structural studies. In our preliminary work, we show that we are able to form NRG1-bound HER4 and HER2/HER4 heterocomplexes. Initial characterization of these receptors by negative-stain and cryo-EM already reveal receptor features that have not been observed in published crystal structures of isolated receptor domains. These results demonstrate feasibility of our approach and its application to understand receptor activation mechanism and its fine tuning by different ligands. We will validate our structure-function findings in cell-proliferation assays via mutagenesis. Our studies will be invaluable for uncovering fundamental principles of ligand-induced HER4 receptor signaling and the design of targeted therapies in cardiovascular and neuronal disorders.