The structural basis of biased signaling through HER4 receptor complexes

以结构相关蛋白EGFR、HER2、HER3和HER4为代表的人表皮生长因子受体(HER)酪氨酸激酶是细胞内稳态的守门人。它们通过将细胞外生长因子结合到细胞内的化学事件来控制细胞的生长、存活和运动。最不典型的家族成员是HER4，尽管它在心脏和神经组织的发育和功能中起着至关重要的作用。在心脏中，HER4与HER2形成复合体，指导胚胎心脏的发育和成年心脏的功能。HER4和HER4/HER2信号的错误调节与心脏病和神经疾病(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可以被神经生长因子家族(NRG1-4)的所有四个成员激活，并具有显著的区分它们的能力，将不同NRG类型的信息传递到细胞内。这种有偏见的激动剂导致对细胞的配体特定的功能后果，并决定细胞是增殖、死亡还是分化。然而，我们目前还不清楚不同的配体与HER4胞外区的结合是如何影响其胞内信号域--激酶的。这是因为没有同时存在胞外区和激酶区的高分辨率结构。在这个提议中，我的目标是通过获得与不同的NeuRegin生长因子和HER2形成的复合体中的近全长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EM和我们最近获得的能力，以稳健的数量纯化HER4和HER2，从而进行结构研究。在我们的初步工作中，我们发现我们能够形成与NRG1结合的HER4和HER2/HER4杂化络合物。通过负染和冷冻-EM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没有观察到的受体特征。这些结果证明了我们方法的可行性及其在了解受体激活机制和不同配体对其微调方面的应用。我们将通过突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受体信号的基本原理以及设计心血管和神经元疾病的靶向治疗将是非常有价值的。

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.