The structural basis of biased signaling through HER4 receptor complexes

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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.