Receptor tyrosine kinases (RTKs) are activated by extracellular ligands to transduce the bulk of the signals that control cellular growth, proliferation and survival. The canonical model of RTK activation defines the role of ligands as dimerizing agents that bring receptors into close proximity to activate the intracellular kinase domains. However, many RTKs form dimers in the absence of ligands and their activation is dependent on the proper association of domains on both sides of the plasma membrane. The molecular mechanisms governing such allosteric effects remain unknown due to the lack of full-length receptor structures. The main goal of this proposal is to understand how these mechanisms operate in the family of human epidermal growth factor (EGFR/HER) receptors by obtaining their high-resolution full-length structures. HERs are unique RTKs because in contrast to other RTKs, their kinase domains are not activated by trans-phosphorylation but by the formation of an asymmetric kinase dimer in which one kinase domain becomes an allosteric activator of another. Through structural work on portions of these receptors, we and others have shown that the asymmetric kinase domain module of HER kinases is coupled to conformation of the adjacent juxtamembrane and transmembrane domains, and those in turn are affected by the orientation of the extracellular domain modules. These relative structures are additionally modulated by different HER receptor ligands which have been shown to elicit different biological outcomes. How all these elements come together at the cell membrane is unknown. The inability to purify stable complexes of HER receptors has impeded full-length structural studies. We have now developed a robust system for expressing and purifying recombinant, nearly full-length HER receptors and routinely collect negative stain EM (NS-EM) and cryo-EM data sets on these samples. Using this pipeline, we will focus on obtaining high resolution structures of full length HER receptors in their inactive and ligand-bound active states. We will focus on three members of the HER receptor family: HER2, HER3 and HER4 which engage in a range of heterodimeric complexes in response to a spectrum of ligands. We hypothesize that the combinatorial power of receptor interactions starts at the level of active complex formation. Using cryo-EM, X-ray crystallography, enzymatic measurements and cell-based testing of structurally-derived models, we will focus on answering the following questions: 1. How do ligand-induced conformational changes propagate in the receptor across the plasma membrane? 2. What are the differences in mechanism between different receptor heterodimers? 3. How is the mechanism of activation fine-tuned by different ligands and by disease mutations? While our studies will be focused on HER receptors, the developed innovative experimental approaches will be applicable to the entire RTK family and other single-pass proteins, thus paving the way for many future discoveries. The biological knowledge we will acquire in the process will also contribute to the development of innovative therapeutics that target selected HER receptor complexes in human diseases.
Receptor tyrosine kinases (RTKs) constitute second largest family of membrane receptors in human genome that transduce the bulk of the signals that control cellular growth, proliferation and survival. Their activation by extracellular growth factors sets in motion a series of poorly understood conformational changes which transduce signals across the plasma membrane. By aiming to solve first high resolution full-length structures of human epidermal growth factor receptor tyrosine kinases (EGFR/HERs), we aim to provide critical information about the mechanism of RTK signal transduction, ultimately aiming to enable the development of new strategies to combat aberrant signaling by these receptor complexes in cancer and other human diseases.
