PROJECT SUMMARY/ABSTRACT
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.