We propose to combine biophysical imaging and biochemical approaches to address two key unanswered questions in the field of signaling by receptor tyrosine kinases (RTKs) ? a principal class of therapeutic targets in cancer where resistance to current therapies necessitates new pharmacological approaches: a. What defines the distinct set of responses, and cell fate, downstream of a particular RTK? b. How can different ligands for a given RTK promote distinct cellular responses? It is well known that epidermal growth factor (EGF) and nerve growth factor (NGF) promote proliferative and differentiative responses respectively in PC12 cells, through EGFR and TrkA, apparently using the same set of signaling pathways. Early studies showed that Erk activation kinetics plays a key role in determining the different cell fates induced by these ligands, with transient Erk activation being associated with proliferation and sustained Erk activation with differentiation. Rather than being defined solely by different feedback ?wiring? in the intracellular MAP kinase cascade, we and others have found that the RTK activation kinetics play a direct role in defining the dynamic properties of the signaling network. Several recent studies further argue that the strength of ligand-induced RTK dimers (and/or their lifetime) dictates signaling specificity, offering the possibility of dynamically-determined biased agonism in RTK signaling. To gain insight into the mechanistic basis for biased agonism in RTK signaling, we propose to elucidate how activating the same RTK intracellular region in different ways can result in dramatically different cellular responses (proliferation vs differentiation). Our previous structural, biophysical, and biochemical data suggest the hypothesis that signaling outcome is determined by RTK dimer stability and dynamics. Using intact and chimeric receptors, we will test this hypothesis by asking how altering RTK dimerization kinetics influences receptor endocytosis, post-endocytic trafficking, and dynamics of the downstream signaling network. We combine single-molecule imaging and microscopy studies of RTK trafficking with mass spectrometry and biochemical studies of downstream signaling networks to yield an integrated picture of this. Our primary motivation is to investigate how modifying RTK signaling dynamics (rather than simply inhibiting RTKs) might be used in future therapeutic approaches in cancer.
Our Specific Aims address the following questions: 1 How can the same RTK intracellular region elicit orthogonal cellular responses depending on how it is activated? 2 What are the lifetimes of ErbB4/HER4 dimers induced by different neuregulin (NRG) ligands, and what is the basis for their sustained signaling? Our overall goal is to understand how dimerization dynamics can define signaling specificity, possibly through a kinetic proofreading similar to that seen in the T-cell receptor.
Receptor tyrosine kinases (RTKs) have been targeted therapeutically in cancer with agents designed to block all activity, and shut down aberrant signaling. The work proposed in this application will investigate whether it is possible to alter the dynamics of receptor signaling, and correct (rather than simply inhibit) aberrant signaling by oncogenic receptors ? causing them to promote death or quiescence of cancer cells.