Diversity of receptor tyrosine kinase signaling mechanisms

Receptor tyrosine kinases (RTKs) are a family of 58 transmembrane proteins in humans that play crucial roles in many biological processes and diseases. Different RTKs utilize subtly (but importantly) distinct molecular mechanisms for transmembrane signaling, and understanding these differences is crucial for devising new ways to intervene pharmacologically when aberrant RTK signaling causes cancer and other diseases. In this thesis, I focus on three RTK families: the ALK/LTK family, the Wnt-binding RTKs, and the EGF receptor – where I concentrate on efforts to understand its C-terminal regulatory region. My studies of ALK, for anaplastic lymphoma kinase, were motivated by the fact that this RTK sub-group has a unique domain architecture in its extracellular region. Little is known about the mechanisms of ligand binding to – and activation of – ALK, and the nature of its ligand(s) is(are) still not completely clear. Using biochemical, biophysical and structural biology approaches, I characterized the low-resolution structure of the ALK extracellular region. I further identified the binding mode of ALK binding to heparin, a recently discovered modulatory ligand for ALK. Based on a low-resolution structural analysis of ALK/heparin complex, I propose a model for ligand-induced ALK dimerization and activation. Ryk is one of the five RTKs that are now known to be Wnt receptors. In this thesis, I studied the Drosophila homolog of Ryk, Derailed (Drl), and its binding to ligand DWnt5. We were able to express and purify milligrams of active DWnt5 – thus overcoming a major obstacle in this field. We further characterized Drl/ DWnt5 interactions. Using hydrogen/deuterium exchange approaches, I identified the DWnt5-binding interface on Drl. My efforts to understand the molecular mechanisms of Drl/DWnt5 binding using experimental and computational approaches suggest that DWnt5 may interact with Drl through a binding mode that differs from Wnt binding to other receptors. Across the RTK family, many receptors contain a long carboxy-terminal tail (C-tail) that harbors autophosphorylation sites for docking of downstream signaling molecules. This region is generally considered to be intrinsically disordered. I studied the dynamics of the EGFR C-tail, and showed that it is highly unstructured – but contains some somewhat ‘structured’ regions. I also showed that phosphorylation of the EGFR C-tail promotes receptor dimerization. Using hydrogen exchange, I identified possible C-tail docking sites on the kinase domain that may be responsible for this effect. I also studied binding of downstream SH2 domain-containing molecules to the EGFR C-tail, with results that indicate that not all features of SH2 domain binding to the C-tail can be recapitulated by simple phosphopeptides; binding of SH2 domains to the C-tail exhibits binding affinities and stoichiometries that are not captured by simple peptide-level studies. Moreover, my binding competition assays suggest that there may be cooperativity in binding of multiple SH2 domains to a single phosphorylated C-tail. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) This dissertation is available at ScholarlyCommons: http://repository.upenn. edu/edissertations/2104 Graduate Group Biochemistry & Molecular Biophysics First Advisor Mark A. Lemmon