Project Summary The research proposed in this MIRA application seeks to understand unexplored mechanisms of transmembrane signaling across the receptor tyrosine kinase (RTK) superfamily, members of which play an important role in human disease – from neurodevelopmental disorders, to bone diseases, cancers, diabetes, and several congenital malformations. In the traditional view of RTK signaling, growth factor ligands induce receptor dimers that become tyrosine autophosphorylated and recruit downstream signaling molecules. As we understand more about the 20 different RTK families (which include 58 RTKs), however, it becomes clearer that this view only applies to a subset of these receptors. The research proposed here focuses on two characteristics that demand a very different mechanistic view – RTKs that bind Wnt proteins (and do not dimerize as a result) and RTKs that have `dead' kinase or pseudokinases in their intracellular regions. New paradigms must be understood in order to appreciate how these important receptors signal. Our goals over the next 5-10 years are: 1. To develop a coherent picture of the role played by RTKs in Wnt signaling (which involves 4 of the 20 RTK families), 2. To understand how RTKs with pseudokinase domains that do not even bind ATP (found in 5 of the 20 RTK families) can signal, and 3. To determine why pseudokinases are over- represented among the Wnt-regulated RTKs. Guided by cellular and in vivo studies of receptor/ligand relationships we will study ligand-induced complexes biochemically, and with high-resolution structural approaches, in order to understand in detail how Wnt protein binding leads to activation of the receptors and co-receptors in the signaling complex. In pursuing these studies, we will investigate the role played by Wnt acylation – requirements for which appear to be different for binding to Frizzled-family and RTK-family Wnt receptors. We also hope to define specificity determinants in the Wnt proteins for distinct modes of signaling. In parallel with these pursuits, we will use a structurally-guided approach, combined with chemical biology and functional analysis of mutated receptors, to explore the mechanism of signaling by pseudokinases in the RTK superfamily. These studies will have important implications for the 10% of the human kinome thought to be pseudokinases, and will systematically test the hypothesis that regulated switching of pseudokinase conformation is required for signaling. Our approaches will also bring new opportunities for therapeutic targeting of pseudokinases such as PTK7, Ror2, and Ror1, which have been implicated in several diseases. Together, our studies will provide important fundamental new insight into signaling by a class of receptors that do not fit into normal paradigms for RTKs or Wnt receptors. Understanding them is crucial for deconvoluting the complexity of Wnt signaling specificity and teasing out its multiple roles. In addition, our findings should open new av...