The parent MIRA grant (R35-GM122485) supports our work to understand transmembrane signaling by an unusual ? but very important ? set of receptor tyrosine kinases (RTKs) that respond to Wnts and have ?dead? pseudokinases (instead of normal tyrosine kinase domains) in their intracellular regions. These ?pseudokinase? RTKs play important roles in human disease ? from neurodevelopmental disorders, to bone diseases, cancers, diabetes, and several congenital malformations. In addition to understanding the signaling pathways/networks that these receptors impinge upon, key questions of the parent grant are: 1). To understand how the extracellular regions of these receptors binds Wnts; and 2). How do their ?dead? pseudokinase domains ? which do not even bind ATP ? function? We have solved crystal structures of both extracellular regions and pseudokinase domains from these receptors, which provides a static view of their architecture. With collaborators, we have also generated small molecules that bind and potentially modulate pseudokinase RTK signaling, and are studying the signaling effects of various mutations. Key questions that we are now trying to tackle require studies of structural dynamics, for which hydrogen/deuterium exchange mass spectrometry (HDX-MS) studies would be ideal. HDX-MS studies were proposed in the original application, but with a priority less than we now appreciate is necessary. HDX-MS studies will be crucial for identifying Wnt binding sites on the extracellular domains of pseudokinase RTKs and, even more importantly, for the receptors on the Wnt surface. Preliminary studies of the latter question have shown that we need very good peptide coverage of the highly disulfide-bonded Wnts in order to locate bound regions. HDX-MS is also invaluable for assessing the conformational effects of small molecule modulators and mutations on the intracellular pseudokinase domains and for mapping their sites of interaction with putative signaling effectors. Our advances with the project to date have revealed the need for high resolution HDX-MS studies that we cannot currently achieve at Yale, so we are requesting funds towards a Synapt G2-Si mass spectrometer that can provide the peptide separation needed to map binding sites to near single amino-acid resolution. Our preliminary data and the work of others suggest that this advance in our technological arsenal will advance this project ? and our understanding of these poorly characterized Wnt receptors ? dramatically. We hope that this understanding will also bring new opportunities for therapeutic targeting of pseudokinases such as PTK7, Ror2, and Ror1, which have been implicated in several orphan diseases. The requested instrument will also allow significant advances in other NIGMS-funded grants whose PIs are submitting cross-referenced Administrative Supplement applications: Titus Boggon and Benjamin Turk (MPI: R01-GM102262), Karen Anderson (R01- GM049551), and Ya Ha (R01-GM112778), supported with funds from the Yale Cancer Biology Institute.
Alterations in Wnt signaling cause a wide variety of diseases from bone diseases to defects in wound healing, to cancer. There are many types of Wnt receptor, including a subset of receptors in the receptor tyrosine kinase superfamily that are very poorly understood. Applying methods that have yielded valuable insight for other receptor tyrosine kinases ? including novel therapeutic approaches ? our proposed research aims to understand how Wnts regulate this class of receptors, adding important knowledge to Wnt signaling and revealing new therapeutic opportunities. The request in this supplement would advance the resolution of a key method for observing structural dynamics.
