The research proposed in this MIRA renewal application seeks to understand mechanisms of transmembrane signaling by members of the receptor tyrosine kinase (RTK) superfamily – and other receptor-like kinases – that do not fit the ‘rules’ that explain most RTKs. Non-canonical receptors in this group play important roles in human disease – from neurodevelopmental disorders, to bone diseases, cancers, and congenital malformations. The traditional model for RTK signaling involves ligand-induced receptor dimerization, which promotes tyrosine autophosphorylation of the receptor and recruitment of downstream signaling molecules. As we understand more about the 58 human RTKs, it has become clear that this mechanism only applies to about half of them. For example, 10% of human RTKs are linked to WNT signaling, and must function differently because their intracellular regions often have only catalytically inactive ‘pseudokinase’ domains. Still other RTKs (such as ALK) have unique extracellular regions for which ligand binding and receptor regulation are poorly understood. We have made significant progress in understanding extracellular and intracellular structures of WNT-regulated pseudokinase RTKs. Their extracellular WNT-binding modules differ significantly from their counterparts in proteins involved in ‘canonical’ WNT signaling, in ways that suggest well-defined hypotheses for how these RTKs might participate as co-receptors with Frizzled receptors in b-catenin independent WNT signaling. The intracellular pseudokinase domains of the RTKs structurally resemble the insulin receptor kinase in its ‘inactive’ conformation, and analysis of their structural dynamics suggests that they can undergo the same ‘inactive-like’ to ‘active-like’ conformational transitions seen for normal tyrosine kinase domains. Indeed, we have found that these transitions can be promoted by small molecule kinase inhibitor-like molecules, despite the fact that the isolated pseudokinase domains do not bind ATP. In the next 5-10 years of this project, we propose to test hypotheses for WNT-induced assembly of receptor complexes that incorporate pseudokinase RTKs, elucidate their specific WNT dependence and reliance on WNT acylation, gain high resolution structural views through crystallography and EM, and analyze their signaling properties. We will test hypotheses for how the pseudokinase domains contribute to signaling – either by acquiring kinase activity or by functioning as allosterically switchable interaction platforms, with precedents in other pseudokinases and in catalytically active kinases such as Aurora A. In parallel with these efforts, we will investigate new opportunities for therapeutic targeting of pseudokinase RTKs such as PTK7, ROR1, ROR2, and RYK, which have all been implicated in numerous diseases. Together, these studies will provide important new insight into signaling by receptors that do not fit normal paradigms for RTKs or WNT receptors. The new lessons should a...