PROJECT SUMMARY/ABSTRACT The Wnt5a-Ror signaling pathway is essential for embryonic tissue morphogenesis. Disruption of Wnt5a-Ror signaling results in birth defects such as Robinow syndrome and Brachydactyly Type B, and is frequently implicated in cancer metastasis and inflammatory disorders. The Wnt5a protein is palmitoleoylated at a conserved serine, a unique feature shared by all the Wnt ligands, which require the GPCR-like protein Wntless to transport their lipid-modified mature forms from the endoplasmic reticulum to the plasma membrane for secretion. In contrast to the canonical Wnts whose mechanisms are relatively well characterized, Wnt5a signals noncanonically through the ROR family of receptor tyrosine kinases (ROR1/2), which is independent of the transcriptional factor β-catenin. Despite its physiological and pathological importance, the mechanisms of Wnt5a- Ror signaling remain poorly understood. The major unanswered questions include how Wnt5a is secreted, how Wnt5a activates RORs and how active RORs transduce Wnt5a signals to the cytoplasm. In preliminary work, we determined a Cryo-EM structure of Wnt5a in complex with Wntless that reveals an unanticipated feature of Wnt5a with important implications for its non-canonical function. We showed that purified extracellular domain (ECD) of ROR2 promotes Wnt5a secretion, together with the recent crystal structure of Drosophila ROR2 ECD bound with palmitoleic acid, suggesting that ROR2 directly recognizes the Wnt5a lipid. In addition, we developed a peroxidase APEX2-catalyzed proximity labeling approach, and by quantitative multiplexed proteomics, we demonstrated that for the first time, the recruitment of the downstream effector protein Dishevelled (DVL) to the ROR receptors is exclusively dependent on Wnt5a stimulation. Based on these results, we propose to combine biochemistry, structural, cell biology and proteomic studies to accomplish the following aims: A) To characterize how Wntless transports Wnt5a, and how Wntless is recycled after Wnt5a secretion; B) To determine how Wnt5a binds and activates the ROR receptors; and C) To elucidate how active ROR receptors trigger the downstream cytoplasmic signaling events. These studies will have high impacts for the following reasons: 1) They will advance our mechanistic understanding of Wnt5a-Ror signaling; 2) They will help explain the pathogenesis of human diseases caused by abnormal Wnt5a-Ror signaling; 3) They will facilitate the therapeutic discoveries by providing novel targets and strategies to treat Wnt5a-associated cancers; and 4) The APEX2-catalyzed proximity labeling and proteomic approach that we developed will have broad applications to study signal transduction pathways beyond Wnt5a-Ror signaling.