Project summary The Hedgehog cell-cell signaling pathway is essential for embryonic development, for adult stem cell maintenance, and is deeply involved in human cancer and birth defects. The pathway is activated by the Hedgehog ligand, a secreted protein uniquely modified with two lipids, which are both essential for function: a fatty acid and cholesterol. From the producing cell, the Hedgehog ligand spreads to distant cells, on the surface of which it binds its receptor, a tumor suppressor membrane protein called Patched, thus triggering a specific set of cellular responses. A puzzling aspect of the Hedgehog ligand is that it spreads and signals at a distance, in spite of the fact that its lipid modifications make it stick strongly to membranes. Many critical aspects of the Hedgehog ligand remain obscure: we do not know how it is released from cells, how it is able to diffuse outside the cell, and how it is delivered to responding cells. We recently discovered that Hedgehog ligand release is accomplished by two synergistic interactions involving its cholesterol modification: one with the membrane protein Dispatched, and the second with the secreted protein Scube. Using novel chemical probes, we found that Dispatched and Scube recognize cholesterol differently, suggesting that the Hedgehog ligand is handed off from Dispatched to Scube. We also discovered that Scube is important for ligand reception, via Cdon and Boc, two proteins critical for Hedgehog signaling; this suggested a novel mechanism for Hedgehog ligand delivery. Finally, we found that Gas1, another protein important for Hedgehog ligand reception, interacts with the ligand in a unique way, dependent on its fatty acid modification; this indicated that Gas1 uses a mechanism distinct from Cdon and Boc. We propose to use a combination of biochemistry, cell biology, and chemical biology to accomplish the following aims: A) To elucidate precisely how the Hedgehog ligand is released form the membrane of producing cells B) To determine how Scube delivers the Hedgehog ligand to responding cells via Cdon and Boc C) To elucidate how Gas1 participates in Hedgehog ligand reception These studies are important to undertake for the following reasons: 1) They will advance our understanding of the Hedgehog pathway, by elucidating the route taken by the Hedgehog ligand during the signaling process; 2) They will identify novel targets for blocking Hedgehog signaling in cancer, based on the mechanisms of Hedgehog ligand release and delivery; and 3) Our novel chemical probes will be broadly applicable to study cholesterol and fatty acids in health and in disease.