Sending and receiving Hedgehog and Wnt signals

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Application for an Administrative Supplement to: 5R01 GM122920-6 “Sending and receiving Hedgehog and Wnt signals” Abstract Embryogenesis and adult physiology are orchestrated by a handful of cell-cell signaling pathways[1]. The two pathways that are the focus of the present proposal, Hedgehog (Hh) and Wnt, play critical roles in development[2] and adult stem cell maintenance[3]. Insufficient Hh or Wnt signaling is the cause of numerous birth defects[4], while excessive activation is involved in many cancers[3a, 5]. A common feature of Hh and Wnt signaling is that they are both triggered by lipid-modified secreted proteins[6]: Hh ligands are modified with cholesterol[7] and palmitate[8], while Wnts are modified with palmitate/palmitoleate[9]. While lipidation is essential for signaling function, it makes Hh and Wnt ligands strongly membrane-attached[10], raising the question of how they spread far from producing cells. We first investigated this question for the Hh ligand, Sonic hedgehog (Shh)[11]. We discovered that Shh is released as a soluble and potent complex with a secreted carrier protein, Scube2[12], and that Scube2-Shh formation is catalyzed by the transporter Dispatched1[13] (Disp1), powered by the plasma membrane Na+ gradient[14]. Scube2-Shh then delivers Shh to its receptor, the tumor suppressor membrane protein Patched1 (Ptch1)[15], triggering Hh pathway activation[16]. We discovered that the Shh co- receptors, comprising the homologous membrane proteins Cdon and Boc and the unrelated GPI-anchored Gas1[17], are essential for moving Shh from Scube2 to Ptch1[18]. Our results defined a pathway whereby Cdon/Boc recruit Scube2-Shh to the cell surface, after which Gas1 promotes Shh transfer to Ptch1. Finally, we recently discovered two families of secreted proteins that function as novel release factors and carriers for Wnt ligands, paralleling the mechanism of Shh release. Our findings open several critical questions about Hh and Wnt signaling. We propose combining biochemistry, structural, chemical and cell biology to accomplish the following aims: 1) To determine precisely how Shh is released form the membrane of producing cells; 2) To elucidate how Gas1 catalyzes Shh-Ptch1 complex formation, to trigger Hh signaling; 3) To determine how Wnt ligands are secreted and then delivered to responding cells. These studies are important to undertake for the following reasons: A) They will elucidate the pathways of Shh secretion and Shh-Ptch1 complex assembly, two crucial but poorly understood steps in Hh signaling; B) They will define novel mechanisms for the release of Wnt ligands from cells and for how they activate signaling in responding cells; C) They will define novel targets for blocking Hh and Wnt signaling in cancer, such as their specific release and delivery pathways; and D) Our novel chemical probes for studying lipids will be broadly applicable to the cell biology of cholesterol and fatty acids.