Sending and receiving Hedgehog and Wnt signals

申请下列项目的行政补编： 5R01 GM122920-6“发送和接收Hedgehog和Wnt信号” 摘要 胚胎发生和成体生理学是由几个细胞-细胞信号通路协调的[1]。两个人 作为本提案重点的通路，Hedgehog(HH)和WNT，在 发展[2]和成人干细胞维持[3]。HH或WNT信号不充分是导致大量 出生缺陷[4]，而过度激活与许多癌症有关[3a，5]。HH和WNT的共同特征 信号是它们都是由脂质修饰的分泌蛋白[6]触发的：HH配体被修饰为 胆固醇[7]和棕榈酸酯[8]，而WNTs用棕榈酸酯/棕榈酸酯[9]修饰。虽然唇化是必不可少的 对于信号功能，它使HH和Wnt配体强烈附着在膜上[10]，提出了一个问题：如何 它们的传播远远不是生产细胞。我们首先研究了HH配体Sonic Hedgehog的这个问题 (嘘)[11]。我们发现Shh以一种可溶的、有效的复合体的形式与分泌的载体蛋白一起释放， Scube2[12]，且Scube2-Shh形成由分派的转运体1[13](Disp1)催化，动力为 质膜Na+梯度[14]。Scube2-Shh然后将Shh传递给其受体，即肿瘤抑制因子 膜蛋白Patched1(Ptch1)[15]，触发HH途径激活[16]。我们发现Shh的合作- 受体，包括同源的膜蛋白Cdon和Boc以及无关的GPI锚定 Gas1[17]，是将Shh从立方体2移动到ptch1[18]所必需的。我们的结果定义了一条路径， CDON/Boc向细胞表面募集Scube2-Shh，之后Gas1促进Shh向ptch1转移。最后，我们 最近发现了两个分泌蛋白家族，它们作为Wnt的新释放因子和载体发挥作用 配体，与Shh的释放机制平行。我们的发现打开了几个关于HH和 WNT信令。我们建议将生物化学、结构、化学和细胞生物学结合起来，以完成 以下目标：1)准确地确定Shh是如何从生产细胞的细胞膜中释放出来的；2) 阐明Gas1如何催化Shh-ptch1复合体的形成，从而触发HH信号；3)确定Wnt如何 配体被分泌，然后被输送到反应细胞。 进行这些研究很重要，原因如下：a)它们将阐明Shh的途径 分泌和Shh-ptch1复合体组装，这是HH信号转导中两个关键但知之甚少的步骤；B)它们 将定义从细胞中释放Wnt配体的新机制以及它们如何激活细胞内的信号 反应细胞；C)他们将定义新的靶点来阻断癌症中的HH和WNT信号，例如他们的 特定的释放和传递途径；以及D)我们用于研究脂质的新型化学探针将被广泛地 适用于胆固醇和脂肪酸的细胞生物学。

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.