Hedgehog Signal Transduction Across the Cell Membrane

描述（由申请人提供）：Hedgehog（Hh）蛋白家族通过在Hh产生细胞周围的细胞中引发分级反应，在许多发育环境中协调细胞生长和分化。异常的Hh通路活性与面部和前脑的发育畸形以及前列腺癌和基底细胞癌（BCC）等流行癌症有关。因此，了解Hh信号如何从细胞外环境传递到细胞内效应物对于成功预防、检测和治疗这些和其他与Hh信号传导相关的疾病至关重要。该建议的重点是允许细胞识别分泌的Hh蛋白在质膜和随后的信号跨膜转导，以诱导特定的细胞内反应的机制。Hh与十二跨膜蛋白Patched（Ptc）的直接相互作用是解除Ptc介导的对靶细胞中七跨膜蛋白Smoothened（Smo）的抑制所必需的。这种相互作用由Dally样蛋白（Dlp）（一种硫酸乙酰肝素蛋白聚糖）和CG 9211（一种免疫球蛋白（IG）受体超家族成员）促进。激活的Smo在一个过程中跨膜转导信号，该过程涉及将包括转录因子Cubitus interruptus（Ci）的大型调节复合物募集到其胞质尾。Dlp、CG 9211和这些细胞质蛋白-蛋白相互作用促进Hh通路应答的机制尚不清楚。为了研究Hh信号在细胞膜上的接收和传递，我们开发了新的工具，包括基于培养细胞的途径响应性测定，用于分离和检测Hh信号复合物的试剂，以及通过RNA介导的干扰（RNAi）抑制果蝇和小鼠中特定基因功能的双链RNA文库。通过将这些工具结合到基于生物化学和遗传学的策略中，我们提出：1）确定Hh信号如何在细胞膜上被感知，2）确定Smo如何将Hh信号转导到细胞质组分，以及3）通过使用基于RNAi的技术系统地测试基因功能来鉴定Hh通路组分。

DESCRIPTION (provided by applicant): The Hedgehog (Hh) family of proteins coordinates cellular growth and differentiation in a number of developmental contexts by eliciting graded responses in the cells surrounding Hh-producing cells. Aberrant Hh pathway activity is associated with developmental deformities of the face and forebrain, and prevalent cancers such as prostate cancer and basal cell carcinoma (BCC). Understanding how the Hh signal is conveyed from the extracellular milieu to intracellular effectors is therefore pivotal to the successful prevention, detection, and treatment of these and other diseases associated with Hh signaling. This proposal focuses on the mechanisms that allow cellular recognition of the secreted Hh protein at the plasma membrane and subsequent transduction of a signal across the membrane to induce specific intracellular responses. Direct interaction of Hh with the twelve transmembrane protein Patched (Ptc) is required for relinquishing Ptc-mediated suppression of the seven transmembrane protein Smoothened (Smo) in target cells. This interaction is facilitated by Dally-like protein (Dlp), a heparan sulfate proteoglycan, and CG9211, a member of the immunoglobulin (Ig) superfamily of receptors. Activated Smo transduces a signal across the membrane in a process that involves recruiting to its cytoplasmic tail a large regulatory complex that includes the transcription factor Cubitus interruptus (Ci). The mechanisms by which Dlp, CG9211, and these cytoplasmic protein-protein interactions contribute to Hh pathway response are unclear. In order to study reception and transmission of the Hh signal at the cell membrane, we have developed novel tools that include cultured cell-based assays for pathway responsiveness, reagents for isolating and detecting Hh signaling complexes, and double-stranded RNA libraries that inhibit specific gene function in Drosophila and mouse by RNA-mediated interference (RNAi). By incorporating these tools in a biochemically- and genetically-based strategy, we propose to: 1) determine how Hh signal is sensed at the cell membrane, 2) determine how Smo transduces the Hh signal to cytoplasmic components, and 3) identify Hh pathway components by systematically testing gene function using RNAi-based technology.