Hedgehog Signal Transduction Across the Cell Membrane

Hedgehog（Hh）蛋白家族在许多细胞中协调细胞生长和分化。 通过在产生Hh的细胞周围的细胞中引发分级反应来调节发育环境。异常 Hh通路活动与面部和前脑的发育畸形有关，并且普遍存在 癌症如前列腺癌和基底细胞癌（BCC）。了解Hh信号是如何 因此，从细胞外环境传递到细胞内效应物的蛋白质是成功的关键。 预防、检测和治疗这些和其他与Hh信号传导相关的疾病。这项建议 集中在机制，使细胞识别分泌的Hh蛋白在血浆中， 细胞膜上的信号转导，并随后跨膜转导信号，以诱导特异性细胞内 应答 Hh与十二跨膜蛋白Patched（Ptc）的直接相互作用是必需的， 解除Ptc介导的对靶细胞中七跨膜蛋白Smoothened（Smo）的抑制 细胞这种相互作用由Dally样蛋白（Dip）（一种硫酸乙酰肝素蛋白聚糖）和CG 9211（一种抗肿瘤药物）促进。 受体免疫球蛋白（IG）超家族成员。激活的Smo将信号传导到 在一个过程中，涉及招募到它的细胞质尾一个大的调节复合物，包括 转录因子Cubitus interruptus（Ci）。Dip、CG 9211和这些药物的作用机制 胞质蛋白质-蛋白质相互作用对Hh通路应答的贡献尚不清楚。 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反应性测定， 分离和检测Hh信号传导复合物，以及抑制特定基因的双链RNA文库， 通过RNA介导的干扰（RNAi）在果蝇和小鼠中发挥作用。通过将这些工具整合到 基于生物化学和遗传学的策略，我们建议：1）确定Hh信号是如何被感知的， 细胞膜，2）确定Smo如何将Hh信号转导至细胞质组分，以及3）鉴定 通过使用基于RNA干扰的技术系统地测试基因功能来研究Hh途径组分。

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 (Dip), 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 Dip, 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.