Hedgehog Signal Transduction Across the Cell Membrane

跨细胞膜的刺猬信号转导

• 批准号: 7019386
• 负责人: LAWRENCE LUM
• 金额: $ 30.22万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-01 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7019386
• 关键词: Drosophilidae; RNA interference; animal tissue; biological signal transduction; cell membrane; cell surface receptors; cytoplasm; double stranded RNA; genetic library; genetic regulation; membrane proteins; molecular dynamics; phosphorylation; protein protein interaction; protein transport; proteoglycan; sonic hedgehog gene /protein; tissue /cell culture; transcription factor

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.

描述（由申请人提供）：刺猬蛋白（Hh）家族通过在产生Hh的细胞周围的细胞中引发分级反应来协调多种发育环境中的细胞生长和分化。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) 等常见癌症有关。因此，了解 Hh 信号如何从细胞外环境传递到细胞内效应器对于成功预防、检测和治疗这些疾病以及与 Hh 信号传导相关的其他疾病至关重要。该提案重点关注细胞识别质膜上分泌的 Hh 蛋白以及随后跨膜转导信号以诱导特定细胞内反应的机制。 Hh 与十二跨膜蛋白 Patched (Ptc) 的直接相互作用是放弃 Ptc 介导的对靶细胞中七跨膜蛋白 Smoothened (Smo) 的抑制所必需的。这种相互作用是由 Dally 样蛋白 (Dlp)（一种硫酸乙酰肝素蛋白聚糖）和 CG9211（免疫球蛋白 (Ig) 受体超家族成员）促进的。激活的 Smo 跨膜转导信号，该过程涉及将包含转录因子 Cubitus Interruptus (Ci) 的大型调节复合物募集到其细胞质尾部。 Dlp、CG9211 和这些细胞质蛋白-蛋白相互作用促进 Hh 通路反应的机制尚不清楚。为了研究 Hh 信号在细胞膜上的接收和传输，我们开发了新的工具，包括基于培养细胞的通路反应性测定、用于分离和检测 Hh 信号复合物的试剂以及抑制特异性 Hh 信号复合物的双链 RNA 文库。通过RNA介导的干扰（RNAi）在果蝇和小鼠中发挥基因功能。通过将这些工具纳入基于生化和遗传学的策略，我们建议：1）确定细胞膜如何感知 Hh 信号，2）确定 Smo 如何将 Hh 信号转导至细胞质成分，以及 3）识别 Hh 通路使用基于 RNAi 的技术系统地测试基因功能。