Deciphering the functional role of actin-spectrin-based membrane skeleton in subcellular compartmentalization of signaling proteins and cell signal transduction

项目概要/摘要： 受体酪氨酸激酶（RTK）、G蛋白偶联受体（GPCR）和细胞粘附分子（CAM） 是所有真核细胞中细胞表面蛋白的三个主要家族，它们共同代表了 细胞应答细胞外刺激，并启动多种信号通路， 随后调节细胞增殖和分化，促进细胞存活，并调节细胞 代谢和细胞间通讯。影响这些信号通路的突变导致许多人类 综合征和疾病，如各种类型的神经退行性疾病和癌症。临床 这些信号蛋白的重要性促使了靶向治疗的发展， 膜受体的激活和下游信号转导。越来越多的证据 这表明，有显着的信号串扰之间的这三个膜蛋白家族在 这些蛋白质可以形成高度组织化的膜微簇或纳米簇， 独特的生物化学和生物物理特性，决定了信号转导的结果。然而，分子 这种膜相关信号蛋白的相互作用和区室化的机制 被启动并维持以调节下游信号传导的敏感性和特异性， 难以捉摸。我们最近发现了一种新鉴定的基于肌动蛋白-血影蛋白的膜相关周期性骨架 (MPS)作为神经元中GPCR和CAM的RTK反式激活的信号平台的结构提供了 这些细胞表面蛋白质之间的协同作用如何协调， 上升到下游信号。该提案的目的是将超分辨率成像、细胞和细胞成像技术联合收割机结合起来， 分子生物学工具和质谱分析，以研究独特的物理分子 通过识别关键的分子相互作用， 负责信号蛋白簇的MPS依赖性组装和分解（即，信令 蛋白质复合物），并检查液-液相分离、受体内吞和接触的作用 位点之间的质膜和细胞内膜结合的细胞内细胞器在MPS- 介导神经元中的细胞信号。由于基于血影蛋白-肌动蛋白的MPS结构可能存在于其他分化的细胞中， 细胞类型，如淋巴细胞，从而控制淋巴细胞的发育和活化，我们的分析将 也可以扩展到检查MPS在免疫应答期间淋巴细胞信号传导中的作用。 拟议的研究不仅将拓宽我们对细胞信号转导的基本理解， 神经元和免疫细胞的膜骨架和信号蛋白的相分离行为， 而且还有助于为包括神经变性疾病在内的人类疾病提供潜在的药物靶点， 癌

Project Summary/Abstract: Receptor tyrosine kinases (RTKs), G-protein-coupled receptors (GPCRs), and cell adhesion molecules (CAMs) are three major families of cell surface proteins in all eukaryotic cells, and together represent the primary first responders for cells to respond an extracellular stimulus and initiates a variety of signaling pathways to subsequently regulate cell proliferation and differentiation, promote cell survival, and modulate cellular metabolism and cell-to-cell communication. Mutations affecting these signaling pathways result in many human syndromes and diseases, such as various types of neurodegenerative disorders and cancer. The clinical importance of these signaling proteins has motivated the development of targeted therapies designed to block the activation of the membrane receptors and the downstream signal transduction. Increasing evidence has suggested that there is significant signaling crosstalk among these three membrane protein families at the plasma membrane level and these proteins can form highly organized membrane micro- or nano-clusters with unique biochemical and biophysical properties, dictating the signaling outcome. However, the molecular mechanisms by which how such crosstalk and compartmentalization of membrane-associated signaling proteins are initiated and maintained to modulate the sensitivity and specificity of the downstream signaling remain largely elusive. Our recent discovery of a newly identified actin-spectrin-based membrane-associated periodic skeleton (MPS) structure being a signaling platform for RTK transactivation by GPCRs and CAMs in neurons provides molecular insights into how the cooperative action among these cell surface proteins can be coordinated to give rise to the downstream signaling. The objective of this proposal is to combine super-resolution imaging, cell and molecular biology tools, and mass spectrometry analyses, to investigate the distinctively physical molecular mechanisms responsible for the MPS-mediated cell signaling, by identifying the key molecular interactions responsible for the MPS-dependent assembly and disassembly of the signaling protein clusters (i.e., signaling protein complexes) and examining the roles of liquid-liquid phase separation, receptor endocytosis, and contact sites between the plasma membrane and intracellular membrane-bound intracellular organelles in the MPS- mediated cell signaling in neurons. As the spectrin-actin based MPS structures likely exist in other differentiated cell types such as lymphocytes and thereby control lymphocyte development and activation, our analyses will also be extended to examine the role of the MPS in lymphocyte signaling during immune responses Our proposed research will not only broaden our fundamental understanding of cell signal transduction controlled by the membrane skeleton and the phase separation behaviors of signaling proteins in neurons and immune cells, but also help suggest potential drug targets for human diseases including neurodegenerative diseases and cancer.