BAR proteins linking membrane and cytoskeleton dynamics

连接膜和细胞骨架动力学的 BAR 蛋白

• 批准号: 8247180
• 负责人: ROBERTO DOMINGUEZ
• 金额: $ 39.6万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-15 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8247180
• 关键词: AMPA Receptors; Actins; Address; Binding; Biochemical; Cells; Complex; Cytoskeleton; Dimerization; Drug Delivery Systems; Epithelial Cells; Family; Feedback; Filopodia; Goals; Guanosine Triphosphate Phosphohydrolases; Health; Human; Intestines; Knowledge; Lead; Learning; Length; Link; Long-Term Depression; Medical; Membrane; Memory; Methods; Molecular; Morphology; Motor; Myosin ATPase; Neurons; Peptides; Pilot Projects; Play; Process; Proteins; Research; Rest; Role; SH3 Domains; Shapes; Signal Transduction; Structure; Synapses; Synaptic plasticity; Tail; Tertiary Protein Structure; Testing; Vertebral column; Vesicle; brush border membrane; link protein; mouse Prkcabp protein; protein structure function; receptor; rho; scaffold; trafficking

Project Summary Actin cytoskeleton dynamics and membrane dynamics are often interconnected and tightly regulated. BAR domain-containing proteins are emerging as a critical linkage between signaling, the cytoskeleton and membranes. The BAR domain is a dimerization, membrane-curvature sensing/inducing module that occurs in modular proteins in association with other domains, including actin cytoskeleton regulatory, auto-inhibitory, and signaling modules. While the study of BAR domain proteins has recently intensified, what is critically lacking is a comprehensive structure-function understanding of the interplay between their membrane-binding, cytoskeleton-regulatory and signaling activities, which is the goal of this proposal. Initially, the focus will be on three proteins: PICK1, IRSp53 and PInB. PICK1 has emerged as a key regulator of AMPA receptor trafficking in neuronal cells, a process linked to synaptic plasticity, learning, and memory. IRSp53 is enriched in synapses, and is implicated in the formation of neuronal spines and cellular protrusions such as lamellipodia and filopodia. PInB had never been characterized, but preliminary studies presented here suggest that it stabilizes the brush border membrane of epithelial cells. IRSp53 and PInB share moderate sequence identity (24%), and will be studied in parallel, because it is anticipated that these two proteins share similar functional mechanisms and binding partners. Aim 1 will test the hypothesis, emerging from preliminary studies, that PICK1 functions as a scaffold linking membrane vesicles and myosin motors for receptor trafficking in neurons. Another hypothesis suggested by the pilot studies that will be tested is that PICK1 is internally auto-inhibited in the resting state, and becomes activated by coordinated interactions of its various domains with receptor tails, membranes and myosin motors. Aim 2 will test the hypothesis that PInB represents a fundamentally new type of BAR domain protein, involved in the formation of planar membrane structures in epithelial cells. The mechanisms of auto-inhibition and activation by Rho-family GTPases of IRSp53 and PInB will be investigated. Binding partners of the SH3 domain of PInB will be identified in cells, and their interactions will be characterized. Extensive preliminary results lay the groundwork for these studies. Nearly all the protein constructs have been expressed and characterized. Full-length PICK1 was crystallized with bound Ca2+ and the GluR2 AMPA receptor tail. The structure of the BAR domain of PInB is nearly finished. Collaborative cellular studies on PICK1 and PInB have already produced important results and, more importantly, the feedback between the cellular and structural/biophysical studies is beginning to generate new hypotheses.

项目摘要 肌动蛋白的细胞骨架动力学和膜动力学往往是相互关联的，并且受到严格的调控。酒吧 含有结构域的蛋白质正在成为信号、细胞骨架和信号之间的关键联系 膜。杆状结构域是一种二聚化、膜曲率感应/诱导模块，它发生在 在与其他结构域相关的模块化蛋白中，包括肌动蛋白细胞骨架调节、自身抑制、 和信令模块。 虽然对BAR结构域蛋白的研究最近有所加强，但严重缺乏的是一种全面的 对它们的膜结合、细胞骨架调节之间相互作用的结构和功能的理解 以及信号传递活动，这是本提案的目标。最初，重点将放在三种蛋白质上：PICK1， IRSP53和PInB。PICK1已成为神经细胞AMPA受体运输的关键调节因子， 与突触可塑性、学习和记忆相关的过程。IRSP53富含突触，是 与神经元棘突和细胞突起的形成有关，如片状和丝状突起。 PInB从未被表征，但这里提出的初步研究表明，它稳定了 上皮细胞刷状缘膜。IRSP53和PInB有中等的序列同源性(24%)，以及 将进行平行研究，因为预计这两种蛋白质具有相似的功能 机制和有约束力的伙伴。 目标1将检验从初步研究中产生的假设，即PICK1起着连接脚手架的作用 神经元中受体运输的膜小泡和肌球蛋白马达。另一种假设是 将被测试的先导研究是PICK1在静息状态下内部自动抑制，并成为 通过其不同结构域与受体尾巴、膜和肌球蛋白的协调作用而激活 摩托公司。 目标2将检验PInB代表一种全新类型的BAR结构域蛋白的假设， 参与上皮细胞中平面膜结构的形成。自我抑制的机制 并将研究IRSP53和PInB的Rho家族GTP酶的激活情况。SH3的结合伙伴 PInB的结构域将在细胞中确定，并将描述它们之间的相互作用。 广泛的初步结果为这些研究奠定了基础。几乎所有的蛋白质结构都有 已被表达和表征。全长PICK1与结合的钙离子和GluR2 AMPA结晶 受体尾巴。PInB的棒状结构域的结构基本完成。协作性细胞研究 PICK1和PINB已经取得了重要成果，更重要的是， 细胞和结构/生物物理研究开始产生新的假说。