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: 8423070
• 负责人: ROBERTO DOMINGUEZ
• 金额: $ 38.02万
• 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/8423070
• 关键词: AMPA Receptors; Actins; Address; Binding; Biochemical; Cells; Complex; Cytoskeleton; Dimerization; Drug Targeting; 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 肌动蛋白细胞骨架动力学和膜动力学通常相互连接并严格调节。酒吧 含域的蛋白质正在成为信号传导，细胞骨架和 膜。条形域是发生的二聚体化，膜外感应/诱导模块 在模块化蛋白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性，自动抑制性， 和信号模块。 虽然对棒域蛋白质的研究最近已加强，但严重缺乏的是一个全面的 对膜结合，细胞骨架调节之间相互作用的结构功能理解 和信号活动，这是该提案的目标。最初，重点将放在三种蛋白质上：pick1， IRSP53和PINB。 Pick1已成为神经元细胞中AMPA受体运输的关键调节剂，A 与突触可塑性，学习和记忆相关的过程。 IRSP53丰富了突触，是 与形成神经元棘和细胞突起的形成，例如层状脂蛋白和丝状菌素。 PINB从未被描述过，但是这里介绍的初步研究表明，它稳定了 刷上皮细胞的边界膜。 IRSP53和PINB具有中等序列身份（24％），并且 将同时研究，因为预计这两种蛋白质具有相似的功能 机制和约束伙伴。 AIM 1将测试来自初步研究的假设，即Pick1充当脚手架连接的假设 膜囊泡和肌球蛋白电机用于神经元中的受体运输。另一个假设由 将要测试的试点研究是，PICS1在静止状态下内部自动抑制，并成为 通过其各个域与受体尾巴，膜和肌球蛋白的协调相互作用激活 电动机。 AIM 2将检验以下假设：PINB代表一种根本上新型的条形域蛋白质，即 参与上皮细胞中平面膜结构的形成。自动抑制的机制 将研究IRSP53和PINB的Rho-Family GTPases的激活。 SH3的约束伙伴 PINB的结构域将在细胞中识别，它们的相互作用将被表征。 广泛的初步结果为这些研究奠定了基础。几乎所有蛋白质构建体都有 被表达和表征。全长pick1用绑定的Ca2+和Glur2 Ampa结晶 受体尾巴。 PINB的条形域的结构几乎完成了。关于协作的细胞研究 Pick1和Pinb已经产生了重要的结果，更重要的是， 细胞和结构/生物物理研究开始产生新的假设。