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: 8010561
• 负责人: ROBERTO DOMINGUEZ
• 金额: $ 39.96万
• 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 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8010561
• 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; public health relevance; receptor; rho; scaffold; trafficking

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: BAR domain-containing proteins are emerging as a critical linkage between signaling, the cytoskeleton and membranes. Two of the proteins studied here play critical roles in neuronal function, including spine morphology and synaptic plasticity, processes underlying learning and memory. The third protein, PInB, appears to stabilize the brush border membrane of intestinal epithelial cells. This research will lead to a better understanding of BAR protein structure-function, and could have medical applications. PICK1, in particular, is a recognized drug target, and has been implicated in long-term depression, such that knowledge of it structure may have a potential impact on human health.

描述（由申请人提供）：肌动蛋白细胞骨架动力学和膜动力学通常相互关联并受到严格调控。含 BAR 结构域的蛋白质正在成为信号传导、细胞骨架和细胞膜之间的关键联系。 BAR 结构域是一种二聚化、膜曲率传感/诱导模块，出现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、自动抑制和信号传导模块。虽然最近对 BAR 结构域蛋白的研究有所加强，但目前严重缺乏对其膜结合、细胞骨架调节和信号传导活动之间相互作用的全面结构功能理解，而这正是本提案的目标。最初，重点将放在三种蛋白质上：PICK1、IRSp53 和 PInB。 PICK1 已成为神经元细胞中 AMPA 受体运输的关键调节因子，这一过程与突触可塑性、学习和记忆有关。 IRSp53 在突触中富集，并参与神经元棘和细胞突起（例如板状伪足和丝状伪足）的形成。 PInB 从未被表征过，但本文提出的初步研究表明它可以稳定上皮细胞的刷状缘膜。 IRSp53 和 PInB 具有中等的序列同一性 (24%)，并将并行研究，因为预计这两种蛋白质具有相似的功能机制和结合伙伴。目标 1 将检验初步研究中提出的假设，即 PICK1 作为连接膜囊泡和肌球蛋白马达的支架，用于神经元中受体运输。将要测试的试点研究提出的另一个假设是，PICK1 在静息状态下是内部自动抑制的，并通过其各个结构域与受体尾部、膜和肌球蛋白马达的协调相互作用而被激活。目标 2 将检验以下假设：PInB 代表一种全新类型的 BAR 结构域蛋白，参与上皮细胞平面膜结构的形成。将研究 IRSp53 和 PInB 的 Rho 家族 GTPases 的自动抑制和激活机制。 PInB SH3 结构域的结合伴侣将在细胞中被鉴定，并且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。几乎所有的蛋白质构建体都已被表达和表征。全长 PICK1 与结合的 Ca2+ 和 GluR2 AMPA 受体尾部一起结晶。 PInB的BAR结构域的结构已接近完成。关于 PICK1 和 PInB 的合作细胞研究已经产生了重要的结果，更重要的是，细胞和结构/生物物理研究之间的反馈开始产生新的假设。 公共健康相关性：含 BAR 结构域的蛋白质正在成为信号传导、细胞骨架和细胞膜之间的关键联系。这里研究的两种蛋白质在神经元功能中发挥着关键作用，包括脊柱形态和突触可塑性，以及学习和记忆的基础过程。第三种蛋白质 PInB 似乎可以稳定肠上皮细胞的刷状缘膜。这项研究将有助于更好地了解 BAR 蛋白质的结构-功能，并可能具有医学应用。尤其是 PICK1，它是公认的药物靶点，并且与长期抑郁症有关，因此了解其结构可能对人类健康产生潜在影响。