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连接并严格调节。含杆结构域的蛋白质正在作为信号传导，细胞骨架和膜之间的关键联系。条形结构域是一个二聚化，膜曲面传感/诱导模块，它与其他结构域（包括肌动蛋白细胞骨架调节，自身抑制性和信号传导模块）相关的模块化蛋白发生。虽然对棒域蛋白的研究最近已经加强了，但严重缺乏的是对它们的膜结合，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的理解，这是该提案的目标。最初，焦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，这是与突触可塑性，学习和记忆有关的过程。 IRSP53富含突触，与神经元棘的形成和细胞突起（例如薄片和丝状虫）的形成有关。 PINB从未被表征过，但是这里介绍的初步研究表明，它稳定了上皮细胞的刷边界。 IRSP53和PINB具有中等序列身份（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伴侣。 AIM 1将测试来自初步研究的假设，即Pick1充当连接膜囊泡和肌球蛋白电机的脚手架，用于神经元中的受体运输。试验研究提出的另一个假设将被测试，即PICS1在静止状态下内部自动抑制，并通过其各个域与受体尾巴，膜和肌球蛋白电机的协调相互作用而激活。 AIM 2将检验以下假设：PINB代表了一种从根本上说明的条形结构蛋白，该假设与上皮细胞中平面膜结构的形成有关。将研究IRSP53和PINB的Rho-Family GTPases自动抑制和激活的机制。 PINB的SH3结构域的结合伙伴将在细胞中鉴定，并将表征它们的相互作用。广泛的初步结果为这些研究奠定了基础。几乎所有蛋白质构建体都已表达和表征。全长pick1用结合的Ca2+和GlUR2 AMPA受体尾巴结晶。 PINB的条形域的结构几乎完成了。 Pick1和PINB的协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。 公共卫生相关性：含棒域的蛋白质正在成为信号，细胞骨架和膜之间的关键联系。这里研究的两个蛋白质在神经元功能中起关键作用，包括脊柱形态和突触可塑性，即学习和记忆的过程。第三个蛋白质PINB似乎稳定了肠上皮细胞的刷子边界膜。这项研究将使对条形蛋白结构功能的了解更好，并可能具有医疗应用。尤其是Pick1是公认的药物靶标，并且已经与长期抑郁症有关，因此对IT结构的了解可能会对人类健康产生潜在的影响。