BAR proteins linking membrane and cytoskeleton dynamics

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是相互关联和严格调控的。含有BAR结构域的蛋白质正在成为信号、细胞骨架和细胞膜之间的关键联系。BAR结构域是一个二聚化的膜曲率感知/诱导模块，与其他结构域结合在一起存在于模块蛋白中，包括肌动蛋白细胞骨架调节、自身抑制和信号传递模块。虽然最近对bar结构域蛋白的研究有所加强，但严重缺乏对其膜结合、细胞骨架调节和信号活动之间相互作用的全面结构和功能的了解，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已经成为神经细胞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富含突触，与神经元棘突和细胞突起的形成有关，如片状脂膜和丝状突起。PInB从未被表征过，但这里提出的初步研究表明，它能稳定上皮细胞的刷状缘膜。IRSP53和PInB有中等的序列同源性(24%)，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结合伙伴。目的1将验证从初步研究中产生的假设，即PICK1作为连接膜小泡和肌球蛋白马达的支架在神经元中进行受体运输。先导研究提出的另一个假说是，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巴、膜和肌球蛋白马达的协调作用而激活。目的2验证PInB代表一种全新的杆状结构域蛋白的假说，该蛋白参与上皮细胞平面膜结构的形成。我们将研究IRSP53和PInB的Rho家族GTP酶自抑制和激活的机制。PInB的SH3结构域的结合伙伴将在细胞中确定，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。几乎所有的蛋白质结构都已被表达和鉴定。全长PICK1与结合的钙离子和GluR2 AMPA受体尾部结晶。PInB的棒状结构域的结构基本完成。关于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。 与公共卫生相关：含有BAR结构域的蛋白质正在成为信号、细胞骨架和膜之间的关键联系。这里研究的两种蛋白质在神经元功能中发挥关键作用，包括脊椎形态和突触可塑性，以及支持学习和记忆的过程。第三种蛋白质PInB似乎稳定了肠上皮细胞的刷状缘膜。这项研究将有助于更好地了解bar蛋白的结构和功能，并有可能在医学上应用。特别是，PICK1是公认的药物靶点，并与长期抑郁症有关，因此对其结构的了解可能会对人类健康产生潜在影响。

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.