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相互关联并受到严格调节。含BAR结构域的蛋白是信号传导、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，发生在模块蛋白中，与其他结构域（包括肌动蛋白细胞骨架调节、自抑制和信号传导模块）相关。虽然BAR结构域蛋白的研究最近得到了加强，但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，这是本提案的目标。最初，重点将放在三个蛋白上：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，并与神经元棘和细胞突起（如板足和丝状足）的形成有关。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边界膜。IRSp53和PInB具有中等的序列同源性（24%），并且将并行研究，因为预计这两个蛋白具有相似的功能机制和结合伙伴。Aim 1将验证从初步研究中得出的假设，即PICK1作为连接膜囊泡和肌球蛋白马达的支架，用于神经元中的受体运输。试点研究提出的另一个有待验证的假设是，PICK1在静息状态下是内部自动抑制的，并通过其与受体尾部、膜和肌球蛋白马达的各种结构域的协调相互作用而被激活。目的2将验证一个假设，即PInB是一种全新的BAR结构域蛋白，参与上皮细胞平面膜结构的形成。IRSp53和PInB的rho家族gtpase的自抑制和激活机制将被研究。将在细胞中鉴定PInB的SH3结构域的结合伙伴，并对它们的相互作用进行表征。广泛的初步结果为这些研究奠定了基础。几乎所有的蛋白质结构都已被表达和表征。全长PICK1与结合的Ca2+和GluR2 AMPA受体尾部结晶。PInB的BAR结构已基本完成。对PICK1和PInB的协同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。

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.