Molecular Function of Palladin's Ig Domains in Cell Adhesion and Motility

描述（由申请人提供）：细胞-细胞和细胞-基质粘附都需要肌动蛋白细胞骨架的参与。肌动蛋白丝交联成与细胞-细胞连接相关的周向环和锚定在细胞-基质连接处的收缩应力纤维。Palladin是最近发现的一种蛋白质，其定位于细胞-细胞和细胞-基质连接处，也定位于应力纤维、Z盘和其他富含肌动蛋白的结构。除了细胞培养研究证明palladin在肌动蛋白细胞骨架组装中的重要作用外，另外两条证据表明palladin在肌动蛋白依赖性细胞行为中起着核心作用：（1）palladin对正常哺乳动物发育至关重要，如Palladin敲除小鼠的胚胎致死表型所证明的和（2）人Palladin基因以遗传性胰腺癌的形式突变，这是一种以细胞-细胞粘附丧失和失调的、肌动蛋白依赖性的侵入性细胞运动为特征的疾病。这个建议的目标是了解palladin在组织肌动蛋白阵列的精确分子功能，具体来说，我们将测试的假设，palladin作为一个肌动蛋白交联蛋白，在体外和体内的功能。Palladin以三种主要同种型存在，含有三至五个拷贝的高度保守的Ig样结构域。已发表的其他三个Ig结构域蛋白的研究表明，帕拉丁的IG结构域可能直接结合到f-肌动蛋白。我们已经产生了初步的结果表明，纯化，全长palladin结合f-肌动蛋白，并在体外产生肌动蛋白束。此外，我们的研究结果表明，指定为Ig 3的结构域是最小的palladin片段，结合到肌动蛋白，而Ig 4和Ig 5不结合，并且含有Ig 3 + Ig 4结构域的片段能够交联肌动蛋白丝成束。为了充分研究这种效应的分子基础，我们建议表达，纯化和表征所有的IG结构域的帕拉丁。我们的目的是（1）利用我们最近确定的Ig 3的溶液结构和合理的诱变方法，最终定位palladin的IG结构域中包含的肌动蛋白结合位点，并研究肌动蛋白结合在palladin生物活性中的作用，（2）在体外和体内验证palladin作为肌动蛋白交联蛋白的假设，并通过荧光和电子显微镜确定肌动蛋白产生的束的几何形状，以及（3）检验palladin形成同源二聚体的假设，绘制二聚化结构域，并确定二聚体形成是否是palladin生物学功能的关键。此外，我们将确定palladin的Ig 4结构域的结构，并研究在胰腺癌细胞系中发现的该结构域中的一种新突变。到目前为止，还没有关于palladin或palladin家族成员的IG结构域的结构信息，因此我们提出的研究代表了第一个详细的palladin的IG结构域的结构/功能分析。 公共卫生相关性：肌动蛋白丝形成复杂的亚细胞阵列，对于建立细胞形状、维持相邻细胞之间的粘附以及产生细胞与其下层结缔组织之间的连接至关重要。Palladin是最近描述的蛋白质，其在组织这些肌动蛋白丝阵列中起关键作用，并且Palladin的突变或过表达已经涉及多种病理学，包括致命的出生缺陷，皮肤、脉管系统和神经系统中的瘢痕形成，以及胰腺癌的侵袭。因此，了解palladin的精确分子功能可能会深入了解控制正常细胞中的基本功能和导致病理细胞行为的功能障碍的细胞机制。

DESCRIPTION (provided by applicant): Both cell-cell and cell-matrix adhesions require the participation of the actin cytoskeleton. Actin filaments are crosslinked into circumferential rings that are associated with cell-cell junctions and contractile stress fibers that are anchored at cell-matrix junctions. Palladin is a recently identified protein that localizes to cell-cell and cell-matrix junctions, and also to stress fibers, Z-discs and other actin-rich structures. In addition to cell culture studies that demonstrate palladin's essential role in the assembly of the actin cytoskeleton, two other lines of evidence suggest that palladin plays a central role in actin-dependent cell behaviors: (1) palladin is critically important for normal mammalian development, as evidenced by the embryonic lethal phenotype of the palladin knockout mouse and (2) the human palladin gene is mutated in a form of inherited pancreatic cancer, which is a disease characterized by a loss of cell-cell adhesion and disregulated, actin-dependent, invasive cell motility. The goal of this proposal is to understand the precise molecular function of palladin in organizing actin arrays; specifically, we will test the hypothesis that palladin functions as an actin-crosslinking protein, both in vitro and in vivo. Palladin exists as three major isoforms, containing between three and five copies of a highly conserved Ig-like domain. Published studies of three other Ig-domain proteins suggest that palladin's Ig domains may bind directly to f-actin. We have generated preliminary results showing that purified, full-length palladin binds to f-actin and generates actin bundles in vitro. In addition, our results show that the domain designated Ig3 is the smallest palladin fragment that binds to actin, while Ig4 and Ig5 do not bind, and that a fragment containing the Ig3+Ig4 domains is able to cross-link actin filaments into bundles. To fully investigate the molecular basis for this effect, we propose to express, purify and characterize all of the Ig domains of palladin. Our aims are (1) to conclusively map the actin binding site contained within palladin's Ig domains, using our recently determined solution structure of Ig3 and rational mutagenesis approaches, and to investigate the role of actin-binding in palladin's biological activity, (2) to test the hypothesis that palladin functions as an actin- crosslinking protein in vitro and in vivo, and to determine the geometry of the actin-generated bundles by both fluorescence and electron microscopy, and (3) to test the hypothesis that palladin forms homodimers, map the dimerization domains, and establish if dimer formation is key to palladin's biological function. In addition, we will determine the structure of palladin's Ig4 domain and investigate a novel mutation in this domain that has been identified in a pancreatic cancer cell line. To date, no structural information has been published regarding the Ig domains of palladin or palladin's close family members, such that our proposed research represents the first detailed structure/function analysis of palladin's Ig domains. Public Health Relevance: Actin filaments form complex subcellular arrays that are essential for establishing cell shape, maintaining adhesion between neighboring cells, and generating connections between cells and their underlying connective tissue. Palladin is a recently described protein that plays a key role in organizing these actin filament arrays, and mutation or over-expression of palladin has been implicated in multiple pathologies, including lethal birth defects, scar formation in the skin, vasculature and nervous system, and the invasion of pancreatic cancer. Thus, understanding the precise molecular function of palladin is likely to yield insights into the cellular mechanisms that control both essential functions in normal cells, and dysfunctions that result in pathological cell behavior.