Cellular Functions of the Human Filamins

人类细丝蛋白的细胞功能

• 批准号: 7091565
• 负责人: SANDOR S SHAPIRO
• 金额: $ 41.56万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-15 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7091565
• 关键词: RNA interference; actin binding protein; actins; binding sites; cell biology; clinical research; cytoskeleton; gene expression; gene mutation; gene targeting; human subject; laboratory mouse; melanoma; microarray technology; microfilaments; neoplastic cell; phenotype; phosphatidylinositols; protein binding; protein isoforms; protein structure function; shear stress; yeast two hybrid system; zebrafish

DESCRIPTION (provided by applicant): Platelet shape change and endothelial and vascular smooth muscle migration are central to the processes of hemostasis and thrombosis, angiogenesis and wound healing. Rapid alterations in cytoskeletal structure are required for these responses. Filamins (FLNs) are important in modulating these responses. The human FLN family consists of three approximtely 280 kD paralogs -A, B and C - each containing a approximately 250 residue N-terminal actin-binding domain (ABD) followed by 24 internally homologous repeats. FLNs induce formation of three-dimensional actin networks and connect the cell membrane to the cytoskeleton by binding to the cytoplasmic tails of several integrins and other membrane proteins. FLNs also bind to a number of cellular components, including several small GTPases, and may serve as scaffolds on which intracellular signaling and protein trafficking pathways are organized; these interactions do not require the presence of an ABD. Cell lines lacking FLNA are abnormal in shape and migration and mutations in FLNA, either in the ABD or in the repeats, are associated with human disease. Very recently, our collaborators and we have found that mutations in FLNB also lead to human diseases whose phenotypes differ from those seen with FLNA mutations. We have also found that knock-down of zebrafish FLNB causes major embryologic abnormalities. Furthermore, using the yeast two-hybrid technique we have identified several new FLNB binding partners with varying FLN-binding characteristics, including Rab22B, that does not bind to FLNA, fibrillin-1, that does not bind to FLNs A or C, and a new proline-rich-domain and LIM-domain protein that we have named FBLP-1 (Filamin-Binding LIM Protein-I), that binds to all three FLNs. Thus it is almost certain that, although FLNs may share some functions, each FLN has unique functional characteristics. The aims of this proposal are: 1) To delineate the roles of the FLN ABD vs. the repeats, in FLN function in vitro; 2) To determine in vivo specificity of FLN functions by a) knockdown of FLNs in zebrafish and b) knockout of FLNB in the mouse (knockout of FLNA has been achieved by others, but not yet published); and 3) To further delineate the function of our newly identified FBLP-1.

描述(申请人提供)：血小板形状变化和血管内皮细胞和血管平滑肌迁移是止血和血栓形成、血管生成和伤口愈合过程的中心。这些反应需要细胞骨架结构的快速改变。细丝蛋白(flamins，Fln)在调节这些反应方面很重要。人类Fln家族由三个约280 kD的对偶A、B和C组成，每个对偶包含约250个残基的N末端肌动蛋白结合结构域(ABD)，其后是24个内部同源重复。Fln通过与几种整合素和其他膜蛋白的细胞质尾巴结合，诱导形成三维肌动蛋白网络，并将细胞膜连接到细胞骨架上。Fln还与许多细胞成分结合，包括几个小的GTP酶，并可作为组织细胞内信号和蛋白质运输途径的支架；这些相互作用不需要ABD的存在。缺乏FLNA的细胞系的形状和迁移异常，以及FLNA的突变，无论是在ABD中还是在重复序列中，都与人类疾病有关。最近，我们的合作者和我们发现，FLNB的突变也会导致人类疾病，其表型与FLNA突变的表型不同。我们还发现，斑马鱼Flb基因的敲除会导致重大的胚胎异常。此外，利用酵母双杂交技术，我们已经鉴定出几个新的具有不同的Fln结合特征的结合伙伴，包括不与Flna结合的Rab22B，不与Flns A或C结合的纤维蛋白-1，以及我们命名为FBLP-1(微丝结合Lim蛋白-I)的新的富含Pro结构域和LIM结构域的蛋白，它可以与所有三个Fln结合。因此，几乎可以肯定的是，尽管FLN可能共享一些功能，但每个FLN都有独特的功能特征。这项建议的目的是：1)描述Fln Abd和重复序列在Fln体外功能中的作用；2)通过a)敲除斑马鱼中的Flns和b)敲除小鼠中的Flb来确定Fln功能的体内特异性(其他人已经实现了Flna的敲除，但尚未发表)；以及3)进一步描述我们新发现的FBLP-1的功能。