MOLECULAR BIOLOGY OF ACTIN-BINDING PROTEIN

肌动蛋白结合蛋白的分子生物学

• 批准号: 3087560
• 负责人: JED B GORLIN
• 金额: $ 8.85万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 1993-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3087560
• 关键词: actins; binding proteins; chemical structure function; complementary DNA; cytoskeleton; gel electrophoresis; genetic library; genetic manipulation; genetic mapping; human genetic material tag; human tissue; laboratory rabbit; molecular cloning; monoclonal antibody; nucleic acid hybridization; nucleic acid probes; nucleic acid sequence; protein sequence; transposon /insertion element; vascular endothelium

Actin-binding protein (ABP), sometimes called filamin, crosslinks actin filaments into an orthogonal array in the cortical cytoplasm, an organelle-excluding area involved in dynamic changes during cell motility. ABP is found in leukocytes, platelets, fibroblasts, epithelial cells, amphibian eggs and cardiac and smooth muscle. Although there has been significant functional and morphologic characterization, the primary structure of this protein has not been determined. ABP is composed of two apparently identical 270 kDa subunits, each containing domains for self-association, actin-binding and transmembrane protein binding (platelet GP1b). The elucidation of the structure of this cytoskeletal protein may facilitate our understanding of cytoplasmic structure and motility. The project begins with the cloning and sequencing of ABP cDNA. Clones are being selected from an endothelial cell expression library using monoclonal antibodies against ABP. Restriction sites within the ABP cDNA and serial deletions will be used to subclone into vectors for sequencing. Since the ABP cDNA should be at least 8.1 kb, most clones obtained will not encompass the entire ABP message. Rescreening the library with DNA probes from the ends of previously identified clones or synthesis of a specific primer-extended library may be required. Tissue and interspecies distribution of ABP will be investigated by probing for ABP message in cellular extracts of RNA. The primary structure of the protein will be analysed to identify structural correlation of functional domains and will be compared with known sequence of other actin binding proteins. Phase two of the project will use ABP cDNA clones to further characterize functional domain structure. Monoclonal antibodies with domain specificity will be used to correlate structure to function. Transfection of unique constructs encoding fragments of ABP into cells (i.e. cause the cell to manufacture ABP subunit pieces that will associate with native subunits generating inactive dimers) may further clarify ABP's role in vivo (e.g. expression of the self- association domain). Characterization of the gene coding for ABP will include chromosomal localization and mapping. Preliminary studies with somatic cell hybrid lines suggest localization of the ABP gene to the X-chromosome, distal to the fragile site of the X- chromosome. In situ hybridization studies will be performed. Restriction fragment length polymorphisms will be established to aid in mapping the gene.

肌动蛋白结合蛋白（ABP），有时称为Filamin，交联 肌动蛋白丝进入皮质细胞质中的正交阵列， 在涉及动态变化的细胞器排放区域 细胞运动。 ABP在白细胞，血小板，成纤维细胞中发现， 上皮细胞，两栖卵，心脏和平滑肌。 尽管功能和形态学有明显的功能 表征，该蛋白的主要结构尚未 已确定。 ABP由两个显然相同的 270 kDa亚基，每个亚基都包含自我关联的域， 肌动蛋白结合和跨膜蛋白结合（血小板GP1B）。 阐明该细胞骨架蛋白的结构可能 促进我们对细胞质结构和 运动。 该项目始于ABP cDNA的克隆和测序。 从内皮细胞表达式选择克隆 使用针对ABP的单克隆抗体的库。 限制 ABP cDNA和串行删除的位置将用于 亚克隆到向量进行测序。 因为ABP cDNA应该 至少为8.1 kb，获得的大多数克隆将不包括 整个ABP消息。 用DNA探针对库进行重新筛选 从先前确定的克隆的末端或合成 可能需要特定的引物扩展的库。 组织和 ABP的种间分布将通过探测研究 对于RNA的细胞提取物中的ABP消息。 主要 将分析蛋白质的结构以识别结构 功能域的相关性，将与 其他肌动蛋白结合蛋白的已知序列。 第二阶段 项目将使用ABP cDNA克隆进一步表征 功能域结构。 具有域的单克隆抗体 特异性将用于将结构与功能相关联。 将ABP的片段编码的独特结构转染到 单元格（即导致细胞制造ABP亚基零件 将与生成非活动二聚体的天然亚基相关联） 可以进一步阐明ABP在体内的作用（例如，自我的表达 关联域）。 ABP的基因编码的表征将包括 染色体定位和映射。 初步研究 体细胞杂交线表明将ABP基因定位到 X染色体，距离X-脆弱位点的远端 染色体。 将进行原位杂交研究。 将确定限制片段长度多态性 有助于映射基因。