MOLECULAR BIOLOGY OF ACTIN-BINDING PROTEIN

肌动蛋白结合蛋白的分子生物学

• 批准号: 3087557
• 负责人: JED B GORLIN
• 金额: $ 6.66万
• 依托单位: MASSACHUSETTS GENERAL 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 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3087557
• 关键词: actins; binding proteins; chemical structure function; complementary DNA; cytoskeleton; gel electrophoresis; genetic library; genetic manipulation; genetic mapping; human tissue; laboratory rabbit; molecular cloning; monoclonal antibody; nucleic acid hybridization; nucleic acid probes; 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），有时称为细丝蛋白， 肌动蛋白丝在皮层细胞质中形成正交阵列， 一个参与动态变化的细胞器排除区， 细胞运动性 ABP存在于白细胞、血小板、成纤维细胞中， 上皮细胞、两栖动物卵、心脏和平滑肌。 尽管在功能和形态上 在表征中，该蛋白质的一级结构没有 确定了 ABP由两个明显相同的 270 kDa亚基，每个亚基含有用于自缔合的结构域， 肌动蛋白结合和跨膜蛋白结合（血小板GP1b）。 这种细胞骨架蛋白的结构的阐明可能 有助于我们理解细胞质结构， 能动性 该项目从ABP cDNA的克隆和测序开始。 克隆是从内皮细胞表达中选择的 使用针对ABP的单克隆抗体构建文库。 限制 ABP cDNA内的位点和连续缺失将用于 亚克隆到载体中用于测序。 由于ABP cDNA应 1kb，则获得的大多数克隆将不包含该基因。 完整的ABP消息。 用DNA探针重新筛选文库 从先前鉴定的克隆的末端或合成 可能需要特异性引物延伸文库。 组织和 ABP的种间分布将通过探测 在RNA的细胞提取物中的ABP信息。 主 将分析蛋白质的结构以鉴定结构 功能域的相关性，并将与 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 单克隆抗体 特异性将用于将结构与功能相关联。 将编码ABP片段的独特构建体转染入 细胞（即使细胞产生ABP亚基片段， 将与产生无活性二聚体的天然亚基缔合） 可能进一步阐明ABP在体内的作用（例如，表达自 关联域）。 编码ABP的基因的表征将包括 染色体定位和作图。 初步研究， 体细胞杂交系表明ABP基因定位于 X染色体，位于X染色体脆性部位的远端， 染色体 将进行原位杂交研究。 将建立限制性片段长度多态性， 帮助绘制基因图谱。