MOLECULAR BIOLOGY OF ACTIN-BINDING PROTEIN

点击翻译按钮获取中文摘要

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.