The endothelium functions in many physiological and pathological processes, such as inflammation, thrombosis, wound repair, and neovascularization. These processes all involve adhesion of the endothelial cell to other cells and to the extracellular matrix. A prerequisite for the ability to undergo shape changes during migration and tube formation is a flexible cytoskeleton. Immunofluorescence studies have demonstrated alterations in cytoskeletal organization during migration and adhesion. However, the molecular events which govern the transformations of the cytoskeleton are currently not well understood. We have preliminary data which suggest that one mechanism for cytoskeletal rearrangement would be via changes in protein 4.1 isoforms. Protein 4.1 is a cytoskeletal protein with widespread tissue distribution. Its erythroid functions are well- characterized and include binding to spectrin and actin as well as transmembrane glycoproteins and lipids. It is expressed as a series of isoforms which arise by alternative splicing of mRNA. This proposal will focus on the elucidation of the changes in the distribution and types of protein 4.1 isoforms which occur upon adhesion to different extracellular matrix components in two and three-dimensional cell cultures and with exposure to TGF-beta. The distribution and types of protein 4.1 isoforms will first be established for the resting endothelial cell in confluent cultures. This will involve both protein and mRNA analysis, including immunoblotting, immunofluorescence, Northern blotting, nuclease protection, metabolic labeling and polymerase chain reaction. The endothelial cells will then by examined with the same techniques under conditions that promote migration and with alteration in the extracellular matrix composition. The question of whether the longer (170 kD) protein 4.1 isoform arise by alternative splicing of as yet unidentified regions of nucleotide sequence will be addressed with techniques such as random amplification of cDNA ends. After the pattern of protein 4.1 isoforms has been identified, the functions of those peptides found to be significant will be examined. Binding of these peptides to other cytoskeletal proteins and to membrane receptors will be analyzed by affinity chromatography, solid phase assay, sedimentation, competition assay, and site directed mutagenesis. Eventually, these experiments could lead to future investigations of the transmembrane signal generated by adhesion to extracellular matrix and the mechanism by which the cell regulates alternative splicing of mRNA. This work should further elucidate the molecular response to processes such as vascular injury secondary to angioplasty and endarterectomy, thrombosis, neovascularization, inflammation and wound repair.
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