The role of carboxy-terminal glycosaminoglycan-binding domain of vitronectin in cytoskeletal organization and migration of endothelial cells.
Vitronectin is a major cell adhesion molecule present in the subendothelial matrix that mediates the attachment and spreading of a variety of cells. The carboxy-terminal end of vitronectin has a consensus sequence for glycosaminoglycan-binding. To define the functional role of this domain, we generated fragments of vitronectin that lack the glycosaminoglycan-binding domain by formic acid cleavage of plasma-derived vitronectin. In addition, we also generated similar recombinant fragments of vitronectin as glutathione S-transferase fusion proteins in E. coli. These fragments were tested for their ability to support the adhesion of human umbilical vein endothelial cells. These fragments promoted endothelial cell adhesion, reaching half maximal activity at 2-5 micrograms/well compared to plasma-derived vitronectin which reached at 0.2 micrograms/well. However, the cells that adhered to these fragments did not develop well-formed focal adhesion plaques and actin stress fibers. In addition, these fragments were poorly chemotactic for endothelial cell migration when compared to intact plasma-derived vitronectin in a modified Boyden chamber assay. The present studies show that carboxy-terminal glycosaminoglycan-binding domain of vitronectin is essential for proper cytoskeletal organization and migration of endothelial cells on vitronectin substratum.
Thiagarajan
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Department of Medicine, University of Texas School of Medicine, Houston 77030, USA. perumal@heart.med.uth.tmc.edu
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Snuggs
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VanWinkle
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D002448:Cell Adhesion; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000648:ultrastructure*; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology*; D006025:Glycosaminoglycans / Q000378:metabolism*; D006801:Humans; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014471:Umbilical Veins / Q000166:cytology; D019096:Vitronectin / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
D006025:Glycosaminoglycans; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D019096:Vitronectin