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Journal of Cell Science, Vol 111, Issue 4 425-433, Copyright © 1998 by Company of Biologists
JOURNAL ARTICLES |
LM Memmo and P McKeown-Longo
Cell and Molecular Biology Program, Albany Medical College, Albany, New York 12208, USA.
Endocytosis and degradation of vitronectin by human skin fibroblasts are regulated by the beta5 integrin. To determine whether the beta5 integrin is directly mediating the internalization of vitronectin, both vitronectin and the beta5 integrin were localized by indirect immunofluorescence during the endocytic process. This analysis showed that both vitronectin and beta5 were found in intracellular vesicles within 5 minutes of the addition of exogenous vitronectin to fibroblast cell layers. By 15 minutes, approximately 20% of the vitronectin-containing vesicles stained positively for beta5. In contrast, the beta3 integrin was not found in any intracellular vesicles. Within 30 minutes, more than 50% of vitronectin-containing vesicles also stained for lamp-1, indicating that internalized vitronectin traveled to lysosomes. Inhibition of clathrin assembly by either potassium depletion or hypertonic buffer inhibited vitronectin internalization, suggesting that vitronectin internalization occurred through coated pits. Confocal analysis confirmed the colocalization of vitronectin and alphavbeta5 in intracellular compartments and further demonstrated that the highest colocalization of the two proteins occurred within 1.8 microm from the ventral surface of the cell, suggesting endocytosis occurred at the substrate level. Pretreatment of cells with the PI-3 kinase inhibitor, wortmannin, resulted in a marked increase in the coincidence of vitronectin and beta5 staining within vesicles and prevented the accumulation of vitronectin within lysosomes. This suggests that following internalization, vitronectin and the alphavbeta5 integrin are segregated to different cellular compartments. This study provides the first evidence that the alphavbeta5 vitronectin receptor directly mediates the internalization of vitronectin.
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