Alpha v beta 5 integrin is localized at focal contacts by HT-1080 fibrosarcoma cells and human skin fibroblasts attached to vitronectin.
In this study we characterized alpha v beta 5 integrin on HT-1080 fibrosarcoma cells. First, alpha v beta 5 integrin was immunoprecipitated by 125I-surface labeled HT-1080 cells using a polyclonal antibody specific for beta 5 subunit (cytoplasmic domain). A heterodimer consisting of a beta 5-chain running at 100 kD (reduced) and 90 kD (non-reduced) associated with an alpha-chain 145 kD (non-reduced) and 125 kD (reduced) was obtained by SDS-PAGE and autoradiography. By double-immunofluorescence labeling, we then investigated alpha v beta 5 distribution on HT-1080 cells. Upon staining with anti-beta 5 subunit antibody, alpha v beta 5 was detected in focal contacts on cells attached to vitronectin (vn), co-localizing with vinculin at the end of actin filaments. Comparative analysis of alpha v beta 5 and alpha v beta 3 showed that both receptors can occupy the same focal contact, although on the same cell mostly they are clustered in independent focal contacts. Focal distribution of alpha v beta 5 was also found on normal human fibroblasts attached to vn, suggesting that this is not a specific feature of HT-1080 cells. Finally, we investigated the role of alpha v beta 5 and alpha v beta 3 integrins in mediating HT-1080 cell adhesion to vn. Inhibition studies using antibodies with function-blocking activity to alpha v beta 5 and alpha v beta 3 suggest a primary role of alpha v beta 5 to support cell adhesion, with a weak contribute of alpha v beta 3. Their activity can be modulated by divalent cations. Our results provide the first evidence of focal distribution of alpha v beta 5 integrin on cells attached to vn.
Conforti
G
G
Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy.
Calza
M
M
Beltrán-Nuñez
A
A
D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D005347:Fibroblasts / Q000378:metabolism*; D005354:Fibrosarcoma / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D016023:Integrins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D011233:Precipitin Tests; D016024:Receptors, Cytoadhesin / Q000378:metabolism; D019030:Receptors, Vitronectin; D014407:Tumor Cells, Cultured / Q000378:metabolism; D019096:Vitronectin
D006023:Glycoproteins; D016023:Integrins; D016024:Receptors, Cytoadhesin; D019030:Receptors, Vitronectin; D019096:Vitronectin; C064660:integrin alphaVbeta5; D008274:Magnesium; D002118:Calcium