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)beta5 integrin on HT-1080 fibrosarcoma cells. First, alpha(v)beta5 integrin was immunoprecipitated by I-125-surface labeled HT-1080 cells using a polyclonal antibody specific for beta5 subunit (cytoplasmic domain). A heterodimer consisting of a beta5-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)beta5 distribution on HT-1080 cells. Upon staining with anti-beta5 subunit antibody, alpha(v)beta5 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)beta5 and alpha(v)beta5 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)beta5 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)beta5 and alpha(v)beta3 integrins in mediating HT-1080 cell adhesion to vn. Inhibition studies using antibodies with function-blocking activity to alpha(v)beta5 and alpha(v)beta3. suggest a primary role Of alpha(v)beta5 to support cell adhesion, with a weak contribute of alpha(v)beta3. Their activity can be modulated by divalent cations. C)ur results provide the first evidence of focal distribution of alpha(v)beta5 integrin on cells attached to vn.