CROSS-LINKING OF THE NH2-TERMINAL REGION OF FIBRONECTIN TO MOLECULES OF LARGE APPARENT MOLECULAR-MASS - CHARACTERIZATION OF FIBRONECTIN ASSEMBLY SITES INDUCED BY THE TREATMENT OF FIBROBLASTS WITH LYSOPHOSPHATIDIC ACID

Cell surface molecules on adherent cells that bind I-125-labeled fibro nectin or its 70-kDa N-terminal fragment were identified by cross-link ing with factor XIIIa and by photoaffinity labeling. Such cross-linkin g caused the 70-kDa fragment to become associated irreversibly to cell layers and was greater in cells treated with lysophosphatidic acid, a n enhancer of fibronectin assembly and strong modulator of cell shape. Cross-linking of the 70-kDa fragment with factor XIIIa was to molecul es that migrated in discontinuous sodium dodecyl sulfate-polyacrylamid e gels at the top of the 3.3% stacking gel and near the top of the sep arating gel. Estimated sizes of these large apparent molecular mass mo lecules (LAMMs) were >>3 MDa and similar to 3 MDa. The label in 70-kDa fragment conjugated with I-125-sulfosuccinimidyl 2-(p-azidosalicylami do)-1,3'-dithiopropionate was associated with >>3-MDa LAMMs without re duction and with similar to 3-MDa LAMMs after reduction and transfer o f the cleavable label. The LAMMs were expressed on monolayer cells sho rtly after adherence, required both 1% Triton X-100 and 2 M urea for e fficient extraction, and were susceptible to digestion with trypsin bu t not to cathepsin D digestion. Complexes of I-125-70-kDa fragment and LAMMs were also susceptible to limited acid digestion and Glu-C prote ase digestion but were not cleaved by chondroitin lyase or heparitinas e. Neither the uncleaved complexes nor the cleavage products were immu noprecipitated with anti-fibronectin antibodies directed toward epitop es outside the 70-kDa region. Thus, cell surface molecules that are ei ther very large or not dissociated in sodium dodecyl sulfate comprise the labile matrix assembly sites for fibronectin.