Detachment of murine Balb/c 3T3 cells (or their oncogenic virus transformants or partially adhesion-defective variants) from the tissue culture substratum (coated with plasma fibronectin [pFn] with EGTA treatment leaves tight-focal adhesion sites and some close-contact adhesion sites on the undersurface of the cell as substratum-attached material (SAM). SAM is a considerable enrichment of the adhesive material of these cells and affords us the opportunity to examine the biochemical requirements for forming tight focal contacts and close contacts. Since SAM contains principally heparan sulfate proteoglycan (HS-PG), hyaluronate, and cell surface fibronectin (cFn), along with some chondroitin sulfate proteoglycan and cytoskeletal elements, we investigated and established evidence for binding of cFn or pFn to HS chains and the binding preferentially of CFn to HA. We will continue to study the properties of the two forms of Fn binding to these glycosaminoglycan-containing elements and the possible importance of these bindings in forming or altering the two types of adhesive contacts described above. Specifically, the binding of the Fn's to HS-PG, rather than free chains, will be examine in more detail. Second, the requirements and properties of binding of cFn to HA will be studied with the eventual goal of better understanding the simultaneous binding of HA and HS-PG to cFn and possible consequences. Third, we will attempt to isolate cFn:HS-Pg and/or cFn:HA complexes from the SAM's of these cells for characterization under various growth and attachment conditions. Finally, we will study the significance of Fn:GAG binding in substratum adhesion of these cells by comparing the adhesive responses of untransformed, transformed, or adhesion-variant cells attaching to substrata coated with pFn (or its specific binding domains) or with specific GAG-binding proteins. (A)
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