Dissecting the mechanisms of cell adhesion modulation

Princeton University, Princeton NJ

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Abstract

[unreadable] DESCRIPTION (provided by applicant): Adhesion modulation of fibronectin involves the action of anti-adhesive proteins that reduce cell interactions with the adhesive matrix, thus allowing cell migration, shape changes and proliferation. Tenascin-C is an adhesion modulating protein that directly interacts with the heparin-binding domain of FN. Recent studies demonstrated that tenascin-C exerts its anti-adhesive effects by the competitive blockade of FN binding to the heparan sulfate proteoglycan receptor, syndecan-4. This proposal assesses the role of the heparin binding site in the heparin-binding (hepll) domain of FN in the adhesion mpdulatory interactions of tenascin- C within a three-dimensional (3-D) provisional matrix. The role of the functional sequence of FN in adhesion modulation will be investigated by using a recombinant FN molecule which lacks heparin-binding activity (hepll-). Mutant protein will be expressed using baculovirus and adenovirus expression systems. Effects of FNhepll-matrices on cytoskeletal organization, Rho and FAK activation, and matrix contraction will be assessed. The mutated protein will be used in binding assays to test the role of the heparin-binding sequence in direct interactions between the hepll domain and tenascin-C as well as with fibulin-1, another adhesion modulating protein that binds to the hepll domain. Recombinant protein constructs of the FN- binding region (TNfnlH3-5) of tenascin-C will be incorporated into fibrin-FN matrices to test their domain- specific function in modulating 3T3 fibroblast behavior. The effects of fibulin-1 -mediated modulation on 3T3 fibroblast morphology, signaling and matrix contraction will be compared to well-characterized aspects of tenascin-C anti-adhesion to determine whether both proteins employ a common modulatory mechanism. [unreadable] [unreadable] [unreadable]