Interactions of cells with their neighbors and the surrounding extracellular matrix are critically important for normal physiological and pathological processes. The major goal of this project is to characterize the role of the newly identified integrin-binding adhesion coreceptor, tissue transglutaminase (tTG), in cell adhesion and adhesion-dependent cellular functions, including signaling and assembly of fibronectin (Fn) matrix. A number of deletion and point mutants, as well as chimeric constructs containing swaps with the a subunit of Factor XIII, a structural homologue of tTG which does not interact with integrins or Fn, will be employed to map the integrin- and Fn-binding sites on tTG. Dominant negative tTG mutants which retain integrin binding, but fail to interact with Fn, will be used in various assays designed to analyze the adhesive function of tTG. We will quantitate an increase in adhesion strength generated by integrin-associated surface tTG. A proposed bridging function of tTG in cell adhesion as a mediator of integrin-Fn interaction will be defined. We will test the ability of surface tTG to alter the overall pattern of cell-ECM recognition. The effects of tTG on integrin activation, clustering and cytoskeletal association will be studied. Since our initial data indicate the ability of tTG to alter activation of Rho and Rac GTPases, we will separate and define the roles of cell surface and cytoplasmic tTG in integrin-mediated and direct activation of Rho and Rac GTPases. Then, the contributions of Rho, Rac and their common downstream target, myosin II, to the unique phenotype of cells that express high levels of surface tTG, will be determined. We will also continue to analyze mechanisms of stimulation of Fn assembly by integrin-bound tTG. A hypothesis will be tested that binding of Fn to tTG or dual interaction of Fn with integrins and tTG exposes a cryptic self-assembly site in modules I9III1 of Fn, and thereby stimulates fibril formation. Finally, the effect of association with integrins on the ability of tTG to crosslink Fn will be examined. Together, the proposed experiments will provide novel insights into the role of tTG in cell adhesion, adhesion-mediated signaling and Fn matrix formation. Furthermore, our studies will define how adhesive and signaling functions of integrins are regulated by their association with tTG. Ultimately, this acquired knowledge will advance our understanding of basic mechanisms of cell-matrix interactions.
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