The extracellular matrix is believed to influence cellular growth through a complex set of interactions between the matrix and the cytoskeleton. Tumor cells have escaped the adhesion requirement for growth and their inability to respond to cues from the matrix is thought to contribute to their transformed phenotype. Integrin receptors are believed to transduce signals from the matrix and these signaling pathways converge with growth factor pathways in focal adhesion structures. The hypothesis to be tested in this grant is that the polymerization of fibronectin affects the nature of cell-matrix contacts and changes signaling pathways within the cell which affect cell growth. Preliminary data on human fibroblast cells indicate that fibronectin polymerization occurs in focal adhesion areas and is up-regulated by the serum mitogen, LPA through a signaling pathway regulated by the cytoplasmic region of the beta1 integrin. Our data also indicate that the matrix assembly domain of fibronectin binds to specific receptors in focal adhesion sites, affects focal adhesion structure and inhibits cell growth. Adhesion to extracellular matrix is known to be a requirement for cell cycle progression, however nothing is known about the relationship of the process of fibronectin polymerization to cell growth. In this application, we propose to recreate the steps of matrix assembly using recombinant proteins containing regions of fibronectin which interact during the polymerization of fibronectin, and then evaluate the effect of these steps on focal adhesion structure and cell cycle progression. Focal adhesion structure will be characterized morphologically by immunostaining and biochemically by comparing the phosphorylation patterns of the focal adhesion proteins FAK, paxillin and tensin. The effect of different steps in matrix assembly on cell cycle progression will be monitored by Northern analysis of marker genes for various stages of G1. The expression of cyclins as will as their effector proteins will be measured by Western blotting. Chemical cross-linking studies will be done to identify the receptor which binds to the matrix assembly domain of fibronectin. beta1 null cells will be transfected with an lL-2 beta1 chimeric receptor containing only the cytoplasmic domain, to define the molecular basis for the beta1-dependent binding of the matrix assembly domain to the cell surface. These studies will define a role for the polymerization of fibronectin in the regulation of signaling pathways which affect cell growth.
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