The long-range goal of these studies is to understand the regulation of human tight junctions. These structures form a continuous intercellular contact between both epithelial and endothelial cells, sealing the paracellular path to the movement of water, solutes and immune cells. Permeability of the barrier varies widely among epithelia, is physiologically regulated and effected by disease processes. Thus, understanding barrier regulation has significant implications for human epithelial pathophysiology, therapeutics and drug delivery. Presently, cellular signalling mechanisms which regulate assembly and sealing are poorly understood. cDNA sequencing of two tight junction proteins, Z0-1 and Z0-2 reveals both are members of the Membrane-Associated Guanylate Kinase Homolog (MAGUK) family of putative signal transduction proteins, the original member of this family is the discs-large tumor suppressor of Drosophila. We have shown that transient transfection, and over- expression, of Z0-1 in MDCK cells induces perijunctional actin accumulation. Further, we observe in the A431 cell line that Epidermal Growth Factor induces actin and Z0-1 to move into tight junctions and both Z0-1/2 to undergo transient phosphorylation on tyrosine residues. In MDCK cells HGF induces increased junctional permeability and actin rearrangements. We hypothesize tyrosine phosphorylation of Z0-1 induced by growth factors creates transient association with an SH2 domain- containing signalling protein which is involved in regulating perijunctional actin. The immediate goals are to identify SH2 domain- containing proteins which associate with tyrosine-phosphorylated Z0-1/2 in cultured MDCK and A431 cells. Phosphorylated tyrosines will be mapped in vivo, on epitope-tagged fragments of Z0-1 expressed in and immunoprecipitated from MDCK and A431 cells. Site directed mutagenesis of epitope tagged Z0-1, expressed in MDCK and A431 cells, will be used to test the requirement for phosphorylation of specific tyrosines in inducing SH2-protein binding, and structural and permeability changes induced by EGF and HGF. All MAGUKs contain a conserved tyrosine, Y621 in Z0-1, and we will perform in vitro binding studies attempting to identity an SH2- containing protein which binds this site. The protein domains of Z0-1 responsible for inducing actin accumulation will be defined using transfection techniques in MDCK cells. Structural changes will be characterized at the light and ultrastructural levels. The role of tyrosine kinases, c-src, c-yes and c-fyn, in regulating Z0-1 tyrosine phosphorylation, junction morphology and sealing will be tested using MDCK cells stably transfected and expressing different levels of these kinases. The conserved nature of phosphorylation-dependent signaling among MAGUKs will be investigated with other Program Project investigators.
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