The expression of protease activity by tumor cells is proposed to facilitate their penetration of basement membranes and capillary walls allowing them to spread to other sites and establish metastases. Several lines of evidence suggest that the invasive properties of tumor cells may at least partly depend on their proteolytic capability. Fundamental to the proteolytic capacity of cells is their ability to regulate conversion of surface-associated plasminogen (Pg) to plasmin (Pm). The main Pg converting enzymes are tPA and uPA. The Ca/2+-binding protein annexin II is present on the cell surface and has been implicated in the regulation of Pg. Our laboratory has demonstrated that in fluid phase, the heterotetrameric form of annexin II, annexin II tetramer (AIIt) is a potent acceleration of tPA- and uPA- dependent plasminogen activation. We therefore propose to characterize the affinity, stoichiometry and modulation of the binding of tPA, uPA, Pg and Pm to fluid phase, solid phase (heparin and phospholipid-bound) and cell- associated AIIt. We will also elucidate the kinetics and mechanism of AIIt-dependent acceleration of tPA and uPA-dependent Pg activation, and examine the potential modulatory role of uPAR in this process. Site directed mutagenesis of p36 and p11 examine the potential modulatory role of uPAR in this process. Site directed mutagenesis of p36 and p11 subunits of AIIt will be used to identify the domains of AIIt involved in the binding of tPA, uPA, Pg and Pm and to identify the domains of AIIt involved in Pg activation. Transfection experiments will be to generate cell lines with either increased (293 epithelial cells transfected with AIIt subunits) or decreased (using an AIIt disrupter mutant with endothelial, RAW117 and U-937 cells) surface-associated AIIt. These cells will be used to access the in vivo role of AIIt in Pg regulation. The transfected cell lines will also be used to examine the possible influence on uPAR on AIIt-dependent Pg regulation. Lastly, immunofluorescence localization studies and immunoprecipitation studies will be used to establish the extracellular distribution of AIIt and also to examine the possible colocalization of uPA, uPAR, Pg and AIIt. Overall, the proposed studies will define the role of AIIt in the regulation of Pg.
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