Binding of urokinase-type plasminogen activator (uPA) to its receptor, uPAR, activates diverse cell-signaling pathways, including the Ras-ERKJMAP kinase pathway, and thereby regulates cell migration, growth, and apoptosis. uPAR-initiated cell-signaling is sensitive to low concentrations of uPA and requires as few as 3x103 copies of cell-surface uPAR. Furthermore, in aggressive cancer cells, an autocrine pathway may be established in which binding of endogeneously-produced uPA to uPAR is responsible for maintaining a high basal level of activated ERK. We hypothesize that uPAR-initiated cell-signaling is a major determinant of cancer cell aggressiveness in vivo. The major goal of this research project is to elucidate the uPA-uPAR cell-signaling system. Specific Aim 1 focuses on the basic biochemistry of uPAR-initiated cell-signaling. Studies are proposed to characterize the kinetics of uPAR-ligation in relation to cell-signaling, determine how the cell integrates uPAR- and integrin-initiated cell-signaling responses, determine the function of complementary pathways such as the Ras-ERK and RhoRho-kinase pathways, and identify feedback-loops that shut-down cell-signaling after uPAR ligation. We will also address the possibility that uPAR may regulate cell-signaling, in part by uPA-independent mechanisms. In Specific Aim 2, we will test our hypothesis that uPA-initiated cell-signaling reflects the activity of a biochemical system that includes plasminogen, plasminogen activator inhibitor-1(PAI-1), other Serpins, and LDL receptor homologues, such as the VLDL receptor (VLDLr). A major goal of Specific Aim 2 is to reconstitute uPAR-initiated cell-signaling in vitro with components that may regulate cell-signaling in vivo. In Specific Aim 3, translational studies are proposed to characterize the uPA-uPAR cell-signaling system as a target for the development of novel anticancer therapeutics. We will study the activity of antibodies that block uPA-binding to uPAR alone and in combination with herceptin, cisplatin or etoposide. We will also develop novel chimeric receptors that may rapidly catabolize uPA, decreasing the amount of uPA available to ligate uPAR. The proposed studies offer the potential to elucidate properties of uPA and PAL-1 that are not currently understood, further our understanding of this key cell-regulatory system, and generate novel strategies for treating cancer.