Urokinase (uPA) contributes to physiologic processes (e.g fibrinolysis, angiogenesis and wound healing) and pathophysiologic processes (e.g. inflammation, atherosclerosis and metastasis) by promoting cell adhesion and proteolysis of extracellular barriers. We observed that the complex of single chain uPA (scuPA) and its receptor (uPAR) is enzymatically active, pro-adhesive and relatively resistant to PAI-1. Our data also indicate that multiple sites of interaction between scuPA and uPAR are required for optimal enzymatic and adhesive activity. However, the molecular basis of these interactions and how they are controlled are incompletely understood. We identified a peptide (K136PSSPPEE143)connecting the amino-terminal fragment and protease domains of scuPA that contributes to the binding of uPA fragments to uPAR, inhibits uPA-mediated cell adhesion and retards uPA-mediated metastases.
In Specific Aim 1 A, we will examine the effects of mutating three residues (K135, K136 and Ser139) in the isolated peptide and in full-length scuPA on binding to uPAR and on cell adhesion. The impact of these mutations on metastasis formation will be examined using tumor cells that co-express human uPAR and plasmin-insensitive scuPA-Glu158. We also observed that Arg137 Arg145 in domain II of uPAR is required for scuPA binding.
In Specific Aim I B, we will determine whether Arg137-Arg144 binds scuPA directly or contributes to a composite binding site that involves domain I.
Specific Aim 2 is predicated on pilot studies which demonstrate that: a) kringle (K)-IV of plasminogen forms a ternary complex with scuPA-uPAR and inhibits its enzymatic activity; b) apo(a), which contains multiple K-IV repeats, binds to uPAR directly and inhibits cell-associated plasminogen activator activity as well; and c) plasma clearance of Lp(a) is delayed in uPAR-/- mice.
In Aim 2 A, we will identify molecular determinants in plasminogen K-IV, scuPA and uPAR required to form the ternary complex.
In Aim 2 B we will examine the binding sites in apo(a) for uPAR and the contribution of uPAR to the deposition of Lp(a) in the vasculature of uPAR uPAR-/-/apo(a)+/+ mice. These studies address newly described interactions between scuPA and its receptor involved in cell adhesion and plasminogen activation and a previous undescribed pathway in which uPAR-mediated enzymatic and adhesive activities are regulated by the kringles of plasminogen and Lp(a).
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