Differential inhibition of soluble and cell surface receptor-bound single-chain urokinase by plasminogen activator inhibitor type 2. A potential regulatory mechanism.
Urokinase (u-PA)-mediated cell surface plasminogen activation is required for cellular tissue invasion. This invasion occurs in environments rich in plasminogen activator inhibitors (PAIs), which efficiently inhibit receptor-bound two-chain u-PA. Single-chain u-PA (scu-PA) was recently found to efficiently initiate cell surface plasminogen activation, and we herein describe the interaction of scu-PA with PAI type 2 (PAI-2). In the fluid phase (no cells) the plasminogen-activating activities of both scu-PA and Glu158-scu-PA (a plasmin non-activatable variant of scu-PA) were inhibited in a concentration-dependent manner by recombinant human PAI-2. This inhibition occurred with both forms of scu-PA remaining as single-chain molecules throughout the interactions. Although scu-PA did not form SDS-stable complexes with PAI-2, preincubation of scu-PA with 125I-PAI-2 demonstrated a dose-dependent inhibition of SDS-stable complex formation between 125I-PAI-2 and subsequently added two-chain u-PA. This indicates that although a "stable intermediate" type complex between scu-PA and PAI-2 was not detected, there was a physical association between the two molecules that shared at least some determinants with the two-chain u-PA-PAI-2 complex. In contrast, Glu158-scu-PA bound to u-PA receptors on monocytes was only minimally inhibited by a large molar excess of PAI-2. These data suggest that the initiation of cell surface plasminogen activation may involve the partitioning of scu-PA between PAI-2 (a "negative modulator") and the u-PA receptor (a "positive modulator") and that the enzymatic activity of receptor-bound scu-PA may allow initiation of cell surface proteolysis even in PAI-2-rich environments. A model along these lines is presented.
Schwartz
B S
BS
Department of Medicine, University of Wisconsin, Madison.
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D014644:Genetic Variation; D005971:Glutamates; D018698:Glutamic Acid; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D010958:Plasminogen / Q000378:metabolism; D017396:Plasminogen Activator Inhibitor 2 / Q000302:isolation & purification / Q000494:pharmacology*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins / Q000494:pharmacology; D012996:Solutions; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*
D005971:Glutamates; C524898:PLAUR protein, human; D017396:Plasminogen Activator Inhibitor 2; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins; D012996:Solutions; D018698:Glutamic Acid; D010958:Plasminogen; D014568:Urokinase-Type Plasminogen Activator