Topological localization of plasminogen activator inhibitor type 2.
BACKGROUND
Plasminogen activator inhibitor type 2 (PAI-2) is a member of the serine protease inhibitor (SERPIN) superfamily and forms stable complexes with urokinase type plasminogen activator (uPA). uPA can be found on the cell surface attached to its specific receptor (uPAR), allowing for controlled degradation of the extracellular matrix by the activation of plasminogen into plasmin. The aim of this study was to evaluate if PAI-2 could also be detected on the cell surface, providing a means of regulating the activity of cell surface uPA.


METHODS
Intact or permeabilized cell lines or human peripheral blood leukocytes were assayed by flow cytometry for cell surface uPA or PAI-2. Plasma membrane-enriched preparations prepared from Jurkat, HaCaT, THP-1, U937, or MM6 cells were assayed by enzyme-linked immunosorbent assay (ELISA) or Western blotting for PAI-2 antigen.


RESULTS
By flow cytometry, cell surface PAI-2 was not detected on monocytes from human peripheral blood, MM6, or HaCaT cells. Cell surface PAI-2 was only detected very weakly on the surface of U937 cells. In contrast, PAI-2 could be detected in all of these cells when fixed and permeabilized. By ELISA, PAI-2 was very abundant in the cytosol-enriched preparations of U937, MM6, and HaCaT cells, but was present in lower amounts in the plasma membrane-enriched preparations. By Western blotting, monomeric nonglycosylated PAI-2, but not uPA/PAI-2 complexes, could be detected in the cytosol and plasma membrane-enriched preparations.


CONCLUSIONS
These results indicate that PAI-2 cannot be detected on the surface of PAI-2-expressing cells, and confirm that PAI-2 is predominantly a cytosolic protein.
Liew
M A
MA
Department of Biological Sciences, University of Wollongong, Wollongong, NSW, Australia. liewma@netzero.net
McPhun
V
V
Baker
M S
MS
D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007124:Immunoenzyme Techniques; D007074:Immunoglobulin G; D009000:Monocytes / Q000737:chemistry / Q000276:immunology; D017396:Plasminogen Activator Inhibitor 2 / Q000032:analysis* / Q000276:immunology; D020298:U937 Cells; D014568:Urokinase-Type Plasminogen Activator / Q000032:analysis / Q000276:immunology
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D017396:Plasminogen Activator Inhibitor 2; D014568:Urokinase-Type Plasminogen Activator