Kinetic analysis of plasminogen activator inhibitor type-2: urokinase complex formation and subsequent internalisation by carcinoma cell lines.
The overexpression of urokinase (uPA), which plays a key role in tumour invasion and metastasis, is an established prognostic marker and potential therapeutic target. Plasminogen activator inhibitor type 2 (PAI-2), an efficient and specific inhibitor of uPA, has been shown to selectively deliver potent cytotoxins to tumour cells. However, a direct quantitative analysis of both the inhibition kinetics and subsequent fate of PAI-2 upon interaction with cell-surface uPA has not been previously undertaken. In this study, we analysed specific PAI-2 binding to receptor-bound uPA on human breast and prostate cancer cell lines to directly measure inhibition kinetics. Cell-surface uPA:PAI-2 complex formation, which is reflective of complete uPA inhibition, was found to be very efficient (inactivation constant [K(I)] = 60-80 pM, depending on cell line used) and rapid (inactivation rate constant [k(inact)] = 0.32-0.47 min(-1) at 37 degrees C, depending on cell line used). To directly quantify and visualise cellular internalisation and localisation, we developed a novel assay based on the use of PAI-2 labelled with Alexa(488) fluorochrome and a polyclonal antibody to quench Alexa(488) fluorescence. The efficient and rapid formation of uPA:PAI-2 complexes was thus shown to be associated with specific and rapid internalisation of PAI-2, which could be localised within endosomes and lysosomes. PAI-2 was subsequently degraded, presumably within lysosomes. This study is the first to provide definitive evidence for uPA/uPAR-mediated PAI-2 endocytosis.
Al-Ejeh
Fares
F
School of Biological Sciences, University of Wollongong, NSW 2522, Australia.
Croucher
David
D
Ranson
Marie
M
D001667:Binding, Competitive / Q000502:physiology; D002277:Carcinoma / Q000188:drug therapy / Q000201:enzymology / Q000378:metabolism*; D045744:Cell Line, Tumor; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology; D011992:Endosomes / Q000187:drug effects / Q000201:enzymology; D005260:Female; D019085:Fluorescent Antibody Technique, Direct / Q000379:methods; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D008247:Lysosomes / Q000187:drug effects / Q000201:enzymology; D046911:Macromolecular Substances; D008297:Male; D017396:Plasminogen Activator Inhibitor 2 / Q000378:metabolism* / Q000493:pharmacokinetics; D011485:Protein Binding / Q000502:physiology; D011930:Reaction Time / Q000187:drug effects / Q000502:physiology; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism; D013997:Time Factors; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors* / Q000378:metabolism*
D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D017396:Plasminogen Activator Inhibitor 2; D011956:Receptors, Cell Surface; D014568:Urokinase-Type Plasminogen Activator