title: Modulation of surface-associated urokinase: binding, interiorization, delivery to lysosomes, and degradation in human keratinocytes.
abstract: Receptor-mediated endocytosis of urokinase-type plasminogen activator (u-PA) was characterized with the human keratinocyte cell line NCTC, by both biochemical and ultrastructural methods. Binding to specific cell surface receptors at low temperature occurs with both catalytically active and inhibited u-PA. At 37 degrees C a single cohort of bound u-PA molecules is rapidly reduced at the surface level by both membrane dissociation and intracellular accumulation of the ligand, with no difference between active and inhibited u-PA. After a short lag period, both intact u-PA and u-PA degradation products are released into the culture medium. In the continued presence of native and inhibited u-PA at 37 degrees C the cumulative ligand uptake largely exceeds the total cellular capacity of binding sites measured at low temperature, consistent with receptor recycling. Catalytically inhibited u-PA shows a reduced interiorization rate, consistent with a requirement of an intact catalytic site which becomes evident in the presence of multiple cycles of endo-exocytosis. In the presence of a molar excess of anti-plasminogen activator inhibitor-type 1 (PAI-1) antibodies the interiorization rate is similar to that observed with catalytically inhibited u-PA, suggesting that PAI-1 molecules can modulate the intracellular accumulation of u-PA in this cell line. Parallel electron microscopy studies of a u-PA-colloidal gold complex have shown that membrane-associated u-PA molecules are concentrated in clusters before invagination of the underlying membrane to form endosomes which then fuse with lysosomes, where at least a part of u-PA degradation is likely to occur. Also, ultrastructural studies have confirmed the decrease in intracellular u-PA accumulation after inhibition of u-PA catalytic site. We conclude that cell surface-associated u-PA modulation in human keratinocytes involves ligand binding, uptake, and degradation, mediated by the classic receptor system for u-PA A chain, which can be modulated by membrane-associated PAI-1 molecules.
authors.affiliation: Istituto di Patologia Generale Università di Firenze, Italia.
authors.forename: M
authors.initials: M
authors.lastname: Del Rosso
authors.forename: G
authors.initials: G
authors.lastname: Fibbi
authors.forename: M
authors.initials: M
authors.lastname: Pucci
authors.forename: G
authors.initials: G
authors.lastname: Dini
authors.forename: C
authors.initials: C
authors.lastname: Grappone
authors.forename: M L
authors.initials: ML
authors.lastname: Nolli
mesh_terms: D002451:Cell Compartmentation; D002478:Cells, Cultured; D004705:Endocytosis; D006801:Humans; D066298:In Vitro Techniques; D015603:Keratinocytes / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism; D008854:Microscopy, Electron; D008970:Molecular Weight; D011956:Receptors, Cell Surface / Q000502:physiology; D055293:Receptors, Urokinase Plasminogen Activator; D013329:Structure-Activity Relationship; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
chemical_list: C524898:PLAUR protein, human; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator