ABSTRACT
The main ligand-binding determinant of the human urokinase receptor (uPAR) is located in the amino terminal domain D1, but when isolated this domain presents a 1500 fold lower affinity than the intact three-domain uPAR (D1D2D3). uPAR mutants missing either domain 2 (D1HD3) or domain 3 (D1D2) were expressed in murine LB6 cells and showed to be properly GPI-anchored to the cell surface. Binding assays with [125I]ATF demonstrated that these mutants possessed a normal (D1D2) or slightly reduced (D1HD3) affinity, indicating that a high ligand-affinity may be achieved by a combination of D1 with domain D2 or D3.
Subject(s)
Receptors, Cell Surface/metabolism , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Clone Cells , Fluorescent Antibody Technique , Glycosylphosphatidylinositols/metabolism , Humans , Kinetics , L Cells , Ligands , Mice , Molecular Sequence Data , Mutagenesis , Mutagenesis, Insertional , Phosphatidylinositol Diacylglycerol-Lyase , Phosphoric Diester Hydrolases/biosynthesis , Phosphoric Diester Hydrolases/metabolism , Phosphoric Diester Hydrolases/pharmacology , Receptors, Cell Surface/biosynthesis , Receptors, Cell Surface/isolation & purification , Receptors, Urokinase Plasminogen Activator , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Transfection , Urokinase-Type Plasminogen Activator/metabolismABSTRACT
The biosynthesis and the surface localization of the urokinase plasminogen activator receptor (uPAR) were analysed in MDCK epithelial cells and in unpolarized fibroblasts. No differences were observed with respect to rate of synthesis, nature of precursors and time of surface appearance. uPAR was localized particularly at the focal and cell-cell contacts when expressed in fibroblasts. On the contrary, in MDCK cells uPAR was found mostly on the apical surface; in agreement with its localization, down-regulation of uPAR by the uPA-PAI-1 complex was observed only from the apical membrane.
