Direct binding of occupied urokinase receptor (uPAR) to LDL receptor-related protein is required for endocytosis of uPAR and regulation of cell surface urokinase activity.
Low-density lipoprotein receptor-related protein (LRP) mediates internalization of urokinase:plasminogen activator inhibitor complexes (uPA:PAI-1) and the urokinase receptor (uPAR). Here we investigated whether direct interaction between uPAR, a glycosyl-phosphatidylinositol-anchored protein, and LRP, a transmembrane receptor, is required for clearance of uPA:PAI-1, regeneration of unoccupied uPAR, activation of plasminogen, and the ability of HT1080 cells to invade extracellular matrix. We found that in the absence of uPA:PAI-1, uPAR is randomly distributed along the plasma membrane, whereas uPA:PAI-1 promotes formation of uPAR-LRP complexes and initiates redistribution of occupied uPAR to clathrin-coated pits. uPAR-LRP complexes are endocytosed via clathrin-coated vesicles and traffic together to early endosomes (EE) because they can be coimmunoprecipitated from immunoisolated EE, and internalization is blocked by depletion of intracellular K(+). Direct binding of domain 3 (D3) of uPAR to LRP is required for clearance of uPA-PAI-1-occupied uPAR because internalization is blocked by incubation with recombinant D3. Moreover, uPA-dependent plasmin generation and the ability of HT1080 cells to migrate through Matrigel-coated invasion chambers are also inhibited in the presence of D3. These results demonstrate that GPI-anchored uPAR is endocytosed by piggybacking on LRP and that direct binding of occupied uPAR to LRP is essential for internalization of occupied uPAR, regeneration of unoccupied uPAR, plasmin generation, and invasion and migration through extracellular matrix.
Czekay
R P
RP
Department of Cellular and Molecular Medicine, San Diego, La Jolla, California 92093-0651, USA. rpczekay@scripps.edu
Kuemmel
T A
TA
Orlando
R A
RA
Farquhar
M G
MG
D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002465:Cell Movement / Q000187:drug effects; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D005354:Fibrosarcoma; D006801:Humans; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D010960:Plasminogen Activators / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
D026503:Low Density Lipoprotein Receptor-Related Protein-1; C524898:PLAUR protein, human; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator