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.