Urokinase-catalysed cleavage of the urokinase receptor requires an intact glycolipid anchor

Urokinase (uPA) has the striking ability to cleave its receptor, uPAR, ther eby inactivating the binding potential of this molecule. Here we demonstrat e that the glycosylphosphatidylinositol (GPI) anchor of uPAR, which is atta ched to the third domain, is an important determinant in governing this rea ction, even though the actual cleavage occurs between the first and second domains. Purified full-length GPI-anchored uPAR (GPI-uPAR) proved much more susceptible to uPA-mediated cleavage than recombinant truncated soluble uP AR (suPAR), which lacks the glycolipid anchor. This was' not a general diff erence in proteolytic susceptibility since GPI-uPAR and suPAR were cleaved with equal efficiency by plasmin. Since the amino acid sequences of GPI-uPA R and suPAR are identical except for the C-terminal truncation, the differe nt cleavage patterns suggest that the two uPAR variants differ in the confo rmation or the flexibility of the linker region between domains 1 and 2. Th is was supported by the fact that an antibody to the peptide AVTYSRSRYLE, a mino acids 84-94 in the linker region, recognizes GPI-uPAR but not suPAR. T his difference in the linker region is thus caused by a difference in a rem ote hydrophobic region. In accordance with this model, when the hydrophobic lipid moiety was removed from the glycolipid anchor by phospholipase C, lo w concentrations. of uPA could no longer cleave the modified GPI-uPAR and t he reactivity to the peptide antibody was greatly decreased. Naturally occu rring suPAR, purified from plasma, was found to have a similar resistance t o uPA cleavage as phospholipase C-treated GPI-uPAR and recombinant suPAR.