REGULATION OF SINGLE-CHAIN UROKINASE BINDING, INTERNALIZATION, AND DEGRADATION BY A PLASMINOGEN-ACTIVATOR INHIBITOR 1-DERIVED PEPTIDE

The internalization and degradation of cell associated urokinase type plasminogen activator (uPA) through the alpha(2)-macroglobulin recepto r/low density lipoprotein-related receptor (alpha(2)MR/LRP) represent important steps in the control of plasmin formation. Complexes between two chain urokinase (tcuPA) and plasminogen activator type 1 are degr aded rapidly whereas single chain urokinase (scuPA) is not, suggesting that alpha(2)MR/LRP requires specific epitopes in the serpin for effe ctive function. We report an alternative mechanism that may contribute to this process. The binding of scuPA to LM-TK- cells that lack the u PA receptor was stimulated by the hexapeptide EEIIMD, corresponding to amino acids 350-355 of plasminogen activator type 1, which contacts t he sequence RHRGGS, corresponding to amino acids 179-184 in uPA. EEIIM D increased the B-max of scuPA binding 4-fold with the half-maximal ef fect achieved at a peptide concentration of 50 mu M. Stimulation was d ependent on the charge on the COOH-terminal amino acid but not on the NH2 terminus of the peptide. EEIIMD also stimulated the internalizatio n and degradation of scuPA. Both the binding and internalization of sc uPA in the presence of EEIIMD were blocked by recombinant, 39-kDa alph a(2)MR/LRP-associated protein as well as by an anti-alpha(2)MR/LRP ant ibody. EEIIMD also stimulated the binding of scuPA to purified alpha(2 )MR/LRP. EEIIMD had no effect on the binding of tcuPA or of complexes between scuPA and its receptor. These results suggest that EEIIMD regu lates the binding of scuPA with alpha(2)MR/LRP. These findings also su ggest a potential mechanism by which scuPA can be cleared which is ind ependent of activation by plasmin or binding to uPA receptor.