CHEMICAL MODIFICATION OF THE UROKINASE-TYPE PLASMINOGEN-ACTIVATOR ANDITS RECEPTOR USING TETRANITROMETHANE - EVIDENCE FOR THE INVOLVEMENT OF SPECIFIC TYROSINE RESIDUES IN BOTH MOLECULES DURING RECEPTOR-LIGAND INTERACTION

The high-affinity interaction between urokinase-type plasminogen activ ator (uPA) and its glycolipid anchored receptor (uPAR) is essential fo r the confinement of plasminogen activation to cell surfaces where it is thought to play an important role in cancer cell invasion and metas tasis. The receptor binding site of uPA is retained within its isolate d growth factor-like module (GFD; residues 4-43). The NH2-terminal dom ain of uPAR has a primary role in uPA binding, although maintenance of its multidomain structure has been shown to be necessary for the high affinity of this interaction [Ploug, M., Ellis, V., & Dano, K. (1994) Biochemistry 33, 8991-8997]. To identify residues engaged in the uPAR -uPA interaction, we have performed a ''protein-protein footprinting'' study on preformed uPAR-GFD complexes by chemical modification with t etranitromethane. All six tyrosine residues in uPAR and the sin,ale ty rosine residue in GFD (Tyr(24)) were susceptible to nitration in the n ative uncomplexed proteins, whereas in the receptor-ligand complexes b oth Tyr(57) of uPAR and Tyr(24) of GFD were protected from modificatio n. Modification of uPAR alone led to a parallel reduction in the poten tial to bind pro-uPA and 8-anilino-1-naphthalenesulfonate, an extrinsi c fluorophore reporting on the accessibility of a hydrophobic site inv olved in uPA binding. These data clearly demonstrate that Tyr(57) in t he NH2-terminal domain of uPAR and Tyr(24) in uPA are intimately engag ed in the receptor-ligand interaction, whereas Tyr(87) positioned in t he Linker region between the first two domains of uPAR does not appear to be shielded by the resulting intermolecular interface.