Chemical modification of the urokinase-type plasminogen activator and its 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 activator (uPA) and its glycolipid anchored receptor (uPAR) is essential for the confinement of plasminogen activation to cell surfaces where it is thought to play an important role in cancer cell invasion and metastasis. The receptor binding site of uPA is retained within its isolated growth factor-like module (GFD; residues 4-43). The NH2-terminal domain 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., & Danø, 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 tetranitromethane. All six tyrosine residues in uPAR and the single tyrosine residue in GFD (Tyr24) were susceptible to nitration in the native uncomplexed proteins, whereas in the receptor-ligand complexes both Tyr57 of uPAR and Tyr24 of GFD were protected from modification. Modification of uPAR alone led to a parallel reduction in the potential to bind pro-uPA and 8-anilino-1-naphthalenesulfonate, an extrinsic fluorophore reporting on the accessibility of a hydrophobic site involved in uPA binding. These data clearly demonstrate that Tyr57 in the NH2-terminal domain of uPAR and Tyr24 in uPA are intimately engaged in the receptor-ligand interaction, whereas Tyr87 positioned in the linker region between the first two domains of uPAR does not appear to be shielded by the resulting intermolecular interface.
Ploug
M
M
Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark.
Rahbek-Nielsen
H
H
Ellis
V
V
Roepstorff
P
P
Danø
K
K
D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000737:chemistry; D055293:Receptors, Urokinase Plasminogen Activator; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013774:Tetranitromethane / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry*; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry*
D008024:Ligands; C524898:PLAUR protein, human; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D014443:Tyrosine; D014568:Urokinase-Type Plasminogen Activator; D013774:Tetranitromethane