Ligand interaction between urokinase-type plasminogen activator and its receptor probed with 8-anilino-1-naphthalenesulfonate. Evidence for a hydrophobic binding site exposed only on the intact receptor.
The cellular receptor for urokinase-type plasminogen activator (uPAR) is a glycolipid-anchored membrane protein thought to play a primary role in the generation of pericellular proteolytic activity, and to be involved in cancer cell invasion and metastasis. This protein is composed of three homologous domains, the NH2-terminal of which is involved in the high-affinity binding (Kd approximately 0.1-1.0 nM) to the epidermal growth factor-like module of urokinase-type plasminogen activator (uPA). Here we report that intact uPAR binds the low molecular weight fluorophore 8-anilino-1-naphthalenesulfonate (ANS) to form a 1:1 stoichiometric complex and that the resulting enhancement of the ANS fluorescence probes the functional state of uPAR as judged by several independent criteria. First, the uPAR-mediated increase in ANS fluorescence can be titrated by uPA as well as by its receptor binding derivatives (the amino-terminal fragment and the growth factor-like module). Second, an anti-uPAR monoclonal antibody, capable of preventing uPA binding, can also titrate the uPAR-dependent ANS fluorescence whereas other antibodies not interfering with uPA binding are unable to exert this effect. Third, the dissociation profile of uPA-uPAR complexes as a function of increasing concentrations of guanidine hydrochloride closely parallels the loss of the ANS binding site in uPAR. Finally, liberation of the NH2-terminal domain from uPAR by limited chymotrypsin cleavage after Tyr87 leads to a loss of both enhanced ANS fluorescence and high-affinity uPA binding.(ABSTRACT TRUNCATED AT 250 WORDS)
Ploug
M
M
Finsen Laboratory, Rigshospitalet, Copenhagen O, Denmark.
Ellis
V
V
Danø
K
K
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism*; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002918:Chymotrypsin; D005456:Fluorescent Dyes; D006868:Hydrolysis; D008024:Ligands; D011956:Receptors, Cell Surface / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D013050:Spectrometry, Fluorescence; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
D000817:Anilino Naphthalenesulfonates; D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D008024:Ligands; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; C027132:1-anilino-8-naphthalenesulfonate; D002918:Chymotrypsin; D014568:Urokinase-Type Plasminogen Activator