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

Abstract

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)

Urokinase Induces Basophil Chemotaxis through a Urokinase Receptor Epitope That Is an Endogenous Ligand for Formyl Peptide Receptor-Like 1 and -Like 2

“…We have extended their observation by showing that even if both basophils and THP-1 cells express mRNA for FPRL1 and FPRL2, uPA and uPAR 84 -95 induce basophil chemotaxis mainly through the engagement of FPRL2. These results suggest that after binding to uPA, uPAR undergoes conformational changes (59), allowing it to interact with FPRL1 in monocytes (9) and with FPRL2 in basophils. We cannot exclude the possibility that FMLP receptor binding of the uPA/uPAR complex triggers additional conformational changes that are important in mediating the transmission of signal from the cell surface to the inner domains involved in basophil chemotaxis.…”

Removal of domain D2 or D3 of the human urokinase receptor does not affect ligand affinity

“…The disulfide bond pattern of D1 conforms to the general structure of the Ly-6/neurotoxins protein family and has suggested overall structural homology to CD59 and snake a-bungarotoxin for which a tertiary structural analysis is available [17][18][19]. The purified amino terminal domain (D1) of uPAR possesses independent ligand-binding activity, although with an affinity 1,500 fold lower than the wild type [1]; the purified carboxy-terminal fragment (D2D3) appears to be devoid of binding activity [1,15]. We have investigated, therefore, the effect of removing domain D2 or D3 on the binding properties of human uPAR.…”

Urokinase/urokinase receptor and vitronectin/αvβ3 integrin induce chemotaxis and cytoskeleton reorganization through different signaling pathways

“…UPA preferentially binds to domain 1, but all three domains are required for high a nity binding. UPAR also binds vitronectin (VN) and again all three domains are required for high a nity binding (Behrendt et al, 1991;Wei et al, 1994;Waltz and Chapman, 1994;Ploug et al, 1994;Kanse et al, 1996;Hùyer-Hansen et al, 1997;Sidenius and Blasi, 2000).…”