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
M. Ploug et al., 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, Biochemistry, 33(30), 1994, pp. 8991-8997
The cellular receptor for urokinase-type plasminogen activator (uPAR)
is a glycolipid-anchored membrane protein thought to play a primary ro
le in the generation of pericellular proteolytic activity, and to be i
nvolved in cancer cell invasion and metastasis. This protein is compos
ed of three homologous domains, the NH2-terminal of which is involved
in the high-affinity binding (K-d approximate to 0.1-1.0 nM) to the ep
idermal growth factor-like module of urokinase-type plasminogen activa
tor (uPA). Here we report that intact uPAR binds the low molecular wei
ght fluorophore 8-anilino-1-naphthalenesulfonate (ANS) to form a 1:1 s
toichiometric complex and that the resulting enhancement of the ANS fl
uorescence probes the functional state of uPAR as judged by several in
dependent criteria. First, the uPAR-mediated increase in ANS fluoresce
nce can be titrated by uPA as well as by its receptor binding derivati
ves (the amino-terminal fragment and the growth factor-like module). S
econd, an anti-uPAR monoclonal antibody, capable of preventing uPA bin
ding, can also titrate the uPAR-dependent ANS fluorescence whereas oth
er antibodies not interfering with uPA binding are unable to exert thi
s effect. Third, the dissociation profile of uPa-uPAR complexes as a f
unction of increasing concentrations of guanidine hydrochloride closel
y parallels the loss of the ANS binding site in uPAR. Finally, liberat
ion of the NH2-terminal domain from uPAR by limited chymotrypsin cleav
age after Tyr(87) leads to a loss of both enhanced ANS fluorescence an
d high-affinity uPA binding. This latter effect, a 1500-fold decrease
in uPA-binding affinity to isolated NH2-terminal domain, demonstrates
that this domain of uPAR does not contain all the determinants necessa
ry for uPA binding, possibly due to a requirement for interdomain inte
ractions to either stabilize an active conformation of this domain or
be directly involved in the binding process.