The Urokinase/PAI-2 Complex: A NEW HIGH AFFINITY LIGAND FOR THE ENDOCYTOSIS RECEPTOR LOW DENSITY LIPOPROTEIN RECEPTOR-RELATED PROTEIN

David Croucher,Darren N Saunders,Marie Ranson

doi:10.1074/jbc.m513645200

Abstract

The efficient inactivation of urokinase plasminogen activator (uPA) by plasminogen activator inhibitor type 2 (PAI-2) at the surface of carcinoma cells is followed by rapid endocytosis of the uPA-PAI-2 complex. We now show that one pathway of this receptor-mediated endocytosis is mediated via the low density lipoprotein receptor-related protein (LRP) in prostate cancer cells. Detailed biochemical analyses using ligand binding assays and surface plasmon resonance revealed a novel and distinct interaction mechanism between native, human LRP and uPA-PAI-2. As reported previously for PAI-1, inhibition of uPA by PAI-2 significantly increased the affinity of the complex for LRP (K(D) of 36 nm for uPA-PAI-2 versus 200 nm for uPA). This interaction was maintained in the presence of uPAR, confirming the validity of this interaction at the cell surface. However, unlike PAI-1, no interaction was observed between LRP and PAI-2 in either the stressed or the relaxed conformation. This suggests that the uPA-PAI-2-LRP interaction is mediated by site(s) within the uPA molecule alone. Thus, as inhibition of uPA by PAI-2 resulted in accelerated clearance of uPA from the cell surface possibly via its increased affinity for LRP, this represents a mechanism through which PAI-2 can clear proteolytic activity from the cell surface. Furthermore, lack of a direct interaction between PAI-2 and LRP implies that downstream signaling events initiated by PAI-1 may not be activated by PAI-2.

Highlights

The efficient inactivation of urokinase plasminogen activator by plasminogen activator inhibitor type 2 (PAI-2) at the surface of carcinoma cells is followed by rapid endocytosis of the uPAPAI-2 complex
Candidate Endocytosis Receptors Involved in the Internalization of PAI-2—We have previously shown efficient inhibition of cell surface uPA by PAI-2 on carcinoma cell lines followed by rapid internalization of the uPAR/uPA-PAI-2 complex and delivery into endosomes and lysosomes through a receptor-associated protein (RAP)-inhibitable mechanism (7), suggesting internalization via receptor-mediated endocytosis
The efficient inactivation of uPA by PAI-2 at the surface of carcinoma cells is followed by rapid internalization of the uPA-PAI-2 complex into endosomes and lysosomes (7)

Summary

EXPERIMENTAL PROCEDURES

Antibodies, and Reagents—Recombinant human PAI-2 (47kDa form) and PAI-2 CD loop mutant (residues 66 –98 deleted) (41) were provided by PAI-2 Proprietary Ltd. (Sydney, Australia). The cells were resuspended at 1 ϫ 106 cells/ml in ice-cold binding buffer containing primary polyclonal antibodies or their irrelevant isotype control antibody (5 ␮g/ml) or biotinylated RAP (10 nM) and incubated for 45 min on ice. Proteins were biotin-labeled using biotin NHS according to the manufacturer’s instructions. After three washes with ice-cold binding buffer, the cells were incubated with goat anti-rabbit IgG-FITC (1:50 dilution) or streptavidin-FITC (1:200 dilution), respectively, for 45 min on ice. In all cases, cell surface fluorescence was analyzed by dual color flow cytometry as described previously (42). In the case of EDTA inhibition, EDTA (5 mM) was present during the incubation with uPA-PAI-2 to prevent the restoration of calcium binding by LRP After another three 10-min washes with TBST, the membranes were incubated with neutravidin-HRP (1:10,000 dilution) or rabbit anti-mouse IgG-HRP (1:50 dilution) for 1 h at room temperature in TBST, 1% gelatin, CaCl2. A one-binding site model with a drifting baseline provided the best fit according to ␹2 values and analysis of residual plots

RESULTS

DISCUSSION

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