Abstract
The urokinase plasminogen activator receptor (uPAR) is a multifunctional, GPI-linked receptor that modulates cell adhesion/migration and fibrinolysis. We mapped the interaction sites between soluble uPAR (suPAR) and high molecular mass kininogen (HK). Binding of biotin-HK to suPAR was inhibited by HK, 56HKa, and 46HKa with an IC50 of 60, 110, and 8 nm, respectively. We identified two suPAR-binding sites, a higher affinity site in the light chain of HK and 46HKa (His477-Gly496) and a lower affinity site within the heavy chain (Cys333-Lys345). HK predominantly bound to suPAR fragments containing domains 2 and 3 (S-D2D3). Binding of HK to domain 1 (S-D1) was also detected, and the addition of S-D1 to S-D2D3 completely inhibited biotin-HK or -46HKa binding to suPAR. Using sequential and overlapping 20-amino acid peptides prepared from suPAR, two regions for HK binding were identified. One on the carboxyl-terminal end of D2 (Leu166-Thr195) blocked HK binding to suPAR and to human umbilical vein endothelial cells (HUVEC). This site overlapped with the urokinase-binding region, and urokinase inhibited the binding of HK to suPAR. A second region on the amino-terminal portion of D3 (Gln215-Asn255) also blocked HK binding to HUVEC. Peptides that blocked HK binding to uPAR also inhibited prekallikrein activation on HUVEC. Therefore, HK interacts with suPAR at several sites. HK binds to uPAR as part of its interaction with its multiprotein receptor complex on HUVEC, and the biological functions that depend upon this binding are modulated by urokinase.
Highlights
Recent investigations indicate that high molecular mass kininogen (HK)1 binds to endothelial cell membranes through an interaction with a multiprotein receptor complex comprising at least cytokeratin 1, gC1qR, and the urokinase plasminogen activator receptor [1,2,3,4,5]
HK, 56HKa, and 46HKa migrated predominantly at 120 kDa, at 64 and 56 kDa, and at 64 and 46 kDa, respectively (Fig. 1A). These results indicate that 56HKa and 46HKa retained their heavy chains and their respective light chains
Interference of HK binding by peptides to the HK-binding region on soluble uPAR (suPAR) blocked PK activation on cells, supporting the relevance of the data obtained using suPAR and indicating an obligatory role for urokinase plasminogen activator receptor (uPAR) in PK activation. These investigations address the mechanism by which the HK and PK complex assembles on the uPAR-containing multiprotein HK receptor complex on endothelial cells
Summary
Proteins and Materials—Single-chain and two-chain HK (specific activity of 13–17 units/mg and 17 units/ml, respectively) in 4 mM sodium acetate-HCl and 0.15 M NaCl, pH 5.3, plasma kallikrein, and PK (specific activity of 22–27 units/mg) in 4 mM sodium acetate-HCl and 0.15 M NaCl, pH 5.3, were purchased from Enzyme Research Laboratories, Inc. (South Bend, IN). Biotin-HK (10 nM) in HEPES carbonate buffer was incubated with monolayers of human umbilical vein endothelial cells (A) or purified suPAR (B) in the absence or presence of increasing concentration of purified HK, 56Hka, or 46HKa for 1 h at 37 °C. Biotin-Protein or -Peptide Binding Studies—Biotinylated proteins or peptides in the absence or presence of competing proteins or peptides in HEPES carbonate buffer, pH 7.4 [8], were added to microtiter plates coated with monolayers of HUVEC or with other proteins or peptides (1 g/ml) coated by an overnight incubation at 4 °C in 0.1 M sodium carbonate, pH 9.6. The bound biotinylated proteins or peptides were measured using ImmunoPure streptavidin horseradish peroxidase conjugate (Pierce) and peroxidase-specific fast reacting substrate, 3,3Ј,5,5Ј-tetramethylbenzidine dihydrochloride (Pierce) by measuring the absorbance of the reaction mixture at 450 nm using a microplate autoreader EL 311 (Bio-Tek Instrument, Winooski, VT), as previously described [14, 21]
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