Mapping the Interaction between High Molecular Mass Kininogen and the Urokinase Plasminogen Activator Receptor

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 (su-PAR) and high molecular mass kininogen (HK). Binding of biotin-HK to suPAR was inhibited by HK, 56HKa, and 46HKa with an IC 50 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 (His 477 -Gly 496 ) and a lower affinity site within the heavy chain (Cys 333 -Lys 345 ). 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. 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 (uPAR) (1-5). The three proteins co-localize on the endothelial cell membrane (6). The same three proteins form a receptor complex for factor XII (7), but binding of factor XII in vivo is likely limited both by the low plasma concentration of free Zn 2ϩ , which is below the requirement for FXII binding and by the much higher plasma concentration of HK (7). Binding of HK to this multiprotein receptor complex predominates, localizing prekallikrein (PK) to the cell surface. The plasma concentration of PK and the ambient free Zn 2ϩ concentration in plasma also prevent FXI from binding to HK under conditions where platelets or other cells are not activated (8). PK bound to HK on endothelial cells is proteolyzed by membrane-expressed prolylcarboxypeptidase to form plasma kallikrein (9, 10). This multiprotein receptor complex thereby regulates the assembly and activation of the plasma kallikrein/ kinin system.The requirements for HK binding to each component of this receptor complex and the effects of other biologically relevant ligands, e.g. urokinase, on this binding have not been well delineated. It is known that both the heavy and light chains of HK interact with a region of cytokeratin 1 coded by exon 1 (2). Antibody to this region completely inhibits HK binding to cytokeratin 1 as well as to the receptor complex (6). Likewise, some antibodies to gC1qR and uPAR completely block HK binding to the proteins individually as well as when they are part of the complex expressed on endothelial cells (6, 8). However, it is unclear whether each component of the cellular complex recognizes discrete or overlapping portions of HK and whether each molecule of HK binds to more than one component of the complex at the same time. To begin to address these issues, we sought to identify the regions in HK and uPAR required for binding. The results indicate the existence of multiple potential sites of interaction between this ligand and receptor and provide insight into the assembly of HK on its multireceptor co...

Domain 2 of uPAR regulates single-chain urokinase-mediated angiogenesis through β1-integrin and VEGFR2

“…Overlapping peptides (LRG20, YLP20, and PGS20) from uPAR domain 2 (amino acids 144 -173) blocked ScuPAinduced expression of pERK1/2 (P Ͻ 0.003; Fig. 3, C and F, and Table 1) (50). A corresponding scrambled peptide (PGS20) had no effect.…”

“…Angiogenesis microscopic images were obtained using a Nikon TE200 microscope with a ϫ20/0.45 lens. (50). The modeling was performed with the Cn3D4.3 program available from the National Center for Biotechnology Information structure website.…”

“…Therefore, it was not clear how human ScuPA induced pERK1/2 in SIN1 Ϫ/Ϫ MEFs. Although murine and human ScuPA share only 60% overall sequence homology, there is 77% amino acid sequence homology in domain 2 of uPAR from Leu 144 to Thr 173 , the region that expresses overlapping binding sites for ScuPA, factor XII, vitronectin, and HK (15,42,43,50,28). Leu 144 to Thr 173 lies on the surface of uPAR, with the potential to contact ScuPA (Fig.…”

“…Although the growth factor domain of urokinase interacts with domain 1 of uPAR, domain 2 of uPAR also is a recognized binding site for ScuPA (4,18,23,50). High-molecular-weight kininogen (HK), vitronectin, and factor XII, uPAR-soluble ligands, also bind to the same region on domain 2 as ScuPA (15,28,42,43,50).…”

High Molecular Weight Kininogen Activates B2 Receptor Signaling Pathway in Human Vascular Endothelial Cells

“…Although previous studies indicated that HK did not bind to urokinase plasminogen activator receptor (uPAR) (41), emerging evidence suggests that the unique regions of HK might serve as urokinase-binding sites (45). Recently, it has been reported that both HK and cleaved HK bound to gC1qR, cytokeratin 1 and the soluble extracellular form of urokinase plasminogen activator receptor (uPAR) (46).…”