MAPPING THE INTERACTION BETWEEN HIGH MOLECULAR WEIGHT KININOGEN AND THE UROKINASE PLASMINOGEN ACTIVATOR RECEPTOR

multifunctional, GPI-linked and We mapped the interaction sites between soluble uPAR (suPAR) and high molecular weight kininogen (HK). 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 (H 477 -G 496 ) and a lower affinity site within the heavy chain (C 333 -K 345 ). to suPAR fragments containing domains 2 and 3 (S-D2D3). to domain was also and addition of S-D1 to S-D2D3 inhibited biotin-HK or -46HKa binding to suPAR. Using sequential and overlapping 20 amino acid peptides prepared from suPAR, two regions for HK were identified. One on the carboxyterminal end of D2 (L 166 -T 195 blocked HK binding to suPAR and to umbilical vein (HUVEC). This site overlapped with the urokinase binding region and urokinase inhibited the binding of HK to suPAR. A second region on the aminoterminal portion of D3 (Q 215 -N 255 also blocked HK binding to HUVEC. Peptides that blocked HK binding to uPAR also inhibited PK activation on HUVEC. Therefore, HK interacts with suPAR at several sites. HK to uPAR as part of its interaction with its multiprotein receptor complex on HUVEC and the biological functions that upon this are modulated by urokinase.


INTRODUCTION
Recent investigations indicate that high molecular weight kininogen (HK 1. ) binds to endothelial cell membranes through an interaction with a multiprotein receptor complex comprising at least cytokeratin 1 (CK1), gC1qR, and the urokinase plasminogen activator receptor (uPAR) (1)(2)(3)(4)(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 2+ , 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 2+ 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 effect of other biologically relevant ligands, e.g. urokinase, on this binding, has not been well delineated. It is known that both the heavy and light chains of HK interact with a region of CK1 coded by exon 1 (2). Antibody to this region completely inhibits HK binding to CK1 as well as corresponding to each domain of suPAR were synthesized at Multiple Peptide Systems (San Diego, CA) (See Table II). All peptides from suPAR are numbered based upon the full-length sequence including the 22 amino acid signal peptide of uPAR. The peptides used were colorless, odorless, and >95% pure as determined by reverse phase HPLC and mass spectrometry. A monoclonal antibody against uPAR (3B10FC) was generously provided by Dr. Robert F. Todd III from the University of Michigan, Ann Arbor, MI (16). Monoclonal anti-uPAR antibodies E180 and E33 were the kind gifts of Dr. A. Mazar (Attenuon, San Diego, CA).

Preparation of wild type and mutant suPAR:
Soluble urokinase plasminogen activator receptor (suPAR), isolated domains of suPAR (1,2,3), a fragment containing recombinant soluble suPAR domains 1 and 2 and 3 (S-D1, S-D2D3) and scuPA were prepared and expressed using the Drosophilia Expression System (Invitrogen, Carlsbad, CA) according to manufacturer's instructions, as described previously (17,18). Isolated domains 1,2, and 3 of suPAR were prepared from wild type protein by sequential digestion with chymotrypsin and pepsin as previously reported (19). Mutagenesis of suPAR in pMT/Bip/V5 (Invitrogen) was performed with the QuikChange Site-directed Mutagenesis Kit (Stratagene, La Jolla, CA) (17). The suPAR domain 2 and 3 mutants are shown in Table III. Wild-type and variant suPARs were purified from the media using a monoclonal (E180 or E33) anti-uPAR antibody affinity column. Wildtype scuPA was purified from the media using a monoclonal anti-uPA antibody column and were analyzed by SDS-PAGE and western blot (17). All suPAR mutants are numbered based upon the full-length sequence of suPAR including the signal peptide.  (17).

Prekallikrein activation on endothelial cells.
The ability of peptides from suPAR to block PK activation on endothelial cells was determined. HUVEC were grown as monolayers in microtiter plate wells. HK and PK (20 nM each) were then added to the wells in the absence or presence of 100 M peptide DLC20, LRG20, YLP20, PGS20, FHN20, TKC19, QCY21, or DCR20 from domains 1 to 3 of suPAR (See Table II

Characterization of HK species:
One g of HK, 46HKa, and 56HKa was analyzed using 8% SDS-PAGE under reduced and non-reduced conditions. Under reduced conditions, HK, 56HKa, and 46HKa migrated predominantly at 120 kDa, 64 and 56 kDa, and 64 and 46 kDa, respectively ( Figure 1A). These results indicate that 56HKa and 46HKa retained their heavy and their respective light chains. Under non-reduced conditions, HK, 56HKa and 46HKa migrated as single bands at 120 kDa, 110 kDa, and 108 kDa, respectively (Figure1B), consistent with previous reports of a change in the molecular mass of plasma HK when it is activated by plasma kallikrein (20).

Binding of biotin-HK to HUVEC and suPAR.
We then determined if these forms of HK differed in their affinity for HUVEC or for suPAR. Initial investigations were with HUVEC.
Native HK, 56HKa, and 46HKa blocked the binding of biotinylated-HK to HUVEC with an IC 50 of 115 nM, 115 nM, and 50 nM, respectively ( Figure 2A). Native HK, 56HKa, and 46HKa blocked biotin-HK binding to suPAR with an IC 50 of 60 nM, 110 nM, and 8 nM, respectively ( Figure 2B). These data indicate that 46HKa is a more potent inhibitor of the binding of native HK to HUVEC and suPAR than are the less activated forms of HK. This outcome was consistent with previously reported information that kallikrein-cleaved HK bound more avidly to uPAR than did intact HK (5). The data also demonstrated that native HK also bound to suPAR, albeit with lower affinity than its cleaved counterparts. This information suggested that cleavage of HK enhanced its binding to suPAR. This increase in affinity of binding for 46HKa was less pronounced on HUVEC which express additional binding sites for the kininogens.

Binding sites for suPAR within domain 5 of HK. It has been shown in previous studies that HK
bound to cells through determinants within both its light and heavy chains (13,15). Therefore, we next sought to identify the region(s) in domain 5 of HK that bound to suPAR and inhibited the binding of native HK and 46HKa (13)(14)(15). Binding of biotin-HK to suPAR was blocked by peptides GKE19, HNL21, GHG19, HKH20, and HVL24 with an IC 50 of 300 M, 20 M, 30 M, 2 M, and 0.7 M, respectively (Table I). HKH20 was a weaker inhibitor of biotin-46HKa binding (Table I). Peptides HKH20, HNL21, and HVL24 inhibited biotin-46HKa binding to suPAR with an IC 50 of 20 M, 10 M and 0.5 M, respectively (Table I). These data indicate that the HVL24 region, corresponding to amino acids 471-494 of domain 5 of HK, has the highest affinity for suPAR, although other portions of D5 also had the capacity to bind.

Binding sites for suPAR within domain 3 of HK. A similar investigation was performed with
peptides within the cell-binding region in domain 3 of HK (13) ( Table I) (Table I), whereas peptide KIC11 from the amino terminal end of this domain was inactive (Table I) (Table I). CM-papain was an equipotent inhibitor of biotin-46HKa and biotin-HK binding (IC 50 =1.5 M) (data not shown). Taken together, these data suggested that HK bound to suPAR through regions found on both its heavy and light chains, although the binding of suPAR to domain 5 peptides was at least 10-fold more avid (compare IC 50 of HVL24 with CNA13). This conclusion was supported by the finding that biotin-HKH20 and biotin-CNA13 bound specifically to suPAR ( Figure 3).

Binding sites for HK in suPAR.
We next investigated the binding sites in suPAR for HK. As a first step, the epitope recognized by a monoclonal anti-uPAR antibody (3B10FC) that blocked HK binding to cultured endothelial cells (6,16) was partially mapped. On immunoblot, 3B10FC bound to suPAR domain 2 and to fragments containing domains 2 + 3 (D2D3, S-D2D3); 3B10FC bound weakly to D3, but not to D1, under the same experimental conditions ( Figure 4).
These data are consistent with previous studies in which it was reported that a polyclonal antibody to domains 2 and 3 of uPAR blocked HK binding to HUVEC (5,6). Additional investigations were then performed to determine if HK bound directly to domains 2 and 3 of suPAR (5). Ten M purified S-D2D3 inhibited the binding of biotin-HK and -46HKa to fulllength suPAR by ~55-60% ( Figures 5A and 5B). At the same concentration, isolated domain 1 of suPAR blocked binding 20-25% ( Figure 5A and 5B). When combined, isolated S-D1 and S-D2D3 blocked biotin-HK and -46HKa binding to an even greater extent than that seen with intact suPAR. Based on these findings, we focused our attention on identifying the HK binding regions with domains 2 and 3 of suPAR.

Binding sites for HK within domains 2 and 3 of suPAR.
To map the region(s) within this fragment of suPAR that recognize HK, a series of 20 amino acid peptides were synthesized starting at the carboxyterminus of domain 1 and spanning domains 2 and 3 (Table II). Peptide PGS20, located in the carboxyterminus of domain 2, inhibited binding of biotin-HK with an IC 50 of 1.8 M (Table II). Additional overlapping peptides that incorporated or flanked the PGS20 region, LRG20, YLP20, and FHN20, inhibited biotin-HK binding to suPAR with an IC 50 of 0.9 M, 20 M, and 1 M, respectively (Table II). These data suggested that full-length suPAR contains a binding site for HK within amino acids 166-195 of domain 2. This finding is biologically important because these peptides also inhibited biotin-HK binding to HUVEC (Table II). Binding of biotin-HK to HUVEC also was inhibited by PGS20, YLP20, LRG20, and FHN20 with an IC 50 of 40 M, 25 M, 7 M, and 4 M, respectively (Table II).
A similar approach was employed to begin to map HK binding site(s) within suPAR domain 3 (Table II). Two peptides from the amino terminal portion of domain 3 of uPAR, QCY21 and DCR20, inhibited biotin-HK binding to HUVEC with an IC 50 of 12 and 22 M, respectively (Table II). These data suggested that an HK binding site also was contained within amino acids 215-255 of domain 3 of uPAR.
Binding of a uPAR-domains 2+3 peptide to HK. Based on its capacity to inhibit HK binding to suPAR, we next determined if biotinylated PGS20 from suPAR bound directly to HK. Biotin-PGS20 bound to HK linked to microtiter plate cuvette wells in a specific, concentrationdependent and saturable manner ( Figure 6A). Binding of biotin-PGS20 to immobilized HK was blocked by HK and PGS20. Biotin-QCY21 or -DCR20 also bound specifically to HK ( Figure   6B) and 100-fold molar excess unlabeled QCY21 or 50-fold molar excess unlabeled DCR20 blocked the binding of their biotinylated forms to HK ( Figure 6B). These data suggest that regions on domains 2 and 3 combined to form an HK binding site.
Mapping the HK binding site in suPAR using site-directed mutagenesis. As a second, independent approach to identify the binding sites for HK in domains 2 and 3 of suPAR, sitedirected mutagenesis was performed on the charged amino acids in those regions that had been implicated in binding based on peptide inhibition. The suPAR mutant H 182 A, N 184 A lost its ability to inhibit biotin-HK binding to suPAR ( Figure 7A, Table III). In contrast, suPAR mutant H 182 A, D 185 A inhibited biotin-HK binding with an IC 50 of 10 M, a value identical to wild type suPAR ( Figure 7A, Table III). These results indicated that position H 182 contributed little to HK binding to suPAR. To examine this interpretation, N184 was then mutated to the more conservative residue glutamine rather than to alanine. SuPAR N 184 Q at 10 M inhibited biotin-HK binding unlike suPAR H 182 A, N 184 A, but less so than the wild type protein ( Figure 7A, Table   III). These data indicated that N184 was important for the suPAR-HK interaction. These data were also consistent with the peptide inhibition data in Table II   Studies were also performed to map the binding site for HK in domain 3 of suPAR. The variant suPAR mutant N 222 Q did not inhibit biotin-HK binding to suPAR ( Figure 7B, Table III).

In contrast, point mutations in neighboring region (suPAR mutant E 230 A, E 231 A) inhibited biotin-
HK binding with an IC 50 =6 M, similar to wild type suPAR. These data pointed to the involvement of amino acid 222 in domain 3 for binding of HK, an amino acid localization consistent with the results of the peptide mapping experiments shown in Figure 9.

Effect of suPAR peptides on prekallikrein activation.
We then asked whether peptides from suPAR that bound HK interfered with PK activation on cultured endothelial cells (21). Peptide FHN20 inhibited PK activation on HUVEC by 78% (Table II) (Figure 9), consistent with its capacity to inhibit HK binding to these cells (Table II). Likewise, peptides LRG20, YLP20, and PGS20 that blocked HK binding to suPAR with an IC 50 of 7, 30, and 70 M, respectively, also inhibited PK activation on HK by 62%, 50%, and 38%, respectively ( Figure 9). Thus, interference of HK binding by peptides to the HK binding region on suPAR blocked PK activation on cells, supporting the relevance of the data obtained using suPAR and indicating an obligatory role for uPAR in PK activation.

DISCUSSION
These investigations address the mechanism by which the HK and PK complex assembles on the uPAR-containing multiprotein HK receptor complex on endothelial cells. Our studies indicate that the binding of HK to uPAR is mediated through regions on both its domain 3 heavy chain and domain 5 light chain. These results are consistent with our previous work, which showed that domains 3 and 5 of HK are involved in cell binding (13,14). It is of interest that the domain 5 region of HK, which contains its anti-angiogenic and anti-proliferative activity, binds to suPAR with a 10-fold higher affinity than does the domain 3 region, suggesting that uPAR may contribute to these biological functions.
It has been reported previously that native HK does not bind to suPAR in contrast to cleaved HK (HKa) (5). These results are at odds with our finding that biotin-HK bound to uPAR on cultured endothelial cells (6). This difference may be explained by findings in the present study which show that intact HK or 56 kDa kallikrein-cleaved HK (56HKa) binds suPAR with 7.5-to 14-fold lower affinity than does 46 kDa kallikrein-cleaved HK (46HKa). Thus, proteolysis of HK liberating the bradykinin moiety and an 11 kDa amino terminal portion from the light chain of HK promotes its binding to uPAR, rather than necessarily invoking the involvement of a novel receptor (24). These findings have implications for the anti-angiogenic and anti-proliferative activity of HKa. Intact HK, which under most physiologic circumstance is in molar excess to HKa, is likely to inhibit HKa binding to uPAR under physiological conditions (24)(25)(26). Bradykinin liberated from HK expresses pro-angiogenic activity (26,27). Thus it is likely that HKa inhibits angiogenesis only in situations where sufficiently large concentrations of these proteolytic fragments have been generated to overcome the competing influence of native plasma HK. Thus, the local ratio of HK to HKa may modulate its pro-and anti-angiogenic activities (24)(25)(26)(27).
The HK binding region within domain 2 of uPAR overlaps with a region implicated in the binding of uPA (23) (Figure 10). In contrast to a previous study (5), we found that scuPA inhibited the binding of HK to suPAR. We attribute this difference to the source of reagents as we found that several commercial sources of uPA showed little or no inhibition as well. The plasma concentration of HK is 700 nM and that of scuPA 1-2 nM, implying that HK may limit the binding of uPA to uPAR in vivo. On the other hand, we, and others, have found that plasma kallikrein is a potent activator of scuPA and that endothelial cell membranes provide a kinetically favorable environment for this process (22,28). This apparent paradox may be explained by the fact that scuPA has a 113-fold tighter affinity for uPAR than does HK. Thus, at physiological plasma concentrations, approximately one-quarter of the uPA receptors would be predicted to be occupied by scuPA, the remainder by HK. Higher occupancy rates of uPAR by uPA may occur when cells have been stimulated to migrate and synthesize uPA locally.           Table II