Mapping Part of the Functional Epitope for Ligand Binding on the Receptor for Urokinase-type Plasminogen Activator by Site-directed Mutagenesis

Abstract

The urokinase-type plasminogen activator receptor (uPAR) 1 is a multifunctional membrane glycoprotein primarily involved in the regulation of pericellular proteolysis due to its high affinity interaction with the growth factor-like module of urokinase-type plasminogen activator (uPA) (1), but which has also been implicated in the promotion of cell adhesion due to its vitronectin and integrin binding properties (2), in signal transduction (3) and chemotaxis (4, 5). However, the uPA-uPAR interaction per se is intimately coupled to the latter "nonproteolytic" functions of uPAR, since this interaction either elicits or modulates these events. Cell surfaces expressing uPAR constitutes favored microenvironments for uPA-mediated plasminogen activation (6). Consequently, the uPA-uPAR interaction represents an attractive molecular target for the development of small receptor binding antagonists that may prove useful during treatment of certain diseases in which uPAR has been implicated, i.e. cancer invasion and metastasis (7-13).uPAR is a glycosylphosphatidylinositol-anchored plasma membrane protein (14) having an extracellular part composed of three homologous domains belonging to the Ly-6/uPAR protein domain family, 2 as reviewed (15). This family is dominated by single domain proteins among which the glycolipid-anchored members are found primarily in mammalians (i.e. CD59, Ly-6, E48, and ThB) whereas the secreted members belong to either reptiles or amphibians (i.e. ␣-neurotoxins, fasiculins, cardiotoxins, and xenoxins). Intriguingly, a similar phylogenetic relationship exists for the few family members identified so far containing two Ly-6/uPAR domains, i.e. the glycolipid anchored RoBo-1 (16) and metastasis-associated C4.4 (17) isolated from rat versus the secreted phospholipase A2 inhibitor isolated from cobra blood (18). Comparison of the three-dimensional protein structures available for several single domain members of this protein family reveals a "three finger" consensus structure consisting of 3 loops, a central 3-stranded ␤-sheet, and a globular, disulfide-rich core (19 -21). The individual domains of uPAR (numbered I, II and III) are thought to adopt a similar "three finger fold" (15).The involvement of uPAR domain I (residues 1-87) 3 in uPA binding is demonstrated by several lines of evidence. First, uPAR domain I can be specifically cross-linked to a receptor binding derivative of uPA using two different chemical crosslinking reagents (23,24). Second, the uPA-uPAR interaction can be inhibited competitively by monoclonal antibodies recognizing epitopes on uPAR domain I (25-27). Third, uPAR domain I is also a target for the specific photoinsertion from a small peptide antagonist of uPA binding (28). Specific proteolytic cleavage after Tyr 87 , situated in the linker region between domains I and II, is, however, also accompanied by a Ͼ1,500-fold reduction in the affinity for uPA (⌬⌬G Ͼ 4 kcal/mol), which clearly emphasizes the necessity of the multidomain structure of uPAR for high affinity ligand binding (2...

Conformational Regulation of Urokinase Receptor Function

“…In contrast, Tyr 57 is located at the bottom of the hydrophobic uPA binding cavity and is completely shielded from solvent in the corresponding uPAR-ATF complex (16,40). The affinity of uPAR Y57A for uPA is accordingly decreased by ϳ7-fold compared with both uPAR wt and uPAR W32A (34,39), whereas vitronectin binding is unaffected.…”

“…Purified mouse anti-uPAR mAbs (1 g/ml) were covalently immobilized on a carboxymethylated dextran matrix (CM5 sensor chip) using N-hydroxysuccinimide/N-ethyl-NЈ- [3-(diethylamino)propyl] carbodiimide, yielding levels of 400 -1000 RU-immobilized mAbs (39). To establish the kinetics for the mAb-uPAR interactions, serial 2-fold dilutions of purified human uPAR (0.4 -200 nM in running buffer) were analyzed at 20°C with 50 l/min flow rates.…”

Sequences within Domain II of the Urokinase Receptor Critical for Differential Ligand Recognition

“…The uPA binding site within uPAR has been studied extensively in the past several years, using a number of different approaches (15)(16)(17)(18). In good agreement with the published data, we found that the uPA binding site occupies a broad region that encompasses all three domains, with the most critical residues located in D1 and D2.…”

“…These domains fold into a central 3-stranded ␤-sheet and a globular, disulfide-rich core (14). The uPA binding site within uPAR has been studied extensively using a number of different approaches, including site-directed photoaffinity labeling, chemical cross-linking, alanine scanning, as well as synthetic peptides, and the functional residues have been located within the ␣-helix and the three connecting loops, especially the second and third loops of all three domains (13,(15)(16)(17)(18). In contrast to the well characterized uPA binding site, the Vn binding site within uPAR remains elusive.…”

Cyclo19,31[D-Cys19]-uPA19-31 Is a Potent Competitive Antagonist of the Interaction of Urokinase-Type Plasminogen Activator with Its Receptor (CD87)

“…Although the N-terminal domain I of uPAR contains major determinants for uPA-binding, high affinity interaction with uPA is dependent on the multidomain structure of the receptor, indicating that all three protein domains are involved in the formation of a composite ligand binding site (Ploug, 1998;Gårdsvoll et al, 1999;Bdeir et al, 2000). In contrast, uPA binds to uPAR via a defined continuous peptide sequence …”