Michael Ploug

Structure—function relationships in the receptor for urokinase-type plasminogen activator Comparison to other members of the Ly-6 family and snake venom α-neurotoxins

Plasminogen activation is regulated by the interaction between urokinase‐type plasminogen activator (uPA) and its specific glycolipid‐anchored cell surface receptor (uPAR). uPAR is composed of three homologous domains and is the only multi‐domain member of the Ly‐6 family of glycolipid‐anchored membrane proteins. Recent evidence has highlighted similarities between the individual domains of uPAR and the large family of secreted, single domain snake venom α‐neurotoxins, suggesting that uPAR may adopt the same gross folding pattern as these structurally well characterized proteins. Structural aspects of the binding between α‐neurotoxins and the acetylcholine receptor may have a major influence on future studies of the interaction between uPA and uPAR.

Cell-induced potentiation of the plasminogen activation system is abolished by a monoclonal antibody that recognizes the NH2-terminal domain of the urokinase receptor

We have raised four monoclonal antibodies recognizing different epitopes within the human cell‐surface receptor for urokinase‐type plasminogen activator (u‐PA). One of these antibodies completely abolishes the potentiation of plasmin generation observed upon incubation of the zymogens pro‐u‐PA and plasminogen with U937 cells. This antibody, which is also the only one to completely inhibit the binding of DFP‐inactivated [125I]‐u‐PA to U937 cells, is directed against the u‐PA binding NH2‐terminal domain of u‐PAR, a well‐defined fragment formed by limited chymotrypsin digestion of purified u‐PAR, demonstrating the functional independence of the u‐PA binding domain as well as the critical role of u‐PAR in the assembly of the cell‐surface plasminogen activation system.

Crystal structure of the human urokinase plasminogen activator receptor bound to an antagonist peptide

We report the crystal structure of a soluble form of human urokinase‐type plasminogen activator receptor (uPAR/CD87), which is expressed at the invasive areas of the tumor‐stromal microenvironment in many human cancers. The structure was solved at 2.7 Å in association with a competitive peptide inhibitor of the urokinase‐type plasminogen activator (uPA)–uPAR interaction. uPAR is composed of three consecutive three‐finger domains organized in an almost circular manner, which generates both a deep internal cavity where the peptide binds in a helical conformation, and a large external surface. This knowledge combined with the discovery of a convergent binding motif shared by the antagonist peptide and uPA allowed us to build a model of the human uPA–uPAR complex. This model reveals that the receptor‐binding module of uPA engages the uPAR central cavity, thus leaving the external receptor surface accessible for other protein interactions (vitronectin and integrins). By this unique structural assembly, uPAR can orchestrate the fine interplay with the partners that are required to guide uPA‐focalized proteolysis on the cell surface and control cell adhesion and migration.

The intact urokinase receptor is required for efficient vitronectin binding: receptor cleavage prevents ligand interaction

The urokinase receptor (uPAR) is a receptor for both urokinase plasminogen activator (uPA) and the adhesion protein vitronectin. There are two forms of cell surface‐bound uPAR; intact uPAR and a cleaved form, uPAR(2+3), which is formed by uPA‐catalyzed cleavage of uPAR. In ligand‐blotting experiments we found that vitronectin binds uPAR but not uPAR(2+3). In real‐time biomolecular interaction analysis using recombinant, soluble uPAR (suPAR) both plasma and multimeric forms of vitronectin bound to intact, antibody‐immobilized suPAR. Monoclonal antibodies against domain 1 of uPAR blocked suPAR binding to vitronectin and vitronectin did not interact with suPAR(2+3). Both suPAR(2+3) and the isolated domain 1 failed to compete with the intact suPAR in binding to vitronectin. We therefore conclude that the intact receptor is required for efficient vitronectin binding.

Plasminogen activation independent of uPA and tPA maintains wound healing in gene-deficient mice

Simultaneous ablation of the two known activators of plasminogen (Plg), urokinase‐type (uPA) and the tissue‐type (tPA), results in a substantial delay in skin wound healing. However, wound closure and epidermal re‐epithelialization are significantly less impaired in uPA;tPA double‐deficient mice than in Plg‐deficient mice. Skin wounds in uPA;tPA‐deficient mice treated with the broad‐spectrum matrix metalloproteinase (MMP) inhibitor galardin (N‐[(2R)‐2‐(hydroxamido‐carbonylmethyl)‐4‐methylpentanoyl]‐L‐tryptophan methylamide) eventually heal, whereas skin wounds in galardin‐treated Plg‐deficient mice do not heal. Furthermore, plasmin is biochemically detectable in wound extracts from uPA;tPA double‐deficient mice. In vivo administration of a plasma kallikrein (pKal)‐selective form of the serine protease inhibitor ecotin exacerbates the healing impairment of uPA;tPA double‐deficient wounds to a degree indistinguishable from that observed in Plg‐deficient mice, and completely blocks the activity of pKal, but not uPA and tPA in wound extracts. These findings demonstrate that an additional plasminogen activator provides sufficient plasmin activity to sustain the healing process albeit at decreased speed in the absence of uPA, tPA and galardin‐sensitive MMPs and suggest that pKal plays a role in plasmin generation.

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

The urokinase-type plasminogen activator receptor (uPAR) is a glycolipid anchored multidomain member of the Ly-6/uPAR protein domain superfamily. Studies by site-directed photoaffinity labeling, chemical cross-linking, and ligand-induced protection against chemical modification have highlighted the possible involvement of uPAR domain I and particularly loop 3 thereof in ligand binding (Ploug, M. (1998) Biochemistry 37, 16494–16505). Guided by these results we have now performed an alanine scanning analysis of this region in uPAR by site-directed mutagenesis and subsequently measured the effects thereof on the kinetics of uPA binding in real-time by surface plasmon resonance. Only four positions in loop 3 of uPAR domain I exhibited significant changes in the contribution to the free energy of uPA binding (ΔΔG ≥ 1.3 kcal mol−1) upon single-site substitutions to alanine (i.e. Arg53, Leu55, Tyr57, and Leu66). The energetic impact of these four alanine substitutions was not caused by gross structural perturbations, since all monoclonal antibodies tested having conformation-dependent epitopes on this domain exhibited unaltered binding kinetics. These sites together with a three-dimensional structure for uPAR may provide an appropriate target for rational drug design aimed at developing new receptor binding antagonists with potential application in cancer therapy.

Quantitation of the receptor for urokinase plasminogen activator by enzyme-linked immunosorbent assay

Binding of the urokinase plasminogen activator (uPA) to a specific cell surface receptor (uPAR) plays a crucial role in proteolysis during tissue remodelling and cancer invasion. An immunosorbent assay for the quantitation of uPAR has now been developed. This assay is based on two monoclonal antibodies recognizing the non-ligand binding part of this receptor, and it detects both free and occupied uPAR, in contrast to ligand-binding assays used previously. In a variant of the assay, the occupied fraction of uPAR is selectively detected with a uPA antibody. To be used as a standard, a soluble variant of uPAR, suPAR, has been constructed by recombinant technique and the protein content of a purified suPAR standard preparation was determined by amino acid composition analysis. The sensitivity of the assay (0.6 ng uPAR/ml) is strong enough to measure uPAR in extracts of cultured cells and cancer tissue. Recent studies have shown that a high uPA level in tumor extracts is in some cancers associated with poor prognosis. The present assay will now allow similar prognostic studies of uPAR levels.

Structure-based engineering of species selectivity in the interaction between urokinase and its receptor: implication for preclinical cancer therapy.

The high affinity interaction between the urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) is decisive for cell surface-associated plasminogen activation. Because plasmin activity controls fibrinolysis in a variety of pathological conditions, including cancer and wound healing, several intervention studies have focused on targeting the uPA·uPAR interaction in vivo. Evaluations of such studies in xenotransplanted tumor models are, however, complicated by the pronounced species selectivity in this interaction. We now report the molecular basis underlying this difference by solving the crystal structure for the murine uPA·uPAR complex and demonstrate by extensive surface plasmon resonance studies that the kinetic rate constants for this interaction can be swapped completely between these orthologs by exchanging only two residues. This study not only discloses the structural basis required for a successful rational design of the species selectivity in the uPA·uPAR interaction, which is highly relevant for functional studies in mouse models, but it also suggests the possible development of general inhibitors that will target the uPA·uPAR interaction across species barriers.

Glycosylation Profile of a Recombinant Urokinase-type Plasminogen Activator Receptor Expressed in Chinese Hamster Ovary Cells

Association of urokinase-type plasminogen activator (uPA) to cells via binding to its specific cellular receptor (uPAR) augments the potential of these cells to support plasminogen activation, a process that has been implicated in the degradation of extracellular matrix proteins during cell migration and tissue remodeling. The uPA receptor is a glycolipid-anchored membrane protein belonging to the Ly-6/uPAR superfamily and is the only multidomain member identified so far. We have now purified the three individual domains of a recombinant soluble uPAR variant, expressed in Chinese hamster ovary cells, after limited proteolysis using chymotrypsin and pepsin. The glycosylation patterns of these domains have been determined by matrix assisted laser desorption ionization and electrospray ionization mass spectrometry. Of the five potential attachment sites for asparagine-linked carbohydrate in uPAR only four are utilized, as the tryptic peptide derived from domain III containing Asn233 was quantitatively recovered without carbohydrate. The remaining four attachment sites were shown to exhibit site-specific microheterogeneity of the asparagine-linked carbohydrate. The glycosylation on Asn52 (domain I) and Asn172 (domain II) is dominated by the smaller biantennary complex-type oligosaccharides, while Asn162 (domain II) and Asn200 (domain III) predominantly carry tri- and tetraantennary complex-type oligosaccharides. The carbohydrate moiety on Asn52 in uPAR domain I could be selectively removed byN-glycanase treatment under nondenaturing conditions. This susceptibility was abrogated when uPAR participitated in a bimolecular complex with pro-uPA or smaller receptor binding derivatives thereof, demonstrating the proximity of the ligand-binding site to this particular carbohydrate moiety. uPAR preparations devoid of carbohydrate on domain I exhibited altered binding kinetics toward uPA (a 4–6-fold increase in K d ) as assessed by real time biomolecular interaction analysis.

Structural analysis and tissue localization of human C4.4A: a protein homologue of the urokinase receptor

C4.4A, a structural homologue of the urokinase-type plasminogen activator receptor (uPAR), was originally identified as a metastasis-associated membrane protein, but little is known about its structural and functional properties. Therefore, we expressed, purified and characterized a soluble truncated form of human C4.4A, and used this protein to produce specific polyclonal anti-C4.4A antibodies. By immunohistochemistry we observed a pronounced surface staining for C4.4A in suprabasal keratinocytes of chronic human wounds and found C4.4A expression markedly upregulated in migrating keratinocytes during re-epithelisation of incisional skin wounds. Phorbol-ester-induced hyperplasia of mouse skin is also accompanied by a significant induction of C4.4A expression in the multilayered, suprabasal keratinocytes. C4.4A contains two Ly-6 (leucocyte antigen 6)/uPAR/alpha-neurotoxin modules. Our recombinant human C4.4A is extensively modified by post-translational glycosylation, which include 5-6 N-linked carbohydrates primarily located in or close to its second Ly-6/uPAR/alpha-neurotoxin module and approximately 15 O-linked carbohydrates clustered in a Ser/Thr/Pro-rich region at the C-terminus. A highly protease-sensitive region (Tyr200-Arg204) is located between these two clusters of N- and O-linked carbohydrates. The natural, glycolipid-anchored C4.4A from amnion membranes of human term placenta exhibits similar properties. Using recombinant, soluble C4.4A or MCF 7 cells, which express significant amounts of GPI-anchored C4.4A, we find no evidence for an interaction between C4.4A and uPA, a property suggested previously for rat C4.4A. Collectively these data indicate that C4.4A, although being a structural homologue of uPAR, is unlikely to have a functional overlap with uPAR.