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M Ploug

Publications and source records attributed to M Ploug.

At least 19 recordsLinked to original sources

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗

Peptide-derived antagonists of the urokinase receptor. affinity maturation by combinatorial chemistry, identification of functional epitopes, and inhibitory effect on cancer cell intravasation.

The high-affinity interaction between urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) plays an important role in pericellular plasminogen activation. Since proteolytic degradation of the extracellular matrix has an established role in tumor invasion and metastasis, the uPA-uPAR interaction represents a potential target for therapeutic intervention. By affinity maturation using combinatorial chemistry we have now developed and characterized a 9-mer, linear peptide antagonist of the uPA-uPAR interaction demonstrating specific, high-affinity binding to human uPAR (K(d) approximately 0.4 nM). Studies by surface plasmon resonance reveal that the off-rate for this receptor-peptide complex is comparable to that measured for the natural protein ligand, uPA. The functional epitope on human uPAR for this antagonist has been delineated by site-directed mutagenesis, and its assignment to loop 3 of uPAR domain III (Met(246), His(249), His(251), and Phe(256)) corroborates data previously obtained by photoaffinity labeling and provides a molecular explanation for the extreme selectivity observed for the antagonist toward human compared to mouse, monkey, and hamster uPAR. When human HEp-3 cancer cells were inoculated in the presence of this peptide antagonist, a specific inhibition of cancer cell intravasation was observed in a chicken chorioallantoic membrane assay. These data imply that design of small organic molecules mimicking the binding determinants of this 9-mer peptide antagonist may have a potential application in combination therapy for certain types of cancer.

Animals↗

The murine receptor for urokinase-type plasminogen activator is primarily expressed in tissues actively undergoing remodeling.

uPAR is a cellular receptor for urokinase plasminogen activator, an enzyme involved in extracellular matrix degradation during processes involving tissue remodeling. We have expressed a recombinant soluble form of murine uPAR and raised rabbit polyclonal antibodies to study the expression of uPAR by immunohistochemistry. The immunohistochemical localization of uPAR was determined in normal mouse organs and in tumors formed by the highly metastatic Lewis lung carcinoma. uPAR immunoreactivity was found in the lungs, kidneys, and spleen, and in endothelial cells in the uterus, urinary bladder, thymus, heart, liver, and testis. No uPAR immunoreactivity was detected in muscle. In general, strong uPAR immunoreactivity was observed in organs undergoing extensive tissue remodeling, as exemplified by trophoblast cells in placenta, and in migrating, but not resting, keratinocytes at the edge of incisional wounds. Staining was not detected in any tissue sections derived from uPAR-deficient mice, thus confirming the specificity of the immunohistochemical staining of uPAR in normal mouse tissues. In Lewis lung carcinoma, uPAR immunoreactivity was found in the tumor cells of the primary tumor and in lung metastases. (J Histochem Cytochem 49:237-246, 2001)

Animals↗

Plasminogen-independent initiation of the pro-urokinase activation cascade in vivo. Activation of pro-urokinase by glandular kallikrein (mGK-6) in plasminogen-deficient mice.

The plasminogen activation (PA) system is involved in the degradation of fibrin and various extracellular matrix proteins, taking part in a number of physiological and pathological tissue remodeling processes including cancer invasion. This system is organized as a classical proteolytic cascade, and as for other cascade systems, understanding the physiological initiation mechanism is of central importance. The attempts to identify initiation routes for activation of the proform of the key enzyme urokinase-type plasminogen activator (pro-uPA) in vivo have been hampered by the strong activator potency of the plasmin, that is generated during the progress of the cascade. Using gene-targeted mice deficient in plasminogen (Plg -/- mice) [Bugge, T. H., Flick, M. J., Daugherty, C. C., and Degen, J. L. (1995) Genes Dev. 9, 794-807], we have now demonstrated and identified a component capable of initiating the cascade by activating pro-uPA. The urine from Plg -/- mice contained active two-chain uPA as well as a proteinase capable of activating exogenously added pro-uPA. The active component was purified and identified by mass spectrometry-based peptide mapping as mouse glandular kallikrein mGK-6 (true tissue kallikrein). The pro-uPA converting activity of the mGK-6 enzyme, as well as its ability to cleave a synthetic substrate for glandular kallikrein, was inhibited by the serine proteinase inhibitor leupeptin but not by other serine proteinase inhibitors such as aprotinin, antithrombin III, or alpha(1)-antitrypsin. We suggest that mouse glandular kallikrein mGK-6 is an activator of pro-uPA in the mouse urinary tract in vivo. Since this kallikrein is expressed in a number of tissues and also occurs in plasma, it can also be considered a candidate for a physiological pro-uPA activator in other locations.

Amino Acid Sequence↗

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 (DeltaDeltaG >/= 1.3 kcal mol(-1)) upon single-site substitutions to alanine (i.e. Arg(53), Leu(55), Tyr(57), and Leu(66)). 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.

Amino Acid Sequence↗

Identification of specific sites involved in ligand binding by photoaffinity labeling of the receptor for the urokinase-type plasminogen activator. Residues located at equivalent positions in uPAR domains I and III participate in the assembly of a composite ligand-binding site.

Plasminogen activation by the urokinase-type plasminogen activator (uPA) is facilitated in the presence of cells expressing the glycolipid-anchored high-affinity receptor for uPA (denoted uPAR). Structures involved in the interaction between human uPAR and a decamer peptide antagonist of uPA binding (SLNFSQYLWS) were previously tagged by specific site-directed photoaffinity labeling [Ploug, M., Ostergaard, S., Hansen, L. B. L., Holm, A., and Dano, K. (1998) Biochemistry 37, 3612-3622]. Replacement of the key functional residues Phe4 and Trp9 with either benzophenone or (trifluoromethyl)aryldiazirine rendered this peptide antagonist photoactivatable, and as a consequence, it incorporated covalently upon photolysis into either uPAR domain I or domain III depending on the actual position of the photophore in the sequence. The residues of uPAR specifically targeted by photoaffinity labeling were identified by matrix-assisted laser desorption mass spectrometry, NH2-terminal sequence analysis, and amino acid composition analysis after enzymatic fragmentation and HPLC purification. According to these data, the formation of the receptor-ligand complex positions Phe4 of the peptide antagonist very close to Arg53 and Leu66 in uPAR domain I and Trp9 of the antagonist in the vicinity of His251 in uPAR domain III. The gross molecular arrangement of the deduced receptor-ligand interface provides a rational structural basis for the observed requirement for the intact multidomain state of uPAR for achieving high-affinity ligand binding, since according to this model ligand binding must rely on a close spatial proximity of uPAR domains I and III. In addition, these data suggest that the assembly of the composite ligand binding site in uPAR may resemble the homophilic interdomain dimerization of kappa-bungarotoxin, a structural homologue of the Ly-6/uPAR domain family.

Amino Acid Sequence↗

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 by N-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 Kd) as assessed by real time biomolecular interaction analysis.

Amino Acid Sequence↗

Photoaffinity labeling of the human receptor for urokinase-type plasminogen activator using a decapeptide antagonist. Evidence for a composite ligand-binding site and a short interdomain separation.

Binding of urokinase-type plasminogen activator (uPA) to its cellular receptor (uPAR) renders the cell surface a favored site for plasminogen activation. Recently, a 15-mer peptide antagonist of the uPA-uPAR interaction, with an IC50 value of 10 nM, was identified using phage display technology [Goodson, R. J., Doyle, M. V., Kaufman, S. E., and Rosenberg, S. (1994) Proc. Natl. Acad. Sci. 91, 7129-7133]. In the present study, the molecular aspects of the interaction between this peptide and uPAR have been investigated. We have characterized the real-time receptor binding kinetics for the antagonist using surface plasmon resonance and identified critical residues by alanine replacements. The minimal peptide antagonist thus derived (SLNFSQYLWS) was rendered photoactivatable by replacing residues important for uPAR binding with photochemically active derivatives of phenylalanine containing either (trifluoromethyl)diazirine or benzophenone. These peptides incorporated covalently into purified soluble uPAR upon photoactivation, and this was inhibited by preincubation with receptor binding derivatives of uPA. The intact three-domain structure of uPAR was essential for efficient photoaffinity labeling. Proteolytic domain mapping using chymotrypsin revealed a specific labeling of both uPAR domain I and domains II + III dependent on the position of the photoprobe in the antagonist. On the basis of these studies, we propose the existence of a composite ligand binding site in uPAR combined of residues located in distinct structural domains. According to this model, a close spatial proximity between uPAR domain I and either domains II or III in intact uPAR is required for the assembly of this composite binding site. Since the receptor binding properties of the peptide antagonist closely mimic those of uPA itself, these two ligands presumably share coincident binding site in uPAR.

Amino Acid Sequence↗

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.

Antibodies, Monoclonal↗

Cell-surface acceleration of urokinase-catalyzed receptor cleavage.

The urokinase-type plasminogen activator (uPA) binds to a specific cell-surface receptor, uPAR. On several cell types uPAR is present both in the full-length form and a cleaved form, uPAR(2+3), which is devoid of binding activity. The formation of uPAR(2+3) on cultured U937 cells is either directly or indirectly mediated by uPA itself. In a soluble system, uPA can cleave purified uPAR, but the low efficiency of this reaction has raised doubts as to whether uPA is directly responsible for uPAR cleavage on the cells. We now report that uPA-catalyzed cleavage of uPAR on the cell surface is strongly favored relative to the reaction in solution. The time course of uPA-catalyzed cleavage of cell-bound uPAR was studied using U937 cells stimulated with phorbol 12-myristate 13-acetate. Only 30 min was required for 10 nM uPA to cleave 50% of the cell-bound uPAR. This uPA-catalyzed cleavage reaction was inhibited by a prior incubation of the cells with uPA inactivated by diisopropyl fluorophosphate, demonstrating a requirement for specific receptor binding of the active uPA to obtain the high-efficiency cleavage of cell-bound uPAR. Furthermore, amino-terminal sequence analysis revealed that uPAR(2+3), purified from U937 cell lysates, had the same amino termini as uPAR(2+3), generated by uPA in a purified system. In both cases cleavage had occurred at two positions in the hinge region connecting domain 1 and 2, between Arg83-Ala84 and Arg89-Ser90, respectively. The uPA-catalyzed cleavage of uPAR is a new negative-feedback regulation mechanism for cell-surface plasminogen activation. We propose that this mechanism plays a physiological role at specific sites with high local concentrations of uPA, thus adding another step to the complex regulation of this cascade reaction.

Amino Acid Sequence↗

Structure, function and expression on blood and bone marrow cells of the urokinase-type plasminogen activator receptor, uPAR.

Several important functions have been assigned to the receptor for urokinase-type plasminogen activator, uPAR. As implied by the name, uPAR was first identified as a high affinity cellular receptor for urokinase plasminogen activator (uPA). It mediates the binding of the zymogen, pro-uPA, to the plasma membrane where trace amounts of plasmin will initiate a series of events referred to as "reciprocal zymogen activation" where plasmin converts pro-uPA to the active enzyme, uPA, which in turn converts plasma membrane-associated plasminogen to plasmin. This is an efficient machinery to generate broad-spectrum proteolytic activity which is spatially restricted to the plasma membrane, since plasmin that diffuses away from the plasma membrane is rapidly inactivated by circulating inhibitors (i.e., alpha 2-antiplasmin). The system is controlled by a series of plasminogen activator inhibitors (PAIs), most importantly PAI-1 and PAI-2, providing means of temporally restricting the process of plasminogen activation. In addition to its role in plasminogen activation, compelling evidence has demonstrated a role for uPAR in cell-cell and cell-extracellular matrix adhesion, both directly and indirectly. uPAR is directly involved in binding to the extracellular matrix molecule, vitronectin, and the affinity of this binding is increased when uPAR is occupied by (pro-)uPA. A more indirect but presumably very important role of uPAR in cell adhesion seems to be mediated through interactions between uPAR and beta 1- or beta 2-integrins. It has been demonstrated that uPAR may bind physically to integrins in a reversible manner. The interaction seems to be of functional importance since the affinity of the integrin for its corresponding ligand is modulated by the association of integrin with uPAR. In some experimental setups uPAR has been shown to reduce the affinity of the associated integrin for certain ligands, while other experimental systems have demonstrated an increased affinity of the interaction between integrin and ligand after binding of uPAR to the integrin. Finally, uPAR has also been shown to participate in signal transduction events. Since uPAR is not a transmembrane molecule but belongs to the group of proteins that are tethered to the plasma membrane via a glycosyl-phosphatidylinositol anchor, association with a transmembrane adaptor is required for transmission of signals via uPAR. Integrins may serve as such signal transducers, and indeed uPAR has been shown to be associated in the plasma membrane with complexes of integrins and (phosphorylated) tyrosin kinases suggesting a role for these complexes in transmembrane transmission of signals via uPAR. In the hematopoietic system it has been shown that urokinase-type plasminogen activator (uPAR) is expressed as a differentiation antigen on cells of the myelomonocytic lineage and as an activation antigen on monocytes and T lymphocytes. Neutrophils contain intracellular reservoirs of uPAR that are translocated to the plasma membrane upon activation, and neutrophils from patients with the rare blood disease paroxysmal nocturnal hemoglobinuria (PNH) that fail to express glycosyl-phosphatidylinositol-anchored proteins including uPAR, show a very significantly reduced transmigration over an endothelial barrier. Cell-associated plasminogen activation by PNH-affected neutrophils is severely impaired, and it has been proposed that this may be causally related to the propensity for thrombosis in PNH. The pattern of expression of uPAR in hematological malignancies mirrors the expression by normal blood and bone marrow counterparts with some exceptions (differentiated myeloid leukemias are positive, undifferentiated myeloid may be negative and the majority of lymphoid leukemias and lymphomas are negative). The potential clinical relevance of uPAR expression in leukemias and lymphomas has not been determined.

Bone Marrow Cells↗

Chemical modification of the urokinase-type plasminogen activator and its receptor using tetranitromethane. Evidence for the involvement of specific tyrosine residues in both molecules during receptor-ligand interaction.

The high-affinity interaction between urokinase-type plasminogen activator (uPA) and its glycolipid anchored receptor (uPAR) is essential for the confinement of plasminogen activation to cell surfaces where it is thought to play an important role in cancer cell invasion and metastasis. The receptor binding site of uPA is retained within its isolated growth factor-like module (GFD; residues 4-43). The NH2-terminal domain of uPAR has a primary role in uPA binding, although maintenance of its multidomain structure has been shown to be necessary for the high affinity of this interaction [Ploug, M., Ellis, V., & Danø, K. (1994) Biochemistry 33, 8991-8997]. To identify residues engaged in the uPAR-uPA interaction, we have performed a "protein-protein footprinting" study on preformed uPAR-GFD complexes by chemical modification with tetranitromethane. All six tyrosine residues in uPAR and the single tyrosine residue in GFD (Tyr24) were susceptible to nitration in the native uncomplexed proteins, whereas in the receptor-ligand complexes both Tyr57 of uPAR and Tyr24 of GFD were protected from modification. Modification of uPAR alone led to a parallel reduction in the potential to bind pro-uPA and 8-anilino-1-naphthalenesulfonate, an extrinsic fluorophore reporting on the accessibility of a hydrophobic site involved in uPA binding. These data clearly demonstrate that Tyr57 in the NH2-terminal domain of uPAR and Tyr24 in uPA are intimately engaged in the receptor-ligand interaction, whereas Tyr87 positioned in the linker region between the first two domains of uPAR does not appear to be shielded by the resulting intermolecular interface.

Amino Acid Sequence↗

Ligand interaction between urokinase-type plasminogen activator and its receptor probed with 8-anilino-1-naphthalenesulfonate. Evidence for a hydrophobic binding site exposed only on the intact receptor.

The cellular receptor for urokinase-type plasminogen activator (uPAR) is a glycolipid-anchored membrane protein thought to play a primary role in the generation of pericellular proteolytic activity, and to be involved in cancer cell invasion and metastasis. This protein is composed of three homologous domains, the NH2-terminal of which is involved in the high-affinity binding (Kd approximately 0.1-1.0 nM) to the epidermal growth factor-like module of urokinase-type plasminogen activator (uPA). Here we report that intact uPAR binds the low molecular weight fluorophore 8-anilino-1-naphthalenesulfonate (ANS) to form a 1:1 stoichiometric complex and that the resulting enhancement of the ANS fluorescence probes the functional state of uPAR as judged by several independent criteria. First, the uPAR-mediated increase in ANS fluorescence can be titrated by uPA as well as by its receptor binding derivatives (the amino-terminal fragment and the growth factor-like module). Second, an anti-uPAR monoclonal antibody, capable of preventing uPA binding, can also titrate the uPAR-dependent ANS fluorescence whereas other antibodies not interfering with uPA binding are unable to exert this effect. Third, the dissociation profile of uPA-uPAR complexes as a function of increasing concentrations of guanidine hydrochloride closely parallels the loss of the ANS binding site in uPAR. Finally, liberation of the NH2-terminal domain from uPAR by limited chymotrypsin cleavage after Tyr87 leads to a loss of both enhanced ANS fluorescence and high-affinity uPA binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Anilino Naphthalenesulfonates↗

Structure-function relationships in the receptor for urokinase-type plasminogen activator. Comparison to other members of the Ly-6 family and snake venom alpha-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 alpha-neurotoxins, suggesting that uPAR may adopt the same gross folding pattern as these structurally well characterized proteins. Structural aspects of the binding between alpha-neurotoxins and the acetylcholine receptor may have a major influence on future studies of the interaction between uPA and uPAR.

Amino Acid Sequence↗

The receptor for urokinase-type plasminogen activator and urokinase is translocated from two distinct intracellular compartments to the plasma membrane on stimulation of human neutrophils.

The cellular receptor for urokinase-type plasminogen activator (uPAR) binds pro-urokinase (pro-uPA) and facilitates its conversion to enzymatically active urokinase (uPA). uPA in turn activates surface-bound plasminogen to plasmin, a process of presumed importance for a number of biologic processes including cell migration and resolution of thrombi. We have previously shown that uPAR is expressed on the plasma membrane of circulating neutrophils, and we now report that stimulation with phorbol myristate acetate (PMA), FMLP, or tumor necrosis factor-alpha results in a rapid increase in the expression of uPAR. This process is accompanied by an increased cell-associated plasminogen activation after preincubation of neutrophils with pro-uPA in vitro. By subcellular fractionation of unstimulated neutrophils, 50% of uPAR is recovered in fractions containing latent alkaline phosphatase, corresponding to an intracellular compartment of easily mobilizable secretory vesicles distinct from both primary and specific granules, whereas the remaining 50% of uPAR is associated with a compartment eluting close to the specific granules. In contrast, the ligand pro-uPA is primarily (approximately 80%) found in the specific granules, but small amounts of pro-uPA/uPA (approximately 20%) coelute with latent alkaline phosphatase. Stimulation of neutrophils with FMLP results in translocation of uPAR as well as of pro-uPA from the secretory vesicles, whereas stimulation with PMA is required to translocate material from specific granules. Flow cytometry of neutrophils saturated with exogenous diisopropyl fluorophosphate-uPA shows a large excess (approximately 90%) of unoccupied uPAR on resting as well as FMLP- and PMA-stimulated neutrophils, suggesting a possible role for exogenous pro-uPA in providing neutrophils with a potential for plasminogen activation. These processes may be important for neutrophil extravasation and migration through extracellular matrix and for the contribution of neutrophils to resolution of thrombi.

Biological Transport↗

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.

Amino Acids↗

Studies on functional and structural role of urokinase receptor and other components of the plasminogen activation system in malignancy.

Using immunohistochemistry and in-situ hybridization, we studied the expression of the components of the plasminogen activation system during progression to malignant melanoma with fresh melanocytic lesions. Expression of these components is confined to late stages of melanoma. t-PA expression is limited to rare cases of metastatic melanoma. The other components are frequently expressed concomitantly in the same tumour. Urokinase (u-PA) is expressed in stromal cells and only in tumour cells at invasive foci, urokinase receptor (u-PAR) in tumour cells, plasminogen activator inhibitor type I (PAI-1) in the intratumoral extracellular matrix and plasminogen activator inhibitor type II (PAI-2) in tumour cells and stromal cells. In order to investigate the role of u-PAR as a prognostic marker, we have developed an assay for quantitation of the receptor. As a first step towards structural investigations, we have determined the disulfide cross-links of the first domain of uPAR.

Biomarkers, Tumor↗

Localization of the disulfide bonds in the NH2-terminal domain of the cellular receptor for human urokinase-type plasminogen activator. A domain structure belonging to a novel superfamily of glycolipid-anchored membrane proteins.

The receptor for human urokinase-type plasminogen activator (uPAR) is synthesized as a 313-residue-long polypeptide containing 28 cysteine residues, the pattern of which defines three homologous repeats within the protein. These entities are believed to represent a novel protein domain structure, of which the NH2-terminal domain of uPAR can be covalently cross-linked to the epidermal growth factor-like module of urokinase after receptor-ligand interaction. The NH2-terminal domain of a recombinant, soluble uPAR derivative, labeled with [35S]cysteine, was isolated after limited proteolysis with chymotrypsin. The four disulfide bonds present within this domain were assigned by a combination of plasma desorption mass spectrometry, amino acid composition, and sequence analyses of peptides generated by trypsin, endoproteinase Asp-N, and thermolysin. The following disulfide bond structure was determined: Cys3-Cys24, Cys6-Cys12, Cys17-Cys45, and Cys71-Cys76. Similar cysteine pairing is likely to be found within other members of this protein superfamily, i.e. the membrane inhibitor of reactive lysis, Ly-6, and the remaining two domains of uPAR. However, an additional pair of cysteines present within these domains probably forms a fifth disulfide bond.

Amino Acid Sequence↗