Inhibition of NF-kappa B-Rel A expression by antisense oligodeoxynucleotides suppresses synthesis of urokinase-type plasminogen activator (uPA) but not its inhibitor PAI-1.

The essential role of urokinase-type plasminogen activator (uPA) in tumor invasion and metastasis stresses the necessity of a fine-tuned cellular control over its expression. It has been shown that changes in uPA directly correlate with changes in cell invasiveness. We examined the role of Rel-related proteins in uPA synthesis by human ovarian cancer cells by inhibiting their expression using the antisense (AS) oligodeoxynucleotide (ODN) technology. Exposure of OV-MZ-6 cells to 10 microM phosphorothioate (PS)-derivatized AS-ODN directed to Rel A led to a maximal 50% decrease of uPA antigen in cell lysates and a 70% reduction in cell cultures supernatants accompanied by a significant transient decline in uPA mRNA levels. Antisense-PS-ODN directed to NF-kappa B1 (p50) or c-rel had no effect on uPA protein expression. AS-PS-ODN directed to Rel A also affected the proteolytic capacity of OV-MZ-6 cells reflected by an approximately 70% decrease in the fibrinolytic capacity of the cells within 24 h compared to untreated controls. AS-PS-ODN directed to I kappa B alpha expression increased uPA in cell culture supernatants up to 50%. uPA receptor (uPAR) production and synthesis of plasminogen activator inhibitor type-1 (PAI-1) were not altered by either AS-PS-ODN applied. Western blot and gel retardation analyses revealed constitutive expression of Rel-related proteins in nuclear protein extracts of OV-MZ-6 cells. Thus these proteins seem to be implicated in uPA regulation and may thereby contribute to tumor spread and metastasis.     

Novel bi- and trifunctional inhibitors of tumor-associated proteolytic systems

Serine proteases, cysteine proteases, and matrix metalloproteinases (MMPs) are involved in cancer cell invasion and metastasis. Recently, a recombinant bifunctional inhibitor (chCys-uPA19-31) directed against cysteine proteases and the urokinase-type plasminogen activator (uPA)/plasmin serine protease system was generated by introducing the uPA receptor (uPAR)-binding site of uPA into chicken cystatin (chCysWT). In the present study, we designed and recombinantly produced multifunctional inhibitors also targeting MMPs. The inhibitors comprise the N-terminal inhibitory domain of human TIMP-1 (tissue inhibitor of matrix metalloproteinase-1) or TIMP-3, fused to chCys-uPA19-31 or chCysWT. As demonstrated by various techniques, these fusion proteins effectively interfere with all three targeted protease systems. In in vitro Matrigel invasion assays, the addition of recombinant inhibitors strongly reduced invasion of ovarian cancer cells (OV-MZ-6#8). Additionally, OV-MZ-6#8 cells were stably transfected with expression plasmids encoding the various inhibitors. Synthesis and secretion of the inhibitors was verified by a newly developed ELISA, which selectively detects the recombinant proteins. Invasive capacity of inhibitor-producing cells was significantly reduced compared to vector-transfected control cells. Thus, these novel, compact, and small-size inhibitors directed against up to three different tumor-associated proteolytic systems may represent promising agents for prevention of tumor cell migration and metastasis.     

Inhibition of intraperitoneal tumor growth of human ovarian cancer cells by bi- and trifunctional inhibitors of tumor-associated proteolytic systems

Several proteolytic systems are involved in (anti)adhesive, migratory, and proteolytic processes, necessary for tumor progression and metastasis. We analyzed whether multifunctional inhibitors of different tumor-associated proteolytic systems reduce tumor growth and spread of human ovarian cancer cells in vivo. Bifunctional inhibitors are composed of the N-terminal domain of either the human matrix metalloproteinase inhibitors TIMP-1 or TIMP-3 and the cysteine protease inhibitor chicken cystatin (chCysWT); trifunctional inhibitors are composed of N-TIMP-1 or -3 and a chicken cystatin variant harboring the uPAR binding site of uPA, chCys-uPA19-31, which in addition to its inhibitory activity toward cysteine proteases interferes with the interaction of the serine protease uPA with its receptor. OV-MZ-6#8 cancer cells, stably transfected with plasmids expressing the multifunctional inhibitors, displayed similar proliferative and adhesive features as the vector-transfected control, but showed significant reduction in their invasive behavior in vitro. The cell lines expressing the multifunctional inhibitors were inoculated into the peritoneum of nude mice. Expression of three of the four inhibitor variants (N-hTIMP-1-chCysWT, N-hTIMP-1-chCys-uPA19-31, and N-hTIMP-3-chCysWT) resulted in a significant reduction of tumor burden compared to the vector-control cell line. These compact and small inhibitors may represent promising agents for gene therapy of solid malignant tumors.     

uPA-silica-Particles (SP-uPA): a novel analytical system to investigate uPA-uPAR interaction and to test synthetic uPAR antagonists as potential cancer therapeutics

The urokinase-type plasminogen activation system, including the serine protease uPA (urokinase-type plasminogen activator) and its cell surface receptor (uPAR, CD87), are important key molecules in tumor invasion and metastasis. Besides its proteolytic function, binding of uPA to uPAR on tumor cells exerts various cell responses such as migration, adhesion, proliferation, and differentiation. Hence, the uPA/uPAR system is a potential target for tumor therapy. We have designed a new generation of uPA-derived synthetic cyclic peptides suited to interfere with the binding of uPA to uPAR and present a new technology involving micro silica particles coated with uPA (SP-uPA) and reacting with recombinant soluble uPAR (suPAR), to rapidly assess the antagonistic potential of uPA-peptides by flow cytofluorometry (FACS). For this, we used silica particles of 10 microm in diameter to which HMW-uPA is coupled using the EDC/NHS method. Soluble, recombinant suPAR was added and the interaction of SP-uPA with suPAR verified by reaction with monoclonal antibody HD13.1 directed to uPAR, followed by a cyan dye (cy5)-labeled antibody directed against mouse IgG. Thereby it was possible to test naturally occurring ligands of uPAR (HMW-uPA, ATF) as well as highly effective, synthetic cyclic uPA-derived peptides (cyclo21,29[D-Cys21Cys29]-UPA21-30, cyclo21,29[D-Cys21Nle28Cys29]-uPA21-30, cyclo21,29[D-Cys(21)2-Nal24Cys29]-uPA21-30, and cyclo21,29[D-Cys21Orn23Thi24Thi25Cys29]-uPA21-30. The results obtained with the noncellular SP-uPA/uPAR system are highly comparable to those obtained with a cellular system involving FITC-uPA and the promyeloid cell line U937 as the source of uPAR.     

RNA interference-directed knockdown of urokinase plasminogen activator and urokinase plasminogen activator receptor inhibits prostate cancer cell invasion, survival, and tumorigenicity in vivo

The invasive ability of tumor cells plays a key role in prostate cancer metastasis and is a major cause of treatment failure. Urokinase plasminogen activator-(uPA) and its receptor (uPAR)-mediated signaling have been implicated in tumor cell invasion, survival, and metastasis in a variety of cancers. This study was undertaken to investigate the biological roles of uPA and uPAR in prostate cancer cell invasion and survival, and the potential of uPA and uPAR as targets for prostate cancer therapy. uPA and uPAR expression correlates with the metastatic potential of prostate cancer cells. Thus, therapies designed to inhibit uPA and uPAR expression would be beneficial. LNCaP, DU145, and PC3 are prostate cancer cell lines with low, moderate, and high metastatic potential, respectively, as demonstrated by their capacity to invade the extracellular matrix. In this study we utilized small hairpin RNAs (shRNAs), also referred to as small interfering RNAs, to target human uPA and uPAR. These small interfering RNA constructs significantly inhibited uPA and uPAR expression at both the mRNA and protein levels in the highly metastatic prostate cancer cell line PC3. Our data demonstrated that uPA-uPAR knockdown in PC3 cells resulted in a dramatic reduction of tumor cell invasion as indicated by a Matrigel invasion assay. Furthermore, simultaneous silencing of the genes for uPA and uPAR using a single plasmid construct expressing shRNAs for both uPA and uPAR significantly reduced cell viability and ultimately resulted in the induction of apoptotic cell death. RNA interference for uPA and uPAR also abrogated uPA-uPAR signaling to downstream target molecules such as ERK1/2 and Stat 3. In addition, our results demonstrated that intratumoral injection with the plasmid construct expressing shRNAs for uPA and uPAR almost completely inhibited established tumor growth and survival in an orthotopic mouse prostate cancer model. These findings uncovered evidence of a complex signaling network operating downstream of uPA-uPAR that actively advances tumor cell invasion, proliferation, and survival of prostate cancer cells. Thus, RNA interference-directed targeting of uPA and uPAR is a convenient and novel tool for studying the biological role of the uPA-uPAR system and raises the potential of its application for prostate cancer therapy.     

Characterization of low-glycosylated forms of soluble human urokinase receptor expressed in Drosophila Schneider 2 cells after deletion of glycosylation-sites

The urokinase-type plasminogen activator receptor (uPAR) is a glycolipid-anchored membrane protein that is thought to play an active role during cancer cell invasion and metastasis. We have expressed a truncated soluble form of human uPAR using its native signal peptide in stably transfected Drosophila Schneider 2 (S2) cells. This recombinant product, denoted suPAR (residues 1–283), is secreted in high quantities in serum-free medium and can be isolated in very high purity. Characterization by SDS–PAGE and mass spectrometry reveals that suPAR produced in this system carries a uniform glycosylation composed of biantennary carbohydrates. In contrast, suPAR produced in stably transfected Chinese hamster ovary (CHO) cells carries predominantly complex-type glycosylation and exhibits in addition a site-specific microheterogeneity of the individual N-linked carbohydrates. Measurement of binding kinetics for the interaction with uPA by surface plasmon resonance reveals that S2-produced suPAR exhibits binding properties similar to those of suPAR produced by CHO cells. By site-directed mutagenesis we have furthermore removed the five potential N-linked glycosylation-sites either individually or in various combinations and studied the effect thereof on secretion and ligand-binding. Only suPAR completely deprived of N-linked glycosylation exhibits an impaired level of secretion. All the other mutants showed comparable secretion levels and retained the ligand-binding properties of suPAR-wt. In conclusion, stable expression of suPAR in Drosophila S2 cells offers a convenient and attractive method for the large scale production of homogeneous preparations of several uPAR mutants, which may be required for future attempts to solve the three-dimensional structure of uPAR by X-ray crystallography.     

Urokinase-type plasminogen activator receptor (CD87) is a ligand for integrins and mediates cell-cell interaction

Binding of urokinase-type plasminogen activator (uPA) to its receptor (uPAR/CD87) regulates cellular adhesion, migration, and tumor cell invasion. However, it is unclear how glycosyl phosphatidylinositol-anchored uPAR, which lacks a transmembrane structure, mediates signal transduction. It has been proposed that uPAR forms cis-interactions with integrins as an associated protein and thereby transduces proliferative or migratory signals to cells upon binding of uPA. We provide evidence that soluble uPAR (suPAR) specifically binds to integrins alpha4beta1, alpha6beta1, alpha9beta1, and alphavbeta3 on Chinese hamster ovary cells in a cation-dependent manner. Anti-integrin and anti-uPAR antibodies effectively block binding of suPAR to these integrins. Binding of suPAR to alpha4beta1 and alphavbeta3 is blocked by known soluble ligands and by the integrin mutations that inhibit ligand binding. These results suggest that uPAR is an integrin ligand rather than, or in addition to, an integrin-associated protein. In addition, we demonstrate that glycosyl phosphatidylinositol-anchored uPAR on the cell surface specifically binds to integrins on the apposing cells, suggesting that uPAR-integrin interaction may mediate cell-cell interaction (trans-interaction). These previously unrecognized uPAR-integrin interactions may allow uPAR to transduce signals through the engaged integrin without a hypothetical transmembrane adapter and may provide a potential therapeutic target for control of inflammation and cancer.     

Down‐regulation of uPA and uPAR by 3,3′‐diindolylmethane contributes to the inhibition of cell growth and migration of breast cancer cells

3,3′‐Diindolylmethane (DIM) is a known anti‐tumor agent against breast and other cancers; however, its exact mechanism of action remains unclear. The urokinase plasminogen activator (uPA) and its receptor (uPAR) system are involved in the degradation of basement membrane and extracellular matrix, leading to tumor cell invasion and metastasis. Since uPA‐uPAR system is highly activated in aggressive breast cancer, we hypothesized that the biological activity of B‐DIM could be mediated via inactivation of uPA‐uPAR system. We found that B‐DIM treatment as well as silencing of uPA‐uPAR led to the inhibition of cell growth and motility of MDA‐MB‐231 cells, which was in part due to inhibition of VEGF and MMP‐9. Moreover, silencing of uPA‐uPAR led to decreased sensitivity of these cells to B‐DIM indicating an important role of uPA‐uPAR in B‐DIM‐mediated inhibition of cell growth and migration. We also found similar effects of B‐DIM on MCF‐7, cells expressing low levels of uPA‐uPAR, which was due to direct down‐regulation of MMP‐9 and VEGF, independent of uPA‐uPAR system. Interestingly, over‐expression of uPA‐uPAR in MCF‐7 cells attenuated the inhibitory effects of B‐DIM. Our results, therefore, suggest that B‐DIM down‐regulates uPA‐uPAR in aggressive breast cancers but in the absence of uPA‐uPAR, B‐DIM can directly inhibit VEGF and MMP‐9 leading to the inhibition of cell growth and migration of breast cancer cells. J. Cell. Biochem. 108: 916–925, 2009. © 2009 Wiley‐Liss, Inc.     

Urokinase plasminogen activator/urokinase-specific surface receptor expression and matrix invasion by breast cancer cells requires constitutive p38alpha mitogen-activated protein kinase activity

Overexpression of urokinase plasminogen activator (uPA) and its receptor (uPAR) has been well documented in a wide variety of tumor cells. In breast cancer, expression of uPA/uPAR is essential for tumor cell invasion and metastasis. However, the mechanism responsible for uPA/uPAR expression in cancer cells remains unclear. In the studies reported here, we show that endogenous p38 MAPK activity correlates well with breast carcinoma cell invasiveness. Treatment of highly invasive BT549 cells with a specific p38 MAPK inhibitor SB203580 diminished both uPA/uPAR mRNA and protein expression and abrogated the ability of these cells to invade matrigel, suggesting that p38 MAPK signaling pathway is involved in the regulation of uPA/uPAR expression and breast cancer cell invasion. We also demonstrated that SB203580-induced reduction in uPA/uPAR mRNA expression resulted from the de- stabilization of uPA and uPAR mRNA. Finally, by selectively inhibiting p38alpha or p38beta MAPK isoforms, we demonstrate that p38alpha, rather than p38beta, MAPK activity is essential for uPA/uPAR expression. These studies suggest that p38alpha MAPK signaling pathway is important for the maintenance of breast cancer invasive phenotype by promoting the stabilities of uPA and uPAR mRNA.     

Novel interactions between urokinase and its receptor

Urokinase-type plasminogen activator (uPA) binds to its receptor (uPAR) with a K(d) of about 1 nm. The catalytic activity of the complex is apparent at uPA concentrations close to K(d). Other functions of the complex, such as signal transduction, are apparent at much higher concentrations (35-60 nm). In the present study, we show that uPA and recombinant soluble uPAR (suPAR), at concentrations that exceed the K(d) and the theoretical saturation levels (10-80 nm), establish novel interactions that lead to a further increase in the activity of the single-chain uPA (scuPA)/suPAR and two-chain uPA (tcuPA)/suPAR complexes. Experiments performed using dynamic light scattering, gel filtration, and electron microscopy techniques indicate that suPAR forms dimers and oligomers. The three techniques provide evidence that the addition of an equimolar concentration of scuPA leads to the dissociation of these dimers and oligomers. Biacore data show that suPAR dimers and oligomers bind scuPA with decreased affinity when compared with monomers. We postulate that uPAR is present in equilibrium between oligomer/dimer/monomer forms. The binding of uPA to suPAR dimers and oligomers occurs with lower affinity than the binding to monomer. These novel interactions regulate the activity of the resultant complexes and may be involved in uPA/uPAR mediated signal transduction.     

上皮性卵巢癌组织中uPA及uPAR的表达(英文)

目的:研究uPA和uPAR在卵巢癌中的表达,分析其与卵巢癌的发展和预后的关系。方法:采用免疫组织化学法检测60例上皮性卵巢癌、20例交界性卵巢肿瘤及20例良性卵巢肿瘤组织中uPA、uPAR及表达,并结合临床病理因素进行分析。结果:(1)uPA、uPAR、在上皮性卵巢癌中的表达率(75%,66.7%)明显高于交界性卵巢肿瘤(40%,30%)及良性卵巢肿瘤组织(30%,25%),差异均有统计学意义(uPAx2=22.078,P0.001;uPARx2=19.510,P0.05),而交界性卵巢肿瘤与良性卵巢肿瘤组织间差异无统计学意义(P0.05)。(2)uPA、uPAR在恶性卵巢肿瘤中阳性表达率与手术病理分期、组织分级和有无淋巴结转移有关;晚期癌组阳性表达率高于早期癌组(uPAx2=14.400,P0.05;uPARx2=9.6,P=0.002);低分化癌组的阳性表达率高于中高分化癌(uPAx2=15.002,P0.05;uPARx2=36.906,P0.05);有淋巴结转移癌组阳性表达率高于无淋巴结转移癌组(uPAx2=13.333,P0.05;uPARx2=15.313,P0.05);差异均有统计学意义;但是与癌症病理类型、病人年龄、肿瘤大小无关。uPA与uPAR的表达存在正相关。结论:在uPA和uPAR可通过促进血管生成和降解细胞外基质(基质)和基底膜,在肿瘤的侵袭和转移中发挥重要作用。uPA及uPAR在上皮性卵巢癌组织中表达上调,支持这些因子在肿瘤浸润和转移中发挥重要作用,二者有可能作为预测上皮性卵巢癌预后的重要参考指标。     

High-affinity urokinase-derived cyclic peptides inhibiting urokinase/urokinase receptor-interaction: effects on tumor growth and spread

Urokinase-type plasminogen activator (uPA) binds with high affinity to its specific cell surface receptor (uPAR) (CD87) via a well-defined sequence within the N-terminal region of uPA (uPA(19-31)). Since this uPA/uPAR-interaction plays a significant role in tumor cell invasion and metastasis, it has become an attractive therapeutic target. Two small peptidic cyclic competitive antagonists of uPA/uPAR-interaction have been developed, based on the uPAR binding site in uPA: WX-360 (cyclo(21,29)[D-Cys21]-uPA(21-30)[S21C;H29C]) and its norleucine (Nle) derivative WX-360-Nle (cyclo(21,29)[D-Cys21]-uPA(21-30)[S21C;K23Nle;H29C]). These peptides display an only five to 10-fold lower affinity to uPAR as compared to the naturally occurring uPAR-ligand uPA. In this study, WX-360 and WX-360-Nle were tested in nude mice for their potency to inhibit tumor growth and intraperitoneal spread of lacZ-tagged human ovarian cancer cells. Intraperitoneal administration of either cyclic peptide (20 mg peptide/kg; 1x daily for 37 days) into the tumor-bearing nude mice resulted in a significant reduction of tumor weight and spread within the peritoneum as compared to the untreated control group. This is the first report demonstrating effective reduction of tumor growth and spread of human ovarian cancer cells in vivo by small synthetic uPA-derived cyclic peptides competitively interfering with uPA/uPAR-interaction. Thus, both WX-360 and WX-360-Nle are promising novel compounds to reduce dissemination of human ovarian carcinoma.     

Inactivation of uPA and its receptor uPAR by 3,3′‐diindolylmethane (DIM) leads to the inhibition of prostate cancer cell growth and migration

3,3′‐Diindolylmethane (DIM) has been studied for its putative anti‐cancer properties, especially against prostate cancer; however, its exact mechanism of action remains unclear. We recently provided preliminary data suggesting down‐regulation of uPA during B‐DIM (a clinically active DIM)‐induced inhibition of invasion and angiogenesis in prostate cancer cells. Since the expression and activation of uPA plays important role in tumorigenicity, and high endogenous levels of uPA and uPAR are found in advanced metastatic cancers, we investigated their role in B‐DIM‐mediated inhibition of prostate cancer cell growth and motility. Using PC3 cells, we found that B‐DIM treatment as well as the silencing of uPA and uPAR by siRNAs led to the inhibition of cell growth and motility. Conversely, over‐expression of uPA/uPAR in LNCaP and C4‐2B cells resulted in increased cell growth and motility, which was effectively inhibited by B‐DIM. Moreover, we found that uPA as well as uPAR induced the production of VEGF and MMP‐9, and that the down‐regulation of uPA/uPAR by siRNAs or B‐DIM treatment resulted in the inhibition of VEGF and MMP‐9 secretion which could be responsible for the observed inhibition of cell migration. Interestingly, silencing of uPA/uPAR led to decreased sensitivity to B‐DIM indicating important role of uPA/uPAR in B‐DIM‐mediated regulation of prostate cancer cell growth and migration. Our data suggest that chemopreventive and/or therapeutic activity of B‐DIM is in part due to down‐regulation of uPA–uPAR leading to reduced production of VEGF/MMP‐9 which ultimately leads to the inhibition of cell growth and migration of aggressive prostate cancer cells. J. Cell. Biochem. 107: 516–527, 2009. © 2009 Wiley‐Liss, Inc.     

Suppression of urokinase receptor expression by bikunin is associated with inhibition of upstream targets of extracellular signal-regulated kinase-dependent cascade.

Our laboratory showed that bikunin, a Kunitz-type protease inhibitor, suppresses 4beta-phorbol 12-myristate 13-acetate (PMA)- or tumor necrosis factor-alpha (TNFalpha)-induced urokinase-type plasminogen activator (uPA) expression in different cell types. In addition to its effects on protease inhibition, bikunin could be modulating other cellular events associated with the metastatic cascade. To test this hypothesis, we examined whether bikunin was able to suppress the expression of uPA receptor (uPAR) mRNA and protein in a human chondrosarcoma cell line, HCS-2/8, and two human ovarian cancer cell lines, HOC-I and HRA. The present study showed that (a) bikunin suppresses the expression of constitutive and PMA-induced uPAR mRNA and protein in a variety of cell types; (b) an extracellular signal-regulated kinase (ERK) activation system is necessary for the PMA-induced increase in uPAR expression, as PD098059 and U0126, which prevent the activation of MEK1, reduce the uPAR expression; (c) bikunin markedly suppresses PMA-induced phosphorylation of ERK1/2 at the concentration that prevents uPAR expression, but does not reduce total ERK1/2 antigen level; (d) bikunin has no ability to inhibit overexpression of uPAR in cells treated with sodium vanadate; and (e) we further studied the inhibition of uPAR expression by stable transfection of HRA cells with bikunin gene, demonstrating that bikunin secretion is necessary for inhibition of uPAR expression. We conclude that bikunin downregulates constitutive and PMA-stimulated uPAR mRNA and protein possibly through suppression of upstream targets of the ERK-dependent cascade, independent of whether cells were treated with exogenous bikunin or transfected with bikunin gene.     

Urokinase regulates vitronectin binding by controlling urokinase receptor oligomerization

Adhesion of monocytes to the extracellular matrix is mediated by a direct high affinity interaction between cell-surface urokinase-type plasminogen activator (uPA) receptor (uPAR) and the extracellular matrix protein vitronectin. We demonstrate a tight connection between uPA-regulated uPAR oligomerization and high affinity binding to immobilized vitronectin. We find that binding of soluble uPAR (suPAR) to immobilized vitronectin is strictly ligand-dependent with a linear relationship between the observed binding and the concentration of ligand added. Nevertheless, a comparison of experimentally obtained binding curves to those generated using a simple equilibrium model suggests that the high affinity vitronectin-binding pro-uPA.suPAR complex contains two molecules of suPAR. In co-immunoprecipitation experiments, using different epitope-tagged suPAR molecules, suPAR/suPAR co-immunoprecipitation displayed a similar uPA dose dependence as that observed for vitronectin binding, demonstrating that the high affinity vitronectin-binding complex indeed contains oligomeric suPAR. Structurally, the kringle domain of uPA was found to be critical for the formation of the vitronectin-binding competent complex because the amino-terminal fragment, but not the growth factor-like domain, behaved as a full-length uPA. Our data represent the first demonstration of functional, ligand-induced uPAR oligomerization having extensive implications for glycosylphosphatidylinositol-anchored receptors in general, and for the biology of the uPA/uPAR system in particular.     

Binding of urokinase-type plasminogen activator receptor (uPAR) to the mannose 6-phosphate/insulin-like growth factor II receptor: contrasting interactions of full-length and soluble forms of uPAR

Urokinase-type plasminogen activator receptor (uPAR) binding by the mannose 6-phosphate/insulin-like growth factor II receptor (Man-6-P/IGF2R) is considered important to Man-6-P/IGF2R tumor suppressor function via regulation of cell surface proteolytic activity. Our goal was to map the uPAR binding site of the Man-6-P/IGF2R by analyzing the uPAR binding characteristics of a panel of minireceptors containing different regions of the Man-6-P/IGF2R extracytoplasmic domain. Coimmunoprecipitation assays revealed that soluble recombinant uPAR (suPAR) bound the Man-6-P/IGF2R at two distinct sites, one localized to the amino-terminal end of the Man-6-P/IGF2R extracytoplasmic domain (repeats 1-3) and the other to the more carboxyl-terminal end (repeats 7-9). These sites correspond with the positions of the two Man-6-P binding domains of Man-6-P/IGF2R. Indeed, the suPAR-Man-6-P/IGF2R interaction was inhibited by Man-6-P, and binding-competent su-PAR species represented a minor percentage (8-30%) of the suPAR present. In contrast, Man-6-P/IGF2R binding of endogenous, full-length uPAR solubilized from plasma membranes of the prostate cancer cell line, PC-3, was not inhibited by Man-6-P. Further studies showed that very little (<5%) endogenous uPAR was Man-6-P/IGF2R binding-competent. We conclude that, contrary to previous reports, the interaction between uPAR and Man-6-P/IGF2R is a low percentage binding event and that suPAR and full-length uPAR bind the Man-6-P/IGF2R by different mechanisms.