Cyclization of the Urokinase Receptor-Derived Ser-Arg-Ser-Arg-Tyr Peptide Generates a Potent Inhibitor of Trans-Endothelial Migration of Monocytes

The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration and uPAR_88-92 is the minimal sequence required to induce cell motility. We and others have previously documented that the uPAR_88-92 sequence, even in the form of synthetic linear peptide (SRSRY), interacts with the formyl peptide receptor type 1 (FPR1), henceforth inducing cell migration of several cell lines, including monocytes. FPR1 is mainly expressed by mammalian phagocytic leukocytes and plays a crucial role in chemotaxis. In this study, we present evidence that the cyclization of the SRSRY sequence generates a new potent and stable inhibitor of monocyte trafficking. In rat basophilic leukaemia RBL-2H3/ETFR cells expressing high levels of constitutively activated FPR1, the cyclic SRSRY peptide ([SRSRY]) blocks FPR1 mediated cell migration by interfering with both internalization and ligand-uptake of FPR1. Similarly to RBL-2H3/ETFR cells, [SRSRY] competes with fMLF for binding to FPR1 and prevents agonist-induced FPR1 internalization in human monocyte THP-1 cells. Unlike scramble [RSSYR], [SRSRY] inhibits fMLF-directed migration of monocytes in a dose-dependent manner, with IC₅₀ value of 0.01 nM. PMA-differentiated THP-1 cell exposure to fMLF gradient causes a marked cytoskeletal re-organization with the formation of F-actin rich pseudopodia that are prevented by the addition of [SRSRY]. Furthermore, [SRSRY] prevents migration of human primary monocytes and trans-endothelial migration of monocytes. Our findings indicate that [SRSRY] is a new FPR1 inhibitor which may suggest the development of new drugs for treating pathological conditions sustained by increased motility of monocytes, such as chronic inflammatory diseases.     

Cross-talk between fMLP and vitronectin receptors triggered by urokinase receptor-derived SRSRY peptide

The urokinase-type plasminogen activator receptor (uPAR) sustains cell migration through its capacity to promote pericellular proteolysis, regulate integrin function, and mediate chemotactic signaling in response to urokinase. We have characterized the early signaling events triggered by the Ser-Arg-Ser-Arg-Tyr (SRSRY) chemotactic uPAR sequence. Cell exposure to SRSRY peptide promotes directional migration on vitronectin-coated filters, regardless of uPAR expression, in a specific and dose-dependent manner, with maximal effect at a concentration level as low as 10 nm. A similar concentration profile is observed in a quantitative analysis of SRSRY-dependent cytoskeletal rearrangements, mostly consisting of filamentous structures localized in a single cell region. SRSRY analogues with alanine substitutions fail to drive F-actin formation and cell migration, indicating a critical role for each amino acid residue. As with ligand-dependent uPAR signaling, SRSRY stimulates protein kinase C activity and results in ERK1/2 phosphorylation. The involvement of the high affinity N-formyl-Met-Leu-Phe receptor (FPR) in this process is indicated by the finding that 100 nm N-formyl-Met-Leu-Phe inhibits binding of D2D3 to the cell surface, as well as SRSRY-stimulated cell migration and F-actin polarization. Moreover, cell exposure to SRSRY promotes FPR-dependent vitronectin release and increased uPAR.alphavbeta5 vitronectin receptor physical association, indicating that alphavbeta5 activity is regulated by the SRSRY uPAR sequence via FPR. Finally, we provide evidence that alphavbeta5 is required for SRSRY-dependent ERK1/2 phosphorylation, whereas it is not required for protein kinase C activation. The data indicate that the ability of uPAR to stimulate cell migration and cytoskeletal rearrangements is retained by the SRSRY peptide alone and that it is supported by cross-talk between FPR and alphavbeta5.     

Expression and functional role of formyl peptide receptor in human bone marrow-derived mesenchymal stem cells

We investigated the expression of formyl peptide receptor (FPR) and its functional role in human bone marrow-derived mesenchymal stem cells (MSCs). We analyzed the expression of FPR by using ligand-binding assay with radio-labeled N-formyl-met-leu-phe (fMLF), and found that MSCs express FPR. FMLF stimulated intracellular calcium increase, mitogen-activated protein kinases activation, and Akt activation, which were mediated by G(i) proteins. MSCs were chemotactically migrated to fMLF. FMLF-induced MSC chemotaxis was also completely inhibited by pertussis toxin, LY294002, and PD98059, indicating the role of G(i) proteins, phosphoinositide 3-kinase, and extracellular signal regulated protein kinase. N-terminal fragment of annexin-1, Anx-1(2-26), an endogenous agonist for FPR, also induced chemotactic migration of MSCs. Thus MSCs express functional FPR, suggesting a new (patho)physiological role of FPR and its ligands in regulating MSC trafficking during induction of injured tissue repair.     

Urokinase receptor (uPAR) regulates complement receptor 3 (CR3)-mediated neutrophil phagocytosis

Urokinase receptor (uPAR) associates in cis with complement receptor 3 (CR3). In the present study, we addressed whether this coupling regulates CR3-mediated phagocytosis. CR3-mediated attachment of iC3b-opsonized sheep red blood cells to human neutrophils and internalization of these cells were reduced by removal of cell-bound uPAR by phosphatidylinositol-specific phospholipase C and reconstituted in the presence of soluble uPAR. The attachment and internalization were suppressed in the presence of anti-uPAR polyclonal antibody, proteolytically inactive urokinase and saccharides that disrupt interaction of uPAR with CR3. Thus, uPAR acts as a cofactor for iC3b binding to CR3 and regulates CR3-mediated phagocytosis.     

The Urokinase Receptor Takes Control of Cell Migration by Recruiting Integrins and FPR1 on the Cell Surface

The receptor (uPAR) of the urokinase-type plasminogen activator (uPA) is crucial in cell migration since it concentrates uPA proteolytic activity at the cell surface, binds vitronectin and associates to integrins. uPAR cross-talk with receptors for the formylated peptide fMLF (fMLF-Rs) has been reported; however, cell-surface uPAR association to fMLF-Rs on the cell membrane has never been explored in detail.We now show that uPAR co-localizes at the cell-surface and co-immunoprecipitates with the high-affinity fMLF-R, FPR1, in uPAR-transfected HEK-293 (uPAR-293) cells. uPAR/β1 integrin and FPR1/β1 integrin co-localization was also observed. Serum or the WKYMVm peptide (W Pep), a FPR1 ligand, strongly increased all observed co-localizations in uPAR-293 cells, including FPR1/β1 integrin co-localization. By contrast, a low FPR1/β1 integrin co-localization was observed in uPAR-negative vector-transfected HEK-293 (V-293) cells, that was not increased by serum or W Pep stimulations.The role of uPAR interactions in cell migration was then explored. Both uPAR-293 and V-293 control cells efficiently migrated toward serum or purified EGF. However, cell treatments impairing uPAR interactions with fMLF-Rs or integrins, or inhibiting specific cell-signaling mediators abrogated uPAR-293 cell migration, without exerting any effect on V-293 control cells.Accordingly, uPAR depletion by a uPAR-targeting siRNA or uPAR blocking with an anti-uPAR polyclonal antibody in cells constitutively expressing high uPAR levels totally impaired their migration toward serum.Altogether, these results suggest that both uPAR-positive and uPAR-negative cells are able to migrate toward serum; however, uPAR expression renders cell migration totally and irreversibly uPAR-dependent, since it is completely inhibited by uPAR blocking.We propose that uPAR takes control of cell migration by recruiting fMLF-Rs and β1 integrins, thus promoting their co-localization at the cell-surface and driving pro-migratory signaling pathways.     

Urokinase induces basophil chemotaxis through a urokinase receptor epitope that is an endogenous ligand for formyl peptide receptor-like 1 and -like 2

Basophils circulate in the blood and are able to migrate into tissues at sites of inflammation. Urokinase plasminogen activator (uPA) binds a specific high affinity surface receptor (uPAR). The uPA-uPAR system is crucial for cell adhesion and migration, and tissue repair. We have investigated the presence and function of the uPA-uPAR system in human basophils. The expression of uPAR was found at both mRNA and protein levels. The receptor was expressed on the cell surface of basophils, in the intact and cleaved forms. Basophils did not express uPA at either the protein or mRNA level. uPA (10(-12)-10(-9) M) and its uPAR-binding N-terminal fragment (ATF) were potent chemoattractants for basophils, but did not induce histamine or cytokine release. Inactivation of uPA enzymatic activity by di-isopropyl fluorophosphate did not affect its chemotactic activity. A polyclonal Ab against uPAR inhibited uPA-dependent basophil chemotaxis. The uPAR-derived peptide 84-95 (uPAR84-95) induced basophil chemotaxis. Basophils expressed mRNA for the formyl peptide receptors formyl peptide receptor (FPR), FPR-like 1 (FPRL1), and FPRL2. The FPR antagonist cyclosporin H prevented chemotaxis induced by FMLP, but not that induced by uPA and uPAR84-95. Incubation of basophils with low and high concentrations of FMLP, which desensitize FPR and FPRL1, respectively, but not FPRL2, slightly reduced the chemotactic response to uPA and uPAR84-95. In contrast, desensitization with WKYMVm, which also binds FPRL2, markedly inhibited the response to both molecules. Thus, uPA is a potent chemoattractant for basophils that seems to act through exposure of the chemotactic uPAR epitope uPAR84-95, which is an endogenous ligand for FPRL2 and FPRL1.     

Oxidized low-density lipoprotein-induced foam cell formation is mediated by formyl peptide receptor 2

The increased level of LDL and its modification into oxLDL has been regarded as an important risk factor for the development of cardiovascular diseases such as atherosclerosis. Although some scavenger receptors including CD36 and RAGE have been considered as target receptors for oxLDL, involvement of other receptors should be investigated for oxLDL-induced pathological responses. In this study, we found that oxLDL-induced foam cell formation was inhibited by formyl peptide receptor 2 (FPR2) antagonist WRW⁴. oxLDL also stimulated calcium signaling and chemotactic migration in FPR2-expressing RBL-2H3 cells but not in vector-expressing RBL-2H3 cells. Moreover, oxLDL stimulated TNF-α production, which was also almost completely inhibited by FPR2 antagonist. Our findings therefore suggest that oxLDL stimulates macrophages, resulting in chemotactic migration, TNF-α production, and foam cell formation via FPR2 signaling, and thus likely contributes to atherogenesis.     

The N-formyl peptide receptors and the anaphylatoxin C5a receptors: an overview

Leukocyte recruitment to sites of inflammation and infection is dependent on the presence of a gradient of locally produced chemotactic factors. This review is focused on current knowledge about the activation and regulation of chemoattractant receptors. Emphasis is placed on the members of the N-formyl peptide receptor family, namely FPR (N-formyl peptide receptor), FPRL1 (FPR like-1) and FPRL2 (FPR like-2), and the complement fragment C5a receptors (C5aR and C5L2). Upon chemoattractant binding, the receptors transduce an activation signal through a G protein-dependent pathway, leading to biochemical responses that contribute to physiological defense against bacterial infection and tissue damage. C5aR, and the members of the FPR family that were previously thought to be restricted to phagocytes proved to have a much broader spectrum of cell expression. In addition to N-formylated peptides, numerous unrelated ligands were recently found to interact with FPR and FPRL1. Novel agonists include both pathogen- and host-derived components, and synthetic peptides. Antagonistic molecules have been identified that exhibit limited receptor specificity. How distinct ligands can both induce different biological responses and produce different modes of receptor activation and unique sets of cellular responses are discussed. Cell responses to chemoattractants are tightly regulated at the level of the receptors. This review describes in detail the regulation of receptor signalling and the multi-step process of receptor inactivation. New concepts, such as receptor oligomerization and receptor clustering, are considered. Although FPR, FPRL1 and C5aR trigger similar biological functions and undergo a rapid chemoattractant-mediated phosphorylation, they appear to be differentially regulated and experience different intracellular fates.     

Single Amino Acid Substitutions in the Chemotactic Sequence of Urokinase Receptor Modulate Cell Migration and Invasion

The receptor for urokinase-type plasminogen activator (uPAR) plays an important role in controlling cell migration. uPAR binds urokinase and vitronectin extracellular ligands, and signals in complex with transmembrane receptors such as Formyl-peptide Receptors (FPR)s and integrins. Previous work from this laboratory has shown that synthetic peptides, corresponding to the uPAR_88–92 chemotactic sequence, when carrying the S90P or S90E substitutions, up- or down-regulate cell migration, respectively. To gain mechanistic insights into these opposite cell responses, the functional consequences of S90P and S90E mutations in full-length uPAR were evaluated. First, (HEK)-293 embryonic kidney cells expressing uPAR^S90P exhibit enhanced FPR activation, increased random and directional cell migration, long-lasting Akt phosphorylation, and increased adhesion to vitronectin, as well as uPAR/vitronectin receptor association. In contrast, the S90E substitution prevents agonist-triggered FPR activation and internalization, decreases binding and adhesion to vitronectin, and inhibits uPAR/vitronectin receptor association. Also, 293/uPAR^S90P cells appear quite elongated and their cytoskeleton well organized, whereas 293/uPAR^S90E cells assume a large flattened morphology, with random orientation of actin filaments. Interestingly, when HT1080 cells co-express wild type uPAR with uPAR S90E, the latter behaves as a dominant-negative, impairing uPAR-mediated signaling and reducing cell wound repair as well as lung metastasis in nude mice. In contrast, signaling, wound repair and in vivo lung metastasis of HT1080 cells bearing wild type uPAR are enhanced when they co-express uPAR^S90P. In conclusion, our findings indicate that Ser⁹⁰ is a critical residue for uPAR signaling and that the S90P and S90E exert opposite effects on uPAR activities. These findings may be accommodated in a molecular model, in which uPAR^S90E and uPAR^S90P are forced into inactive and active forms, respectively, suggesting important implications for the development of novel drugs targeting uPAR function.     

Critical role of N-terminal N-glycosylation for proper folding of the human formyl peptide receptor

The human formyl peptide receptor (FPR) is N-glycosylated and activates phagocytes via G(i)-proteins. The FPR expressed with G(i)alpha(2)beta(1)gamma(2) in Sf9 insect cells exhibits high constitutive activity as assessed by strong inhibitory effects of an inverse agonist and Na(+) on basal guanosine 5(')-O-(3-thiotriphosphate) (GTPgammaS) binding. The aim of our study was to analyze the role of N-glycosylation in FPR function. Site-directed mutagenesis of extracellular Asn residues prevented FPR glycosylation but not FPR expression in Sf9 membranes. However, in terms of high-affinity agonist binding, kinetics of GTPgammaS binding, number of G(i)-proteins activated, and constitutive activity, non-glycosylated FPR was much less active than native FPR. FPR-Asn4Gln/Asn10Gln/Asn179Gln and FPR-Asn4Gln/Asn10/Gln exhibited similar defects. Our data indicate that N-glycosylation of N-terminal Asn4 and Asn10 but not of Asn179 in the second extracellular loop is essential for proper folding and, hence, function of FPR. FPR deglycosylation by bacterial glycosidases could be a mechanism by which bacteria compromise host defense.     

Sequences within domain II of the urokinase receptor critical for differential ligand recognition

The receptor for urokinase-type plasminogen activator (uPAR) plays important roles in a number of physiological and pathological processes by virtue of its interactions with urokinase-type plasminogen activator (uPA), vitronectin (Vn), and several other proteins. The uPA binding site spans all three domains (D1 to D3) of uPAR. However, the nature of the Vn binding site within uPAR is still not clear. In this study, we conducted homolog-scanning mutagenesis on uPAR by switching 14 individual segments of 4-8 residues to their counterpart sequences of a uPAR homolog CD59. All 14 mutants were well expressed, reacted with a panel of monoclonal antibodies, and exhibited correct molecular weights. Of these 14 mutants, six mutants were defective in both uPA and Vn binding. Most importantly, we found two unique mutants uPAR(Asn172-Lys175) and uPAR(Glu183-Asn186) within the D2 domain, which displayed differential ligand binding activity: both had high affinity uPA binding, but completely lost Vn binding, indicating that these two sequences constitute a novel Vn binding site. Indeed, two peptides, P1 (153CPGSNGFHNNDTFHFLKC) and P2 (171CNTTKCNEGPILELENLPQ), derived from the sequences of the identified uPA and Vn binding pockets within D2, respectively, behaved like bona fide ligand binding sites: peptide P1 bound uPA but not Vn, whereas peptide P2 bound Vn and inhibited uPAR-mediated cell adhesion, but did not interact with uPA. Altogether, our data demonstrated that uPAR D2 contains two distinct ligand binding sites for uPA and Vn. Such information will help us better understand the complex roles of uPAR in cell adhesion, migration, and tumor metastasis.     

Domain 2 of the urokinase receptor contains an integrin-interacting epitope with intrinsic signaling activity: generation of a new integrin inhibitor

We investigated the interaction between the urokinase receptor (uPAR) and the integrin alphavbeta3. Vitronectin (VN) induces cell migration by binding to alphavbeta3, but expression of the uPAR boosts its efficacy. Thus, uPAR may regulate VN-induced cell migration by interacting laterally with alphavbeta3. In contrast, cells expressing a uPAR mutant lacking domain 2 do not migrate in response to VN. This effect is overcome by D2A, a synthetic peptide derived from the sequence of domain 2. In addition, D2A has chemotactic activity that requires alphavbeta3 and activates alphavbeta3-dependent signaling pathways such as the Janus kinase/Stat pathway. Moreover, D2A disrupts uPAR-alphavbeta3 and uPAR-alpha5beta1 co-immunoprecipitation, indicating that it can bind both of these integrins. We also identify the chemotactically active epitope harbored by peptide D2A. Mutating two glutamic acids into two alanines generates peptide D2A-Ala, which lacks chemotactic activity but inhibits VN-, FN-, and collagen-dependent cell migration. In fact, the GEEG peptide has potent chemotactic activity, and the GAAG sequence has inhibitory capacities. In summary, we have identified an integrin-interacting sequence located in domain 2 of uPAR, which is also a new chemotactic epitope that can activate alphavbeta3-dependent signaling pathways and stimulate cell migration. This sequence thus plays a pivotal role in the regulation of uPAR-integrin interactions. Moreover, we describe a novel, very potent inhibitor of integrin-dependent cell migration.     

uPAR and cathepsin B shRNA impedes TGF-β1-driven proliferation and invasion of meningioma cells in a XIAP-dependent pathway

Overexpression of transforming growth factor β1 (TGF-β1) has been linked to immune suppression, tumor angiogenesis, tumor cell migration, tumor cell survival, and tumor cell invasion in many cancers. In the present study, we found abundant expression of TGF-β1 in the microenvironment of four different pathological types of meningioma tumors. TGF-β1 induced invasion in malignant meningioma cells with an associated upregulation of urokinase-type plasminogen activator (uPA), uPAR, cathepsin B, and MMP-9, and this increase in proliferation was coupled with the expression of anti-apoptotic and pro-survival signaling molecules. In addition to the intense immunoreactivity of meningioma tumors to X-linked inhibitor to apoptosis (XIAP), its knockdown abolished the TGF-β1-induced proliferation of these cells. The stimulation of XIAP expression and the activation of pSMAD-2 is mediated by phosphatidylinositol 3-kinase (PI3K)- and MEK-dependent pathways, and the addition of anti-TGF-β1 antibodies prevented their expression with a consequent decrease in invasion. Bicistronic shRNA constructs targeting uPAR and cathepsin B (pUC) quenched TGF-β1-driven invasion and survival of meningioma cells by downregulation of XIAP and pSMAD-2 expression. Animal models with intracranial tumors showed elevated levels of TGF-β1, XIAP and pSMAD-2, and pUC treatment prevented this increased expression. Thus, targeted silencing of TGF-β1-induced signaling by pUC in meningioma would provide new treatment approaches for management of meningioma.     

Conformational regulation of urokinase receptor function: impact of receptor occupancy and epitope-mapped monoclonal antibodies on lamellipodia induction

The urokinase-type plasminogen activator receptor (uPAR) is a glycolipid-anchored membrane protein with an established role in focalizing uPA-mediated plasminogen activation on cell surfaces. Distinct from this function, uPAR also modulates cell adhesion and migration on vitronectin-rich matrices. Although uPA and vitronectin engage structurally distinct binding sites on uPAR, they nonetheless cooperate functionally, as uPA binding potentiates uPAR-dependent induction of lamellipodia on vitronectin matrices. We now present data advancing the possibility that it is the burial of the β-hairpin in uPA per se into the hydrophobic ligand binding cavity of uPAR that modulates the function of this receptor. Based on these data, we now propose a model in which the inherent interdomain mobility in uPAR plays a major role in modulating its function. Particularly one uPAR conformation, which is stabilized by engagement of the β-hairpin in uPA, favors the proper assembly of an active, compact receptor structure that stimulates lamellipodia induction on vitronectin. This molecular model has wide implications for drug development targeting uPAR function.     

Structural basis of interaction between urokinase-type plasminogen activator and its receptor

Recent studies indicate that binding of the urokinase-type plasminogen activator (uPA) to its high-affinity receptor (uPAR) orchestrates uPAR interactions with other cellular components that play a pivotal role in diverse (patho-)physiological processes, including wound healing, angiogenesis, inflammation, and cancer metastasis. However, notwithstanding the wealth of biochemical data available describing the activities of uPAR, little is known about the exact mode of uPAR/uPA interactions or the presumed conformational changes that accompany uPA/uPAR engagement. Here, we report the crystal structure of soluble urokinase plasminogen activator receptor (suPAR), which contains the three domains of the wild-type receptor but lacks the cell-surface anchoring sequence, in complex with the amino-terminal fragment of urokinase-type plasminogen activator (ATF), at the resolution of 2.8 A. We report the 1.9 A crystal structure of free ATF. Our results provide a structural basis, represented by conformational changes induced in uPAR, for several published biochemical observations describing the nature of uPAR/uPA interactions and provide insight into mechanisms that may be responsible for the cellular responses induced by uPA binding.     

alpha-2 Macroglobulin receptor/Ldl receptor-related protein(Lrp)- dependent internalization of the urokinase receptor

The GPI-anchored urokinase plasminogen activator receptor (uPAR) does not internalize free urokinase (uPA). On the contrary, uPAR-bound complexes of uPA with its serpin inhibitors PAI-1 (plasminogen activator inhibitor type-1) or PN-1 (protease nexin-1) are readily internalized in several cell types. Here we address the question whether uPAR is internalized as well upon binding of uPA-serpin complexes. Both LB6 clone 19 cells, a mouse cell line transfected with the human uPAR cDNA, and the human U937 monocytic cell line, express in addition to uPAR also the endocytic alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP/alpha 2-MR) which is required to internalize uPAR-bound uPA-PAI-1 and uPA-PN-1 complexes. Downregulation of cell surface uPAR molecules in U937 cells was detected by cytofluorimetric analysis after uPA-PAI-1 and uPA-PN-1 incubation for 30 min at 37 degrees C; this effect was blocked by preincubation with the ligand of LRP/alpha 2-MR, RAP (LRP/alpha 2-MR- associated protein), known to block the binding of the uPA complexes to LRP/alpha 2-. MR. Downregulation correlated in time with the intracellular appearance of uPAR as assessed by confocal microscopy and immuno-electron microscopy. After 30 min incubation with uPA-PAI-1 or uPA-PN-1 (but not with free uPA), confocal microscopy showed that uPAR staining in permeabilized LB6 clone 19 cells moved from a mostly surface associated to a largely perinuclear position. This effect was inhibited by the LRP/alpha 2-MR RAP. Perinuclear uPAR did not represent newly synthesized nor a preexisting intracellular pool of uPAR, since this fluorescence pattern was not modified by treatment with the protein synthesis inhibitor cycloheximide, and since in LB6 clone 19 cells all of uPAR was expressed on the cell surface. Immuno-electron microscopy confirmed the plasma membrane to intracellular translocation of uPAR, and its dependence on LRP/alpha 2-MR in LB6 clone 19 cells only after binding to the uPA-PAI-1 complex. After 30 min incubation at 37 degrees C with uPA-PAI-1, 93% of the specific immunogold particles were present in cytoplasmic vacuoles vs 17.6% in the case of DFP-uPA. We conclude therefore that in the process of uPA-serpin internalization, uPAR itself is internalized, and that internalization requires the LRP/alpha 2-MR.     

Critical role of integrin alpha 5 beta 1 in urokinase (uPA)/urokinase receptor (uPAR,CD87) signaling

Urokinase-type plasminogen activator (uPA) induces cell adhesion and chemotactic movement. uPA signaling requires its binding to uPA receptor (uPAR/CD87), but how glycosylphosphatidylinositol-anchored uPAR mediates signaling is unclear. uPAR is a ligand for several integrins (e.g. alpha 5 beta 1) and supports cell-cell interaction by binding to integrins on apposing cells (in trans). We studied whether binding of uPAR to alpha 5 beta 1 in cis is involved in adhesion and migration of Chinese hamster ovary cells in response to immobilized uPA. This process was temperature-sensitive and required mitogen-activated protein kinase activation. Anti-uPAR antibody or depletion of uPAR blocked, whereas overexpression of uPAR enhanced, cell adhesion to uPA. Adhesion to uPA was also blocked by deletion of the growth factor domain (GFD) of uPA and by anti-GFD antibody, whereas neither the isolated uPA kringle nor serine protease domain supported adhesion directly. Interestingly, anti-alpha 5 antibody, RGD peptide, and function-blocking mutations in alpha 5 beta 1 blocked adhesion to uPA. uPA-induced cell migration also required GFD, uPAR, and alpha 5 beta 1, but alpha 5 beta 1 alone did not support uPA-induced adhesion and migration. Thus, binding of uPA causes uPAR to act as a ligand for alpha 5 beta 1 to induce cell adhesion, intracellular signaling, and cell migration. We demonstrated that uPA induced RGD-dependent binding of uPAR to alpha 5 beta 1 in solution. These results suggest that uPA-induced adhesion and migration of Chinese hamster ovary cells occurs as a consequence of (a) uPA binding to uPAR through GFD, (b) the subsequent binding of a uPA.uPAR complex to alpha 5 beta 1 via uPAR, and (c) signal transduction through alpha 5 beta 1.     

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 A 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.     

Nordy, a synthetic lipoxygenase inhibitor, inhibits the expression of formylpeptide receptor and induces differentiation of malignant glioma cells

We recently found that formylpeptide receptor (FPR), a G-protein-coupled receptor that mediates chemotaxis of phagocytic leukocytes induced by bacterial peptide N-formyl-methionyl-leucyl-phenylalanine, is expressed by malignant human glioma cells and promotes tumor growth and angiogenesis. In this study, we examined the effect of Nordy, a novel chiral lipoxygenase inhibitor which was synthesized based on the structure of a natural nordihydroguaiaretic acid, on the expression of FPR by human glioblastoma cells. We found that FPR was expressed at the protein level by highly malignant human glioma cell lines U87 and BT325, and a rat glioma cell line C6. The expression level of FPR was correlated with the degree of the malignancy of tumor cells. The poorly differentiated glioma cell line U87 expressed the highest level of FPR. In U87 glioma cells, the expression of FPR was attenuated at the protein level by Nordy treatment for 48 (P<0.05). Nordy did not affect FPR mRNA expression in U87 cells. In addition, Nordy treatment seemed to promote glioma cell differentiation, as evidenced by their reduced expression of vimentin and increased expression of GFAP. Our results suggest that Nordy was capable of reducing the level of malignancy of glioma cells.     

Differential roles of the NPXXY motif in formyl peptide receptor signaling

The NPXXY motif (X represents any amino acid) in the seventh transmembrane domain of the chemotactic formyl peptide receptor (FPR) is highly conserved among G protein-coupled receptors. Recent work suggested that this motif contributes to G protein-coupled receptor internalization and signal transduction; however, its role in FPR signaling remains unclear. In this study we replaced Asn(297) and Tyr(301) in the NPXXY motif of the human FPR with Ala (N297A) and Ala/Phe (Y301A/Y301F), respectively, and determined the effects of the substitutions on FPR functions in transfected rat basophilic leukemia cells. Whereas all the mutant receptors were expressed on the cell surface, the N297A receptor exhibited reduced binding affinity and was unable to mediate activation of phospholipase C-beta and the p42/44 mitogen-activated protein kinase (MAP kinase). The Y301F receptor displayed significantly decreased ligand-stimulated internalization and MAP kinase activation, suggesting that the hydrogen bonding at Tyr(301) is critical for these functions. The Y301F receptor showed a chemotactic response similar to that of wild-type FPR, indicating that cell chemotaxis does not require receptor internalization and hydrogen bonding at the Tyr(301) position. In contrast, the Y301A receptor displayed a left-shifted, but overall reduced, chemotaxis response that peaked at 0.1-1 nM. Finally, using a specific MAP kinase kinase inhibitor, we found that activation of MAP kinase is required for efficient FPR internalization, but is not essential for chemotaxis. These findings demonstrate that residues within the NPXXY motif differentially regulate the functions of FPR.