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1.
Carretero J Obrador E Esteve JM Ortega A Pellicer JA Sempere FV Estrela JM 《The Journal of biological chemistry》2001,276(28):25775-25782
The mechanism of NO- and H(2)O(2)-induced tumor cytotoxicity was examined during B16 melanoma (B16M) adhesion to the hepatic sinusoidal endothelium (HSE) in vitro. We used endothelial nitric-oxide synthetase gene disruption and N(G)-nitro-l-arginine methyl ester-induced inhibition of nitric-oxide synthetase activity to study the effect of HSE-derived NO on B16M cell viability. Extracellular H(2)O(2) was removed by exogenous catalase. H(2)O(2) was not cytotoxic in the absence of NO. However, NO-induced tumor cytotoxicity was increased by H(2)O(2) due to the formation of potent oxidants, likely ( small middle dot)OH and (-)OONO radicals, via a trace metal-dependent process. B16M cells cultured to low density (LD cells), with high GSH content, were more resistant to NO and H(2)O(2) than B16M cells cultured to high density (HD cells; with approximately 25% of the GSH content found in LD cells). Resistance of LD cells decreased using buthionine sulfoximine, a specific GSH synthesis inhibitor, whereas resistance increased in HD cells using GSH ester, which delivers free intracellular GSH. Because NO and H(2)O(2) were particularly cytotoxic in HD cells, we investigated the enzyme activities that degrade H(2)O(2). NO and H(2)O(2) caused an approximately 75% (LD cells) and a 60% (HD cells) decrease in catalase activity without affecting the GSH peroxidase/GSH reductase system. Therefore, B16M resistance to the HSE-induced cytotoxicity appears highly dependent on GSH and GSH peroxidase, which are both required to eliminate H(2)O(2). In agreement with this fact, ebselen, a GSH peroxidase mimic, abrogated the increase in NO toxicity induced by H(2)O(2). 相似文献
2.
Ortega AL Carretero J Obrador E Gambini J Asensi M Rodilla V Estrela JM 《The Journal of biological chemistry》2003,278(16):13888-13897
High GSH content associates with high metastatic activity in B16-F10 melanoma cells cultured to low density (LD B16M). GSH homeostasis was investigated in LD B16M cells that survive after adhesion to the hepatic sinusoidal endothelium (HSE). Invasive B16M (iB16M) cells were isolated using anti-Met-72 monoclonal antibodies and flow cytometry-coupled cell sorting. HSE-derived NO and H(2)O(2) caused GSH depletion and a decrease in gamma-glutamylcysteine synthetase activity in iB16M cells. Overexpression of gamma-glutamylcysteine synthetase heavy and light subunits led to a rapid recovery of cytosolic GSH, whereas mitochondrial GSH (mtGSH) further decreased during the first 18 h of culture. NO and H(2)O(2) damaged the mitochondrial system for GSH uptake (rates in iB16M were approximately 75% lower than in LD B16M cells). iB16M cells also showed a decreased activity of mitochondrial complexes II, III, and IV, less O(2) consumption, lower ATP levels, higher O(2) and H(2)O(2) production, and lower mitochondrial membrane potential. In vitro growing iB16M cells maintained high viability (>98%) and repaired HSE-induced mitochondrial damages within 48 h. However, iB16M cells with low mtGSH levels were highly susceptible to TNF-alpha-induced oxidative stress and death. Therefore depletion of mtGSH levels may represent a critical target to challenge survival of invasive cancer cells. 相似文献
3.
The cell adhesion molecules N-CAM and L1 are important for cell-cell recognition and cell migration and so may be involved in the metastatic process. We have studied the biosynthesis of N-CAM and L1 in the B16 melanoma cell lines B16-F1 and B16-F10 which differ in metastatic capacity. N-CAM was synthesised as two glycosylated polypeptides with Mr of 150,000 and 210,000; L1 was synthesised as one polypeptide with Mr of 215,000. In fetal neurons N-CAM is synthesised as a 135,000 and a 200,000 Mr polypeptide and L1 as a 200,000 Mr polypeptide. Thus, the Mr of N-CAM and L1 in tumour cells appeared to be 10,000-15,000 higher than in the normal cells. L1 was phosphorylated in the tumour cells as in neurons. The tumour cells also phosphorylated the 210,000 Mr N-CAM polypeptide, whereas no phosphorylation of the 150,000 Mr polypeptide was observed. In neuronal cells both the corresponding polypeptides are phosphorylated and thus the biosynthesis of N-CAM in tumour cells seem to differ from that in neuronal cells with regard to phosphorylation. No differences in biosynthesis of N-CAM or L1 were apparent between the two tumour cell lines, B16-F1 and B16-F10. 相似文献
4.
We present a model of the cell signalling network based on the generic properties of interactions between protein kinases (PKs) and protein phosphatases (PPs) inside cells. The model is designed to examine the global properties and intrinsic dynamics of the phosphorylation system. A genetic algorithm (GA) is used to evolve populations of "cells". The GA selects cells and ranks them based on an analysis of the dynamics of the proteins within the networks from a series of different random starting conditions. The fittest cells are taken to be those which can generate a variety of different "behaviours" from a series of different initial conditions. During the GA, intracellular protein interactions evolve via mutation and an analogue of domain shuffling between protein types that is thought to occur during biological evolution. The dynamics of the simulated networks are presented and we discuss the hypothesis that changes in the behaviour of a cell may be interpretable as a switch between attractor basins in the intracellular signalling network. 相似文献
5.
D C Anderson O Abbassi T K Kishimoto J M Koenig L V McIntire C W Smith 《Journal of immunology (Baltimore, Md. : 1950)》1991,146(10):3372-3379
To define further the molecular basis for abnormal interactions of cord blood or neonatal neutrophils with endothelial cells in vitro, we studied neutrophil adhesion and migration under experimental conditions specifically designed to evaluate CD18-independent mechanisms. Unstimulated cord blood neutrophils of healthy term neonates demonstrated significantly diminished adhesion to IL-1-stimulated endothelial cell monolayers under conditions of shear stress (congruent to 1.85 dynes/cm2); overall levels of migration by neonatal cells were also significantly diminished, although the adherent subpopulation of these cells migrated relatively normally. A mAb (DREG-56) against the human homologue of the murine MEL-14 antigen (termed lectin-, epidermal growth factor-, complement binding domain-cell adhesion molecule-1 (LECAM-1), a member of the LEC-CAM family of adhesion molecules) markedly inhibited adhesion of healthy adult but not cord blood neutrophils. In additional assessments of endothelial cell adhesion or migration in the absence of shear forces, cord blood neutrophils demonstrated significantly diminished values compared to adult controls. Moreover, mAb DREG-56 significantly diminished adhesion of healthy adult but not cord blood suspensions in the presence or absence of the anti-CD18 mAb R15.7. Immunofluorescence assessments of unstimulated cord blood neutrophils or neutrophils of neonates 12 to 48 h of age showed dramatically diminished levels of surface LECAM-1 compared to adult neutrophils. Chemotactic stimuli (FMLP, 10 nM, 15 min) consistently "down-regulated" surface LECAM-1 on adult neutrophils to levels approximately 10% of unstimulated suspensions and comparable to those of most unstimulated neonatal suspensions. Moreover, FMLP stimuli elicited little or no down-regulation of LECAM-1 on neonatal cells. In comparative studies, endothelial cell adhesion of unstimulated cord blood or adult control neutrophils (assessed under conditions of flow) was directly related to levels of neutrophil surface LECAM-1. Although FMLP stimulation significantly diminished both adhesion and LECAM-1 surface levels of adult control cells, the adhesion and LECAM-1 expression observed with cord blood cells were not significantly influenced by this stimulus. The mechanisms underlying diminished LECAM-1 expression and LECAM-1-dependent adhesion of neonatal neutrophils and the physiologic significance of these abnormalities deserve investigation. 相似文献
6.
Ortega A Ferrer P Carretero J Obrador E Asensi M Pellicer JA Estrela JM 《The Journal of biological chemistry》2003,278(41):39591-39599
B16 melanoma (B16M) cells with high GSH content show high metastatic activity. However, the molecular mechanisms linking GSH to metastatic cell survival are unclear. The possible relationship between GSH and the ability of Bcl-2 to prevent cell death was studied in B16M cells with high (F10) and low (F1) metastatic potential. Analysis of a Bcl-2 family of genes revealed that B16M-F10 cells, as compared with B16M-F1 cells, overexpressed preferentially Bcl-2 (approximately 5.7-fold). Hepatic sinusoidal endothelium-induced B16M-F10 cytotoxicity in vitro increased from approximately 19% (controls) to approximately 97% in GSH-depleted B16M-F10 cells treated with an antisense Bcl-2 oligodeoxynucleotide (Bcl-2-AS). l-Buthionine (S,R)-sulfoximine-induced GSH depletion or Bcl-2-AS decreased the metastatic growth of B16M-F10 cells in the liver. However, the combination of l-buthionine (S,R)-sulfoximine and Bcl-2-AS abolished metastatic invasion. Bcl-2-overexpressing B16M-F1/Tet-Bcl-2 and B16M-F10/Tet-Bcl-2 cells, as compared with controls, showed an increase in GSH content, no change in the rate of GSH synthesis, and a decrease in GSH efflux. Thus, Bcl-2 overexpression may increase metastatic cell resistance against oxidative/nitrosative stress by inhibiting release of GSH. In addition, Bcl-2 availability regulates the mitochondrial GSH (mtGSH)-dependent opening of the permeability transition pore complex. Death in B16M-F10 cells was sharply activated at mtGSH levels below 30% of controls values. However, this critical threshold increased to approximately 60% of control values in Bcl-2-AS-treated B16M-F10 cells. GSH ester-induced replenishment of mtGSH levels (even under conditions of cytosolic GSH depletion) prevented cell death. Our results indicate that survival of B16M cells with high metastatic potential can be challenged by inhibiting their GSH and Bcl-2 synthesis. 相似文献
7.
Ogura T Noguchi T Murai-Takebe R Hosooka T Honma N Kasuga M 《The Journal of biological chemistry》2004,279(14):13711-13720
The adhesion receptor SHPS-1 activates the protein-tyrosine-phosphatase SHP-2 and thereby promotes integrin-mediated reorganization of the cytoskeleton. SHPS-1 also contributes to cell-cell communication through association with CD47. Although functional alteration of SHPS-1 is implicated in cellular transformation, the role of the CD47-SHPS-1 interaction in carcinogenesis has been unclear. A soluble SHPS-1 ligand (CD47-Fc) has now been shown to bind to Melan-a non-tumorigenic melanocytes but not to syngeneic B16F10 melanoma cells. Treatment of B16F10 cells with 1-deoxymannojirimycin, which prevents N-glycan processing, restored the ability of SHPS-1 derived from these cells to bind CD47-Fc in vitro, indicating that aberrant N-glycosylation of SHPS-1 impairs CD47 binding in B16F10 cells. CD47-Fc inhibited the migration of Melan-a cells but not that of B16F10 cells. However, a monoclonal antibody that reacts with SHPS-1 on both Melan-a and B16F10 cells inhibited the migration of both cell types similarly. CD47 binding induced proteasome-mediated degradation of SHPS-1 in a tyrosine phosphorylation-independent manner. Furthermore, overexpression of SHPS-1 reduced the level of tyrosine phosphorylation of focal adhesion kinase, and this effect was reversed by CD47 binding. These results suggest that CD47 binds to and thereby down-regulates SHPS-1 on adjacent cells, resulting in inhibition of cell motility. Resistance to this inhibitory mechanism may contribute to the highly metastatic potential of B16 melanoma. 相似文献
8.
9.
Potential ability of hot water adzuki (Vigna angularis) extracts to inhibit the adhesion, invasion, and metastasis of murine B16 melanoma cells 总被引:1,自引:0,他引:1
The 40% ethanol eluent of the fraction of hot-water extract from adzuki beans (EtEx.40) adsorbed onto DIAION HP-20 resin has many biological activities, for example, antioxidant, antitumorigenesis, and intestinal alpha-glucosidase suppressing activities. This study examined the inhibitory effect of EtEx.40 on experimental lung metastasis and the invasion of B16-BL6 melanoma cells. EtEx.40 was found significantly to reduce the number of tumor colonies. It also inhibited the adhesion and migration of B16-BL6 melanoma cells into extracellular matrix components and their invasion into reconstituted basement membrane (matrigel) without affecting cell proliferation in vitro. These in vivo data suggest that EtEx.40 possesses a strong antimetastatic ability, which might be a lead compound in functional food development. 相似文献
10.
P Valverde J C García-Borrón J H Martínez-Liarte F Solano J A Lozano 《FEBS letters》1992,304(2-3):114-118
Tyrosinase induction in murine malignant melanocytes by alpha MSH is well known, but its molecular basis has not been characterized. Treatment of B16 melanoma cells with theophylline or alpha MSH mediates a larger induction of tyrosine hydroxylase than of dopa oxidase activity in total cell extracts, and in the melanosomal and microsomal fractions. No evidence for the modulation of a tyrosinase effector was found. SDS-PAGE and specific activity stain demonstrated two forms of tyrosinase, with different degrees of induction by theophylline. These results agree with the recent proposal that two tyrosinases, encoded by different genes, are present in murine melanocytes. 相似文献
11.
Over-expression of MSG1 transcriptional co-activator increases melanin in B16 melanoma cells: a possible role for MSG1 in melanogenesis. 总被引:2,自引:0,他引:2
S S Nair V A Chaubal T Shioda K R Coser M Mojamdar 《Pigment cell research / sponsored by the European Society for Pigment Cell Research and the International Pigment Cell Society》2001,14(3):206-209
MSG1 is a 27 kDa nuclear protein that is expressed strongly in melanotic B16 melanoma cells but very weakly in amelanotic B16 cells. Transient expression of B16 cells with an expression vector for MSG1 resulted in an increase in levels of the enzyme dopachrome tautomerase but not tyrosinase, as detected by western blotting. Stable transfection of B16 melanoma cells with plasmids containing the full length MSG1 or its deletion mutants, however, generated cell lines that showed an increase in levels of tyrosinase, dopachrome tautomerase and cellular melanin when compared with control transfected cells. Our results suggest that MSG1 plays an important role in melanogenesis, by regulating the levels of the enzymes of the pigmentary system via tyrosinase and dopachrome tautomerase. 相似文献
12.
Y F Cheng R I Clyman J Enenstein N Waleh R Pytela R H Kramer 《Experimental cell research》1991,194(1):69-77
Fibronectin is a major adhesive glycoprotein of the vascular basement membrane. Since fibronectin is also found in the interstitium, it may be important not only for attachment but also for endothelial cell migration during neovascularization. We have analyzed how human dermal microvascular endothelial cells use their diverse set of integrin receptors to interact with this ligand. Immunofluorescent staining with specific antibodies identified both beta 1 and beta 3 integrin receptor complexes in focal adhesion plaques on cells adhering to immobilized fibronectin. Adhesion assays with blocking monoclonal antibodies implicated both beta 1 and beta 3 complexes, specifically alpha 5 beta 1 and alpha v beta 3, in the initial adhesion of cells to fibronectin. Finally, ligand affinity chromatography of extracts of surface radiolabeled cells established that both alpha 5 beta 1 and alpha v beta 3 could bind to the 110-kDa cell-binding fragment of fibronectin. An additional receptor complex composed of an alpha v subunit and a beta 5-like subunit was also detected. These results provide evidence that microvascular endothelial cells use multiple integrin receptors, from several beta families, to attach to fibronectin surfaces. 相似文献
13.
M H Thornhill S M Wellicome D L Mahiouz J S Lanchbury U Kyan-Aung D O Haskard 《Journal of immunology (Baltimore, Md. : 1950)》1991,146(2):592-598
The adhesion of lymphocytes to vascular endothelium is the first step in their passage from the blood into inflammatory tissues. By modulating endothelial cell (EC) adhesiveness for lymphocytes, cytokines may regulate lymphocyte accumulation and hence the nature and progression of inflammatory responses. IL-1, TNF, IFN-gamma, and IL-4 each increase EC adhesiveness for T cells when used alone in adhesion assays in vitro. As cytokines are more likely to act in combination at sites of inflammation in vivo, we have studied the stimulating effect of different combinations of cytokines on EC adhesiveness for T cells and polymorphonuclear leukocytes (PMN). Acting alone IL-1, TNF, IFN-gamma, and IL-4 each significantly enhanced EC adhesiveness for T cells (p less than 0.005), whereas only IL-1 (p less than 0.005) and TNF (p less than 0.005) but not IFN-gamma or IL-4 significantly enhanced adhesiveness for PMN. When EC were stimulated with optimal concentrations of TNF in combination with IL-4 or IFN-gamma, there was a significant further increase in adhesiveness for T cells (p less than 0.003), but not PMN, over that seen with TNF alone. The additive effect of TNF and IL-4 was more marked than that of TNF and IFN-gamma. Although approximately equal proportions of T cells and PMN bound to TNF-stimulated EC, nearly double the proportion of T cells compared with PMN bound EC preincubated with TNF and IL-4 together. A similar interaction with IL-4 or IFN-gamma was exhibited by lymphotoxin. mAb-inhibition studies indicated that the extra increase in binding caused by stimulating EC with TNF and IL-4 in combination was mediated by VCAM-1 whereas that caused by stimulating with TNF and IFN-gamma in combination was substantially mediated through leukocyte function-associated Ag-1- and VCAM-1-independent mechanisms. These observations suggest that whereas IL-1 and TNF alone are unselective in terms of leukocyte adhesion to EC, the combination of TNF (or LT) with IL-4 or IFN-gamma may be of key importance in determining the recruitment of a lymphocyte-predominant infiltrate in immune mediated inflammation, and in initiating the transition from acute to chronic inflammation. 相似文献
14.
Massimiliano Secchi Qiang Xu Paolo Lusso Luca Vangelista 《Protein expression and purification》2009,68(1):34-41
Development of effective topical microbicides for the prevention of HIV-1 sexual transmission represents a primary goal for the control of the AIDS pandemic. The viral coreceptor CCR5, used by the vast majority of primary HIV-1 isolates, is considered a primary target molecule. RANTES and its derivatives are the most suitable protein-based compounds to fight HIV-1 via CCR5 targeting. Yet, receptor activation should be avoided to prevent pro-inflammatory effects and possibly provide anti-inflammatory properties. C1C5 RANTES is a chemokine mutant that exhibits high anti-HIV-1 potency coupled with CCR5 antagonism. However, the need for the formation of an N-terminal intramolecular disulfide bridge between non-natural cysteine residues at positions 1 and 5 represents a challenge for the correct folding of this protein in recombinant expression systems, a crucial step towards its development as a microbicide against HIV-1. We report here a rare case of superior folding in a prokaryote as compared to an eukaryotic expression system. Production of C1C5 RANTES was highly impaired in CHO cells, with a dramatic yield reduction compared to that of wild type RANTES and secretion of the molecule as disulfide-linked dimer. Conversely, a human vaginal isolate of Lactobacillus jensenii engineered to secrete C1C5 RANTES provided efficient delivery of the monomeric protein. This and other reports on successful secretion of complex proteins indicate that lactic acid bacteria are an excellent system for the expression of therapeutic proteins, which can be used as a platform for the engineering of conceptually novel RANTES mutants with potent anti-HIV-1 activity. 相似文献
15.
Haili Qian Jing Yu Yunfeng Li Haijuan Wang Chongwen Song Xueyan Zhang Xiao Liang Ming Fu Chen Lin 《Biology of the cell / under the auspices of the European Cell Biology Organization》2007,99(10):573-581
BACKGROUND INFORMATION: MTA1 (metastasis-associated gene 1) has been reported to be overexpressed in cancers with high potential to metastasize. Studies of the molecular mechanisms revealed that MTA1 plays an important role in the process of metastasis of many types of cancer. However, the role of MTA1 in melanoma development is unclear. RESULTS: We have investigated the therapeutic value of MTA1 in the B16F10 melanoma cell line with the C57BL/6 mouse model. Studies in vitro showed that MTA1 promoted the metastatic ability of B16F10 cancer cells. MTA1 down-regulation by RNA interference greatly reversed the malignant phenotypes of cancer cells. Immunohistochemical staining of MTA1 in human melanoma samples confirmed the up-regulation of MTA1 in the process of carcinogenesis. Studies in vivo confirmed down-regulation of MTA1 suppressed the growth and experimental metastasis of B16F10 melanoma cells. CONCLUSIONS: MTA1 plays an important role in melanoma development and metastasis. It has a promising potential as a target for in cancer gene therapy or chemotherapy. 相似文献
16.
N Kojima K Handa W Newman S Hakomori 《Biochemical and biophysical research communications》1992,189(3):1686-1694
E-selectin has a "multi-recognition" capability in terms of epitope binding specificity, depending on adhesion conditions (static vs. low- or high-shear stress dynamic systems). Specifically, (i) adhesion based on expression of alpha 2-->3 sialylated Le(x) (SLe(x)) is prominent under static or low shear stress dynamic conditions; (ii) adhesion under high shear stress dynamic conditions does not depend on the known SLe(x) species, but rather on Lex with an adjacent unidentified sialosyl substitution, which shows different susceptibility to sialidases and antibodies compared to known SLe(x). 相似文献
17.
Endoglin is a component of the transforming growth factor-beta receptor system in human endothelial cells. 总被引:34,自引:0,他引:34
S Cheifetz T Bellón C Calés S Vera C Bernabeu J Massagué M Letarte 《The Journal of biological chemistry》1992,267(27):19027-19030
Endoglin, a dimeric membrane glycoprotein expressed at high levels on human vascular endothelial cells, shares regions of sequence identity with betaglycan, a major binding protein for transforming growth factor-beta (TGF-beta) that co-exists with TGF-beta receptors I and II in a variety of cell lines but is low or absent in endothelial cells. We have examined whether endoglin also binds TGF-beta and demonstrate here that the major TGF-beta 1-binding protein co-existing with TGF-beta receptors I and II on human umbilical vein endothelial cells is endoglin, as determined by specific immunoprecipitation of endoglin affinity-labeled with 125I-TGF-beta. Furthermore, endoglin ectopically expressed in COS cells binds TGF-beta 1. Competition affinity-labeling experiments showed that endoglin binds TGF-beta 1 (KD approximately 50 pM) and TGF-beta 3 with high affinity but fails to bind TGF-beta 2. This difference in affinity of endoglin for the TGF-beta isoforms is in contrast to beta-glycan which recognizes all three isoforms. TGF-beta however is binding with high affinity to only a small fraction of the available endoglin molecules, suggesting that some rate-limiting event is required to sustain TGF-beta binding to endoglin. 相似文献
18.
Dominique Thuringer Laurence Maulon Christian Frelin 《The Journal of biological chemistry》2002,277(3):2028-2032
Bradykinin (BK) and vascular endothelial growth factor (VEGF)-165 stimulate vasodilatation, microvascular permeability, and angiogenesis via the activation of the B2-type and KDR/Flk-1 receptors. To delineate the signal transduction pathways distal to the receptor activation in microvascular permeability, we compared their effects on two downstream targets, i.e. endothelial nitric-oxide (NO) synthase (eNOS) and F-actin, in primary cultures of cardiac capillary endothelial cells. The two mediators induced a similar cytoskeletal reorganization and both the translocation and activation of eNOS, leading to NO release within the first minutes of cell exposure. At the same time, BK produced the tyrosine phosphorylation and internalization of KDR/Flk-1 as did VEGF itself. This transactivation was blocked by the selective inhibitor of VEGF receptor tyrosine kinase activity but not by inhibitors of epidermal growth factor receptor or protein kinase C activity. The selective inhibitor of VEGF receptor tyrosine kinase activity totally prevented the effects of VEGF but only partially inhibited NO release induced by BK without affecting the concomitant cytoskeletal reorganization. Thus, BK transactivated KDR/Flk-1 through an intrinsic kinase activity of KDR/Flk-1, resulting in a further eNOS activation in endothelial cells. This represents a novel mechanism whereby a G protein-coupled receptor activates a receptor tyrosine kinase to generate biological response. 相似文献
19.
20.
Roman M Salasznyk Maria Zappala Mingzhe Zheng Lin Yu Cynthia Wilkins-Port Paula J McKeown-Longo 《Matrix biology》2007,26(5):359-370
Vitronectin is a plasma protein which can deposit into the extracellular matrix where it supports integrin and uPA dependent cell migration. In earlier studies, we have shown that the plasma protein, vitronectin, stimulates focal adhesion remodeling by recruiting urokinase-type plasminogen activator (uPA) to focal adhesion sites [Wilcox-Adelman, S. A., Wilkins-Port, C. E., McKeown-Longo, P. J., 2000. Localization of urokinase-type plasminogen activator to focal adhesions requires ligation of vitronectin integrin receptors. Cell. Adhes. Commun.7, 477-490]. In the present study, we used a variety of vitronectin constructs to demonstrate that the localization of uPA to adhesion sites requires the binding of both vitronectin integrin receptors and the uPA receptor (uPAR) to vitronectin. A recombinant fragment of vitronectin containing the connecting sequence (VN(CS)) was able to support integrin-dependent adhesion, spreading and focal adhesion assembly by human microvessel endothelial cells. Cells adherent to this fragment were not able to localize uPA to focal adhesions. A second recombinant fragment containing both the amino-terminal SMB domain and the CS domain was able to restore the localization of uPA to adhesion sites. This fragment, which contains a uPAR binding site, also resulted in the localization of uPAR to adhesion sites. uPAR blocking antibodies as well as phospholipase C treatment of cells inhibited uPA localization to adhesion sites confirming a role for uPAR in this process. The SMB domain alone was unable to direct either uPAR or uPA to adhesion sites in the absence of the CS domain. Our results indicate that vitronectin-dependent localization of uPA to adhesion sites requires the sequential binding of vitronectin integrins and uPAR to vitronectin. 相似文献