Novel protein interactors of urokinase-type plasminogen activator receptor

Novel protein interactors of urokinase-type plasminogen activator receptor

Biochemical and Biophysical Research Communications 399 (2010) 738–743 Contents lists available at ScienceDirect Biochemical and Biophysical Researc...

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Biochemical and Biophysical Research Communications 399 (2010) 738–743

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications journal homepage:

Novel protein interactors of urokinase-type plasminogen activator receptor Ahmed H. Mekkawy a,b,1, Charles E. De Bock b,1,2, Zhen Lin b, David L. Morris a, Yao Wang b,3, Mohammad H. Pourgholami a,* a b

Cancer Research Laboratories, Department of Surgery, St. George Hospital, University of New South Wales, Sydney, NSW 2217, Australia Division of Critical Care and Surgery, St. George Hospital, University of New South Wales, Sydney, NSW 2217, Australia

a r t i c l e

i n f o

Article history: Received 28 July 2010 Available online 7 August 2010 Keywords: Breast cancer Protein–protein interaction Yeast two-hybrid uPAR hSpry1 HAX1

a b s t r a c t The urokinase-type plasminogen activator receptor (uPAR) has been implicated in tumor growth and metastasis. The crystal structure of uPAR revealed that the external surface is largely free to interact with a number of proteins. Additionally, due to absence of an intracellular cytoplasmic protein domain, many of the biological functions of uPAR necessitate interactions with other proteins. Here, we used yeast twohybrid screening of breast cancer cDNA library to identify hSpry1 and HAX1 proteins as putative candidate proteins that interact with uPAR bait constructs. Interaction between these two candidates and uPAR was confirmed by GST-pull down, co-immunoprecipitation assays and confocal microscopy. These novel interactions that have been identified may also provide further evidence that uPAR can interact with a number of other proteins which may influence a range of biological functions. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction The urokinase plasminogen activator receptor (uPAR) has a well described role in extracellular matrix (ECM) proteolysis, and other important functions in cell growth, adhesion, migration and invasion [1]. These latter additional roles are influenced by the ability of uPAR to engage in multiple protein–protein interactions [2]. In addition to the primary ligand uPA that binds to uPAR, there are two further ligands, which have been found to bind uPAR. These are vitronectin (VN) [3], and kininogen [4]. Furthermore, uPAR is linked to the cell membrane via a GPI anchor, and so has no intracellular domain [5]. Hence, many of the biological functions of uPAR necessitate interactions with other proteins on the cell surface, in particular, transmembrane proteins. The high lateral mobility of uPAR on the cell membrane may provide the mechanism by Abbreviations: coIP, co-immunoprecipitation; ECM, extracellular matrix; EGF, epidermal growth factor; FGF, fibroblasts growth factor; GPI, glycosyl phosphatidylinositol; GST, glutathione S-transferase; HAX1, human HIS binding protein; hSpry1, human Sprouty protein 1; HUVECs, human umbilical vein endothelial cells; IP, immunoprecipitation; PBS, phosphate-buffered saline; RGD, Arg-Gly-Asp; RTK, receptor tyrosine kinases; suPAR, soluble uPAR; TLC, total cell lysate; uPA, urokinase-type plasminogen activator; uPAR, uPA receptor; VN, vitronectin; Y2H, yeast two-hybrid. * Corresponding author. Fax: +61 291133456. E-mail address: [email protected] (M.H. Pourgholami). 1 These authors contributed equally to this work. 2 Present address: Cancer Research Unit, School of Biomedical Sciences and Pharmacy, University of Newcastle, NSW 2308, Australia. 3 Present address: The State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, China. 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.08.010

which it associates with other transmembrane receptors [6]. Indeed, the recent crystal structure of the soluble form of uPAR was solved at 2.7 Å in association with a competitive peptide inhibitor of the uPA–uPAR interaction [7]. The proposed model for the uPA/uPAR interaction revealed uPA bound to uPAR via a central cavity. This model left the external receptor surface free to bind and interact with other proteins [7]. In addition to the primary ligand uPA, a number of uPAR specific interactions have also been identified and are consistent with the varied roles regulated by uPAR including cell adhesion, cell migration, angiogenesis, apoptosis and cancer metastasis. Indeed, there is now evidence that uPAR can bind with the cell surface b1, b2, b3, b5 and b6 integrin subfamilies members [8–11]. Other associations and physical binding partners include chemotactic receptors [12], the epidermal growth factor receptor (EGFR) [13], a2-macroglobulin receptor/low density lipoprotein receptor-related protein [14], GP130 [15], the mannose-6-phosphate/insulin-like growth factor 2 receptor [16], and Mrj [17]. However, it is believed that there should be other unidentified proteins that interact with uPAR. The amino acid sequence of uPAR has three repeats, approximately 90 amino acids each, suggesting the existence of three homologous independently folded domains. These have been designated DI, DII and DIII from the N-terminal end [18]. These three domains are homologous to the Ly6/neurotoxin family and have two short linker regions between DI and DII, as well as, DII and DIII. Loop 3 of DI and in particular, the amino acid Arg53, Leu55, Tyr57 and Leu66 are important in the ligand binding of uPA [19,20]. Chymotrypsin can cleave uPAR in the linker region between DI and DII at residue 87 and uPA similarly cleaves at residue 84 to

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result in a cleaved form of uPAR [21]. There is also soluble uPAR (suPAR) that may be either the full-length receptor or a truncated form consisting of only DII and DIII [22]. In the present work, we have used yeast two-hybrid (Y2H) to identify novel interacting proteins by utilizing a number of alternate bait constructs based on uPAR. The screening of a breast cancer cDNA library identified hSpry1 and HAX1 proteins as novel candidate proteins that interact with uPAR. These novel protein–protein interactions were confirmed by GST-pull down, co-immunoprecipitation assays and confocal microscopy. 2. Material and methods 2.1. Materials, vectors and yeast strains The yeast vectors pGBD-B and pACT2-B and yeast strains PJ694A and PJ69-4a were kindly provided by Dr. David Markie (Dunedin School of Medicine, New Zealand). Dr. Dafna Bar-Sagi, (State University of New York at Stony Brook, Stony Brook, NY, USA), kindly provided the pCGN/hSpry1 vector. The pGEMÒ-3Zf (+)HAX1 vector was kindly provided by Dr. Maria Olsson (InsGöteborg University, Sahlgrenska University Hospital, Gothenburg, Sweden). Dr. Ying Wei (University of California, San Francisco, CA, USA) kindly provided the stable uPAR transfected human embryonic kidney (HEK293/uPAR) cells. The anti-uPAR antibodies were purchased from American Diagnostica (#3931, Stamford, CT, USA) and R&D Systems (#AF807, Minneapolis, MN, USA). The mouse monoclonal anti-uPAR antibodies R2 and R3 were kindly provided by Dr. Niels Behrendt, (Copenhagen, Denmark). The mouse antihuman hSpry1 (#AO1) was purchased from Abnova, Taiwan. The rabbit anti-human HAX1 antibody (#sc-28268) was purchased from Santa Cruz Biotechnology Inc., USA. 2.2. Cell culture, plasmid transfection The human breast cancer MDA-MB-231 and HEK293 cell lines were maintained in DMEM medium supplemented with 10% FCS, and 1% antibiotics. HEK293/uPAR cells were cultured in the presence of 0.9 mg/ml G418 (Geneticin). Transfections were performed using Lipofectamine2000 (Invitrogen, Carlsbad, CA, USA). 2.3. Expression of cDNA library construction and cloning of open reading frames of uPAR baits A human breast carcinoma cDNA library [23] was transformed on a large scale into PJ69-4a to construct a pre-transformed library of 4.7  107 independent yeast colonies. Five uPAR baits were designed based on uPAR full-length cDNA sequence and amplified by PCR using gene-specific primers containing short sequence tags [24]. The resulting DNA fragments were co-transformed into yeast


host PJ69-4a with BamHI linearized pGBD-B vector DNA and plated with selection for the TRP1 gene. Individual colonies were tested by PCR. 2.4. Yeast two-hybrid screening A mating strategy described previously was used for the Y2H screening [24]. Briefly, library transformed PJ69-4a was grown in YPAD media. Bait plasmid transformed PJ69-4a yeast was grown overnight in a rich Trp-media (SD + C media supplemented with 100 lg/ml leucine, 20 lg/ml histidine, 20 lg/ml uracil and 32 lg/ ml adenine). A total of 5  108 yeast cells of bait and library were used per screen. In order to direct uPAR domain baits into the yeast nucleus (as both bait and prey proteins must interact in a nuclear environment), those peptides that act to signal uPAR to the cellular membrane and are involved in glycosyl phosphatidylinositol (GPI) linkages were omitted. To decrease the number of false positives, the resulting colonies were tested for three different reporter genes activation (HIS3, ADE2 and LacZ) under the control of different GAL4 promoters (Gal1, Gal2 and Gal7 respectively). Candidate proteins were analyzed by DNA sequencing. 2.5. In vitro transcription and translation and GST-pull down assay The TNTÒ T7 Quick coupled Rabbit Reticulocyte Transcription/ Translation System (Promega) was used to label hSpry1, HAX1 or Fibulin-2 with 35S-methionine. PCR reaction products for DI, DII, DIII, DIIDIII and DIDIIDIII uPAR were used to clone into the N-terminal glutathione S-transferase (GST) containing pDEST™15 plasmid using the GatewayÒ system (Invitrogen, Carlsbad, CA, USA). GST-tagged protein was expressed and purified in BL21-AI™ E. coli using standard protocol and purified over a 50% glutathione-agarose bead slurry. Equivalent amount of GST-tagged uPAR protein was incubated with 35S-methionine labeled proteins and washed with pull down buffer (150 mM NaCl, 20 mM Tris/HCl (pH 7.5), 0.1% NP-40, 2.5 mg/ml BSA, 0.01% 2-ME, 1.2 mM PMSF). Bound protein was eluted with 3X SDS loading buffer and separated by SDS–PAGE. The labeled bound protein was visualized by phosphor imager (Bio-Rad, Hercules, CA, USA) and analyzed using the QUANTITY ONE software (Bio-Rad, Hercules, CA, USA). 2.6. Co-immunoprecipitation (coIP) and immunoblotting Immunoprecipitation (IP) and immunoblotting were performed essentially as described previously [25]. Cells were lysed in IP buffer and then pre-cleared with protein G-agarose before undergoing IP with primary antibodies, and their isotype-matched IgG controls. The complex was collected and subjected to Western blotting. No DTT was added to samples to prevent co-migration of the heavy chain IgG with uPAR band.

Fig. 1. Schematic diagram of uPAR domain constructs used as baits in the yeast two-hybrid screen, compared to the full-length uPAR amino acid sequence, where numbers refer to the amino acid positions. The N-terminal signal peptide (S) and C-terminal pro-peptide (P) were omitted. The glycine residue at amino acid position 305 involved in GPI-anchor attachment was also omitted from the bait construction.


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2.7. Confocal microscopy Cells were seeded onto sterilized glass cover slips, washed with phosphate-buffered saline (PBS) and fixed with 0.5% formaldehyde/PBS/ 0.1% sodium azide for 1 h. Cells were then washed and incubated in 70% ethanol for 1 h at 4 °C, washed with PBS, blocked with 1% BSA, incubated with primary antibodies in 1% BSA followed by Rhodamine-conjugated and FITC-conjugated secondary antibodies in 1% BSA. Cells were analyzed for protein localization using Olympus IX71 laser scanning microscopy with 60 oil immersion lens. 3. Results

231 cells. For hSpry1, the deglycosylated uPAR band (35 kDa) was observed in samples immunoprecipitated with anti-hSpry1 and probed with anti-uPAR antibodies. In a separate membrane, two hSpry1 protein bands were detected after protein IP with anti-uPAR and probing with hSpry1 antibodies. Total cell lysate (TLC) showed the presence of corresponding uPAR and hSpry1 protein bands (Fig. 3A). uPAR and hSpry1 bands were also weakly detected in the respective IgG isotype control lanes. For HAX1, the anti-HAX1 antibody again co-precipitated with deglycosylated form of uPAR protein (35 kDa). This coIP was reciprocated when uPAR was precipitated and blotted for cell-endogenous HAX1 (Fig. 3B). Taken together, these results suggest that at least in MDA-MB-231 cells, uPAR can interact directly with endogenous HAX1 and hSpry1.

3.1. Yeast two-hybrid screening In this study, five different uPAR baits, full-length uPAR-DIDIIDIII, uPAR-DIIDIII, uPAR-DIII, uPAR-DII and uPAR-DI were used in the Y2H screening of a breast carcinoma cDNA library of novel candidate proteins that may interact with uPAR (Fig. 1). All prey clones sequences passed two rounds of screening in an attempt to minimize clonal specific false positives. Positive clones were sequenced and identified using BLASTN ( ) (Table 1). For uPAR-DIIDIII, a prey clone was sequenced and identified and found to encode Homo sapiens novel antagonist of fibroblast growth factor (FGF) signaling (hSpry1) (gi:2827283). For uPAR-DI, a colony passed the stringent screening method and found to encode HS1 binding protein (HAX1) (gi:13435355). These two candidate proteins, hSpry1 and HAX1 were pursued for further evidence of binding. 3.2. uPAR interacts with hSpry1 and HAX1 in GST-pull down assays To verify whether hSpry1 or HAX1 could bind uPAR, an in vitro GST-pull down approach was employed. For hSpry1, alternate GSTtagged uPAR constructs were incubated with 35S-methionine labeled hSpry1. Under the binding conditions used, hSpry1 bound GST-uPAR-DIDIIDIII and GST-uPAR-DIIDIII constructs, while GSTuPAR-DI bound hSpry1 at half the affinity. There was no discernible binding between hSpry1 and GST alone. For the final prey, 35Smethionine labeled HAX1 was again incubated with the three different GST-uPAR proteins as above. There was strong binding to GST-uPAR-DIDIIDIII, GST-uPAR-DI (Fig. 2B), significantly less binding to GST-uPAR-DIIDIII, and again no binding to GST alone. These results showed a preference of HAX1 for uPAR-DIDIIDIII and in particular uPAR-DI reconciles with the original Y2H whereby DI of uPAR appears to be essential in the interaction between uPAR and HAX1. 3.3. Co-immunoprecipitation and co-localization of uPAR with hSpry1 and HAX1 To determine whether endogenous hSpry1 or HAX1 and uPAR interact in cells, coIP analysis was carried out using MDA-MB-

Fig. 2. uPAR interacts with hSpry1 and HAX1 in GST-pull down assays. (A) 35Smethionine labeled hSpry1 protein was incubated with three GST-uPAR domain constructs GST-uPAR-DIDIIDIII, GST-uPAR-DIIDIII and GST-uPAR-DI in addition to GST as control. Densitometric analysis shows binding as a percentage of GST-uPARDIIDIII normalized for GST-tagged protein. The strongest binding was for GSTuPAR-DIDII (100%) and GST-uPAR-DIDIIDIII (91%) followed by GST-uPAR-DI (55%). (B) GST-pull down resulted for radiolabeled full-length HAX1 protein, with the strongest binding for GST-uPAR-D1 (100%) and GST-uPAR-DIDIIDIII (80%) followed by GST-uPAR-DIIDIII (38%). Densitometric analysis showing binding as a percentage of GST-uPAR-DI normalized for the amount of GST-tagged protein.

Table 1 Identification of uPAR-DIIDIII and uPAR-DI interacting prey clones using the Y2H assay and the DNA sequencing. Blast result of nucleotide sequence

uPAR baits

Number of times pulled out

Homo sapiens novel antagonist of FGF signaling (hSpry1) gi:2827283



Homo sapiens HS1 binding protein (HAX1) gi:13435355


Diagrammatic representation of the protein identified and the sequence from the rescued prey (black bar) Sprouty Domain 1




HS1 Binding Domain 1



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Fig. 3. uPAR is able to co-immunoprecipitate and co-localize with hSpry1 and HAX1. (A) Western blot probed with anti-uPAR antibody (upper panel). uPAR protein band (35 kDa) was shown after IP with anti-hSpry1, but not with their antibody isotype controls. Western blot probed with anti-hSpry1 antibody (lower panel). The hSpry1 protein was shown after IP with anti-uPAR, but not with their antibody isotype controls. (B) Western blot probed with anti-uPAR antibody (upper panel). uPAR protein band (35 kDa) was shown after IP with anti-HAX1, but not with their antibody isotype controls. Western blot probed with anti-HAX1 antibody (lower panel). The HAX1 protein was shown after IP with anti-uPAR, but not with their antibody isotype controls. The cell lysate (40 lg) of MDA-MB-231 cells are shown as a control. (C) Co-localization of uPAR and hSpry1 in MDA-MB-231 (a) and HEK293/uPAR (b) cells transfected with pCGN/hSpry1. Cells were fixed then immunostained with antibodies against uPAR (red) and hSpry1 (green), co-localization appeared as yellow color. (D) Co-localization of uPAR and HAX1 in MDA-MB-231 and HEK293/uPAR cells. Low level of endogenous HAX1 in MDA-MB231 cells (a) and relatively high level of HAX1 in MDA-MB-231 (b) and HEK293/uPAR cells (c) cells transfected with pGEMÒ-3Zf(+)-HAX1. Cells were fixed then immunostained with antibodies against uPAR (red) and HAX1 (green). Co-localization of HAX1 with uPAR was shown in both cell lines (yellow). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

To determine whether hSpry1 and HAX1 co-localize with uPAR in HEK293/uPAR and MDA-MB-231 cells, we used immunofluorescent confocal microscopy. For hSpry1 and consistent with other reports, hSpry1 proteins localized predominantly within cytosolic vesicles and a smaller subset translocated to membrane ruffles when cultured in growth media. However, uPAR was predominantly located as extracellular receptor on the cell membrane and to a lesser extent in the vesicles. hSpry1 and uPAR proteins were found to co-localize together in the vesicles of actively proliferating cells (Fig. 3C). To visualize the cellular localization of HAX1 and uPAR, both endogenous expression and HAX1 plasmid transfected MDA-MB-231 and HEK293/uPAR cells were used for confocal microscopy analysis. For endogenous expression, HAX1 showed only low expression throughout the MDA-MD-231 cell cytoplasm and there were no detectable HAX1 in HEK293/uPAR cell line (data not shown). Significant co-localization of HAX1 and uPAR occurred within the cytoplasm at both transfected HEK293/uPAR and MDAMB-231 cells (yellow color) (Fig. 3D). These results demonstrate that HAX1 associates with uPAR predominantly within the cytoplasm. 4. Discussion The Y2H system has been successfully used here to identify a number of putative proteins that interact with different domain

forms of uPAR, in particular for uPAR-DIDIIDIII, uPAR-DIIDIII and uPAR-DI. Of the putative candidates identified, hSpry1 and HAX1 were further investigated. The Sprouty family are negative regulators of receptor tyrosine kinases (RTK), by influencing their duration and intensity of signaling [26]. The diverse role of the Sprouty family has recently been reviewed [27]. Briefly, the mammalian Spry1 protein has been found predominantly in the perinuclear regions and in cytoplasmic vesicular structures of unstimulated human umbilical vein endothelial cells (HUVECs) [28]. However, after stimulation with growth factors, a fraction of the protein moves to the plasma membrane, mainly to the lamellipodia at the leading edge of cells, where it shows a partial overlap with the localization of caveolin-1 [28]. Interestingly, uPAR is also found to co-localize with caveolin [29,30]. In addition, it has been suggested that caveolin and uPAR may operate within adhesion sites to organize kinase rich lipid domains in proximity to integrins, promoting efficient signal transduction [31]. Hence, uPAR and hSpry1 may interact within this compartment. It has been found that both the tyrosine phosphorylation of Spry1 and the inhibition of RTK signaling by Spry1 occurs at the plasma membrane, suggesting that the association with caveolin-1 might enhance Spry1 function [32]. Clinically, Spry1 has been found to be down-regulated in breast cancer, and therefore hypothesized that in light of its putative role in the Ras/MAP kinase pathway, it could be deemed as a potential tumor suppressor gene [33]. Recently, in


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embryonal rhabdomyosarcoma harboring an oncogenic mutant RAS, hSpry1 can shift from an antagonist to an agonist and hSpry1 was essential for both cell proliferation and survival by activating ERK and MEK [34], a signaling pathway also affected by uPAR. The other protein–protein interaction investigated in this report was between uPAR and HAX1. HAX1 was first isolated in a Y2H screen using HS1, a substrate of the Src family tyrosine kinases, as bait [35]. The distribution of HAX1 is found on the outer mitochondrial membrane, the endoplasmic reticulum and the nuclear envelope [36]. HAX1 is implicated in cell migration, apoptosis signaling, and mRNA surveillance [37]. uPAR and uPA have been detected in the cell membrane and cytoplasm of cellular components in normal and neoplastic tissues [38–41]. Localization of uPA and uPAR in the cell membrane and cytoplasm of tumor cells has been linked to tumor progression [41]. Furthermore, uPAR has been found to localize in the cytoplasm after endocytosis of uPA/uPAR complexes [42]. Recently, HAX1 has been found to be over expressed in breast cancer, lung cancer and melanoma although the exact molecular mechanism by which over expression of HAX1 may provide an oncogenic role needs to be further evaluated and we propose that one of these may indeed be via its interaction with uPAR. In summary, it is important to identify the proteins that bind to uPAR in order to fully understand the precise role of uPAR in its myriad of roles. Here, we have identified a number of potential protein interactions using yeast two-hybrid system and different uPAR constructs. We have also confirmed the interaction of uPAR with two of these proteins which are hSpry1 and HAX1 using different assays. The functional consequences of uPAR/hSpry1 and uPAR/HAX1 interactions are currently under investigation in our laboratories. Acknowledgments We acknowledge the Ph.D. scholarships to A.H. Mekkawy from the Ministry of Higher Education, Egypt and to C.E. De Bock from the Foundation for Research Science and Technology, New Zealand. References [1] A.H. Mekkawy, D.L. Morris, M.H. Pourgholami, Urokinase plasminogen activator system as a potential target for cancer therapy, Future Oncol. 5 (2009) 1487–1499. [2] K.T. Preissner, S.M. Kanse, A.E. May, Urokinase receptor: a molecular organizer in cellular communication, Curr. Opin. Cell Biol. 12 (2000) 621–628. [3] S.M. Kanse, C. Kost, O.G. Wilhelm, P.A. Andreasen, K.T. Preissner, The urokinase receptor is a major vitronectin-binding protein on endothelial cells, Exp. Cell Res. 224 (1996) 344–353. [4] R.W. Colman, Role of the light chain of high molecular weight kininogen in adhesion, cell-associated proteolysis and angiogenesis, Biol. Chem. 382 (2001) 65–70. [5] M. Ploug, E. Ronne, N. Behrendt, A.L. Jensen, F. Blasi, K. Danø, Cellular receptor for urokinase plasminogen activator. Carboxyl-terminal processing and membrane anchoring by glycosyl-phosphatidylinositol, J. Biol. Chem. 266 (1991) 1926–1933. [6] H.T. Myöhänen, R.W. Stephens, K. Hedman, H. Tapiovaara, E. Rønne, G. HøyerHansen, K. Danø, A. Vaheri, Distribution and lateral mobility of the urokinasereceptor complex at the cell surface, J. Histochem. Cytochem. 41 (1993) 1291– 1301. [7] P. Llinas, M.H. Le Du, H. Gårdsvoll, K. Danø, M. Ploug, B. Gilquin, E.A. Stura, A. Ménez, Crystal structure of the human urokinase plasminogen activator receptor bound to an antagonist peptide, EMBO J. 24 (2005) 1655–1663. [8] M.V. Carriero, S. Del Vecchio, M. Capozzoli, P. Franco, L. Fontana, A. Zannetti, G. Botti, G. D’Aiuto, M. Salvatore, M.P. Stoppelli, Urokinase receptor interacts with avb5 vitronectin receptor, promoting urokinase-dependent cell migration in breast cancer, Cancer Res. 59 (1999) 5307–5314. [9] A.E. May, F.-J. Neumann, A. Schömig, K.T. Preissner, VLA-4 (a4b1) engagement defines a novel activation pathway for b2 integrin-dependent leukocyte adhesion involving the urokinase receptor, Blood 96 (2000) 506–513. [10] N. Dalvi, G.J. Thomas, J.F. Marshall, M. Morgan, R. Bass, V. Ellis, P.M. Speight, S.A. Whawell, Modulation of the urokinase-type plasminogen activator receptor by the beta6 integrin subunit, Biochem. Biophys. Res. Commun. 317 (2004) 92–99.

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