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A Flexible Multidomain Structure Drives the Function of the Urokinase-type Plasminogen Activator Receptor (uPAR)*

  • Haydyn D.T. Mertens
    Footnotes
    Affiliations
    European Molecular Biology Laboratory (EMBL)-Hamburg Outstation, c/o DESY, Notkestrasse 85, 22603 Hamburg, Germany
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  • Magnus Kjaergaard
    Footnotes
    Affiliations
    Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark
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  • Simon Mysling
    Affiliations
    Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
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  • Henrik Gårdsvoll
    Affiliations
    Finsen Laboratory, Rigshospitalet and Biotech Research and Innovation Centre (BRIC), Copenhagen Biocenter, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark
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  • Thomas J.D. Jørgensen
    Affiliations
    Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
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  • Dmitri I. Svergun
    Affiliations
    European Molecular Biology Laboratory (EMBL)-Hamburg Outstation, c/o DESY, Notkestrasse 85, 22603 Hamburg, Germany
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  • Michael Ploug
    Correspondence
    To whom correspondence should be addressed. Tel.: 45-35456037; Fax: 45-35453797
    Affiliations
    Finsen Laboratory, Rigshospitalet and Biotech Research and Innovation Centre (BRIC), Copenhagen Biocenter, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark
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  • Author Footnotes
    * This work was supported by a fellowship from the EMBL Interdisciplinary Postdocs program (EIPOD) (to H. D. T. M.) and a grant from the Danish National Research Foundation (Danish-Chinese Centre for Proteases and Cancer) (to M. P. and H. G.).
    This article contains supplemental Materials and Methods, Table S1, and Figs. S1–S3.
    1 Both authors contributed equally to this work.
Open AccessPublished:August 15, 2012DOI:https://doi.org/10.1074/jbc.M112.398404
      The urokinase-type plasminogen activator receptor (uPAR) provides a rendezvous between proteolytic degradation of the extracellular matrix and integrin-mediated adhesion to vitronectin. These processes are, however, tightly linked because the high affinity binding of urokinase regulates the binding of uPAR to matrix-embedded vitronectin. Although crystal structures exist to define the corresponding static bi- and trimolecular receptor complexes, it is evident that the dynamic property of uPAR plays a decisive role in its function. In the present study, we combine small angle x-ray scattering, hydrogen-deuterium exchange, and surface plasmon resonance to develop a structural model describing the allosteric regulation of uPAR. We show that the flexibility of its N-terminal domain provides the key for understanding this allosteric mechanism. Importantly, our model has direct implications for understanding uPAR-assisted cell adhesion and migration as well as for translational research, including targeted intervention therapy and non-invasive tumor imaging in vivo.

      Introduction

      The urokinase-type plasminogen activator receptor (uPAR)
      The abbreviations used are: uPAR
      uPA receptor
      uPA
      urokinase-type plasminogen activator
      ATF
      amino-terminal fragment of uPA
      GFD
      growth factor-like domain
      HDX
      hydrogen-deuterium exchange
      LU domains
      Ly6/uPAR-like domains
      SAXS
      small angle x-ray scattering
      SMB
      somatomedin B domain of vitronectin
      EOM
      ensemble optimization method.
      focuses uPA-mediated plasminogen activation to cell surfaces in vivo, which plays an important physiological role in, for example, hepatic fibrin clearance and liver homeostasis by attenuating fibrin-induced chronic inflammation (
      • Connolly B.M.
      • Choi E.Y.
      • Gårdsvoll H.
      • Bey A.L.
      • Currie B.M.
      • Chavakis T.
      • Liu S.
      • Molinolo A.
      • Ploug M.
      • Leppla S.H.
      • Bugge T.H.
      Selective abrogation of the uPA-uPAR interaction in vivo reveals a novel role in suppression of fibrin-associated inflammation.
      ). Several studies have also implicated uPAR in pathological conditions, such as focal segmental glomerulosclerosis (
      • Wei C.
      • Möller C.C.
      • Altintas M.M.
      • Li J.
      • Schwarz K.
      • Zacchigna S.
      • Xie L.
      • Henger A.
      • Schmid H.
      • Rastaldi M.P.
      • Cowan P.
      • Kretzler M.
      • Parrilla R.
      • Bendayan M.
      • Gupta V.
      • Nikolic B.
      • Kalluri R.
      • Carmeliet P.
      • Mundel P.
      • Reiser J.
      Modification of kidney barrier function by the urokinase receptor.
      ,
      • Wei C.
      • El Hindi S.
      • Li J.
      • Fornoni A.
      • Goes N.
      • Sageshima J.
      • Maiguel D.
      • Karumanchi S.A.
      • Yap H.K.
      • Saleem M.
      • Zhang Q.
      • Nikolic B.
      • Chaudhuri A.
      • Daftarian P.
      • Salido E.
      • Torres A.
      • Salifu M.
      • Sarwal M.M.
      • Schaefer F.
      • Morath C.
      • Schwenger V.
      • Zeier M.
      • Gupta V.
      • Roth D.
      • Rastaldi M.P.
      • Burke G.
      • Ruiz P.
      • Reiser J.
      Circulating urokinase receptor as a cause of focal segmental glomerulosclerosis.
      ), paroxysmal nocturnal hemoglobinuria (
      • Ploug M.
      • Plesner T.
      • Rønne E.
      • Ellis V.
      • Høyer-Hansen G.
      • Hansen N.E.
      • Danø K.
      The receptor for urokinase-type plasminogen activator is deficient on peripheral blood leukocytes in patients with paroxysmal nocturnal hemoglobinuria.
      ), rheumatoid arthritis (
      • Serratì S.
      • Margheri F.
      • Chillà A.
      • Neumann E.
      • Müller-Ladner U.
      • Benucci M.
      • Fibbi G.
      • Del Rosso M.
      Reduction of in vitro invasion and in vivo cartilage degradation in a SCID mouse model by loss of function of the fibrinolytic system of rheumatoid arthritis synovial fibroblasts.
      ), and cancer invasion and metastasis (
      • Stephens R.W.
      • Nielsen H.J.
      • Christensen I.J.
      • Thorlacius-Ussing O.
      • Sørensen S.
      • Danø K.
      • Brünner N.
      Plasma urokinase receptor levels in patients with colorectal cancer. Relationship to prognosis.
      ,
      • Heiss M.M.
      • Allgayer H.
      • Gruetzner K.U.
      • Funke I.
      • Babic R.
      • Jauch K.W.
      • Schildberg F.W.
      Individual development and uPA receptor expression of disseminated tumor cells.
      ,
      • Hu J.
      • Jo M.
      • Cavenee W.K.
      • Furnari F.
      • VandenBerg S.R.
      • Gonias S.L.
      Cross-talk between the urokinase-type plasminogen activator receptor and EGF receptor variant III supports survival and growth of glioblastoma cells.
      ). In the latter group of diseases, uPAR is predominantly expressed by cells residing in the tumor-stromal interface of the invasive front, and high levels of uPAR in either resected tumor tissue or circulating in blood are concordantly a general biomarker for poor prognosis in many types of human cancer (
      • Lund I.K.
      • Illemann M.
      • Thurison T.
      • Christensen I.J.
      • Høyer-Hansen G.
      uPAR as anti-cancer target. Evaluation of biomarker potential, histological localization, and antibody-based therapy.
      ). Following this line of evidence, several intervention strategies targeting uPAR have been developed, and some have shown promising therapeutic effects in preclinical mouse model systems (
      • Bauer T.W.
      • Liu W.
      • Fan F.
      • Camp E.R.
      • Yang A.
      • Somcio R.J.
      • Bucana C.D.
      • Callahan J.
      • Parry G.C.
      • Evans D.B.
      • Boyd D.D.
      • Mazar A.P.
      • Ellis L.M.
      Targeting of urokinase plasminogen activator receptor in human pancreatic cancer.
      ,
      • Kenny H.A.
      • Leonhardt P.
      • Ladanyi A.
      • Yamada S.D.
      • Montag A.
      • Im H.K.
      • Jagadeeswaran S.
      • Shaw D.E.
      • Mazar A.P.
      • Lengyel E.
      Targeting the urokinase plasminogen activator receptor inhibits ovarian cancer.
      ,
      • Gondi C.S.
      • Lakka S.S.
      • Dinh D.H.
      • Olivero W.C.
      • Gujrati M.
      • Rao J.S.
      RNAi-mediated inhibition of cathepsin B and uPAR leads to decreased cell invasion, angiogenesis and tumor growth in gliomas.
      ,
      • Kriegbaum M.C.
      • Persson M.
      • Haldager L.
      • Alpizar-Alpizar W.
      • Jacobsen B.
      • Gårdsvoll H.
      • Kjaer A.
      • Ploug M.
      Rational targeting of the urokinase receptor (uPAR). Development of antagonists and non-invasive imaging probes.
      ). To assist their further clinical translation, a non-invasive imaging modality for visualizing uPAR expression in vivo using positron emission tomography has been developed recently (
      • Kriegbaum M.C.
      • Persson M.
      • Haldager L.
      • Alpizar-Alpizar W.
      • Jacobsen B.
      • Gårdsvoll H.
      • Kjaer A.
      • Ploug M.
      Rational targeting of the urokinase receptor (uPAR). Development of antagonists and non-invasive imaging probes.
      ,
      • Persson M.
      • Madsen J.
      • Østergaard S.
      • Jensen M.M.
      • Jørgensen J.T.
      • Juhl K.
      • Lehmann C.
      • Ploug M.
      • Kjaer A.
      Quantitative PET of human urokinase-type plasminogen activator receptor with 64Cu-DOTA-AE105. Implications for visualizing cancer invasion.
      ,
      • Li Z.B.
      • Niu G.
      • Wang H.
      • He L.
      • Yang L.
      • Ploug M.
      • Chen X.
      Imaging of urokinase-type plasminogen activator receptor expression using a 64Cu-labeled linear peptide antagonist using microPET.
      ).
      One complicating factor in targeting uPAR is the dual function this receptor exerts on both degradation of and adhesion to the extracellular matrix (
      • Smith H.W.
      • Marshall C.J.
      Regulation of cell signaling by uPAR.
      ). These distinct functional properties are, however, interrelated because the low affinity binding between uPAR and the somatomedin B domain (SMB) of vitronectin (
      • Gårdsvoll H.
      • Ploug M.
      Mapping of the vitronectin-binding site on the urokinase receptor. Involvement of a coherent receptor interface consisting of residues from both domain I and the flanking interdomain linker region.
      ) is regulated by uPAR occupancy with its high affinity protease ligand uPA (
      • Waltz D.A.
      • Chapman H.A.
      Reversible cellular adhesion to vitronectin linked to urokinase receptor occupancy.
      ,
      • Gårdsvoll H.
      • Jacobsen B.
      • Kriegbaum M.C.
      • Behrendt N.
      • Engelholm L.
      • Østergaard S.
      • Ploug M.
      Conformational regulation of urokinase receptor function. Impact of receptor occupancy and epitope-mapped monoclonal antibodies on lamellipodia induction.
      ). This raised the unexpected conundrum that small molecules targeting the uPA binding site may unleash undesirable agonist effects by stimulating the uPAR·vitronectin interaction (
      • Kriegbaum M.C.
      • Persson M.
      • Haldager L.
      • Alpizar-Alpizar W.
      • Jacobsen B.
      • Gårdsvoll H.
      • Kjaer A.
      • Ploug M.
      Rational targeting of the urokinase receptor (uPAR). Development of antagonists and non-invasive imaging probes.
      ,
      • Gårdsvoll H.
      • Jacobsen B.
      • Kriegbaum M.C.
      • Behrendt N.
      • Engelholm L.
      • Østergaard S.
      • Ploug M.
      Conformational regulation of urokinase receptor function. Impact of receptor occupancy and epitope-mapped monoclonal antibodies on lamellipodia induction.
      ). This should be considered in future drug discovery programs aiming at controlling uPAR function by a given intervention therapy. The molecular mechanisms underpinning this phenomenon therefore define one of the most important challenges in our present understanding of the structure-function relationships of uPAR in cell biology and in pathophysiology.
      The ligand-binding part of the glycolipid-anchored uPAR (
      • Ploug M.
      • Rønne E.
      • Behrendt N.
      • Jensen A.L.
      • Blasi F.
      • Danø K.
      Cellular receptor for urokinase plasminogen activator. Carboxyl-terminal processing and membrane anchoring by glycosylphosphatidylinositol.
      ) comprises three homologous, three-fingered modules designated DI, DII, and DIII, which belong to the Ly6/uPAR/α-neurotoxin (LU) protein domain family as defined by four conserved disulfide bonds (
      • Galat A.
      The three-fingered protein domain of the human genome.
      ,
      • Ploug M.
      Structure-function relationships in the interaction between the urokinase-type plasminogen activator and its receptor.
      ). The structures of several uPAR complexes have been solved by x-ray crystallography, including those with a synthetic antagonist peptide (
      • Llinas P.
      • Le Du M.H.
      • Gårdsvoll H.
      • Danø K.
      • Ploug M.
      • Gilquin B.
      • Stura E.A.
      • Ménez A.
      Crystal structure of the human urokinase plasminogen activator receptor bound to an antagonist peptide.
      ), the amino-terminal fragment of uPA (ATF) (
      • Barinka C.
      • Parry G.
      • Callahan J.
      • Shaw D.E.
      • Kuo A.
      • Bdeir K.
      • Cines D.B.
      • Mazar A.
      • Lubkowski J.
      Structural basis of interaction between urokinase-type plasminogen activator and its receptor.
      ,
      • Huai Q.
      • Mazar A.P.
      • Kuo A.
      • Parry G.C.
      • Shaw D.E.
      • Callahan J.
      • Li Y.
      • Yuan C.
      • Bian C.
      • Chen L.
      • Furie B.
      • Furie B.C.
      • Cines D.B.
      • Huang M.
      Structure of human urokinase plasminogen activator in complex with its receptor.
      ,
      • Lin L.
      • Gårdsvoll H.
      • Huai Q.
      • Huang M.
      • Ploug M.
      Structure-based engineering of species selectivity in the interaction between urokinase and its receptor. Implication for preclinical cancer therapy.
      ), and the ternary complex with ATF and the SMB domain from vitronectin (
      • Huai Q.
      • Zhou A.
      • Lin L.
      • Mazar A.P.
      • Parry G.C.
      • Callahan J.
      • Shaw D.E.
      • Furie B.
      • Furie B.C.
      • Huang M.
      Crystal structures of two human vitronectin, urokinase and urokinase receptor complexes.
      ). In these structures, the β-hairpin of the growth factor-like domain (GFD) of uPA is buried deeply within a large hydrophobic cavity assembled by all three LU domains in uPAR (
      • Huai Q.
      • Mazar A.P.
      • Kuo A.
      • Parry G.C.
      • Shaw D.E.
      • Callahan J.
      • Li Y.
      • Yuan C.
      • Bian C.
      • Chen L.
      • Furie B.
      • Furie B.C.
      • Cines D.B.
      • Huang M.
      Structure of human urokinase plasminogen activator in complex with its receptor.
      ,
      • Lin L.
      • Gårdsvoll H.
      • Huai Q.
      • Huang M.
      • Ploug M.
      Structure-based engineering of species selectivity in the interaction between urokinase and its receptor. Implication for preclinical cancer therapy.
      ). In contrast, the SMB domain binds a small hydrophobic patch located at the interface between uPAR DI and DII (
      • Gårdsvoll H.
      • Ploug M.
      Mapping of the vitronectin-binding site on the urokinase receptor. Involvement of a coherent receptor interface consisting of residues from both domain I and the flanking interdomain linker region.
      ,
      • Huai Q.
      • Zhou A.
      • Lin L.
      • Mazar A.P.
      • Parry G.C.
      • Callahan J.
      • Shaw D.E.
      • Furie B.
      • Furie B.C.
      • Huang M.
      Crystal structures of two human vitronectin, urokinase and urokinase receptor complexes.
      ). We consider that the overall architectures of these two ligand binding sites on uPAR open the possibility that prior uPA binding may prime a subsequent SMB binding event by altering its composite interdomain binding site. Circumstantial cell biological (
      • Waltz D.A.
      • Chapman H.A.
      Reversible cellular adhesion to vitronectin linked to urokinase receptor occupancy.
      ,
      • Gårdsvoll H.
      • Jacobsen B.
      • Kriegbaum M.C.
      • Behrendt N.
      • Engelholm L.
      • Østergaard S.
      • Ploug M.
      Conformational regulation of urokinase receptor function. Impact of receptor occupancy and epitope-mapped monoclonal antibodies on lamellipodia induction.
      ,
      • Madsen C.D.
      • Ferraris G.M.
      • Andolfo A.
      • Cunningham O.
      • Sidenius N.
      uPAR-induced cell adhesion and migration. Vitronectin provides the key.
      ) and biochemical (
      • Gårdsvoll H.
      • Ploug M.
      Mapping of the vitronectin-binding site on the urokinase receptor. Involvement of a coherent receptor interface consisting of residues from both domain I and the flanking interdomain linker region.
      ) evidence exists to favor this scenario, including our design of a constitutively active receptor mutant (uPARH47C/N259C), where DI is tethered to DIII via an interdomain disulfide located distant from to the SMB binding site (
      • Gårdsvoll H.
      • Kjaergaard M.
      • Jacobsen B.
      • Kriegbaum M.C.
      • Huang M.
      • Ploug M.
      Mimicry of the regulatory role of urokinase in lamellipodia formation by introduction of a non-native interdomain disulfide bond in its receptor.
      ). This constrained uPAR mutant promotes lamellipodia formation on vitronectin-rich matrices in the absence of uPA (
      • Gårdsvoll H.
      • Kjaergaard M.
      • Jacobsen B.
      • Kriegbaum M.C.
      • Huang M.
      • Ploug M.
      Mimicry of the regulatory role of urokinase in lamellipodia formation by introduction of a non-native interdomain disulfide bond in its receptor.
      ), which suggests that a regulatory structural transition occurs at the SMB binding site when uPA binds.
      The unoccupied receptor has so far evaded structural determination despite prolonged efforts aimed at crystallizing it. Only recently has the structure of a constitutively active receptor mutant (uPARH47C/N259C) (
      • Gårdsvoll H.
      • Kjaergaard M.
      • Jacobsen B.
      • Kriegbaum M.C.
      • Huang M.
      • Ploug M.
      Mimicry of the regulatory role of urokinase in lamellipodia formation by introduction of a non-native interdomain disulfide bond in its receptor.
      ) been solved by x-ray crystallography in the absence of bound ligands (
      • Xu X.
      • Gårdsvoll H.
      • Yuan C.
      • Lin L.
      • Ploug M.
      • Huang M.
      Crystal structure of the urokinase receptor in a ligand-free form.
      ). As alluded to above, this structure is, nevertheless, unlikely to represent the prevalent conformation populated by unoccupied uPARwt because the constraints we engineered into uPARH47C/N259C render it a structural and functional surrogate for the receptor conformation selected in the uPA·uPAR complex (
      • Gårdsvoll H.
      • Kjaergaard M.
      • Jacobsen B.
      • Kriegbaum M.C.
      • Huang M.
      • Ploug M.
      Mimicry of the regulatory role of urokinase in lamellipodia formation by introduction of a non-native interdomain disulfide bond in its receptor.
      ,
      • Xu X.
      • Gårdsvoll H.
      • Yuan C.
      • Lin L.
      • Ploug M.
      • Huang M.
      Crystal structure of the urokinase receptor in a ligand-free form.
      ).
      To better understand the structural transition(s) regulating the interaction between uPAR and vitronectin, we therefore in this study embark on a more dynamic approach by probing the conformation(s) of the ligand-free state of uPAR with small angle x-ray scattering (SAXS) and hydrogen-deuterium exchange (HDX). These data are then integrated with functional data obtained by surface plasmon resonance. We find that ligand-free uPARwt is highly extended in solution, where uPAR DI is markedly flexible compared with the rest of the protein. This open structure presents a suboptimal vitronectin binding site and explains how uPA regulates the affinity for vitronectin without sharing a direct contact surface. Trapping uPAR in the open state therefore provides a new avenue for therapeutic inhibition of the proteolytic activity of the plasminogen activation system without stimulating cellular migration.

      DISCUSSION

      The role of uPAR in cell surface-associated proteolysis and adhesion is increasingly well described in molecular terms due to extensive biochemical studies (
      • Gårdsvoll H.
      • Jacobsen B.
      • Kriegbaum M.C.
      • Behrendt N.
      • Engelholm L.
      • Østergaard S.
      • Ploug M.
      Conformational regulation of urokinase receptor function. Impact of receptor occupancy and epitope-mapped monoclonal antibodies on lamellipodia induction.
      ,
      • Gårdsvoll H.
      • Kjaergaard M.
      • Jacobsen B.
      • Kriegbaum M.C.
      • Huang M.
      • Ploug M.
      Mimicry of the regulatory role of urokinase in lamellipodia formation by introduction of a non-native interdomain disulfide bond in its receptor.
      ,
      • Gårdsvoll H.
      • Gilquin B.
      • Le Du M.H.
      • Ménèz A.
      • Jørgensen T.J.
      • Ploug M.
      Characterization of the functional epitope on the urokinase receptor. Complete alanine scanning mutagenesis supplemented by chemical cross-linking.
      ,
      • Ellis V.
      Functional analysis of the cellular receptor for urokinase in plasminogen activation. Receptor binding has no influence on the zymogenic nature of pro-urokinase.
      ) and several published crystal structures of ligand-bound states of the receptor (
      • Llinas P.
      • Le Du M.H.
      • Gårdsvoll H.
      • Danø K.
      • Ploug M.
      • Gilquin B.
      • Stura E.A.
      • Ménez A.
      Crystal structure of the human urokinase plasminogen activator receptor bound to an antagonist peptide.
      ,
      • Barinka C.
      • Parry G.
      • Callahan J.
      • Shaw D.E.
      • Kuo A.
      • Bdeir K.
      • Cines D.B.
      • Mazar A.
      • Lubkowski J.
      Structural basis of interaction between urokinase-type plasminogen activator and its receptor.
      ,
      • Huai Q.
      • Mazar A.P.
      • Kuo A.
      • Parry G.C.
      • Shaw D.E.
      • Callahan J.
      • Li Y.
      • Yuan C.
      • Bian C.
      • Chen L.
      • Furie B.
      • Furie B.C.
      • Cines D.B.
      • Huang M.
      Structure of human urokinase plasminogen activator in complex with its receptor.
      ,
      • Lin L.
      • Gårdsvoll H.
      • Huai Q.
      • Huang M.
      • Ploug M.
      Structure-based engineering of species selectivity in the interaction between urokinase and its receptor. Implication for preclinical cancer therapy.
      ,
      • Huai Q.
      • Zhou A.
      • Lin L.
      • Mazar A.P.
      • Parry G.C.
      • Callahan J.
      • Shaw D.E.
      • Furie B.
      • Furie B.C.
      • Huang M.
      Crystal structures of two human vitronectin, urokinase and urokinase receptor complexes.
      ,
      • Xu X.
      • Gårdsvoll H.
      • Yuan C.
      • Lin L.
      • Ploug M.
      • Huang M.
      Crystal structure of the urokinase receptor in a ligand-free form.
      ). Important challenges, nevertheless, still remain to be solved. These concern the structure of the ligand-free state of uPAR, which is essentially undefined, and the molecular basis underlying the allosteric regulation of the protein. Our current study reveals these two questions to be closely interrelated. Because traditional methods for high resolution structure determination for various reasons have failed for unoccupied uPARwt, we have in the present study adopted an untraditional approach to define the conformation(s) of uPARwt in solution by combining SAXS with hydrogen-deuterium exchange monitored by mass spectrometry. These two technologies are essentially complementary because SAXS data provide information on the overall molecular shapes and domain arrangements, whereas the HDX data allow us to assess solvent accessibility of the backbone amides. Comparison of HDX patterns of several states of uPAR allows us to discriminate between different models consistent with the SAXS data, thus buttressing the conclusions from molecular modeling. The present combination of SAXS and HDX may therefore represent a generally applicable strategy for defining the structures of multidomain proteins that fail to crystallize due to dynamics.
      It is becoming increasingly evident that non-proteolytic roles of uPAR in cell migration and adhesion (
      • Madsen C.D.
      • Ferraris G.M.
      • Andolfo A.
      • Cunningham O.
      • Sidenius N.
      uPAR-induced cell adhesion and migration. Vitronectin provides the key.
      ,
      • Fazioli F.
      • Resnati M.
      • Sidenius N.
      • Higashimoto Y.
      • Appella E.
      • Blasi F.
      A urokinase-sensitive region of the human urokinase receptor is responsible for its chemotatic activity.
      ,
      • Resnati M.
      • Pallavicini I.
      • Wang J.M.
      • Oppenheim J.
      • Serhan C.N.
      • Romano M.
      • Blasi F.
      The fibrinolytic receptor for urokinase activates the G protein-coupled chemotactic receptor FPRL1/LXA4R.
      ,
      • Wei Y.
      • Eble J.A.
      • Wang Z.
      • Kreidberg J.A.
      • Chapman H.A.
      Urokinase receptors promote β1 integrin function through interactions with integrin α3β1.
      ) to a large extent are regulated by receptor occupancy with uPA (
      • Smith H.W.
      • Marshall C.J.
      Regulation of cell signaling by uPAR.
      ,
      • Waltz D.A.
      • Chapman H.A.
      Reversible cellular adhesion to vitronectin linked to urokinase receptor occupancy.
      ,
      • Gårdsvoll H.
      • Jacobsen B.
      • Kriegbaum M.C.
      • Behrendt N.
      • Engelholm L.
      • Østergaard S.
      • Ploug M.
      Conformational regulation of urokinase receptor function. Impact of receptor occupancy and epitope-mapped monoclonal antibodies on lamellipodia induction.
      ). Recently, we showed that constraining uPAR to a structure resembling the one selected by complex formation with uPA (
      • Xu X.
      • Gårdsvoll H.
      • Yuan C.
      • Lin L.
      • Ploug M.
      • Huang M.
      Crystal structure of the urokinase receptor in a ligand-free form.
      ) has the same effect on adhesion and lamellipodia formation on vitronectin as uPA-binding (
      • Gårdsvoll H.
      • Kjaergaard M.
      • Jacobsen B.
      • Kriegbaum M.C.
      • Huang M.
      • Ploug M.
      Mimicry of the regulatory role of urokinase in lamellipodia formation by introduction of a non-native interdomain disulfide bond in its receptor.
      ). This demonstrates that it is the structural transition induced in uPAR per se and not direct contributions from the bound uPA that drives this regulatory potential. In this study, we show that DI of uPAR is flexible relative to the rest of the receptor. Upon uPA binding, our data suggest that DI docks onto DII, and this consequently enables an allosteric regulation of the affinity for secondary ligands engaging the interface between these domains, as illustrated by vitronectin binding (
      • Gårdsvoll H.
      • Ploug M.
      Mapping of the vitronectin-binding site on the urokinase receptor. Involvement of a coherent receptor interface consisting of residues from both domain I and the flanking interdomain linker region.
      ,
      • Huai Q.
      • Zhou A.
      • Lin L.
      • Mazar A.P.
      • Parry G.C.
      • Callahan J.
      • Shaw D.E.
      • Furie B.
      • Furie B.C.
      • Huang M.
      Crystal structures of two human vitronectin, urokinase and urokinase receptor complexes.
      ). Beyond a mere docking event, our HDX data also imply that uPA binding is accompanied by a stabilization of the three-finger fold, suggesting that DI in unoccupied uPAR exists in a partially unfolded state. Such local unfolding is not likely to be detectable by SAXS because this technique is most sensitive to the overall shape properties of the molecule. Ligand-induced folding of DI is furthermore concordant with our previous observation demonstrating that GFD binding protects the N-linked glycan on Asn52 against N-glycanase cleavage (
      • Ploug M.
      • Rahbek-Nielsen H.
      • Nielsen P.F.
      • Roepstorff P.
      • Dano K.
      Glycosylation profile of a recombinant urokinase-type plasminogen activator receptor expressed in Chinese hamster ovary cells.
      ). This glycosylation is confined to the outer convex surface of the central β-sheet in DI (βIE) and is thus not facing the central uPA binding cavity, as illustrated in Fig. 2d. In contrast, there seems to be little ligand-induced structural change in the orientation/stability of DII and DIII as disclosed by their almost invariant HDX patterns. Although a plethora of ligands have been proposed for uPAR, uPA binding will most likely only influence those binding sites that localize to DI and the interface between DI and DII in particular. uPAR is encoded by a small gene cluster containing at least six homologous proteins containing 2–4 LU-domains (
      • Kriegbaum M.C.
      • Persson M.
      • Haldager L.
      • Alpizar-Alpizar W.
      • Jacobsen B.
      • Gårdsvoll H.
      • Kjaer A.
      • Ploug M.
      Rational targeting of the urokinase receptor (uPAR). Development of antagonists and non-invasive imaging probes.
      ,
      • Galat A.
      The three-fingered protein domain of the human genome.
      ). Interestingly, these multidomain homologs all possess an atypical DI, which is lacking one particular consensus disulfide that is essential for the stability of the corresponding single domain proteins. From an evolutionary perspective, this suggests that the multidomain structure precedes gene duplication within this gene locus and that the allosteric switch between DI and DII could potentially be shared among the family members.
      Protein binding reactions that are coupled to a large structural transition generally follow one of two mechanisms, conformational selection or induced fit, depending on whether the structural changes take place before or after protein binding (
      • Boehr D.D.
      • Nussinov R.
      • Wright P.E.
      The role of dynamic conformational ensembles in biomolecular recognition.
      ). Site-directed mutagenesis studies show that a large proportion of the thermodynamic driving force for the uPA·uPAR complex formation is provided by the defined interaction between GFD and uPAR DI (
      • Lin L.
      • Gårdsvoll H.
      • Huai Q.
      • Huang M.
      • Ploug M.
      Structure-based engineering of species selectivity in the interaction between urokinase and its receptor. Implication for preclinical cancer therapy.
      ,
      • Gårdsvoll H.
      • Gilquin B.
      • Le Du M.H.
      • Ménèz A.
      • Jørgensen T.J.
      • Ploug M.
      Characterization of the functional epitope on the urokinase receptor. Complete alanine scanning mutagenesis supplemented by chemical cross-linking.
      ). Accordingly, the uPA derivatives GFD and ATF do bind, albeit weakly, to isolated uPAR DI as opposed to DIIDIII, where no specific binding is detected. Given that DI is detached from the rest of the protein in the unoccupied receptor, it is likely that DI acts as bait, forming a transient intermediate complex with GFD. After the association reaction, the receptor closes and forms the final complex in a mechanism resembling induced fit. The constrained uPARH47C/N259C mutant nonetheless binds uPA with kinetics similar to those of uPARwt, although it is trapped in the closed conformation and hence is forced to bind by a “conformational selection”-dominated mechanism (
      • Gårdsvoll H.
      • Kjaergaard M.
      • Jacobsen B.
      • Kriegbaum M.C.
      • Huang M.
      • Ploug M.
      Mimicry of the regulatory role of urokinase in lamellipodia formation by introduction of a non-native interdomain disulfide bond in its receptor.
      ). This demonstrates that the binding mechanism between uPA and uPAR is plastic and can proceed through both routes. This is in keeping with recent suggestions that the two schematic mechanisms are only extreme cases and that most binding reactions proceed as a composite of the two (
      • Hammes G.G.
      • Chang Y.C.
      • Oas T.G.
      Conformational selection or induced fit. A flux description of reaction mechanism.
      ).
      The involvement of uPAR in cancer invasion and metastasis has prompted several academic institutions and pharmaceutical companies to embark on drug development programs aimed at targeting and controlling uPAR function (
      • Ploug M.
      • Østergaard S.
      • Gårdsvoll H.
      • Kovalski K.
      • Holst-Hansen C.
      • Holm A.
      • Ossowski L.
      • Danø K.
      Peptide-derived antagonists of the urokinase receptor. Affinity maturation by combinatorial chemistry, identification of functional epitopes, and inhibitory effect on cancer cell intravasation.
      ,
      • Schmiedeberg N.
      • Schmitt M.
      • Rölz C.
      • Truffault V.
      • Sukopp M.
      • Bürgle M.
      • Wilhelm O.G.
      • Schmalix W.
      • Magdolen V.
      • Kessler H.
      Synthesis, solution structure, and biological evaluation of urokinase type plasminogen activator (uPA)-derived receptor binding domain mimetics.
      ,
      • Mazar A.P.
      • Ahn R.W.
      • O'Halloran T.V.
      Development of novel therapeutics targeting the urokinase plasminogen activator.
      ,
      • Khanna M.
      • Wang F.
      • Jo I.
      • Knabe W.E.
      • Wilson S.M.
      • Li L.
      • Bum-Erdene K.
      • Li J.
      • W Sledge G.
      • Khanna R.
      • Meroueh S.O.
      Targeting multiple conformations leads to small molecule inhibitors of the uPAR·uPA protein-protein interaction that block cancer cell invasion.
      ). The majority of the small molecules developed so far target the central uPAR cavity to antagonize uPA binding. The Achilles heel of this approach is, nevertheless, that these inhibitors may induce and stabilize the closed form of the receptor and thus stimulate non-proteolytic functions of uPAR (
      • Gårdsvoll H.
      • Jacobsen B.
      • Kriegbaum M.C.
      • Behrendt N.
      • Engelholm L.
      • Østergaard S.
      • Ploug M.
      Conformational regulation of urokinase receptor function. Impact of receptor occupancy and epitope-mapped monoclonal antibodies on lamellipodia induction.
      ,
      • Gårdsvoll H.
      • Kjaergaard M.
      • Jacobsen B.
      • Kriegbaum M.C.
      • Huang M.
      • Ploug M.
      Mimicry of the regulatory role of urokinase in lamellipodia formation by introduction of a non-native interdomain disulfide bond in its receptor.
      ). Alternative strategies to circumvent this agonistic effect would be to either target both sites simultaneously or to stabilize the open unoccupied state of the receptor. Recently, a number of monoclonal anti-uPAR antibodies sharing epitopes located in the proximity of the DI-DIII interface were found to prevent both uPA and vitronectin binding, presumably because the bulkiness of the bound antibodies impedes closure of the receptor by steric hindrance (
      • Gårdsvoll H.
      • Jacobsen B.
      • Kriegbaum M.C.
      • Behrendt N.
      • Engelholm L.
      • Østergaard S.
      • Ploug M.
      Conformational regulation of urokinase receptor function. Impact of receptor occupancy and epitope-mapped monoclonal antibodies on lamellipodia induction.
      ,
      • Gårdsvoll H.
      • Kjaergaard M.
      • Jacobsen B.
      • Kriegbaum M.C.
      • Huang M.
      • Ploug M.
      Mimicry of the regulatory role of urokinase in lamellipodia formation by introduction of a non-native interdomain disulfide bond in its receptor.
      ). This mode of action is particularly evident for the inhibitory anti-uPAR mAb H2 (
      • Kroon M.E.
      • Koolwijk P.
      • van Goor H.
      • Weidle U.H.
      • Collen A.
      • van der Pluijm G.
      • van Hinsbergh V.W.
      Role and localization of urokinase receptor in the formation of new microvascular structures in fibrin matrices.
      ), which has Asn-259 in uPAR DIII as its primary hot spot (
      • Gårdsvoll H.
      • Kjaergaard M.
      • Jacobsen B.
      • Kriegbaum M.C.
      • Huang M.
      • Ploug M.
      Mimicry of the regulatory role of urokinase in lamellipodia formation by introduction of a non-native interdomain disulfide bond in its receptor.
      ). In the uPA·uPAR complexes, this residue stabilizes the DI-DIII interface by hydrogen bonding to His-47 in DI (
      • Huai Q.
      • Mazar A.P.
      • Kuo A.
      • Parry G.C.
      • Shaw D.E.
      • Callahan J.
      • Li Y.
      • Yuan C.
      • Bian C.
      • Chen L.
      • Furie B.
      • Furie B.C.
      • Cines D.B.
      • Huang M.
      Structure of human urokinase plasminogen activator in complex with its receptor.
      ), which is the rational basis for our design of the closed and constitutively active uPARH47C/N259C. Our present demonstration of a flexible and temporarily detached DI opens yet another intervention strategy where the domain interface between DI and DII serves as the targeting site for inhibitors preventing uPA and vitronectin binding by trapping the open conformation of uPAR. This proposition is excellently aligned with the circumstantial observation that a monoclonal anti-uPAR antibody, denoted IIIF10, recognizing a linear epitope in unfolded uPAR (residues 52–60, which is equivalent to βIE), also binds the native glycolipid-anchored uPAR on the cell surface and that this inhibits subsequent uPA binding (
      • Luther T.
      • Magdolen V.
      • Albrecht S.
      • Kasper M.
      • Riemer C.
      • Kessler H.
      • Graeff H.
      • Müller M.
      • Schmitt M.
      Epitope-mapped monoclonal antibodies as tools for functional and morphological analyses of the human urokinase receptor in tumor tissue.
      ). Previously, this reactivity of IIIF10 was not easily reconciled with the limited surface exposure of βIE in the x-ray structures available for intact uPAR (
      • Huai Q.
      • Mazar A.P.
      • Kuo A.
      • Parry G.C.
      • Shaw D.E.
      • Callahan J.
      • Li Y.
      • Yuan C.
      • Bian C.
      • Chen L.
      • Furie B.
      • Furie B.C.
      • Cines D.B.
      • Huang M.
      Structure of human urokinase plasminogen activator in complex with its receptor.
      ,
      • Huai Q.
      • Zhou A.
      • Lin L.
      • Mazar A.P.
      • Parry G.C.
      • Callahan J.
      • Shaw D.E.
      • Furie B.
      • Furie B.C.
      • Huang M.
      Crystal structures of two human vitronectin, urokinase and urokinase receptor complexes.
      ,
      • Xu X.
      • Gårdsvoll H.
      • Yuan C.
      • Lin L.
      • Ploug M.
      • Huang M.
      Crystal structure of the urokinase receptor in a ligand-free form.
      ), where this β-strand is buried in the domain interface with βIID. This observation thus lends further credit to our proposition to explore this particular domain interface as a new druggable target site in uPAR.

      Acknowledgments

      We thank Gitte Juhl-Funch, Haldis Egholm Mønsted, and John Post for excellent technical assistance. Molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by National Institutes of Health Grant P41 RR001081). All plots were created using the software package GRACE.

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