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Cysteine-rich Domain 1 of CD40 Mediates Receptor Self-assembly*

  • Cristian R. Smulski
    Correspondence
    Recipient of grants from the Agence Nationale de la Recherche and the University of Strasbourg. To whom correspondence may be sent at the present address: Dept. of Biochemistry, University of Lausanne, Boveresses 155, CH-1066 Epalinges, Switzerland. Tel.: 41-216925743; Fax: 41-216925705;
    Affiliations
    Institut de Biologie Moléculaire et Cellulaire, Immunologie et Chimie Thérapeutiques, CNRS UPR 9021, 15 rue René Descartes, 67084 Strasbourg, France
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  • Julien Beyrath
    Footnotes
    Affiliations
    Institut de Biologie Moléculaire et Cellulaire, Immunologie et Chimie Thérapeutiques, CNRS UPR 9021, 15 rue René Descartes, 67084 Strasbourg, France
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  • Marion Decossas
    Affiliations
    Institut de Biologie Moléculaire et Cellulaire, Immunologie et Chimie Thérapeutiques, CNRS UPR 9021, 15 rue René Descartes, 67084 Strasbourg, France
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  • Neila Chekkat
    Footnotes
    Affiliations
    Institut de Biologie Moléculaire et Cellulaire, Immunologie et Chimie Thérapeutiques, CNRS UPR 9021, 15 rue René Descartes, 67084 Strasbourg, France
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  • Philippe Wolff
    Affiliations
    Institut de Biologie Moléculaire et Cellulaire, Plateforme Protéomique Strasbourg Esplanade, 15 rue René Descartes, 67084 Strasbourg, France
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  • Karine Estieu-Gionnet
    Affiliations
    Institut Européen de Chimie et de Biologie CBMN, Université de Bordeaux I, CNRS UMR 5248, 2 Rue Robert Escarpit, 33607 PESSAC, France
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  • Gilles Guichard
    Affiliations
    Institut Européen de Chimie et de Biologie CBMN, Université de Bordeaux I, CNRS UMR 5248, 2 Rue Robert Escarpit, 33607 PESSAC, France
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  • Daniel Speiser
    Affiliations
    Department of Oncology and Ludwig Center for Cancer Research, University of Lausanne, Av. P.-Decker 4, CH-1011 Lausanne, Switzerland
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  • Pascal Schneider
    Affiliations
    Department of Biochemistry, University of Lausanne, Boveresses 155, CH-1066 Epalinges, Switzerland
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  • Sylvie Fournel
    Correspondence
    To whom correspondence may be sent at the present address. Tel.: 33-3-68854173; Fax: 33-3-68854306;
    Footnotes
    Affiliations
    Institut de Biologie Moléculaire et Cellulaire, Immunologie et Chimie Thérapeutiques, CNRS UPR 9021, 15 rue René Descartes, 67084 Strasbourg, France
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  • Author Footnotes
    * This work was supported by the Centre National de la Recherche Scientifique (to S. F. and G. G.), the Swiss National Science Foundation (to P. S.), and the Agence Nationale Recherche Grant ANR-08-PCVI-0034-01) (to G. G.).
    This article contains supplemental Figs. 1–7.
    2 Present address: Centre for Systems Biology and Bioenergetics, Radboud University Nijmegen Medical Centre, 6500HB Nijmegen, The Netherlands.
    3 Recipient of a grant from the French Ministère de la Recherche.
    4 Present address: Laboratoire de Conception et Application de Molécules Bioactives, Equipe de Biovectorologie, UMR 7199 CNRS-Université de Strasbourg, Faculté de Pharmacie, 74 Route du Rhin, 67401 Illkirch, France.
Open AccessPublished:March 05, 2013DOI:https://doi.org/10.1074/jbc.M112.427583
      The activation of CD40 on B cells, macrophages, and dendritic cells by its ligand CD154 (CD40L) is essential for the development of humoral and cellular immune responses. CD40L and other TNF superfamily ligands are noncovalent homotrimers, but the form under which CD40 exists in the absence of ligand remains to be elucidated. Here, we show that both cell surface-expressed and soluble CD40 self-assemble, most probably as noncovalent dimers. The cysteine-rich domain 1 (CRD1) of CD40 participated to dimerization and was also required for efficient receptor expression. Modelization of a CD40 dimer allowed the identification of lysine 29 in CRD1, whose mutation decreased CD40 self-interaction without affecting expression or response to ligand. When expressed alone, recombinant CD40-CRD1 bound CD40 with a KD of 0.6 μm. This molecule triggered expression of maturation markers on human dendritic cells and potentiated CD40L activity. These results suggest that CD40 self-assembly modulates signaling, possibly by maintaining the receptor in a quiescent state.

      Introduction

      Ligand-induced oligomerization is an important process for activating cell surface receptors like cytokine receptors, G protein-coupled receptors, protein-tyrosine kinase receptors, Toll-like receptors, and tumor necrosis factor receptor superfamily members (TNFRSF)
      The abbreviations used are: TNFRSF
      TNF receptor superfamily
      BS3
      (bis)sulfosuccinimidyl suberate
      CRD
      cysteine-rich domain
      EDAC
      N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide
      TRAIL
      TNF-related apoptosis-inducing ligand.
      in a number of biological events (
      • Tetsch L.
      • Jung K.
      How are signals transduced across the cytoplasmic membrane? Transport proteins as transmitter of information.
      ,
      • Heldin C.H.
      Dimerization of cell surface receptors in signal transduction.
      ). TNF family ligands form noncovalent homotrimers that can bind three receptor molecules (
      • Bodmer J.L.
      • Schneider P.
      • Tschopp J.
      The molecular architecture of the TNF superfamily.
      ). Although this ligand-receptor complex is generally accepted to be the minimal requirement for signaling, some members of the family are only efficiently triggered by at least two adjacent trimeric ligands (
      • Holler N.
      • Tardivel A.
      • Kovacsovics-Bankowski M.
      • Hertig S.
      • Gaide O.
      • Martinon F.
      • Tinel A.
      • Deperthes D.
      • Calderara S.
      • Schulthess T.
      • Engel J.
      • Schneider P.
      • Tschopp J.
      Two adjacent trimeric Fas ligands are required for Fas signaling and formation of a death-inducing signaling complex.
      ,
      • Haswell L.E.
      • Glennie M.J.
      • Al-Shamkhani A.
      Analysis of the oligomeric requirement for signaling by CD40 using soluble multimeric forms of its ligand, CD154.
      ). The form under which ligand-free TNFRSF members exist is still not fully understood. Indeed, unliganded TNFR1 crystallized as a parallel dimer (
      • Naismith J.H.
      • Devine T.Q.
      • Brandhuber B.J.
      • Sprang S.R.
      Crystallographic evidence for dimerization of unliganded tumor necrosis factor receptor.
      ,
      • Naismith J.H.
      • Devine T.Q.
      • Kohno T.
      • Sprang S.R.
      Structures of the extracellular domain of the type I tumor necrosis factor receptor.
      ) whereas cell-based experiments rather proposed that receptors may preassemble as trimers in the absence of ligand (
      • Chan F.K.
      • Chun H.J.
      • Zheng L.
      • Siegel R.M.
      • Bui K.L.
      • Lenardo M.J.
      A domain in TNF receptors that mediates ligand-independent receptor assembly and signaling.
      ). Interestingly, DcR3 (TNFRSF6B) assembled as parallel dimers when crystallized alone, but as trimers when co-crystallized with its ligand TL1A, each receptor in this trimeric form contacting a different ligand (
      • Zhan C.
      • Patskovsky Y.
      • Yan Q.
      • Li Z.
      • Ramagopal U.
      • Cheng H.
      • Brenowitz M.
      • Hui X.
      • Nathenson S.G.
      • Almo S.C.
      Decoy strategies: the structure of TL1A:DcR3 complex.
      ). Thus, receptors may exist both as dimers or trimers depending on the context. Gathering more structural information on unliganded TNF receptors will help to better understand the mechanism of ligand-induced receptor signaling.
      The extracellular region of TNFRSF members is characterized by the presence of cysteine-rich domains (CRDs) which typically contain six cysteine residues engaged in the formation of three disulfide bonds. The number of CRDs in a given receptor usually varies from one to four (
      • Bodmer J.L.
      • Schneider P.
      • Tschopp J.
      The molecular architecture of the TNF superfamily.
      ). TNFR1 and CD40 possess four CRDs, of which only CRD2 and 3 are directly involved in ligand binding. Crystallography of unliganded TNFR1 (TNFRSF1A) (
      • Naismith J.H.
      • Devine T.Q.
      • Brandhuber B.J.
      • Sprang S.R.
      Crystallographic evidence for dimerization of unliganded tumor necrosis factor receptor.
      ) showed homodimerization in either parallel or anti-parallel manners (
      • Naismith J.H.
      • Devine T.Q.
      • Kohno T.
      • Sprang S.R.
      Structures of the extracellular domain of the type I tumor necrosis factor receptor.
      ). Anti-parallel dimers associate through an interface that overlaps with CRD1 and 2, whereas parallel dimers associate mainly via CRD1 and leave ligand binding sites accessible. Cell-based studies identified a preligand-binding assembly domain at the N terminus of TNFR1 and 2 that is required for preassembly of TNFR complexes in a fully ligand-responsive conformation (
      • Chan F.K.
      • Chun H.J.
      • Zheng L.
      • Siegel R.M.
      • Bui K.L.
      • Lenardo M.J.
      A domain in TNF receptors that mediates ligand-independent receptor assembly and signaling.
      ). In any case, TNF receptors appear to exist as preformed complexes and not as monomeric receptors that would only oligomerize upon ligand binding. In line with these results, the CRD1 of Fas (TNFRSF6) was shown to be essential for the formation of homotypic, ligand-independent receptor complexes (
      • Siegel R.M.
      • Frederiksen J.K.
      • Zacharias D.A.
      • Chan F.K.
      • Johnson M.
      • Lynch D.
      • Tsien R.Y.
      • Lenardo M.J.
      Fas preassociation required for apoptosis signaling and dominant inhibition by pathogenic mutations.
      ). The situation is more complex for the TNF-related apoptosis-inducing ligand (TRAIL), another TNF family member that binds five different receptors: two fully functional death receptors, TRAIL-R1 (TNFRSF10A) and TRAIL-R2 (TNFRSF10B), two membrane bound “decoy” receptors, TRAIL-R3 (TNFRSF10C) and TRAIL-R4 (TNFRSF10D), and a soluble decoy receptor, OPG (TNFRSF11B). Decoy receptors inhibit cell death by competing for TRAIL binding. However, inhibition of TRAIL-induced apoptosis by TRAIL-R4 critically depends on its association with TRAIL-R2 via the N-terminal domain containing the first partial CRD of both receptors. Thus, in contrast to homotypic TNFR1 or Fas complexes, TRAIL-R2 and TRAIL-R4 form mixed complexes as a means to regulate TRAIL-induced apoptosis (
      • Clancy L.
      • Mruk K.
      • Archer K.
      • Woelfel M.
      • Mongkolsapaya J.
      • Screaton G.
      • Lenardo M.J.
      • Chan F.K.
      Preligand assembly domain-mediated ligand-independent association between TRAIL receptor 4 (TR4) and TR2 regulates TRAIL-induced apoptosis.
      ).
      The present study focuses on CD40 (TNFRSF5). Engagement of CD40 by CD40L leads to CD40 clustering, recruitment of TNFR-associated factors, and activation, among others, of the transcription factor NF-κB (
      • Pullen S.S.
      • Miller H.G.
      • Everdeen D.S.
      • Dang T.T.
      • Crute J.J.
      • Kehry M.R.
      CD40-tumor necrosis factor receptor-associated factor (TRAF) interactions: regulation of CD40 signaling through multiple TRAF binding sites and TRAF hetero-oligomerization.
      ,
      • Pullen S.S.
      • Labadia M.E.
      • Ingraham R.H.
      • McWhirter S.M.
      • Everdeen D.S.
      • Alber T.
      • Crute J.J.
      • Kehry M.R.
      High-affinity interactions of tumor necrosis factor receptor-associated factors (TRAFs) and CD40 require TRAF trimerization and CD40 multimerization.
      ). CD40 is expressed mainly on professional antigen-presenting cells, i.e. dendritic cells, macrophages, and B cells (
      • van Kooten C.
      • Banchereau J.
      CD40-CD40 ligand.
      ), and its activation is essential for the development of humoral and cellular immune responses. Thus, selective blockade or activation of CD40 is of pharmacological interest. The crystal structure of CD40-CD40L was recently solved showing that CRD2 and CRD3 are both involved in CD40L binding (
      • An H.J.
      • Kim Y.J.
      • Song D.H.
      • Park B.S.
      • Kim H.M.
      • Lee J.D.
      • Paik S.G.
      • Lee J.O.
      • Lee H.
      Crystallographic and mutational analysis of the CD40-CD154 complex and its implications for receptor activation.
      ); however, little is known about the function of CRD1 and CRD4.
      Here, we show that CD40 exists as preformed, noncovalent dimers on the cell surface and that dimerization is dependent on the extracellular region. Mutation of K29 in CRD1 impaired CD40 self-assembly. Recombinant CD40-CRD1 bound the extracellular domain of CD40 with a KD of approximately 0.6 μm and displayed an agonist-like activity on CD40-expressing cells such as human dendritic cells. Co-incubation of CD40-CRD1 with CD40L potentiated the NF-κB response induced by CD40L alone, suggesting that CRD1 interactions participate in CD40 signaling. These observations provide a novel means to manipulate CD40 signaling.

      DISCUSSION

      Although it is well established that CD154 (CD40L) assembles as a trimer, not much research has been conducted on CD40 oligomerization before ligand engagement and on the potential functional effect of this oligomerization. Reports from Mourad and colleagues suggested that CD40 at the cell surface exist as disulfide-linked dimers and that disulfide-linked homodimerization was increased by CD40L (
      • Reyes-Moreno C.
      • Girouard J.
      • Lapointe R.
      • Darveau A.
      • Mourad W.
      CD40/CD40 homodimers are required for CD40-induced phosphatidylinositol 3-kinase-dependent expression of B7.2 by human B lymphocytes.
      ,
      • Girouard J.
      • Reyes-Moreno C.
      • Darveau A.
      • Akoum A.
      • Mourad W.
      Requirement of the extracellular cysteine at position six for CD40/CD40 dimer formation and CD40-induced IL-8 expression.
      ,
      • Reyes-Moreno C.
      • Sharif-Askari E.
      • Girouard J.
      • Léveillé C.
      • Jundi M.
      • Akoum A.
      • Lapointe R.
      • Darveau A.
      • Mourad W.
      Requirement of oxidation-dependent CD40 homodimers for CD154/CD40 bidirectional signaling.
      ). However, all cysteine residues in the extracellular region of CD40 and other crystallized TNFR family members are engaged in intrachain disulfide bridges, making it unlikely that CD40 would dimerize through interchain extracellular covalent bonds.
      In the present study, we show that CD40 can self-assemble at the cell surface in a noncovalent manner, probably as dimers. Soluble CD40 also dimerizes, suggesting that the extracellular domain plays an important role in dimer formation. In addition, in silico modelization and site-directed mutagenesis confirmed the role of CD40-CRD1 in receptor-receptor interactions. Until now, only TNFR1 and DcR3 have been crystallized as ligand-free receptors. TNFR1 crystallized as both parallel and anti-parallel dimers (
      • Naismith J.H.
      • Devine T.Q.
      • Brandhuber B.J.
      • Sprang S.R.
      Crystallographic evidence for dimerization of unliganded tumor necrosis factor receptor.
      ). The parallel dimer was stabilized by interactions involving CRD1 and exposed fully accessible TNF binding sites in CRD2 and CRD3. In contrast, the anti-parallel dimer was stabilized by interactions involving CRD1 and CRD2 that occluded the TNF binding sites. Parallel TNFR dimers are favored at neutral pH, whereas anti-parallel TNFR dimers are favored at acidic pH (
      • Naismith J.H.
      • Devine T.Q.
      • Kohno T.
      • Sprang S.R.
      Structures of the extracellular domain of the type I tumor necrosis factor receptor.
      ). This observation led to the hypothesis that parallel dimers may occur at the cell surface and could serve to create an activation network in the presence of ligand, in contrast to anti-parallel dimers that may form upon acidification of endocytic vesicles and release the ligand from the complex (
      • Naismith J.H.
      • Devine T.Q.
      • Kohno T.
      • Sprang S.R.
      Structures of the extracellular domain of the type I tumor necrosis factor receptor.
      ). Unliganded DcR3 was also crystallized as parallel dimers, with the main contact area in CRD1 and 4 and an accessible ligand binding site to its ligand TL1A (
      • Zhan C.
      • Patskovsky Y.
      • Yan Q.
      • Li Z.
      • Ramagopal U.
      • Cheng H.
      • Brenowitz M.
      • Hui X.
      • Nathenson S.G.
      • Almo S.C.
      Decoy strategies: the structure of TL1A:DcR3 complex.
      ). Sedimentation equilibrium analyses of DcR3 showed weak self-association, with an estimated equilibrium dissociation constant greater than 2.4 × 10−3 m, a low value if we consider that the physiological concentration of this soluble receptor is in the range of 10−12 pm (
      • Wu Y.
      • Han B.
      • Sheng H.
      • Lin M.
      • Moore P.A.
      • Zhang J.
      • Wu J.
      Clinical significance of detecting elevated serum DcR3/TR6/M68 in malignant tumor patients.
      ). Interestingly, the CRD1-CRD1 interaction surface modeled for CD40 in our study superimposed relatively well with the interaction surface found between the CRD1 of the TNF receptor HVEM and the immunomodulatory protein BTLA in the HVEM-BTLA complex (
      • Compaan D.M.
      • Gonzalez L.C.
      • Tom I.
      • Loyet K.M.
      • Eaton D.
      • Hymowitz S.G.
      Attenuating lymphocyte activity: the crystal structure of the BTLA-HVEM complex.
      ). Therefore, a CRD1 seems to use the same interface to interact with another CRD1 (as in the TNFR1 dimer) or with a regulatory protein. The CRD1 could therefore be considered as an interaction domain that regulates receptor activity by interacting either with itself or with other proteins such as BTLA.
      Using surface plasmon resonance, an equilibrium dissociation constant of 5.7 × 10−7 m was determined for the interaction between CD40 and recombinant CD40-CRD1. Although this equilibrium constant is rather low, it may still be relevant because local protein concentrations on the cell membrane can be high, especially in some microdomains, and because other interaction sites outside CRD1 may also participate to dimerization. These results raise the question of whether the active form of the receptor is the dimeric or the monomeric form and whether receptor dimers persist after ligand engagement. Further analyses are warranted to address these questions.
      The CRD1 of TNFR1 fused to glutathione S-transferase as been described previously as an inhibitor of TNFR1-mediated signaling (
      • Deng G.M.
      • Zheng L.
      • Chan F.K.
      • Lenardo M.
      Amelioration of inflammatory arthritis by targeting the pre-ligand assembly domain of tumor necrosis factor receptors.
      ). This molecule could block the effects of TNF in vitro and was shown to inhibit arthritis in mice (
      • Deng G.M.
      • Zheng L.
      • Chan F.K.
      • Lenardo M.
      Amelioration of inflammatory arthritis by targeting the pre-ligand assembly domain of tumor necrosis factor receptors.
      ). Unfortunately, this study was not designed to address the molecular basis of these effects, nor did it test the potential impact of glutathione S-transferase-induced dimerization on CRD1 activity. In the case of Fas, the study of dominant interfering mutations associated with autoimmune lymphoproliferative syndrome revealed that Fas CRD1 was fully preserved in all dominant interfering mutations, including mutations that truncate Fas after 57 or 62 amino acids of the mature Fas protein and that would be unable to bind FasL (
      • Siegel R.M.
      • Frederiksen J.K.
      • Zacharias D.A.
      • Chan F.K.
      • Johnson M.
      • Lynch D.
      • Tsien R.Y.
      • Lenardo M.J.
      Fas preassociation required for apoptosis signaling and dominant inhibition by pathogenic mutations.
      ). In addition, deletion of CRD1 in dominant negative inhibitory receptors restored susceptibility to FasL-induced apoptosis, indicating that mutant proteins must physically interact with wild-type proteins to create a nonfunctional complex. Although the phenotype of patients provides strong genetic evidence for a dominant negative function of the N-terminal portion of Fas, results obtained with N-terminal Fas deletion mutants must be taken with caution in view of our results demonstrating that CRD1 disruption prevents CD40 surface expression. At least for CD40, N-terminal truncation or severe perturbations of CRD1 structures are not appropriate to study CRD1 function. In any case, the studies mentioned above indicate that the N-terminal portions of TNFR1 and Fas taken alone antagonize receptor signaling.
      Because of the described inhibitory activity of TNFR1 and Fas CRD1, the ability of recombinant CD40-CRD1 to induce a weak CD40 activation rather than an inhibition of NF-κB signaling was unexpected. A gross artifact can probably be excluded because NF-κB activation was CD40-dependent and because CRD1 disruption mutants were ineffective. The agonist activity of CD40-CRD1 could be explained in several ways: (i) if CD40 is inhibited by dimerization, then recombinant CRD1 may activate CD40 by disrupting the dimers; (ii) recombinant CRD1 could oligomerize CD40 on the cell surface, inducing an agonist-like signaling; and (iii) recombinant CRD1 may induce conformational changes in the receptor similar to those induced by the ligand upon signal activation. Importantly, the agonist activity of CD40-CRD1 was not restricted to cells overexpressing CD40, but was also observed in primary, monocyte-derived dendritic cells. However, the observation that CD40-CRD1 was unable to induce the full set of activation markers in dendritic cells probably indicates a weak agonist activity compared with CD40L.
      Altogether, our result have revealed the ability of CD40 to self-assemble through its cysteine-rich domain 1 and the ability of recombinant CD40-CRD1 to potentiate CD40L in HEK CD40 cells and to induce a ligand-like activity in human dendritic cells. Our data indicate that CD40 homo-dimerization controls signaling, possibly by maintaining the receptor in a quiescent state, although further studies are needed to elucidate mechanisms mediating receptor activation.

      Acknowledgments

      We thank Dr. Sylviane Muller (UPR 9021 Strasbourg, France) for constant support and encouragement; Laure Willen (University of Lausanne) for expert technical assistance in plasmid cloning; Marie-Christine Rio and Fabien Alpy for kindly providing the ERBB2-EYFP construct; and Pedro Romero and Petra Baumgaertner (University of Lausanne) for helpful discussions, advice, and assistance in monocyte-derived dendritic cell assay.

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