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Structure and Function of the Intracellular Region of the Plexin-B1 Transmembrane Receptor*

  • Yufeng Tong
    Footnotes
    Affiliations
    Structural Genomics Consortium, University of Toronto, Toronto, Ontario M5G 1L7, Canada
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  • Prasanta K. Hota
    Footnotes
    Affiliations
    Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106
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  • Junia Y. Penachioni
    Footnotes
    Affiliations
    Institute for Cancer Research and Treatment, University of Torino, I-10060 Candiolo (Torino), Italy
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  • Mehdi B. Hamaneh
    Footnotes
    Affiliations
    Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106
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  • SoonJeung Kim
    Affiliations
    Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106
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  • Rebecca S. Alviani
    Affiliations
    Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106
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  • Limin Shen
    Affiliations
    Structural Genomics Consortium, University of Toronto, Toronto, Ontario M5G 1L7, Canada
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  • Hao He
    Affiliations
    Structural Genomics Consortium, University of Toronto, Toronto, Ontario M5G 1L7, Canada
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  • Wolfram Tempel
    Affiliations
    Structural Genomics Consortium, University of Toronto, Toronto, Ontario M5G 1L7, Canada
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  • Luca Tamagnone
    Correspondence
    To whom correspondence may be addressed; IRCC, University of Torino, Strada Prov. 142, I-10060 Candiolo, Italy. Tel.: 39-011-993-3204;
    Footnotes
    Affiliations
    Institute for Cancer Research and Treatment, University of Torino, I-10060 Candiolo (Torino), Italy
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  • Hee-Won Park
    Correspondence
    To whom correspondence may be addressed: Structural Genomics Consortium, MaRS South Tower, 101 College St., Toronto, Ontario M5G 1L7, Canada. Tel.: 416-946-3867
    Affiliations
    Structural Genomics Consortium, University of Toronto, Toronto, Ontario M5G 1L7, Canada

    Department of Pharmacology, University of Toronto, Toronto, Ontario M5G 1L7, Canada
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  • Matthias Buck
    Correspondence
    To whom correspondence may be addressed: Case Western Reserve University, School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106. Tel.: 216-36-8651
    Affiliations
    Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106

    Department of Neuroscience, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106

    Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106
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  • Author Footnotes
    * This work was supported in part by National Institutes of Health Grants R01GM73071 and K02HL084384 (to M. B.) and in part by the Structural Genomics Consortium, a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck & Co., Inc., the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research, and the Wellcome Trust.
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S8.
    3 Supported by grants from the Italian Association for Cancer Research and Regione Piemonte.
    2 Postdoctoral fellow of the American Heart Association, Ohio Valley/Great Rivers Affiliate.
    1 These authors contributed equally to this work.
Open AccessPublished:December 18, 2009DOI:https://doi.org/10.1074/jbc.M109.056275
      Members of the plexin family are unique transmembrane receptors in that they interact directly with Rho family small GTPases; moreover, they contain a GTPase-activating protein (GAP) domain for R-Ras, which is crucial for plexin-mediated regulation of cell motility. However, the functional role and structural basis of the interactions between the different intracellular domains of plexins remained unclear. Here we present the 2.4 Å crystal structure of the complete intracellular region of human plexin-B1. The structure is monomeric and reveals that the GAP domain is folded into one structure from two segments, separated by the Rho GTPase binding domain (RBD). The RBD is not dimerized, as observed previously. Instead, binding of a conserved loop region appears to compete with dimerization and anchors the RBD to the GAP domain. Cell-based assays on mutant proteins confirm the functional importance of this coupling loop. Molecular modeling based on structural homology to p120GAP·H-Ras suggests that Ras GTPases can bind to the plexin GAP region. Experimentally, we show that the monomeric intracellular plexin-B1 binds R-Ras but not H-Ras. These findings suggest that the monomeric form of the intracellular region is primed for GAP activity and extend a model for plexin activation.

      Introduction

      Plexins are single transmembrane receptors for guidance cues, called semaphorins, which regulate the motility and positional maintenance of certain cells. With this function, the receptors play critical roles in many developmental processes, including axon guidance, angiogenesis, and bone formation (
      • Tamagnone L.
      • Comoglio P.M.
      ,
      • Kruger R.P.
      • Aurandt J.
      • Guan K.L.
      ). Moreover, plexins and their ligands are also involved in the regulation of the immune response, in cancer progression, and are thought to restrain tissue regeneration after injury (
      • Neufeld G.
      • Shraga-Heled N.
      • Lange T.
      • Guttmann-Raviv N.
      • Herzog Y.
      • Kessler O.
      ,
      • Pasterkamp R.J.
      • Verhaagen J.
      ).
      Plexins are unusual receptors in that they interact directly with Rho and Ras family small GTPases (
      • Oinuma I.
      • Ishikawa Y.
      • Katoh H.
      • Negishi M.
      ,
      • Toyofuku T.
      • Yoshida J.
      • Sugimoto T.
      • Zhang H.
      • Kumanogoh A.
      • Hori M.
      • Kikutani H.
      ,
      • Uesugi K.
      • Oinuma I.
      • Katoh H.
      • Negishi M.
      ). An intracellular region that has high homology to Ras GTPase-activating proteins (GAPs)
      The abbreviations used are: GAP
      GTPase-activating protein
      RBD
      Rho GTPase binding domain.
      facilitates the hydrolysis of R-Ras-bound GTP. This deactivation of R-Ras leads to functional inhibition of integrins and to a loss of cell adhesion in response to semaphorins (
      • Oinuma I.
      • Ishikawa Y.
      • Katoh H.
      • Negishi M.
      ,
      • Toyofuku T.
      • Yoshida J.
      • Sugimoto T.
      • Zhang H.
      • Kumanogoh A.
      • Hori M.
      • Kikutani H.
      ,
      • Uesugi K.
      • Oinuma I.
      • Katoh H.
      • Negishi M.
      ,
      • Oinuma I.
      • Katoh H.
      • Negishi M.
      ). Interestingly, no GAP activity of plexin-B1 was detected toward the R-Ras-homologous H-Ras (
      • Oinuma I.
      • Ishikawa Y.
      • Katoh H.
      • Negishi M.
      ), suggesting greater substrate specificity compared with the GAP protein p120GAP (
      • Ohba Y.
      • Mochizuki N.
      • Yamashita S.
      • Chan A.M.
      • Schrader J.W.
      • Hattori S.
      • Nagashima K.
      • Matsuda M.
      ). How the plexin receptor is activated and specifically how the GAP function is regulated have been questions of considerable interest (
      • Rohm B.
      • Rahim B.
      • Kleiber B.
      • Hovatta I.
      • Püschel A.W.
      ,
      • Pasterkamp R.J.
      ,
      • Püschel A.W.
      ). A number of studies have pointed to a sequence segment that interrupts the GAP-homologous region and is capable of binding small Rho family GTPases. In the case of plexin-B1, this Rho GTPase binding domain (RBD) can associate with Rnd1, Rac1, and RhoD, which are thought to regulate plexin function. Specifically, in vitro studies in a number of laboratories have used the intracellular region of plexins expressed as two fragments, named C1 (containing the RBD and an N-terminal GAP-homologous segment) and C2 (C-terminal GAP segment). The studies suggest that such fragments are loosely associated. Moreover, the interaction between the RBD and Rnd1 or Rac1 appears to separate the two fragments (
      • Oinuma I.
      • Ishikawa Y.
      • Katoh H.
      • Negishi M.
      ,
      • Toyofuku T.
      • Yoshida J.
      • Sugimoto T.
      • Zhang H.
      • Kumanogoh A.
      • Hori M.
      • Kikutani H.
      ,
      • Uesugi K.
      • Oinuma I.
      • Katoh H.
      • Negishi M.
      ,
      • Oinuma I.
      • Katoh H.
      • Negishi M.
      ,
      • Turner L.J.
      • Nicholls S.
      • Hall A.
      ).
      Structural biology has had a tremendous impact on our understanding of GTPase function and regulation (e.g. see Ref.
      • Vetter I.R.
      • Wittinghofer A.
      ). Representative structures for all of the major families of small GTPase-activating proteins are known, and also by using mutagenesis, the catalytic residues involved have been identified (
      • Bos J.L.
      • Rehmann H.
      • Wittinghofer A.
      ). However, the GAP domain is often surrounded by other protein segments that are known to participate in cell signaling events, such as an SH2 domain in chimerins (
      • Canagarajah B.
      • Leskow F.C.
      • Ho J.Y.
      • Mischak H.
      • Saidi L.F.
      • Kazanietz M.G.
      • Hurley J.H.
      ), C2 in SynGAP (
      • Pena V.
      • Hothorn M.
      • Eberth A.
      • Kaschau N.
      • Parret A.
      • Gremer L.
      • Bonneau F.
      • Ahmadian M.R.
      • Scheffzek K.
      ), and a pleckstrin homology/lipid binding domain in p120GAP (
      • Drugan J.K.
      • Rogers-Graham K.
      • Gilmer T.
      • Campbell S.
      • Clark G.J.
      ). Our understanding of how GAP activity is controlled is still limited, because not many structures that include regulatory domains have been determined to date.
      Characterizing the structure of the intracellular region of human plexin-B1 promises to elucidate the mechanism by which the RBD can control receptor signaling and the function of the GAP domain. The NMR solution conformation (
      • Tong Y.
      • Hughes D.
      • Placanica L.
      • Buck M.
      ,
      • Tong Y.
      • Hota P.K.
      • Hamaneh M.B.
      • Buck M.
      ) and x-ray structure of the RBD of human plexin-B1 show that this domain forms a dimeric ubiquitin-like structure (
      • Tong Y.
      • Chugha P.
      • Hota P.K.
      • Alviani R.S.
      • Li M.
      • Tempel W.
      • Shen L.
      • Park H.W.
      • Buck M.
      ). GTPase association with the RBD domain occurs at a common interface that is adjacent to the dimerization region. These observations combined with biophysical studies suggest that Rho GTPase binding can destabilize a dimeric form of the intracellular region of plexins. On the extracellular side, it has been proposed from the dimeric crystal structure of semaphorin-3A that ligand binding to the semaphorin-homologous region of plexin would cause a conformational rearrangement in the dimeric form of the receptor (
      • Antipenko A.
      • Himanen J.P.
      • van Leyen K.
      • Nardi-Dei V.
      • Lesniak J.
      • Barton W.A.
      • Rajashankar K.R.
      • Lu M.
      • Hoemme C.
      • Püschel A.W.
      • Nikolov D.B.
      ). It is also known that Rac1 binding to the cytoplasmic plexin-B1 RBD increases ligand binding on the cell surface (
      • Vikis H.G.
      • Li W.
      • Guan K.L.
      ). Together, these studies led to the refinement of a model for plexin activation that involves the destabilization of a RBD-mediated intracellular region dimer and explained the observed synergy between ligand binding and GTPase-dependent regulation of these receptors (
      • Tong Y.
      • Chugha P.
      • Hota P.K.
      • Alviani R.S.
      • Li M.
      • Tempel W.
      • Shen L.
      • Park H.W.
      • Buck M.
      ).
      Here, we present the 2.4 Å x-ray structure of the entire intracellular region of plexin-B1 (residues 1511–2135). The role of the different domains is investigated by a combination of biophysical, computational, and functional studies. The protein is monomeric and has a single GAP domain fold with the RBD placed on its side. A detailed comparison also with the structure of the isolated dimeric RBD region and with the RBD bound to Rnd1 suggests that intradomain conformational changes induced by Rho GTPase binding are small in this system. Furthermore, the structures of the RBD dimers provide a model for a dimeric intracellular structure. This model is incompatible with Rho GTPase binding, thus supporting the role of these interactions for interdomain changes. Functional studies in cells confirm the importance of a newly discovered protein segment (the “coupling loop”) that is designed to oppose dimerization of the RBD region as part of the receptor activation mechanism. The plexin GAP fold has high structural similarity to that of p120GAP. Our data suggest that the intracellular domain of plexin-B1, when in the monomeric state, is primed for GAP function even in the absence of Rho GTPases, leading to a critical extension of the current model for plexin activation.

      Addendum

      While this paper was under review (and following the release of the plexin-B1 coordinates in late May 2009), the structure of the plexin-A3 intracellular region also became available. It should be noted that the structures are very similar (Cα root mean square deviation of 0.95 Å for 430 matching residues). However, the interpretation of the structures, drawn in terms of possible molecular mechanisms by He et al. (
      • He H.
      • Taehong Y.
      • Terman J.R.
      • Zhang X.
      ) is substantially different from that presented here.

      Acknowledgments

      Drs. Christina Kiel and Luis Serrano (EMBL-CRG, Barcelona) helped us early in this project with a homology model of plexin-B1 based on p120GAP. Crystallographic data shown in this report were derived from work performed at Argonne National Laboratory, Structural Biology Center, beam line 19ID, at the Advanced Photon Source. Data collection at GM/CA CAT, beam line 23ID-B, has been funded by NCI Grant Y1-CO-1020 and NIGM Grant Y1-GM-1104 from the National Institutes of Health. Use of the Advanced Photon Source was supported by the United States Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract DE-AC02-06CH11357. Calculations were carried out at the Case Western Reserve University High Performance Cluster, at the Ohio Supercomputer Center (Columbus, OH), and at LoneStar (Austin, TX) via a TeraGrid Award (to M. B.).

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