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

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. 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. IntroductionPlexins 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 (1.Tamagnone L. Comoglio P.M. Trends Cell Biol. 2000; 10: 377-383Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 2.Kruger R.P. Aurandt J. Guan K.L. Nat. Rev. Mol. Cell Biol. 2005; 6: 789-800Crossref PubMed Scopus (398) Google Scholar). 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 (3.Neufeld G. Shraga-Heled N. Lange T. Guttmann-Raviv N. Herzog Y. Kessler O. Front. Biosci. 2005; 10: 751-760Crossref PubMed Scopus (95) Google Scholar, 4.Pasterkamp R.J. Verhaagen J. Brain Res. Brain Res. Rev. 2001; 35: 36-54Crossref PubMed Scopus (114) Google Scholar).Plexins are unusual receptors in that they interact directly with Rho and Ras family small GTPases (5.Oinuma I. Ishikawa Y. Katoh H. Negishi M. Science. 2004; 305: 862-865Crossref PubMed Scopus (316) Google Scholar, 6.Toyofuku T. Yoshida J. Sugimoto T. Zhang H. Kumanogoh A. Hori M. Kikutani H. Nat. Neurosci. 2005; 8: 1712-1719Crossref PubMed Scopus (190) Google Scholar, 7.Uesugi K. Oinuma I. Katoh H. Negishi M. J. Biol. Chem. 2009; 284: 6743-6751Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). An intracellular region that has high homology to Ras GTPase-activating proteins (GAPs) 7The abbreviations used are: GAPGTPase-activating proteinRBDRho 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 (5.Oinuma I. Ishikawa Y. Katoh H. Negishi M. Science. 2004; 305: 862-865Crossref PubMed Scopus (316) Google Scholar, 6.Toyofuku T. Yoshida J. Sugimoto T. Zhang H. Kumanogoh A. Hori M. Kikutani H. Nat. Neurosci. 2005; 8: 1712-1719Crossref PubMed Scopus (190) Google Scholar, 7.Uesugi K. Oinuma I. Katoh H. Negishi M. J. Biol. Chem. 2009; 284: 6743-6751Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 8.Oinuma I. Katoh H. Negishi M. J. Neurosci. 2004; 24: 11473-11480Crossref PubMed Scopus (101) Google Scholar). Interestingly, no GAP activity of plexin-B1 was detected toward the R-Ras-homologous H-Ras (5.Oinuma I. Ishikawa Y. Katoh H. Negishi M. Science. 2004; 305: 862-865Crossref PubMed Scopus (316) Google Scholar), suggesting greater substrate specificity compared with the GAP protein p120GAP (9.Ohba Y. Mochizuki N. Yamashita S. Chan A.M. Schrader J.W. Hattori S. Nagashima K. Matsuda M. J. Biol. Chem. 2000; 275: 20020-20026Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). How the plexin receptor is activated and specifically how the GAP function is regulated have been questions of considerable interest (10.Rohm B. Rahim B. Kleiber B. Hovatta I. Püschel A.W. FEBS Lett. 2000; 486: 68-72Crossref PubMed Scopus (158) Google Scholar, 11.Pasterkamp R.J. Trends Cell Biol. 2005; 15: 61-64Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 12.Püschel A.W. Adv. Exp. Med. Biol. 2007; 600: 12-23Crossref PubMed Scopus (55) Google Scholar). 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 (5.Oinuma I. Ishikawa Y. Katoh H. Negishi M. Science. 2004; 305: 862-865Crossref PubMed Scopus (316) Google Scholar, 6.Toyofuku T. Yoshida J. Sugimoto T. Zhang H. Kumanogoh A. Hori M. Kikutani H. Nat. Neurosci. 2005; 8: 1712-1719Crossref PubMed Scopus (190) Google Scholar, 7.Uesugi K. Oinuma I. Katoh H. Negishi M. J. Biol. Chem. 2009; 284: 6743-6751Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 8.Oinuma I. Katoh H. Negishi M. J. Neurosci. 2004; 24: 11473-11480Crossref PubMed Scopus (101) Google Scholar, 13.Turner L.J. Nicholls S. Hall A. J. Biol. Chem. 2004; 279: 33199-33205Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar).Structural biology has had a tremendous impact on our understanding of GTPase function and regulation (e.g. see Ref. 14.Vetter I.R. Wittinghofer A. Science. 2001; 294: 1299-1304Crossref PubMed Scopus (1349) Google Scholar). 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 (15.Bos J.L. Rehmann H. Wittinghofer A. Cell. 2007; 129: 865-877Abstract Full Text Full Text PDF PubMed Scopus (1270) Google Scholar). 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 (16.Canagarajah B. Leskow F.C. Ho J.Y. Mischak H. Saidi L.F. Kazanietz M.G. Hurley J.H. Cell. 2004; 119: 407-418Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), C2 in SynGAP (17.Pena V. Hothorn M. Eberth A. Kaschau N. Parret A. Gremer L. Bonneau F. Ahmadian M.R. Scheffzek K. EMBO Rep. 2008; 9: 350-355Crossref PubMed Scopus (71) Google Scholar), and a pleckstrin homology/lipid binding domain in p120GAP (18.Drugan J.K. Rogers-Graham K. Gilmer T. Campbell S. Clark G.J. J. Biol. Chem. 2000; 275: 35021-35027Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). 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 (19.Tong Y. Hughes D. Placanica L. Buck M. Structure. 2005; 13: 7-15Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 20.Tong Y. Hota P.K. Hamaneh M.B. Buck M. Structure. 2008; 16: 246-258Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) and x-ray structure of the RBD of human plexin-B1 show that this domain forms a dimeric ubiquitin-like structure (21.Tong Y. Chugha P. Hota P.K. Alviani R.S. Li M. Tempel W. Shen L. Park H.W. Buck M. J. Biol. Chem. 2007; 282: 37215-37224Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). 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 (22.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. Neuron. 2003; 39: 589-598Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). It is also known that Rac1 binding to the cytoplasmic plexin-B1 RBD increases ligand binding on the cell surface (23.Vikis H.G. Li W. Guan K.L. Genes Dev. 2002; 16: 836-845Crossref PubMed Scopus (103) Google Scholar). 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 (21.Tong Y. Chugha P. Hota P.K. Alviani R.S. Li M. Tempel W. Shen L. Park H.W. Buck M. J. Biol. Chem. 2007; 282: 37215-37224Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar).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.AddendumWhile 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. (48.He H. Taehong Y. Terman J.R. Zhang X. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 15610-15615Crossref PubMed Scopus (69) Google Scholar) is substantially different from that presented here. IntroductionPlexins 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 (1.Tamagnone L. Comoglio P.M. Trends Cell Biol. 2000; 10: 377-383Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 2.Kruger R.P. Aurandt J. Guan K.L. Nat. Rev. Mol. Cell Biol. 2005; 6: 789-800Crossref PubMed Scopus (398) Google Scholar). 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 (3.Neufeld G. Shraga-Heled N. Lange T. Guttmann-Raviv N. Herzog Y. Kessler O. Front. Biosci. 2005; 10: 751-760Crossref PubMed Scopus (95) Google Scholar, 4.Pasterkamp R.J. Verhaagen J. Brain Res. Brain Res. Rev. 2001; 35: 36-54Crossref PubMed Scopus (114) Google Scholar).Plexins are unusual receptors in that they interact directly with Rho and Ras family small GTPases (5.Oinuma I. Ishikawa Y. Katoh H. Negishi M. Science. 2004; 305: 862-865Crossref PubMed Scopus (316) Google Scholar, 6.Toyofuku T. Yoshida J. Sugimoto T. Zhang H. Kumanogoh A. Hori M. Kikutani H. Nat. Neurosci. 2005; 8: 1712-1719Crossref PubMed Scopus (190) Google Scholar, 7.Uesugi K. Oinuma I. Katoh H. Negishi M. J. Biol. Chem. 2009; 284: 6743-6751Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). An intracellular region that has high homology to Ras GTPase-activating proteins (GAPs) 7The abbreviations used are: GAPGTPase-activating proteinRBDRho 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 (5.Oinuma I. Ishikawa Y. Katoh H. Negishi M. Science. 2004; 305: 862-865Crossref PubMed Scopus (316) Google Scholar, 6.Toyofuku T. Yoshida J. Sugimoto T. Zhang H. Kumanogoh A. Hori M. Kikutani H. Nat. Neurosci. 2005; 8: 1712-1719Crossref PubMed Scopus (190) Google Scholar, 7.Uesugi K. Oinuma I. Katoh H. Negishi M. J. Biol. Chem. 2009; 284: 6743-6751Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 8.Oinuma I. Katoh H. Negishi M. J. Neurosci. 2004; 24: 11473-11480Crossref PubMed Scopus (101) Google Scholar). Interestingly, no GAP activity of plexin-B1 was detected toward the R-Ras-homologous H-Ras (5.Oinuma I. Ishikawa Y. Katoh H. Negishi M. Science. 2004; 305: 862-865Crossref PubMed Scopus (316) Google Scholar), suggesting greater substrate specificity compared with the GAP protein p120GAP (9.Ohba Y. Mochizuki N. Yamashita S. Chan A.M. Schrader J.W. Hattori S. Nagashima K. Matsuda M. J. Biol. Chem. 2000; 275: 20020-20026Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). How the plexin receptor is activated and specifically how the GAP function is regulated have been questions of considerable interest (10.Rohm B. Rahim B. Kleiber B. Hovatta I. Püschel A.W. FEBS Lett. 2000; 486: 68-72Crossref PubMed Scopus (158) Google Scholar, 11.Pasterkamp R.J. Trends Cell Biol. 2005; 15: 61-64Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 12.Püschel A.W. Adv. Exp. Med. Biol. 2007; 600: 12-23Crossref PubMed Scopus (55) Google Scholar). 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 (5.Oinuma I. Ishikawa Y. Katoh H. Negishi M. Science. 2004; 305: 862-865Crossref PubMed Scopus (316) Google Scholar, 6.Toyofuku T. Yoshida J. Sugimoto T. Zhang H. Kumanogoh A. Hori M. Kikutani H. Nat. Neurosci. 2005; 8: 1712-1719Crossref PubMed Scopus (190) Google Scholar, 7.Uesugi K. Oinuma I. Katoh H. Negishi M. J. Biol. Chem. 2009; 284: 6743-6751Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 8.Oinuma I. Katoh H. Negishi M. J. Neurosci. 2004; 24: 11473-11480Crossref PubMed Scopus (101) Google Scholar, 13.Turner L.J. Nicholls S. Hall A. J. Biol. Chem. 2004; 279: 33199-33205Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar).Structural biology has had a tremendous impact on our understanding of GTPase function and regulation (e.g. see Ref. 14.Vetter I.R. Wittinghofer A. Science. 2001; 294: 1299-1304Crossref PubMed Scopus (1349) Google Scholar). 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 (15.Bos J.L. Rehmann H. Wittinghofer A. Cell. 2007; 129: 865-877Abstract Full Text Full Text PDF PubMed Scopus (1270) Google Scholar). 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 (16.Canagarajah B. Leskow F.C. Ho J.Y. Mischak H. Saidi L.F. Kazanietz M.G. Hurley J.H. Cell. 2004; 119: 407-418Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), C2 in SynGAP (17.Pena V. Hothorn M. Eberth A. Kaschau N. Parret A. Gremer L. Bonneau F. Ahmadian M.R. Scheffzek K. EMBO Rep. 2008; 9: 350-355Crossref PubMed Scopus (71) Google Scholar), and a pleckstrin homology/lipid binding domain in p120GAP (18.Drugan J.K. Rogers-Graham K. Gilmer T. Campbell S. Clark G.J. J. Biol. Chem. 2000; 275: 35021-35027Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). 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 (19.Tong Y. Hughes D. Placanica L. Buck M. Structure. 2005; 13: 7-15Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 20.Tong Y. Hota P.K. Hamaneh M.B. Buck M. Structure. 2008; 16: 246-258Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) and x-ray structure of the RBD of human plexin-B1 show that this domain forms a dimeric ubiquitin-like structure (21.Tong Y. Chugha P. Hota P.K. Alviani R.S. Li M. Tempel W. Shen L. Park H.W. Buck M. J. Biol. Chem. 2007; 282: 37215-37224Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). 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 (22.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. Neuron. 2003; 39: 589-598Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). It is also known that Rac1 binding to the cytoplasmic plexin-B1 RBD increases ligand binding on the cell surface (23.Vikis H.G. Li W. Guan K.L. Genes Dev. 2002; 16: 836-845Crossref PubMed Scopus (103) Google Scholar). 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 (21.Tong Y. Chugha P. Hota P.K. Alviani R.S. Li M. Tempel W. Shen L. Park H.W. Buck M. J. Biol. Chem. 2007; 282: 37215-37224Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar).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.

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