Different Requirement for Rnd GTPases of R-Ras GAP Activity of Plexin-C1 and Plexin-D1

Plexins, comprising Plexin-A, -B, -C, and -D subfamilies, are receptors for semaphorins governing cell adhesion, migration, and axon guidance. Among plexin subfamilies, Plexin-A1 and Plexin-B1 have been shown to function as an R-Ras GAP, inducing repulsive responses, and the expression of R-Ras GAP activity requires the binding of Rnd1, a member of Rnd subfamily of Rho GTPases. However, signaling pathways of Plexin-D1 and Plexin-C1 still remain obscure. Here, we found that Plexin-D1 displayed R-Ras GAP activity and inhibited migration of COS-7 cells, and these actions required Rnd2, another Rnd subfamily GTPase. Rnd2 bound to Plexin-D1 in cortical neurons, and Sema3E/Plexin-D1-induced inhibition of axon outgrowth of cortical neurons required Rnd2 and down-regulation of R-Ras activity. On the other hand, Plexin-C1 displayed R-Ras GAP activity and inhibited cell migration of COS-7 cells without Rnd proteins. Therefore, R-Ras GAP activity is a common function of plexin subfamilies but the regulation of R-Ras GAP activity of plexins by Rnd proteins is different among plexin subfamilies. Plexins, comprising Plexin-A, -B, -C, and -D subfamilies, are receptors for semaphorins governing cell adhesion, migration, and axon guidance. Among plexin subfamilies, Plexin-A1 and Plexin-B1 have been shown to function as an R-Ras GAP, inducing repulsive responses, and the expression of R-Ras GAP activity requires the binding of Rnd1, a member of Rnd subfamily of Rho GTPases. However, signaling pathways of Plexin-D1 and Plexin-C1 still remain obscure. Here, we found that Plexin-D1 displayed R-Ras GAP activity and inhibited migration of COS-7 cells, and these actions required Rnd2, another Rnd subfamily GTPase. Rnd2 bound to Plexin-D1 in cortical neurons, and Sema3E/Plexin-D1-induced inhibition of axon outgrowth of cortical neurons required Rnd2 and down-regulation of R-Ras activity. On the other hand, Plexin-C1 displayed R-Ras GAP activity and inhibited cell migration of COS-7 cells without Rnd proteins. Therefore, R-Ras GAP activity is a common function of plexin subfamilies but the regulation of R-Ras GAP activity of plexins by Rnd proteins is different among plexin subfamilies. Semaphorins (Sema) 2The abbreviations used are: Sema, semaphorin; DMEM, Dulbecco's modified Eagle's medium; ECM, extracellular matrix; En, embryonic day n; FBS, fetal bovine serum; FN, fibronectin; GAP, GTPase-activating protein; GFP, green fluorescent protein; GST, glutathione S-transferase; HA, hemagglutinin; Myr, myristoylated; PBS, phosphate-buffered saline; Pn, n days of postnatal age; RBD, Ras-binding domain of c-Raf-1; shRNA, short hairpin RNA; WT, wild-type. are a large family of secreted or membrane-bound molecules that play central roles in axon guidance in developing nervous system (1Kolodkin A.L. Matthes D.J. Goodman C.S. Cell. 1993; 75: 1389-1399Abstract Full Text PDF PubMed Scopus (774) Google Scholar, 2Tamagnone L. Artigiani S. Chen H. He Z. Ming G.I. Song H. Chedotal A. Winberg M.L. Goodman C.S. Poo M. Tessier-Lavigne M. Comoglio P.M. Cell. 1999; 99: 71-80Abstract Full Text Full Text PDF PubMed Scopus (920) Google Scholar, 3Luo Y. Raible D. Raper J.A. Cell. 1993; 75: 217-227Abstract Full Text PDF PubMed Scopus (998) Google Scholar). In addition to the nervous system, they are widely expressed in embryonic and adult tissues and mediate diverse biological processes such as cardiac and skeletal development (4Behar O. Golden J.A. Mashimo H. Schoen F.J. Fishman M.C. Nature. 1996; 383: 525-528Crossref PubMed Scopus (502) Google Scholar), tumor growth, and metastasis (5Christensen C.R. Klingelhofer J. Tarabykina S. Hulgaard E.F. Kramerov D. Lukanidin E. Cancer Res. 1998; 58: 1238-1244PubMed Google Scholar), and the immune response (6Kumanogoh A. Kikutani H. J. Cell Sci. 2003; 116: 3463-3470Crossref PubMed Scopus (74) Google Scholar). The functions of semaphorins are mediated by plexins, which can be classified into four subfamilies: Plexin-A1–4, PlexinB1–3, Plexin-C1, and Plexin-D1 (2Tamagnone L. Artigiani S. Chen H. He Z. Ming G.I. Song H. Chedotal A. Winberg M.L. Goodman C.S. Poo M. Tessier-Lavigne M. Comoglio P.M. Cell. 1999; 99: 71-80Abstract Full Text Full Text PDF PubMed Scopus (920) Google Scholar, 7Nakamura F. Kalb R.G. Strittmatter S.M. J. Neurobiol. 2000; 44: 219-229Crossref PubMed Scopus (243) Google Scholar). PlexinA1–4, together with ligand-binding neuropilins, transduce repulsive signaling for class3 semaphorins (8Takahashi T. Fournier A. Nakamura F. Wang L.H. Murakami Y. Kalb R.G. Fujisawa H. Strittmatter S.M. Cell. 1999; 99: 59-69Abstract Full Text Full Text PDF PubMed Scopus (691) Google Scholar). Plexin-B1 is a receptor for Sema4D and mediates the Sema4D-induced growth cone collapse of hippocampal neurons and inhibition of various cell migration (9Swiercz J.M. Kuner R. Behrens J. Offermanns S. Neuron. 2002; 35: 51-63Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 10Barberis D. Artigiani S. Casazza A. Corso S. Giordano S. Love C.A. Jones E.Y. Comoglio P.M. Tamagnone L. Faseb. J. 2004; 18: 592-594Crossref PubMed Scopus (100) Google Scholar). Plexin-C1, also named VESPR due to its viral origin, serves as a receptor for the virally encoded SemaVA, and Plexin-C1 stimulation induces inhibition of integrin-mediated adhesion and chemokine-induced migration of dendritic cells (2Tamagnone L. Artigiani S. Chen H. He Z. Ming G.I. Song H. Chedotal A. Winberg M.L. Goodman C.S. Poo M. Tessier-Lavigne M. Comoglio P.M. Cell. 1999; 99: 71-80Abstract Full Text Full Text PDF PubMed Scopus (920) Google Scholar, 11Comeau M.R. Johnson R. DuBose R.F. Petersen M. Gearing P. VandenBos T. Park L. Farrah T. Buller R.M. Cohen J.I. Strockbine L.D. Rauch C. Spriggs M.K. Immunity. 1998; 8: 473-482Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 12Walzer T. Galibert L. Comeau M.R. Smedt T.D. J. Immunol. 2005; 174: 51-59Crossref PubMed Scopus (73) Google Scholar). Although it has been proposed that Sema7A is a ligand for Plexin-C1, the effect of Sema7A on neuronal cells is independent of Plexin-C1, and anti-Plexin-C1 monoclonal antibody did not affect Sema7A binding to Plexin-C1, suggesting that Sema7A is not likely to be a physiological binding partner of Plexin-C1 (13Holmes S. Downs A. Fosberry A. Hayes P. Michalovich D. Murdoch P. Moores K. Fox J. Deen K. Pettman G. Wattam T. Lewis C. Scand. J. Immunol. 2002; 56: 270-275Crossref PubMed Scopus (127) Google Scholar). Plexin-D1 was recently found to bind to Sema3E (14Gu C. Yoshida Y. Livet J. Reimert D.V. Mann F. Merte J. Henderson C.E. Jessell T.M. Kolodkin A.L. Ginty D.D. Science. 2005; 307: 265-268Crossref PubMed Scopus (417) Google Scholar). Plexin-D1 is expressed in vascular endothelium, and Sema3E acts as a repulsive cue for the endothelial cells (14Gu C. Yoshida Y. Livet J. Reimert D.V. Mann F. Merte J. Henderson C.E. Jessell T.M. Kolodkin A.L. Ginty D.D. Science. 2005; 307: 265-268Crossref PubMed Scopus (417) Google Scholar). Plexin-D1 is also highly expressed in ventrolateral cortical neurons, and Sema3E induces axonal repulsion of the neurons, guiding corticofugal and stritonigral projection (15Chauvet S. Cohen S. Yoshida Y. Fekrane L. Livet J. Gayet O. Segu L. Buhot M.C. Jessell T.M. Henderson C.E. Mann F. Neuron. 2007; 56: 807-822Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). The Rho family of small GTPases are signal transduction molecules that remodel the actin cytoskeleton and play fundamental roles in numerous cellular processes initiated by extracellular stimuli (16Negishi M. Katoh H. J. Biochem. 2002; 132: 157-166Crossref PubMed Scopus (104) Google Scholar, 17Etienne-Manneville S. Hall A. Nature. 2002; 420: 629-635Crossref PubMed Scopus (3768) Google Scholar). Activation of Rho family GTPases requires GDP-GTP exchange catalyzed by guanine nucleotide exchange factors, whereas the activation of GTPases is down-regulated by GTPase-activating proteins (GAPs), which stimulate the intrinsic GTPase activities. The Rnd proteins, Rnd1, Rnd2, and Rnd3/RhoE, comprise a new branch of Rho family in that they lack intrinsic GTPase activities (18Nobes C.D. Lauritzen I. Mattei M.G. Paris S. Hall A. Chardin P. J. Cell Biol. 1998; 141: 187-197Crossref PubMed Scopus (305) Google Scholar). Rnd1 and Rnd3 are known to inhibit RhoA activity through p190-RhoGAP activation (19Katoh H. Harada A. Mori K. Negishi M. Mol. Cell. Biol. 2002; 22: 2952-2964Crossref PubMed Scopus (70) Google Scholar, 20Wennerberg K. Forget M.A. Ellerbroek S.M. Arthur W.T. Burridge K. Settleman J. Der C.J. Hansen S.H. Curr. Biol. 2003; 13: 1106-1115Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar), while Rnd2 stimulates RhoA activity through binding to its effector, Pragmin (21Tanaka H. Katoh H. Negishi M. J. Biol. Chem. 2006; 281: 10355-10364Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), showing that Rnd1 and Rnd2 oppositely regulate RhoA activity. In addition, Rnd1 has been shown to stimulate signaling of Plexin-A1 and Plexin-B1 through direct binding to the cytoplasmic domains of the plexins (22Oinuma I. Katoh H. Harada A. Negishi M. J. Biol. Chem. 2003; 278: 25671-25677Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 23Zanata S.M. Hovatta I. Rohm B. Puschel A.W. J. Neurosci. 2002; 22: 471-477Crossref PubMed Google Scholar, 24Tong 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 (99) Google Scholar). We recently reported that Plexin-B1 encodes a GAP for R-Ras in the cytoplasmic domain, and the Plexin-B1-Rnd1 complex mediates the Sema4D-induced repulsive response in hippocampal neurons and inhibition of cell migration by stimulating the intrinsic GTPase activity of R-Ras (25Oinuma I. Ishikawa Y. Katoh H. Negishi M. Science. 2004; 305: 862-865Crossref PubMed Scopus (312) Google Scholar, 26Oinuma I. Katoh H. Negishi M. J. Cell Biol. 2006; 173: 601-613Crossref PubMed Scopus (77) Google Scholar, 27Ito Y. Oinuma I. Katoh H. Kaibuchi K. Negishi M. EMBO Rep. 2006; 7: 704-709Crossref PubMed Scopus (106) Google Scholar). Furthermore, Plexin-A1 has also been shown to exhibit R-Ras GAP activity, and Plexin-A1 and Rnd1 complex induces repulsive response through R-Ras GAP activity (28Toyofuku T. Yoshida J. Sugimoto T. Zhang H. Kumanogoh A. Hori M. Kikutani H. Nat. Neurosci. 2005; 8: 1712-1719Crossref PubMed Scopus (187) Google Scholar). The cytoplasmic domains of plexins are highly conserved among all plexin subfamilies. In contrast to Plexin-A1 and Plexin-B1, showing R-Ras GAP activity in the presence of Rnd1, signaling pathways of Plexin-C1 and Plexin-D1 remain to be elusive. We here demonstrate that Plexin-C1 exhibits R-Ras GAP activity without Rnd subfamily while Plexin-D1 requires Rnd2 for displaying R-Ras GAP activity. Plasmid Constructs and Antibodies-cDNAs encoding full-length of mouse Plexin-D1, cytoplasmic domains of mouse Plexin-C1 (Plexin-C1-cyt; amino acids 972–1575), and Plexin-D1 (Plexin-D1-cyt; amino acids 1293–1926) were obtained by PCR from E16 mouse brain. Cytoplasmic domains of Plexin-B1 and Plexin-A1 were obtained as described (22Oinuma I. Katoh H. Harada A. Negishi M. J. Biol. Chem. 2003; 278: 25671-25677Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). N-cyt and C-cyt fragments of Plexin-C1 (amino acids 972–1360 and 1361–1894, respectively) were amplified by PCR from the cytoplasmic domain of Plexin-C1. N-cyt and C-cyt fragments of Plexin-D1 (amino acids and respectively) were also amplified by of plexins were by of Plexin-D1 with signal and the was into cytoplasmic domains of Plexin-C1 and were into of and they were to signal glutathione proteins, these fragments were into or encoding extracellular domain of Plexin-B1 (amino acids and cytoplasmic domain of Plexin-C1 (amino acids was obtained by Rnd1, Rnd2, and green fluorescent Rnd1, Rnd2, and R-Ras were obtained as described (19Katoh H. Harada A. Mori K. Negishi M. Mol. Cell. Biol. 2002; 22: 2952-2964Crossref PubMed Scopus (70) Google Scholar, J. Katoh H. Mori K. Negishi M. Biochem. Res. 2000; 278: PubMed Scopus Google Scholar). Ras-binding domain of amino acids and Sema4D to were obtained as described (25Oinuma I. Ishikawa Y. Katoh H. Negishi M. Science. 2004; 305: 862-865Crossref PubMed Scopus (312) Google Scholar). and were obtained by PCR from and mouse and they were into Plexin-D1 cytoplasmic domain was obtained by PCR from and into The short hairpin for Rnd2 and Plexin-D1 were to of and were expressed by an expression The for Rnd2 and Plexin-D1 are as Rnd2 Rnd2 Plexin-D1 Plexin-D1 The for Rnd1 was obtained as described (25Oinuma I. Ishikawa Y. Katoh H. Negishi M. Science. 2004; 305: 862-865Crossref PubMed Scopus (312) Google Scholar). used were as a mouse monoclonal antibody a antibody a a mouse monoclonal antibody a monoclonal antibody a mouse monoclonal antibody a antibody Rnd2 has been described H. Katoh H. Ishikawa Y. Mori K. Negishi M. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). Cell and cells and were in modified fetal bovine and in and were with to the neurons from ventrolateral were from and with the neurons were and in or in in FBS, and the was with with and of axon growth, mouse or was to were by or and were a The was with in and with The were with and a or of Rnd2 and Plexin-D1, and were in of of of activity of family GTPases in cells was as described (25Oinuma I. Ishikawa Y. Katoh H. Negishi M. Science. 2004; 305: 862-865Crossref PubMed Scopus (312) Google Scholar). of GAP activity of full-length of Plexin-D1, COS-7 cells were on and in cells were with mouse or for of family GAP activity of or COS-7 cells and with were in for were with cell and of cells with were with phosphate-buffered and with cell and Cell were for The were for with of proteins and with for the were with the cell without the bound proteins were in and by and cells with were with cell bovine and Cell were for The were with for and with for The were with and bound proteins were by and the binding ventrolateral from was in cell the were with antibody for and with for The were with the and bound proteins were by and Cell were with in PBS, and in bovine The cells were of cells the of of with of In the mouse or was the of cells on the of the were with and cells on the were with The of cells through the were by the of the the cells were to the of cells cell migration was by the of cells to the of cells were in a of days in were with and in PBS, and with and growth was by the of the of and were with a system with a and axon was of Plexin-C1 and Plexin-D1 with Rnd and other (22Oinuma I. Katoh H. Harada A. Negishi M. J. Biol. Chem. 2003; 278: 25671-25677Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 24Tong 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 (99) Google Scholar, T. Yoshida J. Sugimoto T. Zhang H. Kumanogoh A. Hori M. Kikutani H. Nat. Neurosci. 2005; 8: 1712-1719Crossref PubMed Scopus (187) Google have shown that Plexin-A1 and Plexin-B1 with Rnd1 and that the are required for the expression of R-Ras GAP activity of R-Ras GAP activity of plexins and of Rnd1 are common signaling among plexin subfamilies, we the plexin subfamilies and Rnd GTPases by a with cytoplasmic domains of plexin subfamilies. reported S.M. Hovatta I. Rohm B. Puschel A.W. J. Neurosci. 2002; 22: 471-477Crossref PubMed Google Scholar, B. B. B. Hovatta I. Puschel A.W. 2000; PubMed Scopus Google Scholar), Plexin-A1 bound to Rnd1 and Rnd3 but not to Plexin-B1 was to bind to all Rnd subfamily GTPases as we have (22Oinuma I. Katoh H. Harada A. Negishi M. J. Biol. Chem. 2003; 278: 25671-25677Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Plexin-C1 and Plexin-D1 with Rnd subfamily GTPases full-length of Plexin-D1 with Rnd subfamily GTPases in cells, full-length of Plexin-D1 and Rnd proteins were in COS-7 shown in Plexin-D1 bound to Rnd2 among Rnd subfamily suggesting that Plexin-D1 Rnd2 among Rnd subfamilies as a binding of Rnd for the of R-Ras GAP of Plexin-D1 and Plexin-D1 and Plexin-C1 R-Ras GAP activity, we R-Ras by with in COS-7 COS-7 cells, Plexin-D1, Rnd proteins, and were and with Sema3E stimulation R-Ras activity in COS-7 cells Plexin-D1 and Rnd2, but the down-regulation of R-Ras activity was not in cells Plexin-D1 or with Rnd1 or Rnd3 and of Plexin-A1 and Plexin-B1 have been known to be an domain T. Strittmatter S.M. Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar), and of Plexin-B1 has been shown to exhibit R-Ras GAP activity in the of Sema4D I. Katoh H. Negishi M. J. Neurosci. 2004; PubMed Scopus (100) Google Scholar). We of cytoplasmic domain of Plexin-D1, signal shown in and expressed with Rnd2 R-Ras activity in the of but which has of and domains for GAP activity effect on the R-Ras activity. We Plexin-C1 R-Ras GAP activity. physiological ligand for Plexin-C1 has not been we extracellular domain of Plexin-B1 and domain of Plexin-C1, to Sema4D as a ligand The R-Ras GAP activity in response to Sema4D of the presence of Rnd GTPase and The of cytoplasmic domain of Plexin-C1, signal R-Ras activity, but to R-Ras activity, that Plexin-C1 R-Ras GAP activity without Rnd proteins and that R-Ras GAP activity is a common function among plexin subfamilies, while the regulation of R-Ras GAP activity by Rnd proteins is different among plexin subfamilies. the and the Cytoplasmic of Plexin-C1 and to the and domains of Plexin-B1 and and binding is required for the expression of R-Ras GAP activity of In addition, we have shown that the of the cytoplasmic of Plexin-B1 domain with the domain, and Rnd1 I. Katoh H. Negishi M. J. Neurosci. 2004; PubMed Scopus (100) Google Scholar). We the the cytoplasmic domain of COS-7 cells were with Rnd2 and used in a with amino acids and amino acids of Plexin-D1 cytoplasmic domain, which and shown in Rnd2 with N-cyt but not with We the the and N-cyt of Plexin-D1 was expressed in COS-7 cells with or without Rnd2, and a was with of N-cyt with in the of Rnd2, but was by Rnd2 We also Rnd2 the N-cyt and C-cyt of Plexin-C1. Although Rnd2 bound to N-cyt of Plexin-C1, N-cyt of Plexin-C1 did not with C-cyt of Plexin-C1 of Rnd2 binding and Plexin-D1 and Plexin-C1 Cell through R-Ras GAP signaling migration of a of cells, and we have reported that Sema4D the integrin-mediated cell migration through R-Ras GAP activity of Plexin-B1 I. Katoh H. Negishi M. J. Cell Biol. 2006; 173: 601-613Crossref PubMed Scopus (77) Google Scholar). We Plexin-D1 and Plexin-C1 the cell migration through R-Ras GAP activity. COS-7 cells Plexin-D1 and Rnd with R-Ras were on the of which were on the with of and Rnd2 the cell migration in the presence of Sema3E the whereas or with Rnd1 or Rnd3 did which R-Ras GAP activity, to the inhibition of cell In addition, Sema3E did not the cell migration in the presence of and We also the effect of Plexin-C1 on the cell migration and the cell migration without Rnd proteins, while did did not the cell migration in the presence of that Plexin-D1 and Plexin-C1 inhibit the cell migration through R-Ras GAP activity. Rnd2 for of the biological of Plexin-D1 and Rnd2, we the of Plexin-D1 with Rnd2 in Plexin-D1 was with Rnd2 by antibody but not by antibody used as a in ventrolateral of has been reported that signaling axon of neurons in the ventrolateral of the (15Chauvet S. Cohen S. Yoshida Y. Fekrane L. Livet J. Gayet O. Segu L. Buhot M.C. Jessell T.M. Henderson C.E. Mann F. Neuron. 2007; 56: 807-822Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). R-Ras GAP activity of is in of axon we the expression of Rnd2 in neurons by We to different of Rnd2, Rnd2 and Rnd2 and that Rnd2 and Rnd2 expression of Rnd2 in neurons The Rnd2 were with and the cells were in the presence or of Sema3E for Rnd2 and Rnd2 but not Rnd1 the inhibition of axon by Sema3E and the of the Plexin-D1 in inhibition of axon we Plexin-D1 and Plexin-D1 and the of these on inhibition of axon the inhibition of axon by that the inhibition is mediated by Plexin-D1 of R-Ras activity is required for the Sema3E/Plexin-D1-induced inhibition of axon cortical neurons were with or and in the presence or of Sema3E for of the of axon outgrowth and that induces the inhibition of axon outgrowth through R-Ras GAP activity, and inhibition requires Plexin-C1 and Plexin-D1 GAP subfamily and We GAP activity of Plexin-C1 and Plexin-D1 for and by a with in COS-7 cells and a GAP activity for but not for while GAP activity was not with its also a GAP activity for in the presence of Rnd2 but not for while GAP activity was not with its Plexin-C1 and Plexin-D1 GAP activity for R-Ras subfamily and Plexin-A1 and Plexin-B1 function as an R-Ras GAP, inducing repulsive responses, and R-Ras GAP activity of these plexins requires Rnd1 binding to the cytoplasmic domains of the plexins (25Oinuma I. Ishikawa Y. Katoh H. Negishi M. Science. 2004; 305: 862-865Crossref PubMed Scopus (312) Google Scholar, T. Yoshida J. Sugimoto T. Zhang H. Kumanogoh A. Hori M. Kikutani H. Nat. Neurosci. 2005; 8: 1712-1719Crossref PubMed Scopus (187) Google Scholar). In the we have that Plexin-C1 and Plexin-D1 R-Ras GAP activity, and that the expression of R-Ras GAP activity of Plexin-D1 requires Rnd2, another member of Rnd while that of Plexin-C1 not Rnd proteins. The cytoplasmic domains of plexins have highly conserved and which and (25Oinuma I. Ishikawa Y. Katoh H. Negishi M. Science. 2004; 305: 862-865Crossref PubMed Scopus (312) Google Scholar). are for GAP activity, and of the to in the of Plexin-A1 and Plexin-B1 the receptors in R-Ras GAP activities (25Oinuma I. Ishikawa Y. Katoh H. Negishi M. Science. 2004; 305: 862-865Crossref PubMed Scopus (312) Google Scholar). I. H. and M. are also conserved in the cytoplasmic domains of Plexin-C1 and We here that of Plexin-C1 and Plexin-D1 have to inhibit not R-Ras activity but also cell Plexin-C1 and Plexin-D1 R-Ras GAP activity and inhibit cell these and of Plexin-A1 and Plexin-B1, the highly conserved cytoplasmic domains of plexins R-Ras GAP and down-regulation of R-Ras activity through R-Ras GAP domains is likely to be a common signaling for repulsive R-Ras GAP activity of Plexin-A1 and Plexin-B1 requires Rnd1 binding to cytoplasmic domains and the binding domains are and We reported that the of the cytoplasmic domain of Plexin-B1 domain with the domain, and Rnd1 by binding to the and In addition, of R-Ras GAP and is required for the expression of R-Ras GAP activity I. Katoh H. Negishi M. J. Neurosci. 2004; PubMed Scopus (100) Google Scholar). the is a domain for the expression of R-Ras GAP activity. We here that among Rnd subfamily GTPases Plexin-D1 requires Rnd2 for R-Ras GAP activity and inhibition of cell In addition, we that Rnd2 to the cytoplasmic domain of Plexin-D1 and the of the domain and these it is that Rnd2 to the the and domains of Plexin-D1 and induces of R-Ras GAP inducing R-Ras GAP activity and inhibition of cell On the other hand, we that Plexin-C1 not Rnd subfamily for R-Ras GAP activity and inhibition of cell migration, and that the domain not with the domain, that R-Ras GAP domains of Plexin-C1 in the of Rnd proteins. is to R-Ras GAP activity of Plexin-C1. Therefore, the regulation of R-Ras GAP activity of plexin subfamilies is different in on Rnd subfamily Plexin-A1 and Plexin-B1 Rnd1, Plexin-D1 requires Rnd2, and Plexin-C1 not Rnd proteins. In contrast to highly conserved and the and domains are not conserved among plexin subfamilies. of the diverse regulation of R-Ras GAP activity of Plexin-D1 is expressed in the developing central nervous system B. H. 2002; PubMed Scopus (77) Google Scholar, P. T. M. Neuron. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar), and Rnd2 is also expressed in and an in developing central nervous system K. Y. Katoh H. T. Negishi M. Neurosci. Res. 2006; PubMed Scopus Google Scholar, M. H. T. K. T. Sugimoto T. Mol. Res. 1999; PubMed Scopus Google Scholar, B. Y. D. A. Res. 2005; PubMed Scopus Google Scholar). was recently reported to regulate axon in in (15Chauvet S. Cohen S. Yoshida Y. Fekrane L. Livet J. Gayet O. Segu L. Buhot M.C. Jessell T.M. Henderson C.E. Mann F. Neuron. 2007; 56: 807-822Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Sema3E cortical axon through Plexin-D1 (15Chauvet S. Cohen S. Yoshida Y. Fekrane L. Livet J. Gayet O. Segu L. Buhot M.C. Jessell T.M. Henderson C.E. Mann F. Neuron. 2007; 56: 807-822Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). We here that Rnd2 and R-Ras the inhibition of axon growth of cortical neurons by of Rnd2 with Plexin-D1 is for the R-Ras GAP activity of Plexin-D1 in COS-7 that of R-Ras by the complex is for the inhibition of axon outgrowth in cortical In we have that Plexin-D1 R-Ras GAP activity in with Rnd2 while Plexin-C1 the activity without Rnd proteins. In addition, the of Rnd1 for R-Ras GAP activity of Plexin-A1 and of Rnd subfamily GTPases be for the expression of R-Ras GAP activity of plexin subfamilies. We L. H. and H. Kikutani for Plexin-B1, and the of Sema4D expression We also Y. and G. of of for