Rapostlin Is a Novel Effector of Rnd2 GTPase Inducing Neurite Branching

Rho family GTPases are central regulators of neuronal morphology. Recently, Rnd proteins, Rnd1, Rnd2, and Rnd3/RhoE, have been identified as new members of Rho family GTPases. Of these, Rnd2 is specifically expressed in neurons in brain; however, the signaling pathways of Rnd2 are not known. Here we have performed a yeast two-hybrid screen using Rnd2 as a bait and identified a novel Rnd2-effector protein, expressed predominantly in brain. We named it Rapostlin (apostle of Rnd2). Rapostlin has two functional domains, Fer-CIP4 homology (FCH) domain at the amino terminus and SH3 (Src homology 3) domain at the carboxyl terminus. In in vitro binding assays, Rapostlin specifically binds to Rnd2 among the Rho family GTPases in a GTP-dependent manner, and the Rnd2-binding domain of Rapostlin is localized between FCH and SH3 domains. Rapostlin directly binds to microtubules, and the amino-terminal region containing the FCH domain of Rapostlin is essential for this interaction. In PC12 cells, Rapostlin induces neurite branching in response to Rnd2, and at least the amino-terminal region of Rapostlin is necessary for this activity. Therefore, Rapostlin is the first effector of Rnd2, regulating neurite branch formation. Rho family GTPases are central regulators of neuronal morphology. Recently, Rnd proteins, Rnd1, Rnd2, and Rnd3/RhoE, have been identified as new members of Rho family GTPases. Of these, Rnd2 is specifically expressed in neurons in brain; however, the signaling pathways of Rnd2 are not known. Here we have performed a yeast two-hybrid screen using Rnd2 as a bait and identified a novel Rnd2-effector protein, expressed predominantly in brain. We named it Rapostlin (apostle of Rnd2). Rapostlin has two functional domains, Fer-CIP4 homology (FCH) domain at the amino terminus and SH3 (Src homology 3) domain at the carboxyl terminus. In in vitro binding assays, Rapostlin specifically binds to Rnd2 among the Rho family GTPases in a GTP-dependent manner, and the Rnd2-binding domain of Rapostlin is localized between FCH and SH3 domains. Rapostlin directly binds to microtubules, and the amino-terminal region containing the FCH domain of Rapostlin is essential for this interaction. In PC12 cells, Rapostlin induces neurite branching in response to Rnd2, and at least the amino-terminal region of Rapostlin is necessary for this activity. Therefore, Rapostlin is the first effector of Rnd2, regulating neurite branch formation. The organization of the nervous system is a complex and orchestrated process. Neurons migrate to their characteristic locations, extend axons and dendrites toward proper target regions, and form synaptic connections with appropriate partners. These dynamic morphological changes of neurons are largely decided by the cytoskeletal organization. Rho family GTPases, consisting of Rho, Rac, and Cdc42, have been implicated in the regulation of the cytoskeleton and subsequent morphological changes in various cells, such as formations of actin stress fibers, lamellipodia, and filopodia, respectively in fibroblasts (1Hall A. Science. 1998; 279: 509-514Google Scholar). These actions of Rho family GTPases are mediated by a variety of downstream effectors (2Bishop A.L. Hall A. Biochem. J. 2000; 348: 241-255Google Scholar). In neuronal cells, Rho family GTPases have been shown to be involved in the regulation of neuronal cell morphology including neuritogenesis. Rho activation induces the inhibition of neuritogenesis, the collapse of the growth cone, and the retraction of neurites (3Jalink K. Van Corven E.J. Hengeveld T. Morii N. Narumiya S. Moolenaar W.H. J. Cell Biol. 1994; 126: 801-810Google Scholar, 4Kozma R. Sarner S. Ahmed S. Lim L. Mol. Cell. Biol. 1997; 17: 1201-1211Google Scholar, 5Yamaguchi Y. Katoh H. Yasui H. Mori K. Negishi M. J. Biol. Chem. 2001; 276: 18977-18983Google Scholar). These morphological actions of Rho have been shown to be mediated by Rho-kinase, a downstream effector of Rho (6Hirose M. Ishizaki T. Watanabe N. Uehata M. Kranenburg O. Moolenaar W.H. Matsumura F. Maekawa M. Bito H. Narumiya S. J. Cell Biol. 1998; 141: 1625-1636Google Scholar, 7Katoh H. Aoki J. Ichikawa A. Negishi M. J. Biol. Chem. 1998; 273: 2489-2492Google Scholar). On the other hand, Rac and Cdc42 are involved in the formation of filopodia and lamellipodia of the growth cone and in the outgrowth of neurites (4Kozma R. Sarner S. Ahmed S. Lim L. Mol. Cell. Biol. 1997; 17: 1201-1211Google Scholar). Effectors of Rac and Cdc42, PAK1 and neural Wiskott-Aldrich syndrome protein (N-WASP), have been shown to play roles in these actions (8Banzai Y. Miki H. Yamaguchi H. Takenawa T. J. Biol. Chem. 2000; 275: 11987-11992Google Scholar, 9Daniels R.H. Hall P.S. Bokoch G.M. EMBO J. 1998; 17: 754-764Google Scholar). The less studied members of Rho family GTPases, such as RhoG and TC10, are also involved in the regulation of neuritogenesis (10Katoh H. Yasui H. Yamaguchi Y. Aoki J. Fujita H. Mori K. Negishi M. Mol. Cell. Biol. 2000; 20: 7378-7387Google Scholar, 11Tanabe K. Tachibana T. Yamashita T. Che Y.H. Yoneda Y. Ochi T. Tohyama M. Yoshikawa H. Kiyama H. J. Neurosci. 2000; 20: 4138-4144Google Scholar), suggesting that a variety of Rho family GTPases participate in the regulation of neuronal morphology. Recently, a new branch of Rho family GTPases, the Rnd subfamily, consisting of Rnd1, Rnd2, and Rnd3, has been identified (12Nobes C.D. Lauritzen I. Mattei M.G. Paris S. Hall A. Chardin P. J. Cell Biol. 1998; 141: 187-197Google Scholar). Rnd1 is mainly expressed in brain and liver, whereas Rnd3 is expressed ubiquitously (12Nobes C.D. Lauritzen I. Mattei M.G. Paris S. Hall A. Chardin P. J. Cell Biol. 1998; 141: 187-197Google Scholar, 13Foster R. Hu K.Q. Lu Y. Nolan K.M. Thissen J. Settleman J. Mol. Cell. Biol. 1996; 16: 2689-2699Google Scholar). Unlike other Rho family GTPases, Rnd1 and Rnd3 possess very low intrinsic GTPase activity and constitutively bind to GTP (12Nobes C.D. Lauritzen I. Mattei M.G. Paris S. Hall A. Chardin P. J. Cell Biol. 1998; 141: 187-197Google Scholar, 13Foster R. Hu K.Q. Lu Y. Nolan K.M. Thissen J. Settleman J. Mol. Cell. Biol. 1996; 16: 2689-2699Google Scholar, 14Guasch R.M. Scambler P. Jones G.E. Ridley A.J. Mol. Cell. Biol. 1998; 18: 4761-4771Google Scholar). Expression of Rnd1 or Rnd3 in fibroblasts results in loss of actin stress fibers and focal adhesions, indicating the antagonistic effect on the Rho-regulated signaling pathway (12Nobes C.D. Lauritzen I. Mattei M.G. Paris S. Hall A. Chardin P. J. Cell Biol. 1998; 141: 187-197Google Scholar). We have demonstrated that Rnd1 induced neurite process formation, probably due to the inhibition of the Rho-regulated signaling pathway (15Aoki J. Katoh H. Mori K. Negishi M. Biochem. Biophys. Res. Commun. 2000; 278: 604-608Google Scholar), and then we have identified a novel Rnd GTPase-interacting protein, Socius, which is involved in the Rnd1-induced disassembly of actin stress fibers (16Katoh H. Harada A. Mori K. Negishi M. Mol. Cell. Biol. 2002; 22: 2952-2964Google Scholar). In contrast to Rnd1 and Rnd3, little is known about Rnd2, although Rnd2 is expressed specifically in neurons in brain (17Nishi M. Takeshima H. Houtani T. Nakagawara K. Noda T. Sugimoto T. Brain Res. Mol. Brain Res. 1999; 67: 74-81Google Scholar), suggesting that Rnd2 plays an important role in neuronal functions. We have recently demonstrated that Vps4-A, involved in the endosomal vesicle trafficking, is the first Rnd2-binding protein but is not an effector of Rnd2 since it binds to both active and negative forms of Rnd2 (18Tanaka H. Fujita H. Katoh H. Mori K. Negishi M. Biochem. J. 2002; 365: 349-353Google Scholar). To dissect the molecular mechanism of neuronal functions of Rnd2, it is urgent to identify downstream effectors of Rnd2. Here we performed a yeast two-hybrid screen to identify effectors of Rnd2 and found a novel effector protein of Rnd2, Rapostlin. Rapostlin specifically binds to Rnd2 among the Rho family GTPases in a GTP-dependent manner. Rapostlin directly binds to microtubules, and the amino-terminal region of Rapostlin is essential for this interaction. In PC12 cells, Rapostlin in concert with constitutively active Rnd2 induces neurite branching, and at least the amino-terminal region of Rapostlin is necessary for this activity. Therefore, this is the first protein to be identified as an effector of Rnd2. Wild-type and mutant forms of Rnd2, wild-type Rnd1, Rnd3, RhoA, Rac1, and Cdc42 were obtained as described previously (10Katoh H. Yasui H. Yamaguchi Y. Aoki J. Fujita H. Mori K. Negishi M. Mol. Cell. Biol. 2000; 20: 7378-7387Google Scholar, 16Katoh H. Harada A. Mori K. Negishi M. Mol. Cell. Biol. 2002; 22: 2952-2964Google Scholar, 19Fujita H. Katoh H. Hasegawa H. Yasui H. Aoki J. Yamaguchi Y. Negishi M. Biochem. J. 2000; 346: 617-622Google Scholar). For the yeast two-hybrid screen, the cDNA encoding Rnd2Ser-224, lacking a farnesylation site in the carboxyl-terminal CAAX motif by a substitution of cysteine to serine, was fused to the GAL4-DNA binding domain in the yeast expression vector pAS2-1 (Clontech). For purification of recombinant proteins, cDNA of Rnd2 was subcloned into pAcG2T (Pharmingen), whereas cDNAs of Rnd1, Rnd3, RhoA, Rac1, Cdc42, and various deletion mutants of Rapostlin, ΔN1 (amino acids 335–620), ΔN2 (amino acids 399–620), and ΔN3 (amino acids 552–620) were subcloned into pGEX-4T-2 (Amersham Biosciences). For the mammalian two-hybrid screen, cDNAs encoding small GTPases, lacking a prenylation site in the carboxyl-terminal CAAX motif by a substitution of cysteine to serine, were fused to the GAL4-DNA binding domain in the mammalian expression vector pBIND (Promega), whereas Rapostlin was fused to VP16 in mammalian expression vector pACT (Promega). For expression in mammalian cells, cDNAs encoding green fluorescent protein (GFP) 1The abbreviations used are: GFP, green fluorescent protein; GST, glutathione S-transferase; GTPγS, guanosine 5′-3-O-(thio)triphosphate; pAb, polyclonal antibody; mAb, monoclonal antibody; PBS, phosphate-buffered saline; FCH, Fer-CIP4 homology; SH3, Src homology 3; F-actin, filamentous actin. and mutant forms of Rnd2 were subcloned into mammalian expression vector pcDNA3 (Invitrogen), whereas cDNAs encoding the full-length Rapostlin (amino acids 2–620) and Rapostlin ΔN1 (amino acids 335–620) were subcloned into pcDNA3 encoding an initiating Met followed by the Myc epitope tag sequence at the amino terminus. A yeast two-hybrid screen was performed in yeast strain Y190 (Clontech) using a rat brain Matchmaker cDNA library fused to the GAL4 activation domain in the pACT2 (Clontech). Approximately 1.5 × 107 yeast colonies were screened for their ability to grow on selective medium lacking histidine, leucine, tryptophan, and uracil and containing 10 mm3-aminotriazole. Colonies that grew successfully were replated and screened by β-galactosidase assay. From positive yeast colonies, prey plasmids were isolated, and then yeast Y190 was cotransformed with these cDNAs and pAS2-1-Rnd2Ser-224 to confirm the interaction. Positive clones were sequenced using an ABI PRISM 310 DNA sequencer. Northern blot analysis was performed as described previously (10Katoh H. Yasui H. Yamaguchi Y. Aoki J. Fujita H. Mori K. Negishi M. Mol. Cell. Biol. 2000; 20: 7378-7387Google Scholar). Total RNA from various rat tissues was isolated by using an Isogen RNA isolation kit (Nippon-gene, Tokyo, Japan), and 20 μg of total RNA was separated by electrophoresis on a 1.5% agarose gel and transferred onto a nylon membrane (Biodyne; Pall Biosupport Division). The membrane was hybridized at 65 °C for 18 h in a mixture containing 6× SSC (1× SSC is 150 mm NaCl and 15 mm sodium citrate), 0.5% SDS, 5× Denhardt's solution, 100 μg/ml denatured salmon sperm DNA, and a32P (PerkinElmer Life Sciences, NEG-513Z, 6000 Ci/mmol)-labeled probe encoding Rapostlin. The membrane was dried and autoradiographed with an x-ray film for 10 h. Antibody for Rnd2 was raised against bacterially expressed carboxyl-terminal Rnd2 insert region (GHRQLRRTDSRRGMQRSAQLSGRPDRGNEGEI), and the specific antibody was purified with a peptide (GRPDRGNEGEI)-conjugated affinity column. GlutathioneS-transferase (GST)-fused Rnd2 protein was prepared from Sf9 cells, whereas GST, GST fused with other small GTPases (Rnd1, Rnd3, RhoA, Rac1, and Cdc42), and various GST-fused deletion mutants of Rapostlin proteins (Rapostlin ΔN1, ΔN2, and ΔN3) were prepared from Escherichia coli, as described previously (7Katoh H. Aoki J. Ichikawa A. Negishi M. J. Biol. Chem. 1998; 273: 2489-2492Google Scholar, 20Self A.J. Hall A. Methods Enzymol. 1995; 256: 3-10Google Scholar). Recombinant GST or GST-fused small GTPases were loaded with 1 mm GDP or GTPγS in buffer A (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 10 mm MgCl2, and 1 mm dithiothreitol) at 30 °C for 30 min, and the reaction was stopped by addition of MgCl2 to a final concentration of 20 mm (21Manser E. Leung T. Lim L. Methods Enzymol. 1995; 256: 130-139Google Scholar). Dot-blot assays were performed as described previously (22Joberty G. Perlungher R.R. Macara I.G. Mol. Cell. Biol. 1999; 19: 6585-6597Google Scholar). GST-Rapostlin ΔN1 was cleaved with thrombin. Rapostlin ΔN1 (5 μg), bovine serum albumin (10 μg), GST (7 μg), or GST-fused deletion mutants of Rapostlin (GST-Rapostlin ΔN1, ΔN2, or ΔN3) (7 μg) was spotted onto nitrocellulose membranes and to for 1 h at The membranes were in buffer A containing low for 1 h at The membranes were then for 1 h at °C in buffer A containing μg of GST or GST-fused small GTPases with GDP or The membranes were with buffer A and then with low in containing polyclonal antibody or to These were by using and an kit and were in medium containing bovine mm 100 and containing at PC12 were in medium containing bovine mm 100 and containing at were with cDNAs using for and PC12 cells, for cells, or for to the PC12 were with growth in medium the in at a of × were with μg of pBIND vector encoding both for and small GTPases, μg of pACT vector encoding Rapostlin, and μg of the vector encoding the binding in a h the were in 100 of buffer (Promega). and were with of cell by using the system and a activity was as the of activity activity. of Rapostlin and was performed as described previously Moolenaar W.H. Kranenburg O. J. Biol. Chem. 2001; 276: Scholar). in at a of × were with Rapostlin or Rapostlin ΔN1 and with buffer containing and 0.5% sodium the were at × for 1 h at °C in a were for 30 at °C with microtubules, which were using the kit were then by at × for 1 h. and were to and transferred onto a membrane The membrane was with low in and with monoclonal antibody or These were by using and an and PC12 were in at a of × onto mm in and in at a of × onto 18 mm in h the on were with phosphate-buffered and then with in for 15 been with mm in for 10 min, were with in for 10 and with bovine serum in for 30 For of Rnd2, were with in for 1 h followed by with a in for 1 h. For of Rapostlin or Rapostlin ΔN1, were with or in for 1 h followed by with a and a in for 1 h. were with followed by with a in for 1 h. actin was with in for 1 h. on were in containing in and by an system with a and a × activity was as described previously G. K. J. Neurosci. 1998; 18: Scholar). PC12 neurites were from the neurites were as The branching activity was as the of the total of the to the total of the neurites from 20 cells, that the of We this neurites as neurites were To into the downstream signaling of Rnd2, we a yeast two-hybrid screen using Rnd2 as a positive clones were isolated from a rat brain cDNA that of these a protein to protein In the yeast two-hybrid this specifically with Rnd2 the Rnd and with the studied Rho GTPases, RhoA, Rac1, and we named it Rapostlin (apostle of The of the Met of Rapostlin is to the sequence M. J. Biol. Chem. Scholar). Rapostlin and proteins sequence and have regions, a Fer-CIP4 homology (FCH) domain at the amino terminus and an SH3 domain at the carboxyl terminus Rapostlin sequence with which was isolated previously as a Cdc42 effector protein P. Biol. 1997; Scholar). Rapostlin has a insert which is in A region is found between FCH and the insert region of Rapostlin. To the of Rapostlin, we a Northern Rapostlin were predominantly expressed in and expression was in and blot analysis of were isolated from the rat and 20 μg of RNA was in was cDNA encoding the of Rapostlin as a The Rapostlin and are by The of 18 and are by To confirm the of Rapostlin and Rnd2, we Rapostlin lacking the amino-terminal region (Rapostlin was spotted onto a nitrocellulose and the membrane was with or to Rapostlin ΔN1, but not We the of Rapostlin ΔN1 with GST-fused Rnd and known Rho family GTPases and with GTPγS, and we found that specifically to Rapostlin ΔN1 These results that Rnd2 specifically binds to Rapostlin among the Rho family GTPases in a GTP-dependent manner. To the Rnd2-binding domain of Rapostlin, we a by using various GST-fused deletion mutants of Rapostlin (GST-Rapostlin ΔN1, GST-Rapostlin ΔN2, and GST-Rapostlin Rnd2 to Rapostlin ΔN1, to Rapostlin ΔN2, but not bind to Rapostlin ΔN3 These results that Rapostlin is a novel effector of Rnd2 and that the region of Rapostlin (amino acids is important for the binding of Rnd2. to the in of Rapostlin with Rnd2 in mammalian cells, we a mammalian two-hybrid assay. that Rapostlin binds to but not to Rnd2, suggesting that Rnd2 active or We then a constitutively active Rnd2 to a constitutively active in and a negative Rnd2 to a negative in We with pBIND vector encoding small GTPases, pACT vector encoding Rapostlin, and vector encoding the Rapostlin to and to wild-type Rnd2, whereas it not bind to wild-type Rnd1, or wild-type To Rnd2 and Rapostlin are we expressed Rapostlin, or both in In the cells, to be localized to including membrane In of and prepared from the that was mainly localized in the not In the cells, Rapostlin was localized in the were with and Rapostlin, were in a region with These results that Rapostlin binds to constitutively active Rnd2 in mammalian We the of Rnd2 and Rapostlin on in In the cells, Rapostlin was localized in whereas Rapostlin with induced a in the were with and Rapostlin, induced of to the and Rapostlin were These results that the of with Rapostlin induces the of The amino-terminal region of has been to bind to L. J. Biol. Chem. 2000; 275: Scholar). To the amino-terminal region of Rapostlin is essential for the of Rapostlin to microtubules, we expressed Rapostlin or Rapostlin ΔN1 in and binding assays be by binding of Rapostlin to be in these In the of microtubules, Rapostlin to but Rapostlin ΔN1 not These results that Rapostlin binds to microtubules, directly or and that the amino-terminal region of Rapostlin is essential for the interaction. Rnd2 and Rapostlin are predominantly expressed in and in that Rnd2 was expressed specifically in neurons in brain (17Nishi M. Takeshima H. Houtani T. Nakagawara K. Noda T. Sugimoto T. Brain Res. Mol. Brain Res. 1999; 67: 74-81Google Scholar). To Rnd2 and Rapostlin were involved in the regulation of neuronal cell we expressed Rnd2, Rapostlin, or both in the growth PC12 a neurites Expression of a but neurites Expression of Rapostlin or Rapostlin ΔN1 induces or from neurites and On the other hand, of and Rapostlin induced from neurites the of the of Rnd2 and Rapostlin on the branching activity. the branching activity. be mediated by Rapostlin since we the expression of Rapostlin in PC12 by not Rnd2, with Rapostlin the branching but negative Rnd2, to the activity in the of Rapostlin and Therefore, with Rapostlin and induces the branching activity. to the of the amino-terminal region of Rapostlin, we expressed with Rapostlin ΔN1 and These not neurite branching, suggesting that the amino-terminal region of Rapostlin was at least necessary for the neurite Rnd2 is a new of Rho family GTPases, which is specifically expressed in neurons in brain. To identify downstream effectors of Rnd2, we performed a yeast two-hybrid screen with Rnd2 as a bait and isolated a novel effector of Rnd2, Rapostlin, which induces neurite branching in neuronal Rapostlin has a protein, which was identified previously as of the proteins P. EMBO J. 1996; Scholar). Recently, was to with G. R. M. S. R.M. J. A. S. A. 2001; Scholar), suggesting that and Rapostlin are involved in Rapostlin to a Cdc42 effector protein P. Biol. 1997; Scholar), and has domain to an FCH domain at the amino terminus and SH3 domain at the carboxyl terminus. A that the region of for binding to Cdc42 is localized to amino acids L. J. Biol. Chem. 2000; 275: Scholar). has a domain (amino acids with homology to the Rho binding of and and this domain is also in Rapostlin. In this we that Rapostlin not with Cdc42 or and that the insert region is for the Rnd2 binding to Rapostlin. These results that Rapostlin is a specific effector of Rnd2 but not of Cdc42 or The SH3 domain of with Wiskott-Aldrich syndrome protein, a of the actin whereas the amino-terminal region including the FCH domain binds to microtubules, suggesting that is a between the actin cytoskeleton and L. J. Biol. Chem. 2000; 275: Scholar). has been shown to be involved in formation of in S. K. M. J. Cell 2000; Scholar). Rapostlin has two functional domains, amino-terminal region and SH3 of involved in the of both and actin In this we that Rapostlin binds to in the and that Rapostlin induces of in response to Rnd2 in Rapostlin as a between the actin cytoskeleton and Rho family GTPases are central regulators of neuronal including neurite formation, and branching L. Neurosci. 2000; Scholar). The process of neurite branching is a dynamic of branch addition and branch as as branch and In the of branch formation, filopodia and lamellipodia are from the from the and and then are the are with microtubules, indicating that branch formation is by the of both the actin cytoskeleton and has been shown that negative forms of and Cdc42 to a in the of S. L. J. Neurosci. 1999; 19: Scholar). In constitutively active to branch Van L. Neurosci. 2000; Scholar). In contrast to and Cdc42, activation to a in branching L. J. Neurosci. 2000; 20: Scholar). Therefore, Rac and Cdc42 a positive effect on branching and whereas Rho is a negative for branch formation. molecular of branch formation by these Rho family GTPases are not In this we that Rapostlin induces neurite branching in response to Rnd2 in neuronal The binding of active Rnd2 to Rapostlin the branch formation negative Rnd2, to the branch formation. Therefore, Rapostlin is a downstream effector of Rnd2 for the branch formation. we that the amino-terminal region of Rapostlin is necessary for the neurite branching induced by Rapostlin in concert with Rnd2 in PC12 In the this amino-terminal region of Rapostlin is essential for the binding of Rapostlin to microtubules, suggesting that the of Rapostlin with is for the neurite branching induced by Rnd2 and Rapostlin. of Rho family GTPases between and active Unlike Rho family GTPases, Rnd1 and Rnd3 have been shown to be constitutively active forms due to at the of to and serine, intrinsic GTPase activity (12Nobes C.D. Lauritzen I. Mattei M.G. Paris S. Hall A. Chardin P. J. Cell Biol. 1998; 141: 187-197Google Scholar, 13Foster R. Hu K.Q. Lu Y. Nolan K.M. Thissen J. Settleman J. Mol. Cell. Biol. 1996; 16: 2689-2699Google Scholar, 14Guasch R.M. Scambler P. Jones G.E. Ridley A.J. Mol. Cell. Biol. 1998; 18: 4761-4771Google Scholar). In this of Rnd2 is by Here we that Rapostlin binds to Rnd2, but not to Rnd2, in a and that Rapostlin binds to to a constitutively active in whereas Rapostlin not bind to to a negative in we that Rapostlin induces neurite branching in response to but not to in PC12 Therefore, Rnd2 has an ability to active or negative and the active form of Rnd2 binds to Rapostlin, neurite of GTPase activity of Rnd2 is in in In we have identified a novel downstream effector of Rnd2, Rapostlin, involved in the neurite branch formation. Rapostlin is the first to be identified as an effector of Rnd2. have not been about Rnd2 for participate in the signaling of Rnd2. on the of proteins to of the pathway and the regulation of neuronal morphology.