a1atvto aha novn h p75NTR the involving novel pathway a define activation and activation Rac1 GTPase role small crucial in a cleavage demonstrate p75NTR for results Rac1 novel Our a spreading. p75NTR-dependent cell as and mediates , activation that adaptor family partner Cas NRAGE-binding a NEDD9, identify screen, Ato o orsodne([email protected]) correspondence for *Author Canada. Canada. 2B4, 2B4, H3A H3A Quebec Quebec Montreal, Street, University 3801 University, McGill n ciaino nNAEadND9sgaigcascade signaling NEDD9 Zeinieh and Michele NRAGE an of cleavage activation receptor and requires activation Rac1 p75NTR-dependent ARTICLE RESEARCH ß eevd2 eray21;Acpe 1Nvme 2014 November 21 Accepted 2014; February 28 Received 2 signaling p75NTR in 2003, 2012), 1 al., Tohyama., et al., et Sun and Coulson 2010, 2008, al., al., Yamashita et et Park Harrington 2002, 2008, 2005, 2002, al., al., al., et et Domeniconi notably accessory Yamashita et 1999, most al., an GTPases, et Harrington (Yamashita have ligand-binding small Rac1 as studies of and other RhoA Several importance acts 2006). for the to Reichardt., demonstrated p75NTR binding component in (reviewed neurotrophin signaling others, receptors the and in by receptor activated 2010). and, Bothwell., are and p75NTR these superfamily, Schecterson of in Some (TNFR) (reviewed growth apoptosis, and collapse include receptor outgrowth that cone neurite events differentiation, cellular factor signaling, of survival array of necrosis an member in a participates tumor tumor 16), as member known superfamily the also receptor (p75NTR, factor receptor necrosis neurotrophin p75 The INTRODUCTION CasL, , RhoA, NEDD9 Rac1, ADAM17, NGF, WORDS: KEY intracellular p75NTR the of generation (p75NTR and domain COS7 p75NTR p75NTR-dependent and of the ADAM17- cleavage of on activation that dependent the is demonstrate on phenotype relies we that area and surface Rac1, cell causes expression in p75NTR increase by cells, large activated COS7 a in cascades that signaling show We characterize p75NTR. signaling to established bioassay have its we novel of this, a address aspects in To unclear. fundamental implicated remain tumor but is mechanisms as events, 16) known cellular member also superfamily diverse receptor (p75NTR, factor receptor necrosis neurotrophin p75 The ABSTRACT NEDD9. RG as nw sMGD)at ontemo the of downstream acts MAGED1) as known p75NTR (also NRAGE eateto ahlg,MGl nvriy 81Uiest tet Montreal, Street, University 3801 University, McGill Pathology, of Institute, Department Neurological Montreal Neurosurgery, and Neurology of Department 05 ulse yTeCmayo ilgssLd|Junlo elSine(05 2,4749doi:10.1242/jcs.152173 447–459 128, (2015) Science Cell of Journal | Ltd Biologists of Company The by Published 2015. ICD nti acd n,truhayattwo-hybrid yeast a through and, cascade this in ICD 1 .W hwta h 7NRaatrprotein adaptor p75NTR the that show We ). mrSalehi Amir , 2 iih Rajkumar Vijidha , c -secretase-dependent ICD RG and NRAGE , 1 n hlpA Barker A. Philip and oul p75NTR soluble 2011). al., et in (Kumar and epithelia mammary 2005) transitions in al., mesenchymal to (EMT) et epithelial role include (Xue that a adhesion movements al., play cell cell et to homotypic shown Bertrand been regulating 2003, also al., in has et NRAGE Bronfman Interestingly, 2002, 2008). al., et Salehi 2000, Hrigo ta. 08.Ti,i un cst nii Rho-GDI inhibit Rho-GDI to to acts turn, kalirin-9 in This, from 2008). al., preference shift et MBIs binding (Harrington that 2012). proposed p75NTR a been al., has share it the et and (Rho-GDI) factor p75NTR, Sun exchange inhibitor on site guanine 2010, GDP-dissociation RhoA binding Rac 2005, Rho and al., Rho and induces al., et dual (GEF), et a Park that is Domeniconi 2008, which p75NTR Kalirin-9, 2002, component al., et al., and receptor Harrington et (MBIs), (Yamashita the inhibitors activation as -based Lingo1 functions and to receptor reticulon-4) as responds known (also Nogo other p75NTR, and uncertain. these remain which activated by are mechanisms elements precise downstream the but cascades nrclua oan(C)fo t rnmmrn ehr The tether. transmembrane p75NTR its from untethered (ICD) the domain the through intracellular domain which ADAM17, transmembrane by the in c of cleaved cleavage is by (RIP), followed domain proteolysis juxtamembrane extracellular intramembrane promote regulated thereby to and Par3 2012). interface with al., et –glial collaborates (Tep the p75NTR myelination others, to in Rac1 and localize (Harrington 2002), apoptosis al., promotes mediate and et receptor cascade also activated signaling the JNK can links the this p75NTR to settings, some action. In RhoA regulation. Rac1 promote and activity pedn sa htpoie outotu o nlzn a analyzing for output robust a cell COS7-based provides novel that a assay describe spreading we Here, cascades. signaling sap5T neatrta ciae N n -3- and JNK a activates that pathway interactor apoptotic MAGED1) dependent p75NTR as known a adaptor also as cytosolic homolog, (neurotrophin MAGE with NRAGE interacting identified interaction receptor have its studies Previous on . rely events 2005). signaling al., et (Domeniconi morphology al., cell et 2013), in Kenchappa changes 2006, al., and al., et 2010) et Matusica (Kenchappa 2010, apoptosis of of al., induction enhancement et 2008), (Ceni al., signaling et (Wang pro-survival migration cell in implicated ciaino itntsgaigptsrmi neti,and the by uncertain, activated remain selectively are paths that p75NTR signaling cascades signaling distinct specific the for of allow that mechanisms activation The contexts. different in functions freliable of sceaecmlx hs eunilcevg vnsrlaethe release events cleavage sequential These complex. -secretase ae oehr vial aasgetta 7NRadthe and p75NTR that suggest data available together, Taken nnuoa rwhihbto,arcpo ope containing complex receptor a inhibition, growth neuronal In 7NRudrosatose laaeeet nw as known event, cleavage two-step a undergoes p75NTR 7NRi o aayial cieadteeoedownstream therefore and active catalytically not is p75NTR ICD nvitro in aentbe dniid hsi,i at u oalack a to due part, in is, This identified. been not have 1, ICD * sasfraayigp5T n p75NTR and p75NTR analyzing for assays ICD rgetcnpriiaei iearyof array wide a in participate can fragment rgetta sgnrtdhsbeen has generated is that fragment nvitro in and nvivo in Slh tal., et (Salehi 447 ICD

Journal of Cell Science eosrt rca oefrp5T laaei small in pathway cleavage activation results Rac1 p75NTR novel Our p75NTR- p75NTR for a the spreading. define for involving role and cell required activation crucial GTPase also and a a is activation demonstrate as that Rac1 NEDD9 cofactor identify dependent we NRAGE screening, novel hybrid yeast-two through fe DM7dpnetcevg fp5T n liberation and occurs p75NTR p75NTR only the of activation of cleavage Rac1 ADAM17-dependent p75NTR-dependent assay, after that this Using show events. we signaling p75NTR-dependent of subset ARTICLE RESEARCH 448 full-length p75NTR of cell the visualize overexpression After or to that Cy3 p75NTR laminin. shows to wild-type 1C,D exposed with conjugated glass Fig. and agglutinin on non-permeabilized surfaces. pre-coated germ left p75NTR density wheat fixed, been low with were to cells at had together hours, 24 plated that EGFP then with coverslips 1A,B), (Fig. COS7 transfected 2002). constructs al., et were Mitsui and 2002, Takahashi cells al., 1999, et Zanata al., a 2001, et semaphorin- Strittmatter, on (Takahashi study events loosely to signaling previously based proximal induced used bioassay and assay a cleavage contractility p75NTR established COS7 of we role events, the signaling into insight gain spreading To cell COS7 induces p75NTR RESULTS h p75NTR the ICD ICD eiso nNAEdpnetptwyand, pathway NRAGE-dependent an on relies edmntaeta ciaino a1by Rac1 of activation that demonstrate We . ICD RG n NEDD9. and NRAGE , ICD eutdi significant in resulted laaerssatfr fp5T i o rmt cell on promote dependent provide are that not might cleavage. activities spreading signaling receptor did p75NTR cell for a bioassay COS7 p75NTR a almost of that area of overexpression suggesting surface spreading, Interestingly, form cells. cell control mean cleavage-resistant of a that giving double spreading, cell COS7 O7clstasetdwt ullnt 7NRo the or p75NTR full-length with transfected p75NTR. p75NTR by cells induced spreading cognate cell COS7 its of with amount Rac1 the of reduced interaction GEFs, the blocks that inhibitor Rac1 was constructs RacN17. p75NTR expressing the cells by in evoked blocked spreading cell that shows ihfl-eghp5T rwt h p75NTR the with or spreading p75NTR cell and full-length the isoform, Rac1 were with dominant-negative for cells a COS7 RacN17, required this, with do transfected was To overexpression. 2006), p75NTR Rac1 al., and by et induced whether fibroblast Guo for 2004, asked al., necessary et we is (Wells spreading Rac1 in cell Because macrophage activate role Rac1. to prominent shown and been a RhoA actin previously the has plays to p75NTR receptors and GTPases cytoskeleton, membrane plasma small from of signals transducing spreading family cell Rho promote to The Rac1 through acts p75NTR aii n,atr2 or,wr rae ihNC23766 NSC with treated were hours, 24 after (100 and, laminin sa lentv prah eakdwehrNC276 a 23766, NSC whether asked we approach, alternative an As m )fr6hus i.2, hw htteRc inhibitor Rac1 the that shows 2C,D Fig. hours. 6 for M) ICD ora fCl cec 21)18 4–5 doi:10.1242/jcs.152173 447–459 128, (2015) Science Cell of Journal eepae ngascvrlp rcae with precoated coverslips glass on plated were i.1 p75NTR 1. Fig. cl a:25 bar: ICD). Scale and CR p75NTR (p75FL, different constructs the or (CT) EGFP alone overexpressing cells COS7 of assay spreading Representative (C) products. show cleavage to the epoxomicin without and cells with COS7 in constructs p75NTR different the of the overexpression showing blot Western (B) (p75ICD). p75 domain and intracellular cleaved, be to is unable that Fas the of with domain replaced transmembrane is black) in (shown domain transmembrane the which in (p75CR), p75 cleavage-resistant (p75FL), p75 full-length cells: COS7 in overexpressed constructs p75NTR different the showing spreading. cell ** mean the ( as shown controls are of and that to compared in area change cell percent the are as Values expressed cells. p75CR control and the (CT) of that with compared cells p75ICD-expressing in and area p75FL- cell in a increase showing significant assay spreading the of n P 5 , needn experiments); independent 3 0.01. m .()Quantification (D) m. ICD A Schematic (A) eitsCOS7 mediates ICD i.2A,B Fig. . 6 s.e.m.

Journal of Cell Science ** xrse stepretcag ncl racmae ota fcnrl n r hw stemean the are as Values shown spreading. are cell and on controls effect of no that showing to (RhoAN19), compared RhoA area dominant-negative cell without in or change with percent ICD the and as p75FL expressed overexpressing cells with performed EERHARTICLE RESEARCH eraei h elae fp5L n 7IDepesn el rae ihNC276ta a o bevdi Tcls ausaeepesda h per the as expressed are mean Values the cells. CT as in shown observed are not 25 and was that controls bars: that to of 23766 Scale compared NSC that hours. area with to 6 cell treated compared cells in for area p75ICD-expressing change cell 23766 and percent in p75FL- NSC the change of inhibitor as area ce Rac cell expressed the the the are in in Values without decrease decrease cells. significant or CT a of with showing that p75ICD A mean in in or the not assay as but spreading shown the are RacN17, of and overexpressing Quantification controls after (B) cells (RacN17). p75ICD Rac spreading. and dominant-negative cell p75FL without or induce with to p75ICD Rac1 or through (FL) acts p75NTR 2. Fig. P , .1 .. o significant. not n.s., 0.01; 6 ...( s.e.m. n 5 needn xeiet) C ersnaiesraigasyo O7clsepesn GP(T,p75FL (CT), EGFP expressing cells COS7 of assay spreading Representative (C) experiments). independent 5 A ersnaiesraigasyo O7clsoeepesn GP(T,fl-eghp75 full-length (CT), EGFP overexpressing cells COS7 of assay spreading Representative (A) 6 ...( s.e.m. m n .()Qatfcto ftesraigasyi hwn significant a showing C in assay spreading the of Quantification (D) m. 5 needn xeiet) E uniiaino h pedn assay spreading the of Quantification (E) experiments). independent 3 ora fCl cec 21)18 4–5 doi:10.1242/jcs.152173 447–459 128, (2015) Science Cell of Journal 6 ...( s.e.m. n 5 needn xeiet) * experiments); independent 2 P , lae of area ll 0.05; 449 cent of

Journal of Cell Science omo hA(1)atrdsraigidcdb full-length by induced spreading altered (N19) RhoA of form n ytep75NTR p75NTR the full-length by by and evoked spreading cell blocked effectively ARTICLE RESEARCH 450 3. Fig. nprle xeiet,w etdwehradominant-negative a whether tested we experiments, parallel In e etpg o legend. for page next See ICD . 7NRo h p75NTR the or p75NTR a1 u o hA ly rca oei vkn p75NTR- evoking in role cells. crucial COS7 in a that spreading plays demonstrate cell RhoA, data induced not these together, but Taken Rac1, 2E). (Fig. assay this ora fCl cec 21)18 4–5 doi:10.1242/jcs.152173 447–459 128, (2015) Science Cell of Journal ICD u efudta thdn fetin effect no had it that found we but ,

Journal of Cell Science opud a oefc ncl pedn nue ythe by induced these spreading that cell and on in p75NTR effect spreading no p75NTR full-length reduced had sharply expressing compounds BB94 cells and COS7 GM6001 that shows DM7mN xrsini oto O7clso nclsoverexpressing cells in constructs. or p75NTR different cells the COS7 control in the expression as mRNA shown ADAM17 are and controls the of as that expressed to mean are compared Values area cells. cell CT ADAM17 in after or change cells cells percent p75FL p75ICD of in area not cell but knockdown, the in decrease significant a showing as used 25 was bars: (NSsi) Scale siRNA Non-specific control. p75ICD siRNA. or ADAM17 p75FL without (CT), or EGFP with overexpressing cells COS7 of a assay spreading Representative as (D) well knockdown. ADAM17 as upon generation (Ad17si) p75ICD siRNA in reduction ADAM17 expressing ADAM17 cells in COS7 decrease in a expression showing blot Western (C) experiments). independent oehr hs aadmntaeta yiiitn p75NTR initiating p75NTR by the that of generation demonstrate allowing and data cleavage these together, DM7lvl nCS el vrxrsigfull-length overexpressing cells COS7 p75NTR endogenous in and reduce p75NTR to levels used spreading, was cell ADAM17 (siRNA) on p75NTR RNA of interfering effect small the for required is ADAM17 racmae ota fcnrl n r hw stemean the as shown cell are in and change controls percent in of the not that as but to expressed BB94, compared are and area Values GM6001 cells. with CT treated or when cells cells p75ICD p75FL in of decrease significant area a cell showing the A in assay hour. spreading the 1 of Quantification for matrix(B) (GM/BB) broad BB94 the and without GM6001 or inhibitors metalloprotease with spreading. p75ICD and cell (FL) for p75 (CT), full-length necessary EGFP overexpressing cells is COS7 of ADAM17 assay spreading by Representative (A) cleavage p75NTR 3. Fig. ARTICLE RESEARCH euae nrmmrn rtoyi RP,t eeaethe as generate known to event, (RIP), cleavage proteolysis p75NTR two-step intramembrane a regulated undergoes p75NTR for prerequisite spreading a cell is cleavage p75NTR ADAM17-mediated otdwt aii n,atr2 or,wr rae with treated were hours, 24 after and, (10 laminin GM6001 with coated nsraigidcdb h p75NTR the by area effect no cell induced cell had blocked spreading but for overexpression effectively on p75NTR assessed with knockdown by induced on together ADAM17 and spreading siRNA, later. knockdown alone, control hours EGFP ADAM17 48 with transfected or of or were p75NTR+EGFP siRNA cells effect ADAM17 COS7 the with spreading, test cell p75NTR-induced To 3D). (Fig. ffl-eghp5T.CS el rnfce ihfull-length with p75NTR transfected the cells or overexpression COS7 p75NTR by p75NTR. ADAM and induced full-length targeting spreading GM6001 of cell of whether the capable blocked determined proteases, inhibitors first metalloprotease we BB94, morphology, cell xrclua laaeo 7NR(omdie l,21,Ceni 2011, al., et (Kommaddi p75NTR of cleavage cell extracellular the generating in role crucial phenotype. a spreading plays ADAMs cell-surface p75NTR the of eetdi O7clsadislvl r o lee by altered not p75NTR are the cleavage-resistant levels or readily the its p75NTR cell is p75NTR, of and on mRNA full-length form effect cells ADAM17 of its COS7 that overexpression characterized in shows therefore detected 3C and Fig. 2010), spreading. al., et ly rca oei lbrtn h elsraigphenotype spreading cell the overexpression. elaborating p75NTR in by role induced crucial a plays DM7epeso nCS el,adacmlto fthe of accumulation and cells, COS7 p75NTR in expression ADAM17 ehv rvosysonta DM7mdae h initial the mediates ADAM17 that shown previously have We 6 ...( s.e.m. ICD ICD ICD hssget htp5T laaemdae by mediated cleavage p75NTR that suggests This . generation RIP-dependent whether determine To . a lce hnAA1 eeswr suppressed were levels ADAM17 when blocked was n 5 m needn xeiet) * experiments); independent 3 )adB9 20n)fr1hu.Fg 3A,B Fig. hour. 1 for nM) (200 BB94 and M) ICD m .()Qatfcto ftesraigasyi D in assay spreading the of Quantification (E) m. srqie o h hne nCOS7 in changes the for required is ICD ICD DM7sRAefcieyreduced effectively siRNA ADAM17 . eepae ngascvrlp pre- coverslips glass on plated were ICD odtriewhether determine To . P ICD , .5 F TPRof RT-PCR (F) 0.05. Fg EF.Taken 3E,F). (Fig. ICD 6 ADAM17 , ...( s.e.m. n 5 3 Fg A ae2.Svrlsuishv ugse htthe that suggested have p75NTR studies the Several and 2). CTF lane p75NTR of 4A, (CTF) fragment (Fig. C-terminal 25-kDa a a of accumulation XXI, results, is previous compound as with fragment Consistent BB94 by and this as GM6001 of inhibitors well metalloprotease production the The by blocked overexpressed. is receptor hte h T ol eit 7NRdpnetcell p75NTR-dependent mediate p75NTR could p75FL, overexpressing cells addressed CTF therefore COS7 we Underwood spreading. and the 2006, 2010), al., al., et whether et Kenchappa Kenchappa 2008, al., 2005, et al., et (Domeniconi elto lee elsraigidcdb vrxrsinof overexpression by p75NTR NRAGE the induced or whether spreading p75NTR asked full-length we cell protein 5A), altered NRAGE After (Fig. depletion cells suppressed spreading. COS7 siRNAs in cell 2005, expression NRAGE is human al., COS7 NRAGE that whether et p75NTR-dependent confirming asked (Xue transformed therefore for we cells and required 2011), U2OS in al., and et Kumar cells changes epithelial mammary morphological NRAGE, to termed adaptors, proteins these mediates binding of One cytosolic effects. with downstream elicit interaction on relies therefore cuuain hsidctsta h p75NTR the that indicates this accumulation, p75NTR The p75NTR The h p75NTR The cell mediate to p75NTR spreading of downstream acts NRAGE h nraei elsraigidcdb 7NRo the or p75NTR by induced spreading eliminated it cell but cells in control p75NTR by increase occupied area the the on effect no DM7 and ADAM17- htcmon X a oefc nclsepesn GPalone EGFP expressing p75NTR cells the on expressing with effect or treated no had and XXI compound coverslips that glass (10 on XXI plated compound were control EGFP el.Teeoe h p75NTR the COS7 Therefore, in spreading cells. cell p75NTR-dependent for required fragment ybnigt h p75NTR the to binding by cuuaino h 7NRCFwiepeetn p75NTR preventing while CTF causes p75NTR XXI the of compound accumulation Because p75NTR. full-length expressing ontemsgaigo yfclttn h eeainor generation the facilitating p75NTR by the of or maintenance signaling downstream eurdt rv hne nCS elshape. cell COS7 in changes drive to required bevdi hsln n htcntttv,rte hnligand- than rather phenotype. constitutive, spreading cell that the drives and cleavage line p75NTR induced, this phenotype spreading in cell the blocksobserved for required neurotrophin not that that is conclude p75NTR We to domain 1994). binding al., extracellular et (Clary p75NTR binding ligand the antibody an against significantly 4F), (Fig. not directed REX of in was maintained capacity spreading were cells The cell when changed 4E). four drive (Fig. the to line of p75NTR this each full-length in were encoding present mRNA mRNA was receptor but neurotrophins Trk shown) not the (data nor effect detected p75NTR whether no Neither in determine expressed cells. had are to COS7 receptors RT-PCR To hours, their used or 24 we neurotrophins 4D). whether role, for (Fig. establish a or play spreading might hour neurotrophins cell endogenous 1 for p75NTR ng/ml), on 25 at (BDNF) factor (each neurotrophic brain-derived nerve or of addition (NGF) ligands The factor assay. these growth spreading whether COS7 the addressed on impact we an experiments, have next our in and 7NRbnst h ornuorpispeeti mammals in present neurotrophins four the to binds p75NTR ICD ora fCl cec 21)18 4–5 doi:10.1242/jcs.152173 447–459 128, (2015) Science Cell of Journal Fg BC.NAEcudmdaeteeeffects these mediate could NRAGE 5B,C). (Fig. ICD ICD ICD rvscl spreading cell drives sgnrtdi O7clswe h full-length the when cells COS7 in generated is osnthv nrni nyai ciiyand activity enzymatic intrinsic have not does c sceaedpnetp5T laae is cleavage, p75NTR -secretase-dependent m )o eil o or i.4, shows 4B,C Fig. hour. 1 for vehicle or M) ICD ICD ICD aedsic ilgclactivities biological distinct have ICD e tbokdtesraigo cells of spreading the blocked it yet ICD oee,lvl ffull-length of levels However, . c c eeae hog sequential through generated , sceaeihbto eutdin resulted inhibition -secretase n ietrl in role direct a playing and sceaeihbtr(i.4A). (Fig. inhibitor -secretase ICD RG ncdw had knockdown NRAGE . ICD sterelevant the is ICD ran or 451 ICD

Journal of Cell Science EERHARTICLE RESEARCH 452 by cleaved is p75NTR 4. Fig. MOwsue svhcecnrl B ersnaiesraigasyo O7clsoeepesn GP(T,fl-eghp5(L rp5C,wt or with p75ICD, or (FL) p75 full-length (CT), EGFP overexpressing cells COS7 of assay spreading Representative (B) control. the vehicle without as used was DMSO fetp5T-nue pedn.I ,, ausaeepesda h ecn hnei elae oprdt hto otosadso h mean the show and signific controls not of did that addition to Antibody compared control. area as cell used in cells was change COS7 (NSR) percent p75NTR-ind of the serum affect assay as rabbit significantly spreadin spreading expressed Non-specific of not a are Quantification binding. did of (F) values addition ligand cells. ( Quantification C,D,F Neurotrophin blocks COS7 In (D) hours. in that spreading. NT-4) cells. 24 antibody and CT p75NTR-induced for NT-3 p75NTR BDNF, affect or (NGF, ng/ml) a cells neurotrophins (25 REX, different p75ICD BDNF the to in or of exposed not expression NGF the without but showing or XXI, RT-PCR with (E) compound spreading. p75FL, with or treated (CT) when EGFP cells overexpressing p75FL of area cell O7clswe rae ihcmon X ln )adteihbto fbt p75NTR both of inhibition the and 2) (lane XXI compound with treated when cells COS7 n 5 needn xeiet) * experiments); independent 3 c sceaeihbtrcmon X o or cl a:25 bar: Scale hour. 1 for XXI compound inhibitor -secretase c sceaet nuecl spreading. cell induce to -secretase P , .5** 0.05 P , .1ad*** and 0.01 P , 0.001. A etr ltsoigteihbto ftegnrto ftep75NTR the of generation the of inhibition the showing blot Western (A) m .()Qatfcto ftesraigasyi hwn infcn eraei the in decrease significant a showing B in assay spreading the of Quantification (C) m. ICD n T eeaini el rae ihG60 n B4(ae3). (lane BB94 and GM6001 with treated cells in generation CTF and ora fCl cec 21)18 4–5 doi:10.1242/jcs.152173 447–459 128, (2015) Science Cell of Journal ICD u o T in CTF not but ncells in g 6 s.e.m. uced antly

Journal of Cell Science rgetta tre taioai 3 n ne tthe at ended and 637 acid amino at started 2014). al., that et Miao Little fragment 2009, 2012, al., al., et et and Izumchenko Kondo lung 2006, 2013, melanoma, al., al., recently in et et has EMT (Kim and directing in cancer player migration player breast key key cell a a and is as shape a NEDD9 emerged cell 6A). NEDD9, of (Fig. of regulation family the regions Cas the overlapping of distinct from member binding emerge encoded to potential myristoylated clones screen positive of a this the to of source four fused a Interestingly, cDNAs partners. as of signal library with bait brain membrane-localization as fetal protein human fusion NRAGE–SOS a an using screen, hybrid EERHARTICLE RESEARCH oietf RG-neatn rtista ol ikthe link could that proteins NRAGE-interacting p75NTR NEDD9 identify with functionally To and physically interacts NRAGE p75NTR the or p75NTR eteeoefcsdorefrso h yohssta NRAGE possibility. that hypothesis latter the p75NTR the on the efforts out links our ruling focused therefore shown), We not (data knockdown l oro h RG-idn lnscnandaNEDD9 a contained clones NRAGE-binding the of four All ICD oRc ciiy epromdactslcyattwo- yeast cytosolic a performed we activity, Rac1 to ICD odwsra inln partners. signaling downstream to ICD eeucagdb NRAGE by unchanged were h eodhlxo hsdmi ly rca oei their in role crucial a plays that domain and this NRAGE to of bind association. helix to proteins required second helix-loop- is two the the NEDD9 that the in indicate present data between these helix together, interaction Taken G744P 1 6C). no the the (Fig. had helix whereas substitution abolished in interaction, G722P NRAGE–NEDD9 residues mutation The the (G744P). proline on 2 substituting effect helix by in or each helices disrupted (G722P) two the we 62-amino-acid region, test the this this in To of domains domain. helical of the helix-loop-helix of sequence putative relevance a primary revealed 6B). (Fig. the region were domain interaction of NRAGE pulldowns the NEDD9 acids Examination as amino from emerged of stretching 765 binding, NEDD9 to fragments of 704 fragment a smaller NRAGE this, within from progressively and, region for using minimal the performed required identify To NEDD9 fragment. NEDD9 this expressed 198-amino- NRAGE with this shows associating by containing of 6B capable Fig. protein was and fragment fusion acid 834), GST–NEDD9 acid a (amino that protein the of C-terminus nvitro in elae oprdt hto otosadaesona the as shown are and controls in change of percent that mean the to as compared expressed but are area depletion Values NRAGE cell cells. CT after of cells that p75ICD in and p75FL not decrease of significant area cell 25 a the showing bar: in assay Scale spreading (NRsi). control. of siRNA as Quantification NRAGE used (C) without was or (NSsi) with siRNA full- p75ICD Nonspecific (CT), or EGFP (FL) overexpressing p75 cells length COS7 of in Representative assay (B) used spreading siRNA. was nonspecific 1 NS, using siRNA experiments. cells NRAGE subsequent COS7 Only in spreading. siRNAs. knockdown different cell NRAGE in three showing p75NTR blot of Western downstream (A) acts NRAGE 5. Fig. ora fCl cec 21)18 4–5 doi:10.1242/jcs.152173 447–459 128, (2015) Science Cell of Journal 6 rnlto,idctn htNAEdrcl id to binds directly NRAGE that indicating translation, ...( s.e.m. n 5 needn xeiet) * experiments); independent 3 P , 0.05. m m. 453

Journal of Cell Science EERHARTICLE RESEARCH 454 NRAGE. for immunoblotting HLH NRAGE by of the detected interaction of the was outside panel, fusions lower regions GST–NEDD9 the and In with gray, gray. in dark light schematic in in the represented represented are In region is domain. region NRAGE-binding HLH the the as HLH NEDD9 C, NEDD9 the of of 2 2 helix helix and 1 identifies helix in residues proline two mutants. GST–NEDD9 of Introduction different (C) the Coomassie-Blue-stained of expression a the shows confirms that panel gel lower The respectively. fusions interaction, GST–NEDD9 are of with region immunoblot. NRAGE HLH NRAGE of the by detected interaction of was The outside gray. Regions dark gray. in light represented in represented is region with HLH interaction their and by generated, produced NRAGE proteins were region fusion 198-amino-acid GST–NEDD9 HLH the Different the containing (B) of 637–834. regions acids overlapping amino contain spanning identified clones (HLH) helix four loop The helix domain, domain. a SH3 and an domain serine-rich contains which a NEDD9. substrate-binding domain, NEDD9, of a of domain structure C-terminal the showing the Schematic with (A) interacts NRAGE 6. Fig. nvitro in rnlto a nlzd nteshmtci ,the B, in schematic the In analyzed. was translation ! and 2 niaetepeec rabsence or presence the indicate atcptsi hscsae hs u aasgetthat suggest data generation that our on protein dependent Thus, p75NTR is the NRAGE-binding cascade. of spreading novel cell this p75NTR-dependent a in as participates NEDD9 identify RG n ED ucindwsra fp5T to p75NTR of downstream using function depleted Rac1. NEDD9 activate were that demonstrate and NEDD9 findings these NRAGE together, or Taken 8D). NRAGE (Fig. siRNA when in increase blocked p75NTR-dependent increase the was robust significantly, a and, activity produced Rac1 colleagues overexpression energy and Hodgson p75NTR resonance by fluorescence (2010). described the biosensor using (FRET) cells transfer Rac1 assessed COS7 we in pathway, activity NEDD9-dependent and NRAGE- an phenotype. spreading cell pathway the p75NTR–NRAGE mediates a of that effector downstream a is NEDD9 eedn ciaino a1 lhuhgnrto fthe of generation Although Rac1. of p75NTR activation dependent 7NRms ecevdi rtoyi rcs involving process that proteolytic demonstrate a We in through Rac1. cleaved and spreading GTPase ADAM17 be cell small must induces the p75NTR p75NTR of that activation show we Here, DISCUSSION P,NthadDla I fp5T scaatrzdb the by characterized and is ADAM17 as by p75NTR receptor such of the of RIP proteins, cleavage Delta. dual membrane-anchored and diverse Notch APP, of function the events these links 2012, that cascade. al., study et single first Tep the a is 2010, in al., this al., et 2013), et al., Wang al., Ceni et 2008, 2010, et Matusica al., al., Domeniconi et et 2002, Bertrand Kenchappa 2006, al., 2008, p75NTR al., et et of Harrington Kenchappa downstream 2002, 2005, roles al., important et play (Salehi to proposed been a oefc nclstasetdwt GPaoebtstrongly but full-length alone of EGFP overexpression by with mediated transfected spreading cell cells inhibited on knockdown its effect NEDD9 Interestingly, no reduce 8A). had to (Fig. with cells siRNA COS7 interaction in NEDD9-specific levels using robust spreading, a induced sustain to fragment required acids 110-amino-acid is NEDD9. amino this NRAGE 48 of NRAGE–NEDD9 of much C-terminal the abrogate, Therefore, the completely interaction. or not domain. did the domain but Deletion domain. reduced, 666– coiled-coil that coiled-coil homology acids MAGE the amino the revealed to MAGE of within putative C-terminal region mutants is a the that on 775 deletion a relied domain, interaction NRAGE NEDD9 and repeat the of interspersed section Screening domain this in the present homology motifs are alone structural GST to NRAGE main not The but robust 7B). deletion protein cells showed fusion (Fig. HEK293T NRAGE GST–NEDD9 NRAGE the in Full-length to downs. 7A), binding FLAG-tagged Fig. pull performed in FLAG-tagged several and schematically with in this (shown with expressed mutants domain 637–834 then or acids the and amino GST NRAGE, identify fused to we to NEDD9 NEDD9, of and to bound cells that NRAGE mammalian in occurred 7NRo ytep75NTR the by or p75NTR otx.W eosrt htNAEpriiae downstream participates p75NTR NRAGE the that of demonstrate We context. hwta h p75NTR the that show ial,t eemn hte 7NRatvtsRc through Rac1 activates p75NTR whether determine to Finally, I sacnevdadwl-salse ehns htaffects that mechanism well-established and conserved a is RIP ete se hte ED srqie o p75NTR- for required is NEDD9 whether asked then We interaction NRAGE–NEDD9 the whether determine To ICD ora fCl cec 21)18 4–5 doi:10.1242/jcs.152173 447–459 128, (2015) Science Cell of Journal RG inln n a1atvto aeall have activation Rac1 and signaling NRAGE , ICD ICD c sceaefrti fett emnfse,and manifested, be to effect this for -secretase hc ntr rvsNAE n NEDD9- and NRAGE- drives turn in which , npouigtecl pedn hntp and phenotype spreading cell the producing in ICD sterlvn inln oeyi this in moiety signaling relevant the is ICD Fg BC.W ocuethat conclude We 8B,C). (Fig. c sceae which -secretase,

Journal of Cell Science inln aha ognrt h p75NTR the Erk-dependent generate an to activates activation pathway cell NTRK1) signaling as and known (also activation Rac1 for required spreading. component signaling EERHARTICLE RESEARCH 7NR u i o lc elsraigo el rnfce with transfected cells of p75NTR full-length spreading cell the with block transfected not cells did but in p75NTR, spreading cell prevented and p75NTR the releases then ugsigta hsefc ih elgn needn.In independent. ligand induced be is exogenous mRNA might ADAM17 of of effect addition spreading, expression neurons, cell the this sympathetic p75NTR-induced that that on show effect suggesting we no (Wang has Here, migration neurotrophin glioma 2008). for al., al., and et 2005) et al., (Kenchappa et collapse Domeniconi 2006; cone for growth necessary myelin-associated- for glycoprotein-induced is death, cleavage neuron p75NTR sympathetic 2013). that BDNF-induced al., shown et have (Matusica recent signaling works neurons survival Akt Other sympathetic another facilitates in this, signaling cleavage cell Erk p75NTR with and that and Consistent shown has 2011). signaling study al., et Akt (Kommaddi NGF-dependent facilitates ncdw fAA1 niie rdcino h p75NTR the of production inhibited ADAM17 of knockdown n hwta hsi rrqiiefrsubsequent for prerequisite cells a COS7 in is p75NTR this of that cleavage show initial and ADAM17 the that for demonstrate responsible data Our is 2005). al., et Zampieri 2003, eedn rtoyi n ees ftep75NTR the of release and proteolysis dependent npeiu ok ehv hw htNFdpnetTrkA NGF-dependent that shown have we work, previous In ICD ecnld httep75NTR the that conclude we , ICD notectsl(ann tal., et (Kanning cytosol the into ICD ICD hc nturn in which , ICD steactive the is c -secretase- Because . ICD 01 n ED Kn ta. 01 h ta. 02 nthe in 2012) al., et Ahn 2011, al., al., et et (Kumar (Kong NRAGE implicated NEDD9 have and interesting works is 2011) other it but that to NEDD9 2012) note study and al., to first NRAGE the et between is Kondo link This a 2013, 2006). establish Little al., al., 2009, et et (Natarajan al., (Miao et cancer and lung has (Izumchenko 2014), cancer and 2006, al., breast 2013), al., et 2012), al., et al., (Kim et melanoma et can Baquiran adhesion in Ahn 2012, (Bradshaw 2012), factor al., pro-metastasis spreading cell al., et as cell emerged et Zhong and induce 2011, Guerrero al., and migration 2001, et adhesions al., cell focal et stabilize in Bouton in NEDD9 HEPL roles respectively). physiological (reviewed and CASS4, important Efs and p130Cas, Sin plays BCAR1, includes under as also known that (also proteins cleavage adaptor of p75NTR challenge. interesting mechanisms we an precise remains the regulate 2010), circumstances the for required Determining al., not receptor. et that is the p75NTR (Ceni of to results cleavage binding Therefore, neurotrophin ligand previous spreading. that that blocks cell our found conclude on that effect with we no antibody consistent However, had p75NTR, an setting. to and REX, binding this p75NTR to with in bind alter they cleavage incubation that not its conceivable does is in induce it expressed overexpression are and neurotrophins cells, p75NTR four COS7 found All cells, levels. we mRNA but COS7 ADAM17 cascade in signaling that, p75NTR-dependent a through ED as aldHF rCs)blnst h a family Cas the to belongs CasL) or HEF1 called (also NEDD9 e htcnim h xrsino S n S–ED.F,full- FL, GST–NEDD9. and GST of p75. expression length the Coomassie-Blue-stained confirms a that shows gel NRAGE panel using lower fragments detected The NRAGE was immunoblotting. of proteins interaction fusion The GST–NEDD9 (B) with B. from avidity domain. interaction coiled-coil Mage CCD, MHD, domain; domain; that homology repeat mutants interspersed deletion NRAGE. ISP, NRAGE of generated. different domain were the C-terminal showing the Schematic with (A) interacts NEDD9 7. Fig. ora fCl cec 21)18 4–5 doi:10.1242/jcs.152173 447–459 128, (2015) Science Cell of Journal 2 o++rpeetthe represent +++ to 455

Journal of Cell Science iN.Rslsso htNAEo ED elto nue infcn eraei a1atvt nclsoeepesn 7F u o nC el.Va cells. NEDD9 CT in or not NRAGE mean but without p75FL the or overexpressing as cells with shown in p75FL are activity or Rac1 and (CT) in signal EGFP decrease FRET significant overexpressing normalized a cells the induced COS7 as depletion in NEDD9 represented or analysis are FRET NRAGE that by show performed Results assay siRNA. activation Rac1 (D) experiments). Tcls ausaeepesda h ecn hnei elae oprdt hto otosadaesona h mean the as shown are and in not controls but of depletion that NEDD9 after to cells compared p75ICD area and cell p75FL of in area change cell percent the in the decrease as significant expressed a are showing B Values in cells. assay CT spreading the of Quantification (C) eaoamgain(azMrn ta. 2008). collapse al., Rac1-dependent et been activate (Sanz-Moreno cone has and migration NEDD9 DOCK3, melanoma to GEF, growth bind Rac1 directly Another to hippocampal and reported 2011). al., in proNGF and in et activity results of (Deinhardt turn inactivation complex association in Rac1 which the dissociation, Rac1 Trio facilitate scenario, p75NTR–SorCS2 important. in results this p75NTR to to be with In intact reported to growth. and been but neuronal an likely has neurons known Trio seems hippocampal with termed not GEF GEF is associate Rac1 Rac1 activity a a Interestingly, Rac1 of induces activation cascade NEDD9 EERHARTICLE RESEARCH 456 contexts. similar 2012), or NRAGE–NEDD9– al., EMT an et in whether Zhu functions determine 2010, cascade al., to Rac1 et important Frasa be 2008, will al., it et Wu E-cadherin– 2003, essential al., the an et plays at Rac1 role that demonstrated regulatory have studies other that E-cadherin– of regulation negative 25 bar: Scale control. as cells used COS7 was of (NSsi) assay siRNA spreading Nonspecific Representative siRNA. (B) NEDD9 band. without background or a with represents p75ICD asterisk or an (FL) with p75 marked full-length is (CT), that EGFP NEDD9 above overexpressing spreading. cell band in prominent p75NTR The of (N9si). downstream acts NEDD9 8. Fig. h rcs ehns ywihtep75NTR the which by mechanism precise The b b ctnnadi M.Given EMT. in and -catenin ctnncmlx(Pujuguet complex -catenin ICD –NRAGE– A etr ltsoigkokono ED nCS el rnfce ihND9siRNA NEDD9 with transfected cells COS7 in NEDD9 of knockdown showing blot Western (A) 6 ...(he needn xeiet) * experiments); independent (three s.e.m. rie A.TeRXatbd a rvddb oi Reichardt Louis were BDNF by and NGF provided CA). was Francisco, San antibody at California REX of The (University monoclonal CA). The ON, was Irvine, Canada). epitope) ON, Ottawa, FLAG Oakville, against the antibody (F3165, (MA1-5784, against Sigma were (directed from Scientific antibody NEDD9 obtained M2 Fisher the against Ltd and Thermo antibodies Laboratories Canada) from Canada), Cedarlane ON, from obtained antibodies Burlington, purchased 2000), as al., were (E8404, were et Salehi ADAM17 NRAGE 1994, and al., against et p75NTR (Barker previously against described directed antibodies Polyclonal plasmids and Reagents METHODS AND MATERIALS oefc opooia changes. morphological effect to fRc,ietf ED saNAEitrcigpoenand protein NRAGE-interacting p75NTR a a as that NEDD9 show identify Rac1, of h inln vnsligdwsra fp5T.W hwthat show p75NTR We the p75NTR. of of generation downstream lying events signaling the ncnlso,w aeue oe elbsdasyt dissect to assay cell-based novel a used have we conclusion, In ora fCl cec 21)18 4–5 doi:10.1242/jcs.152173 447–459 128, (2015) Science Cell of Journal b atnwsprhsdfo PBoeias(691001, Biomedicals MP from purchased was -actin ICD NAE–ED ope srequired is complex –NEDD9 –NRAGE ICD ly rca oei h activation the in role crucial a plays P , .5 *** 0.05; 6 ...( s.e.m. P , n 5 0.001. independent 3 htof that lues m m.

Journal of Cell Science oto ncdws el eemitie o 8husa 37 at hours 48 for for maintained used were was as siRNA Cells CA), non-specific knockdowns. and, A Carlsbad, control antibiotic- instructions. (Invitrogen, in manufacturer’s transfection 2000 siRNAs the with Lipofectamine different per phosphate the using transfected with calcium medium transfected free were were the later, cells hours using 48 experiments, constructs knockdown p75NTR For request). aaomleyei B o 5mntsa 37 at minutes 15 for PBS in paraformaldehyde ltdo oesis(00clssi)te nuae t37 at incubated then cells/slip) (4000 coverslips on plated eodr nioiswr ucae rmJcsnImmunoResearch horseradish-peroxidase-conjugated Jackson from and purchased were Canada) antibodies BD Laboratories secondary Vector ON, from from was Rhodamine-conjugated purchased was Canada). (PDL) (Burlington, purchased (WGA) ON, Tocris agglutinin Poly-D-lysine germ (Oakville, was from wheat Canada). Sigma were from Laminin ON, 23766 obtained (San (Mississauga, MO). NSC Calbiochem and Biosciences (Ellisville, from (BB94) were batimastat Bioscience epoxomicin CA); and Diego, (GM6001) XXI, Compound Israel). ilomastast (Jerusalem, laboratories Alomone from obtained ARTICLE RESEARCH aaa eecae o 0mntswt oyDlsn (0.5 poly-D-lysine (0.5 with laminin with ON, minutes 37 coated 30 Ottawa, water, sterile for Scientific, with washed coated Fisher Fisherbrand, were mm, Canada) (12 coverslips Glass assay spreading Cell CO 5% under L- mM penicillin-streptomycin, 2 serum, mg/ml 100 bovine fetal and 10% modified glutamine with Dulbecco’s supplemented in maintained medium were Eagle’s cells HEK293T and cells COS7 transfection and culture Cell Addgene al., from p75NTR cleavage- et the obtained p75NTR, (Hodgson and full-length previously was p75NTR encoding described resistant Wisent plasmids biosensor as Glutathione was from Rac 2010). and (Burlington, were MA) The Ltd mounting (Cambridge, Laboratories reagents anti-fading Canada). Cedarlane Dako culture from ON, Canada). purchased cell QC, was medium All Bruno, (Saint PA). Bioproducts Grove, (West epciesma RA eedsge sn h nirgnStealth Invitrogen the using designed were RNAi mRNAs simian respective sample immunoblotting. Laemmli in by lysing analysis or for assay spreading buffer the for coverslips on plating 37 ltdo oesisfrtesraigasyo ye nLemisample Laemmli in immunoblotting. lysed by or analysis assay spreading for the buffer for coverslips on plated eeknl rvddb rPtrMPesn(cilUiest,QC, University, RhoAN19 (McGill and McPherson Peter RacN17 Dr encoding Canada). by Plasmids provided kindly 2011). were al., et (Kommaddi tiig o G rBN,clswr eu tre o or then hours 2 for starved serum were and cells fixation BDNF, to prior or hour NGF 1 For for compounds staining. different the with each treated in were condition per NIH counted the were with cells quantified 100 was The experiment. least inverted camera. cells At Rev.3 GFP-expressing fluorescent software. MRm of ImageJ AxioCam observer area an surface 40 Axio with cell (Zeiss) a Zeiss captured software were using Zen images a and using performed illumination, on Xenon with was equipped 1.4) microscope Imaging (NA performed. objective was imaging until B tre5mnt ahs,clswr nuae ihRhodamine- with incubated were cells (5 washes), WGA tagged 5-minute (three PBS CO bec rpeec fpamd noigtevrosp5T isoforms p75NTR various the encoding plasmids (2 of presence or absence eemutdi nifdn onigmda(ao,adkp t4 at kept and (Dako), media Coverslips mounting wash). anti-fading 5-minute (one in water mounted with were and washes) 5-minute (two ifrn osrcswr anandfr4 or t37 at hours 48 or for with maintained absence GTPases, were the constructs dominant-negative different in the plasmids (0.5 encoding EGFP p75NTR plasmids with of presence co-transfected COS7 were (RhoAN19), RhoA cells or (RacN17) Rac dominant-negative involving RG,ND9adAA1 iN eune ietdaantthe against directed sequences siRNA ADAM17 and NEDD9 NRAGE, ntecs ftetetwt M01 B4adcmon X,cells XXI, compound and BB94 GM6001, with treatment of case the In ˚ ˚ m n gi ahdwt trl ae.TasetdCS el were cells COS7 Transfected water. sterile with washed again and C (0.5 cDNA EGFP with co-transfected were cells COS7 C. 2 )uigteclimpopaetaseto ehd o experiments For method. transfection phosphate calcium the using g) o 4hus eimwste eoe n elcdwt 4% with replaced and removed then was Medium hours. 24 for TM iNspeito loih seii eune vial on available sequences (specific algorithm prediction siRNAs m )c-rnfce nalcss el rnfce ihthe with transfected Cells cases. all in co-transfected g) m /l nPSfr1 iue n hnwse ihPBS with washed then and minutes 10 for PBS in g/ml) ICD eea ecie previously described as were ˚ .Atrwsigwith washing After C. m /l o or at hours 2 for g/ml) ˚ eoeeither before C ˚ ne 5% under C m )i the in g) ˚ m then C g/ml), 2 ˚ 6 at C h el.Frp75NTR For cells. the rnfrt ircluoe ebae eerne nPSte lce in blocked and then SDS-PAGE PBS After in 2010). rinsed were al., membranes et nitrocellulose, to (Ceni transfer previously described as ICD, (1 aml apebfe,adicbtda 100 at incubated and buffer, sample Laemmli notepO etrt rdc RG–O uinta a then was that fusion NRAGE–SOS a produce cloned to was frame vector reading yeast pSOS open the cytosolic NRAGE the into a this, through For screen. identified two-hybrid were proteins NRAGE-interacting screening two-hybrid Yeast was run PCR The 55 request. of upon temperature annealing available an ADAM17. at are simian performed sequences against primers primer with using reagent The performed then mix and was untransfected, master PCR left green above. or GoTaq described ICD, as or extracted COS7 CR was expression, p75FL, mRNA mRNA with NGF, p75NTR, ADAM17 transfected of receptors, neurotrophins were case neurotrophin the cells the In simian TrkC. as and the well TrkB as Ottawa, TrkA, targeting NT-4, (Fisher, and primers reagent NT-3 then mix BDNF, with was master PCR Canada) green primers. GoTaq ON, hexamers as the random Canada) using with ON, performed (Qiagen) Mississauga, kit Healthcare, RT Kit was (GE Ominscript cDNA Mini CA). the RNeasy (Valencia, using Qiagen instructions produced using manufacturer’s cells the to COS7 according from extracted was mRNA RT-PCR and extraction RNA ro olaigo D-AEgl.I h aeo DM7detection, ADAM17 of case the In gels. (0.2 SDS-PAGE BB94 on loading to prior 2 in harvested were immunoblotting Cells and analysis blot Western 20 with incubated then 10% NP40, and 1% NaCl, hours mM 24 150 8.0, after pH Tris-HCl glycerol) mM in (10 mutants buffer lysed NP40 were deletion Cells in transfection. phosphate NRAGE calcium the using FLAG-tagged cells, with HEK293T co-expressed or and NRAGE vector expression FLAG-tagged mammalian 637–834) a acids to (amino transferred fragment was NEDD9 its 198-amino-acid the or containing NRAGE antisera. of anti-NRAGE were with fragment Levels were immunoblotting GST–NEDD9 the by 2000). methodology with determined pulldowns al., associated the that et fragments using and deletion (Salehi fragments an previously WI) GST–NEDD9 using described the produced (Promega, using using was produced performed kit were fusion NRAGE region GST Full-length transcription-translation this a of overlap. produce mutants PCR to and site-directed helix-loop-helix, two- pGEX4-1 putative and a yeast a contained deletion NEDD9 the into of region cloned from This protein. was obtained screen fragment hybrid NEDD9 198-amino-acid The vitro In the 37 per at strain as yeast 2.5 cdc25H membrane, the of From plasma growth supported CA). yeast 70 analyzed, (Stratagene/Agilent, which the clones vector, a instructions to pMyr using the proteins manufacturer’s performed into cloned was fusion cDNAs Screening brain anchors strain. fetal human yeast of cdc25H library a in expressed RG risdlto rget htascae ihteGST–NEDD9 M2. with the immunoblotting with by associated determined that were fragments fragment deletion its or NRAGE rae ihteRXatbd 110 o 4huspirt iainand fixation to prior hours 24 were for cells (1:100) treatment, REX antibody staining. the REX hours of the 24 case with or the hour In treated staining. 1 and for fixation ng/ml) to (25 prior neurotrophins different the with treated grs e n ad eevsaie ihehdu bromide. 1.5% a ethidium on with run visualized were were products bands PCR and control. gel internal ADAM17. agarose an for as cycles used 28 was and Actin receptors neurotrophin and neurotrophins of emnl18aioaiso NEDD9. the of represents which acids 637–834, amino acids 198 cDNAs. amino terminal NEDD9 encoded human these of of fragments longest distinct The contained these of four and ojgtdbas(EHatcr,Bi ’re C aaa o orat hour 1 for Canada) QC, 4 d’Urfe, Baie Healthcare, (GE beads conjugated ˚ .Baswr ahdtretmsi P0bfe,rsseddin resuspended buffer, NP40 in times three washed were Beads C. oietf h eino RG htbudt ED,teGTfusion GST the NEDD9, to bound that NRAGE of region the identify To m )fr6husbfr yi obokpoesmldgaaino the of degradation proteasomal block to lysis before hours 6 for M) rnlto n ulonexperiments pulldown and translation ora fCl cec 21)18 4–5 doi:10.1242/jcs.152173 447–459 128, (2015) Science Cell of Journal m )wsaddt h 2 the to added was M) ICD 6 eeto,clswr r-rae ihepoxomicin with pre-treated were cells detection, aml apebfe n oldfr5minutes 5 for boiled and buffer sample Laemmli 6 aml apebfe eoelysing before buffer sample Laemmli ˚ ˚ o 5cce ntecase the in cycles 35 for C o iue.Lvl of Levels minutes. 5 for C m glutathione- l nvitro in 6 457 10 ˚ C 6

Journal of Cell Science hr ersnsteitniyadB ersnstebedthrough bleed the represents BT and 2005). intensity al., et the (Feige represents I where RTitniyadcluae h omlzdFE NRT value (NFRET) FRET formula: the normalized following from the the into them to calculates according takes subtracts and (YPet–PBD), program intensity donor acceptor the FRET This i.e the chain software. and each using from (CyPet–Rac1) ImageJ done intensities was bleed-through NIH the analysis consideration in FRET plugin (emission). sets 540/40 PixFRET 500/ filter and YFP, 470/20 (emission); (excitation) and following 535/30 20 (excitation) and the (excitation) 430/24 three 430/24 imaging, CFP, FRET, the (emission); used: same ratio the for were at emission Technology) taken imaged (Chroma were For were FRET time. and Images CFP exposure FRET. camera. and with CFP Rev.3 YFP, (Zeiss) Xenon channels software MRm Zen with AxioCam using equipped captured an were microscope images inverted and illumination, fluorescent observer Axio wv re kme-ikpwe lcigslto,wt primary with 4 solution, at blocking overnight powder performed skimmed-milk 2.5% incubations containing TBST dried and in was Primary performed (w/v) that powder. was 20) skimmed-milk incubation Tween dried antibody 2% secondary NaCl, (w/v) mM 5% 150 with 8.0, supplemented pH Tris-HCl mM (10 TBST ARTICLE RESEARCH 458 interaction yeast related the and for mutagenesis except overall MAGE-D1 experiments the and the designed NEDD9 of M.Z. screen, all and two-hybrid performed A.S. P.A.B., M.Z. and plan. project, experimental the of conceived P.A.B. contributions Author interests. financial or competing no declare authors The interests Competing quantification. for the and Ermeichouk Inna on imaging and comments in transfections and help with FRET help for with Dorval assistance Genevieve for manuscript, Soubannier Vincent thank We mean Acknowledgements the as expressed Tukey are and values ANOVA All one-way analysis. using post-hoc performed were analyses Statistical analysis Statistical 37 at hours 48 for incubated then were (2 pYPet–PBD signal. and FRET Rac1 a in results interaction this and Rac1 activated, is with Rac1 interacts Once PBD domain). EYFP binding and biosensor (PAK (CyPet–Rac1) pYPet–PBD CyPet by FLAIR replaced by on is replaced are described YFP Rac is acceptor the ECFP biosensors and the The CFP chains. donor FRET Briefly, different the which in Rac 2010). chain dual a al., the of consists et using (Hodgson assessed previously was activity Rac FRET using activity Rac ihpl--yiefr3 iue n aii o or tadniyof 37 density at a hours at 24 hours for kept 2 were for Cells laminin cells/ml. and 10,000 minutes 30 for poly-D-lysine with ah n one nsie.Sie eekp t4 at kept were 5-minute Slides (one slides. water on sterile mounted and and washes) wash) 5-minute (two PBS with washed ahdi BTatrec nuain muoeciebnswere instructions. manufacturer’s the bands per (Perkin-Elmer as Immunoreactive kit CT), Norwalk, incubation. solution Sciences, chemiluminescence Life each enhanced after extensively using were detected TBST Membranes temperature. in room at washed hour 1 for performed ie ih4 aaomleyefr1 iue t37 at minutes 15 for paraformaldehyde 4% with fixed ioetmn 00i niitcfe eim el eekp for kept were using cells Cells into medium. 37 transfected antibiotic-free at hours siRNA in 48 knocked NEDD9 were 2000 and NEDD9 Lipofectamine and NRAGE NRAGE with later, hours the down as 48 well and, as NEDD9 p75FL biosensors with and Rac not NRAGE or transfected For were cells pYPet–PBD). of transfection, siRNA either or with (CyPet–Rac1 transfected were biosensors cells through, the bleed acceptor and donor for O7clswr rnfce rntwt 7F,tgte ihCyPet– with together p75FL, with not or transfected were cells COS7 mgn a efre sn 40 a using performed was Imaging NFRET ~ IFRET ˚ ne %CO 5% under C { BTDonor m )uigclimpopaetaseto and transfection phosphate calcium using g) 2 p hnpae ngascvrlp precoated coverslips glass on plated then , | IDonor ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi IDonor ˚ ne %CO 5% under C 6 | ˚ n eodr incubations secondary and C betv N .)o Zeiss a on 1.4) (NA objective { IAcceptor BTAcceptor ˚ ne %CO 5% under C ˚ ni imaging. until C 2 ˚ nodrt correct to order In . .Te eethen were They C. 6 | s.e.m. IAcceptor 2 then , ecap,R . apei . ho .V,Bre,P . eg .K., H. Teng, A., P. Barker, V., M. Chao, N., Zampieri, and S., M. R. Bothwell, Kenchappa, C., J. Wiley, S., P. Amieux, M., Hudson, C., K. Kanning, rnmn .C,Thrao,M,Jvn .M n anibr M. Fainzilber, and M. T. Jovin, M., Tcherpakov, C., F. L., Bronfman, J. Smith, M., Mahmassani, P., Bradbury, J., Zhong, N., L. Bradshaw, J. P. Bruce-Staskal, and B. R. M., Riggins, H., Leclercq-Smekens, A. Bouton, D., Andrieu, S., R. Kenchappa, J., M. Bertrand, M. E. Shooter, and P. T. Misko, G., Barbee, A., P. Barker, M. G. O’Neill, and P. Bradbury, B., J. Baquiran, rs,M . aiin,F . mlrzk . rni,R . esn .E., M. Betson, E., R. L. Francis, K., Gelman, Smolarczyk, and C., F. B. Maximiano, A., Desvergne, M. Frasa, W., Wahli, D., Sage, N., J. Feige, V. M. Chao, W., Mellado, M., Hilaire, T., Spencer, N., Zampieri, M., Domeniconi, A., K., T. Neubert, A., C. B. Eipper, E., Underwood, R. Mains, S., K., D. Spellman, T., Kim, Reid, K., Deinhardt, L., S. Osborne, M., L. May, J., E. Coulson, F. L. Reichardt, and R. L. Austin, G., Weskamp, O., D. Clary, S., P. McPherson, X., Liu, E., Vereker, R., Thomas, P., R. Kommaddi, C., Ceni, zmhno . ig,M . ltioa .V,Tkmaoa . ite .L., J. Little, N., Tikhmyanova, V., O. Plotnikova, K., M. Singh, E., Izumchenko, K. Hahn, and F. Shen, L., Hodgson, h,J,Sn-oeo .adMrhl,C J. C. Marshall, and V. Sanz-Moreno, J., Ahn, References [grant Research Health of Institute Canadian the MOP62827]. by number supported was project This Funding arntn .W,L,Q . e,C,Pr,J . e .adYo,S O. S. Yoon, and Z. He, B., J. Park, C., Tep, M., Q. Li, W., A. Harrington, O. S. Yoon, and Y. J. Kim, W., Y. A. Harrington, Zheng, and A. D. Williams, L., Yang, M., Debidda, H. F., A. Guo, Bouton, and T. J. Parsons, S., M. Guerrero, irsoydt.MZ n ...woetemanuscript. quantified the and wrote analyzed P.A.B. collected, and V.R. M.Z. 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Nguyen, through formation thioester 279. by cysteine palmitoylated is p75LNTR, receptor, neurotrophin with adhesion conserved focal and is migration NEDD9 NEDD9-mediated protein regulates dynamics. docking and adhesion Y253 the p130Cas of domain substrate motility. amoeboid inhibit and motility oao . odnig . eba .C,Rk . hain .R tal. et R. E-cadherin M. regulates and Ahmadian, Rab7 A., inactivates Rak, that effector degradation. C., Rac1 accommodate a M. is Seabra, Armus can (2010). J., Goldenring, that E., calculation Lozano, FRET for bleed-throughs. spectral plug-in in variations ImageJ an PixFRET, outgrowth. neurite inhibit to receptor neurotrophin p75 the T. M. Filbin, and Rac. through through signaling receptor channels proneurotrophin on L. B. GIRK Hempstead, and V. M. activating Chao, by death P. 4,5-bisphosphate. cell Sah, phosphatidylinositol and neuronal F. P. mediates Bartlett, A., F. Meunier, factor. growth nerve 563. to responses neuronal mimics linking signaling. 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