APLP1 and APLP2, Members of the App Family of Proteins, Behave

Accepted Article E-mail: [email protected] FAX: 753 5964 207 44 Telephone: 44207753 5877 be *addressed should To correspondence whom Kingdom United WC1NLondon 1AX Square Brunswick 29/39 Pharmacy of School London College University Wei and Dai F. Anne SarahStephenson* L. Cousins, EXPRESSION SURFACE RECEPTOR NMDA ENHANCE AND RECEPTORS NMDA WITH ASSOCIATE THEY THAT IN APP TO SIMILARLY BEHAVE PROTEINS, OF FAMILY APP THE OF MEMBERS APLP2, AND APLP1 Article Original Type: Article This article This protectedis by Allcopyright. rights reserved. an 'Accepted Article', doi: 10.1111/jnc.13063 lead to differences betweenversionthis and the Versionof Record. Please cite this article as throughthebeen typesetting,copyediting, pagination and process proofreading which may This article hasbeen accepted for publicationand undergone fullpeer review but has not both APLP1and cells, mammalian in expression Following receptors. NMDA with interactions their to determineimportant if the related APP proteins other two members,contains precursor-like amyloid afforded new insightsinto functionalits significance.Since APPis a member of agene family that has receptors (NMDA) N-methyl-D-aspartate of expression surface of regulation tothe contributes The ofamyloidfunction precursor protein (A ABSTRACT neuropilin tolloid-like Neto1, 1; NMDA,N-methyl-D-aspartate. subunit; NR2B NR2A subunit, assay; GluN1, immunoadsorbent enzyme linked Abbreviations: APP, amyloid precursor protein; a APLP, protein; precursor APP, amyloid PP) although is unknown, the discovery that it GluN2A, GluN2B, NMDA receptor NR1 subunit, NR1subunit, NMDA receptor GluN2B, GluN2A, possess the same properties as APP with respect to respect APP with as properties same the possess proteins 1and 2 (APLP1 and itAPLP2), is myloid precursor-like protein; ELISA, protein; precursor-like myloid

Accepted Article implicated in pathogenic mechanisms leading to Alzheimer’s Disease (AD) due to its known its known to due (AD) Alzheimer’s Disease to leading mechanisms inpathogenic implicated (APP)isaubiquitously precursorprotein Amyloid INTRODUCTION precursor-like amyloid 1;amyloid protein; precursor receptors;amyloid NMDA Key words: APLP1 Title: Running homeostasis. receptor NMDA surface of cell the regulation to contribute they each that in similarly all Thus surfaceexpression. cell GluN1/GluN2B toAPP,APLP1Furthermore, similarly a receptors. NMDA and GluN2B-containing GluN2A- with APLP1and APLP2 of immunoprecipitation Immunoprecipitations from subunit. detergent showedadult mammalian of extracts brain co- GluN1 the with obligatory via interaction andGluN1/GluN2B, GluN1/GluN2A subtypes, receptor NMDA major thetwo with co-immunoprecipitated both they that in APP to similarly behaved APLP2 This article This protectedis by Allcopyright. rights reserved. amyloid- neurotoxic the generate to amyloidogenic pathway via the processing proteolytic important contributor to the proper functioning of of the functioning contributor to proper important Hoeby also reported were findings Similar expression. receptor surface NMDA cell an enhanced in resulted APP of Over-expression complexes (Cousins and GluN1/GluN2B/Neto1 (Neto1) tolloid-like 1 also and GluN1/GluN2A/neuropilin and GluN1/GluN2B, indeed GluN1/GluN2A i.e. brain, adult in expressed receptors NMDA major the two with co-associated APP that found was it isbecause This homeostasis. receptor (NMDA) N-methyl-D-aspartate surface cell of a regulator These amyloid- (reviewed in Muller and Zheng, 2012; Hoe Zheng, 2012; (reviewed inMuller and and matrix with migration repair extracellular neuronal via outgrowth, proteins neurite interaction promotingfactor a trophic aroleas support evidence does although is unclear, APP itself of function precursor-like 2; Alzheimer’s disease Alzheimer’s 2; precursor-like β peptides form the extracellular amyloid plaques, key pathological traits of AD. The AD. of traits pathological key plaques, amyloid extracellular formthe peptides AND APLP2 ASSOCIATE WITH NMDA RECEPTORS WITHAND NMDA RECEPTORS APLP2 ASSOCIATE et al. (2009), thus both groups surmised groups that APPmaysurmised (2009),thusboth an be et al nd APLP2 both enhanced GluN1/GluN2A and enhancedGluN1/GluN2A nd APLP2 both ., 2012). We recently proposed a new role for APP as APP for newrole a proposed We 2012). ., recently excitatory post-synaptic mechanisms. This may be may This mechanisms. excitatory post-synaptic three members of the APP gene family behave of theAPPgene behave family three members

expressed type I transmembrane protein. It is It protein. I transmembrane type expressed et al et ., 2009; 2013; Innocent 2013; 2009; ., et al β peptides. peptides. ., 2012). .,

Accepted Article previously described (Cik, (Cik, described previously (Rockville, Maryland, USA). Each clone generates APLP1 and APLP2 with a C-terminal c-Myc tag. c-Myc tag. a C-terminal with andAPLP2 APLP1 generates Each USA). clone Maryland, (Rockville, OriGene from werepurchased NM_001142276) number 2accession (variant pCMV6APLP2 and NM_005166) number accession 2, (variant variant. denotethe splice pCMV6APLP1 to GluNR1-1a the GluN1-1a splice variant was pCISGluN1 andare GluN1 used thus usedthroughout the manuscript and receptor cell surface trafficking. The results are reported in this paper. investigate ifAPLP1 andAPLP2 similarly behaved to APP with regard to their respectiveassociation To gain further insight into the significance of APP/NMDA receptor interactions, it was of interest to Aydin in system nervous central (reviewed the the of physiology APLP2 in importance for a particular significantly, and, three members family the between differences functional suggests mice knock-out APLP1/2 and APP study of The 1995). prim found is APLP1 whereas tissues, non-neuronal APP toAPP,and APLP1 APLP2, similarly APLP2. or isubiquitous and is expressed and neuronal in amyloid- APP; however, to the similarly processing proteolytic Walsh in (reviewed identity sequence acid% amino - 51 38 ~ sharing are homologous, highly The proteins three and APLP2). 2 (APLP1 contains ofagene family APP isamember that oftheAPP gene haveduplications identified (Cabrejo been particularly important for familial AD formsearly dominant where of gene onsetautosomal This article This protectedis by Allcopyright. rights reserved. and pCI-neoAPP695 pCISGluN2B pCISGluN2A, pCISGluN1-1a, The constructs, Constructs andantibodies PROCEDURES EXPERIMENTAL et al et ., 1993; Hawkins 1993; ., et al ., 2007; Ali 2007;., et al., two other members, amyloid precursor proteins 1 and 1 and proteins precursor amyloid members, other two 2012). 2012). et al et arily incellsarily of the nervous system (Lorent ., 1999; Perkinton, 1999; ., et al ., ., 2013). APLP1 and APLP2 both undergo et al., β sequence is not conserved between between conserved not is sequence 2006; McNaughton McNaughton 2006; et al et ., 2004). Note that only that Note ., 2004). et al., al., et FLAG were as were 2012). et al., et al., Accepted Article 1 procedures of the Animals (Scientific Procedures) Act with full institutional approval, extracted extracted institutional Procedures)full approval, Actwith theAnimals(Scientific of 1 procedures Schedule to according culled London, College University Services, Biological from rats Dawley male adultSprague form from membranes prepared or, brain X-100 (v/v)1 Triton either % extracted with 293transfected from HEK cells described were aspreviously out carried Immunoprecipitations assaysImmunoprecipitation assays. immunoprecipitation andused in were they harvested or expression receptor NMDA surface assayedfor cell and either post-transfection 24h for incubated were cells Transfected cytotoxicity. were inth cultured post-transfection GluN1/GluN2 described (Chazot, (Chazot, described thefor respective immature proteins) were raised numbering to correspond sequences amino acid antibody (all antibodies (679–695) andanti-APP 60) recobut 1454-1464 NR2A the amino sequence acid (raised against anti-GluN2A/2B anti-GluN2A (1381–1394), (44–58), C2, anti-GluN2A Anti-GluN1 This article This protectedis by Allcopyright. rights reserved. with 10 µg APP695 µg 10 with 1:3 of aratio used with were DNA µg 20 of total al USA) were tran Virginia, (ATCC), Human embryonickidney 293 (HEK) cells (purchased Mammalian cell transfections human against APLP2and anti-APLP2 raised Anti-APLP1 VA). (Charlottesville, Upstate from Scotland). Anti-c LtdTechnologies (Paisley, Life from were antibodies Anti-His USA). (Massachusetts, Millipore from were antibodies GluN2B ., 1993). For double transfections 10 µg DNA were used at a 1:1 ratio. For triple transfections a transfections For triple ratio. a1:1 at used DNAwere µg 10 double transfections For 1993). ., et al et FLAG ., 1992; Hawkins Hawkins 1992; ., , 10 µg APLP1 10µg , sfected using the calcium phosphate method as described (Cik (Cik described method as phosphate calcium the using sfected et al c-Myc 262-491 were from AbCam (Cambridge, UK). were 262-491 fromAbCam UK). (Cambridge, ., 1999; 1999; ., Groc, -Myc clone 4A6 mouse monoclonal antibodies were antibodies 4A6mouse monoclonal -Myc clone or 10 µg APLP2 or 10 µg GluN1: GluN2A or GluN1: GulN2B (10 µg total) total) µg (10 or GluN1: GluN1: GulN2B GluN2A antibodies raised against human APLP1 200-300 APLP1 raisedantibodies against human gnizes GluN2A and GluN2B), anti-GluN2B (46– anti-GluN2B GluN2B), and GluN2A gnizes , generated and affinity-purified as previously previously as affinity-purified generated and , e presence of 1mMcell prevent ketamine to for the American Type Culture fortheAmericanCollection Type Culture et al ., 2006; Cousins Cousins 2006; ., c-Myc . HEK 293 cells expressing expressing cells 293 HEK . et al ., 2009). Anti- 2009). ., et Accepted Article Femto ECL substrate (Perbio Science UK Ltd., Northumberland, UK). For all the immunoblots of the immunoblots all For UK). Northumberland, Ltd., Science UK (Perbio ECL substrate Femto West the SuperSignal using development andsubsequent dilution 1:10,000 a at antibodies secondary above the used tissue with brain Immunoblots UK). Bucks., Chalfont, Little Ltd., Biosciences system (Amersham Western blotting theECL using detected and immunoreactivities were 1:2000 seconda or mouseRabbit anti-c-Myc. horseradish-linked anti-APLP2and anti-APLP1; anti-APP (679–695); 1435–1445; anti-GluN2A (1381–1394); GluN2A (Papadakis previously wasImmunoblotting carried 7.5%using out (w/v) as all minigels slab polyacrylamide described Immunoblotting immunoblotting. by followed SDS-PAGE by analysed were pellets immune resulting The and anti-GluN2A/2B. et al., supernat and the resultant xg 100,000 at centrifuged X-100 Triton deoxycholate. sodium % 1 (w/v) with This article This protectedis by Allcopyright. rights reserved. or GluN2A with co-expressed is GluN1-1a when occurs expression surface cell NMDA receptors, antibodi (46–60) (44–58) andanti-GluN2B GluN2A Gl and GluN1/GluN2A of expression surface The cel receptor NMDA of Determination of for the 42% eachof the pellets antibodies. specificity two additional and antibody primary precipitating bythe analysed were thepellets immune of 14% immune pellets, sample brain andin transfections primaryprecipitating antibody and with 50% secothe transfections, 50 double for immunoprecipitates, 2009). Immunoprecipitating antibodies used used we antibodies Immunoprecipitating 2009). et al ., 2004). Primary antibodies used were as follows: anti-GluN1 C2;anti- anti-GluN1 as follows: Primary antibodies usedwere ., 2004). s where three different antibodies were used for immunoblotting of immunoblotting usedfor were antibodies s wherethree different l surface expression by ELISA byELISA expression surface l uN1/GluN2B receptors was measured anti- measured using was receptors uN1/GluN2B % of immune pellets were analysed by the the % of wereby pellets analysed immune ants used for the immunoprecipitations (Cousins (Cousins immunoprecipitations for the used ants or sodium deoxycholate detergent extracts were were extracts detergent deoxycholate or sodium es respectively since for GluN1-1a-containing GluN1-1a-containing for since respectively es re anti-GluN1 C2, anti-GluN2A (1381–1394) C2, anti-GluN1 re ry antibodies were used at a final dilution of dilution a at final wereused antibodies ry nd different specificity antibody; for triple specificity antibody; different nd Accepted Article All results are results themeans All ± pairedt SEM test. tailed two Student’s and werethe by analysed Analysis of results APP695 The single result was investigated. subunits GluN2B GluN2A and GluN1, with ofAPLP1APLP2 and first theinstance, co-immunoprecipitation the In system. expression heterologous amodel in initially investigated was members family these other th association, receptor ofAPP/NMDA significance sequence acid amino %, 57 least at i.e. significant, contains which family agene of amember APP is subunits with APLP1 andAPLP2associate AND RESULTS DISCUSSION Myc APLP1 with were co-expressed GluN1/GluN2B combinations or GluN1/GluN2A either receptors, NMDA GluN1/GluN2 assembled with APLP2co-immunoprecipitate if APLP1and To determine compartment. subcellular this in GluN1 with associate toAPP,similarly that APLP1 andAPLP2 suggests this endoplasmic retained in reticulum, the is GluN1 alone, expressed Since when subunits. single and 1F) (Figure 1C GluN2B 1E) nor and (Figur subunits GluN1 single with immunoprecipitate Myc previously described (Papadakis (Papadakis described previously immunoprecipitations carried with anti-GluN2A or anti-GluN2A/B antibodies. Since neither APLP1 Since neither antibodies. anti-GluN2A/B or anti-GluN2A with carried immunoprecipitations immunoprecipitates, it implies that they would associate with GluN1/GluN2 assembled heteromeric assembled GluN1/GluN2 with they associate that itimplies would immunoprecipitates, GluN2B receptor subunits (McIlhinney subunits receptor GluN2B This article This protectedis by Allcopyright. rights reserved. or APLP2 or nor APLP2 nor FLAG , both APLP1 , both c-Myc c-Myc in HEK 293 cells, homogenates were collected, detergent solubilised and solubilised detergent collected, were homogenates cells, 293 HEK in co-immunoprecipitate with with subunits, either co-immunoprecipitate if GluN2 is detected in c-Myc (M et al et r = 87± = =18) 1 kDa andAPLP2 n GluN1/GluN2 NMDA association GluN1/GluN2 GluN1 receptors via with ., 2004; Cousins 2004;Cousins ., et al . 1998). Cell surface Cell . 1998). as ELISA was carried out two other members, APLP1 and APLP2, that share thatshare APLP2, and members, APLP1 other two similarity. Thus to gain insight into the functional into insight gain similarity.to Thus e potential interaction of NMDA receptors with with NMDA of receptors interaction e potential et al et e 1A and 1D) but not with GluN2A (Figure 1B 1B (Figure GluN2A with not but 1D) and e 1A s shown in Figure 1 reveal that similarly to 1 Figure reveal that in s shown ., 2008). ., 2008). c-Myc (141 (141 ± co- 3kDan=18) c- c- Accepted Article APLP2 antibodies recognized APP M APP recognized antibodies = Mr bandwith major antibodies APLP1;predictedanti-APLP2 recognize a the size of with consistent Myc antibodies to precipitate GluN2B subunits, results in the detection of GluN1 and APLP1 GluN1 of detection the in results subunits, GluN2B precipitate to antibodies with anti-GluN2A/2B and Similarly, 2A 2C). immunoprecipitation (Figure pellets immune respective the appropriate knock-out mice. knock-out the appropriate control beoptimum the Ultimately, for antibodies. the would the ofthesespecificity use antibodies of apossibility thereisstill Thus APLP2 andAPP. APLP1, i.e. arefolded native, targeting antibodies in the of in assays, the co-immunoprecipitation that molecular weights distinct with species immunoreactive recognize antibodies thethree that thefact Despite 3A). (Figure distinct are and APLP2 APLP1 APP, of sizes since themolecular ofAPP therecognition for valid is theantibody However, This isnot between identity unexpectedthe given Figure 3A shows that anti-APLP1 antibodies recognize a major band with M band with a recognize major antibodies anti-APLP1 that shows Figure 3A anti-APP (679–695), anti-APLP1 with (2 probed similarity with similarity with the other family which acid amino had protein a fusion respective raised against since each was APLP2 antibodies However inorder to do was it this, necessary thespecificity anti-APLP1of to andvalidate anti- with APLP1 APLP2 and of co-association The tissues brain innative NMDA receptors with associate and APLP2 APLP1 with anti-GluN2A antibodies results in the detection of GluN1 and APLP1 and ofGluN1 the detection in antibodies results anti-GluN2A with immunoprecipitation case. Thus is this the indeed that 2show shown inFigure The results receptors. This article This protectedis by Allcopyright. rights reserved. 134 kDa ± 5 (n = 3) consistent with the predicted size of APLP2. Interestingly, anti-APP (679-695)APLP2. anti-APP Interestingly, sizeof predicted with kDathe ± consistent 5(n= 134 3) were each expressed alone in HEK 293 cells, and the resultant transfected solubilized lysates lysates solubilized transfected theresultant and cells, inHEK 293 alone expressed each were C-Myc inthe respective immune pellets and (Figure 2B 2D). r = 103 kDa ± 2 (n = 3) but in addition, also both APLP1 and APLP2. and APLP2. APLP1 both also addition, in but 3) = ±2 (n kDa 103 = members (see Methods). Thus APP695 Thus Methods). (see members native NMDA receptors was also investigated. wasalsoinvestigated. native receptors NMDA 00-300) and anti-APLP2 (262-491) antibodies. antibodies. and anti-APLP2 (262-491) 00-300) of cross-immunoreactivity betweenthe three of cross-immunoreactivity the three isoforms at the respective three isoforms the C-termini. in immunoblots, there is a caveat to aware be to acaveat there is inimmunoblots, FLAG , APLP1 c-Myc r = 99 kDa ± 3)= = 99 2(n or APLP2 c-Myc and APLP2 and c-Myc c-Myc in the or c- Accepted Article GluN1 was always immunoprecipitated as GluN was immunoprecipitated wasalways GluN1 that found It was APP members. family with co-immunoprecipitation possible antibodiesdetermine to and,anti-APLP2 anti-APP, anti-APLP1 and NMDA receptor GluN1/GluN2 an assembled of pelleting forGluN2A an was successful; immunoprecipitation prove thatthe to immunoreactivity forGluN1 were analysed pellets and immune antibodies using C2 were carried out frombrain rat adult anti-GluN1 Immunoprecipitations detergentextracts of This article This protectedis by Allcopyright. rights reserved. (Figure 4). subunits GluN2B GluN2A or of GluN1, expression total the in change observed any by accompanied not was expression cell surface in APPthe similarly increase to Again, 4). observed (Figure was expression surface cell increase in two-fold an each,approximate for and i.e. APLP2 APLP1 by elicited tothat compared whenAPPwas enhancement the fold in difference significant was no GluN1/GluN and enhanced GluN1/GluN2A APLP2 both APLP1 Similarly APP, and to 24hpost-transfection. ELISA by measured was expression receptor APLP1, either with A APLP2 parallel and with in were co-expressed receptors NMDA andGluN1/GluN2B GluN1/GluN2A APLP1 andAPLP2, determine wasTo if this case the for ofNMDA receptors. expression surface thecell enhanced (Hoe others we and NMDA receptors, with to interacting addition In noexpression with concomitant change inreceptor subunits surface cell receptor NMDA GluN1/GluN2B and GluN1/GluN2A enhance APLP1 andAPLP2 3). (Figure samples inthese found were immunoreactivities APLP2 APP, APLP1 nor andno control anegative as used immune Igwas non- control always, As C2immunoprecipitates. anti-GluN1 in found was species immunoreactive aclear Nevertheless, brain. adult in expression APLP2 of level a low reflecting extracts detergent AP APPand for strongest were bands immunoreactive C2antibodies (Figure The 3). anti-GluN1 by APLP1 andAPLP2 alsoallco-immunopreicpitated were PP as a positive control and cell surface NMDA NMDA surface andcell control PP a positive as d GluN2B immunoreactivities to demonstrate the the demonstrate to immunoreactivities GluN2B d 2A and GluN2B (FigureGluN2B 2A and LP1. It was difficult LP1.It wasdifficult detect to APLP2 in 2B cell surface receptor expression. There expression. surface2B cell receptor et al ., APP2009) showed., that 3). Furthermore, APP, Accepted Article properties as APP with respect to their interactions with NMDA receptors. Any observed differences differences observed Any receptors. NMDA with interactions their to respect APPwith as properties asceto important is it members, other two contains Hoe interaction,APP/NMDA receptor beitdirect orindi the thre mustbe between conserved the association reportedthe findings heres is Whichever case, the protein. anintermediary via mediated or direct is receptors APPandNMDA between association co-immunoprecipitation4), hetero-oligomerisationdue to is unlikely. unclearIt isstill ifthe (Figure weight molecular a distinct has each Because antibodies. anti-APLP2 and anti-APLP1 APP, anti- by detected are species immunoreactive single tissue, frombrain native immunoprecipitations APLP2 virtue ofthe areinimmune by found pellets APP APLP1and heteroligomers with APLP1 that APLP2 form either and/or and/or excluded that hetero-oligomers (Kaden can homo-and all threeform both members is that known it that tothis however, is Acaveat APLP2. inthe intera apparent were Thus nodifferences similar to those induced byAPP. enhanced both APLP2 and APLP1 Furthermore, subunit. GluN1 obligatory the with interaction via GluN1/GluN2B, and GluN1/GluN2A subtypes, with receptor major NMDA the two immunoprecipitated APPinthatthey co- to both similarly behaved andAPLP2 APLP1 both In summary, in yieldfurther or similaritiesinsight thus would receptors (Hoe (Hoe receptors NMDA of expression surface of regulation contributesthe to it that thediscovery through been gained recently have significance functional its into newinsights although APP unknown, is of function The remarks Concluding This article This protectedis by Allcopyright. rights reserved. et al. (2009) suggested that APP enhanced GluN2B-containing NMDA receptors by decreasing bydecreasing NMDAreceptors GluN2B-containing APPenhanced that suggested (2009)

GluN1/GluN2A and GluN1/GluN2B cell surface expression to fold-increases that are are that fold-increases to expression surface cell andGluN1/GluN2B GluN1/GluN2A et al ., 2009;Cousins et al ., 2012). Since HEK 293 cells express endogenous APP, it cannot be cannot APP,it endogenous ., Since HEK 293cellsexpress 2012). et al et ., 2009). Since APPisa member of a gene that family ctions between NMDA receptors and APP, APLP1 and and andAPP,APLP1 receptors NMDA between ctions to APP/NMDA receptor protein/protein interactions. protein/protein receptor APP/NMDA to how that the protein binding domain responsible for responsible domain binding protein the that how rtain if the related APP proteins possess the same same the possess proteins relatedAPP the if rtain interaction of APP with GluN1. However, in thein However, GluN1. APP with of interaction rect, results anenhanced surface in expression. e family members. It is also unclear why the the why unclear It isalso members. e family Accepted Article Neurochem. and inratbrain. cells 293 kidney human embryonic in expressed subunit receptor glutamate NMDAR1 of detection Immunological the F.A. (1992) Stephenson, M.and Cik, P.L., Chazot, 2966–2976. and Hannequin, D. (2006) Phenotype associated with APP duplication in five families. fivefamilies. in APPduplication with associated Phenotype (2006) D. and Hannequin, F F., Pasquier, Letournel, C., Verny, C., Anterion, Thomas- F., Fourniere, La De M., Vercelletto, A., Laquerriere, L., Guyant-Marechal, L., Cabrejo, models. mouse from insights system: nervous the familyin gene of the APP Functions (2012) U.C. Muller, S.W. and Weyer, D., Aydin, family. protein precursor amyloid the Redundancy in and divergence De B. (2013) S.and Strooper, S.,Shariati, Ali, REFERENCES declare. interest to of conflicts have no authors The UK. Research, antibody prod help with Pharmacy for of School King’s College London, UK) for the gift of pCI-neoAPP695 of gift the UK) for London, College King’s pGW1PSD95ProfessorM. Sheng for Mishina and M. S. Nakanishi WeProfessors thank ACKNOWLEDGEMENTS disease. to Alzheimer’s relationship and thepossible homeostasis receptor NMDA surface ofcell more therole theAPPfamily thegain studies into necessary are in of insight to regulation Further resolved. be yetto is mechanism the again, but, APP, by enhanced is both of expression surface the since and GluN1/GluN2B GluN1/GluN2A between distinguish does not APP that in differ findings Our elusive. occurs remains this which by mechanism the but internalization, receptor This article This protectedis by Allcopyright. rights reserved.

59 , 1176-1178. , FEBS Lett.

587, 587, 2036-2045. Exp. Brain. Brain. Res.Exp. α c-Myc, Professor Chris C.J. Miller (Institute of Psychiatry, ., Vital, A., Checler, F., Frebourg, T., Campion D. Frebourg, Campion T., A., F., Checler, Vital, ., for the gifts of the original NMDA receptor clones, clones, receptor NMDA original the of gifts the for uction. This work was funded by Alzheimer’s by wasfunded work This uction.

217, 217, 423-434. FLAG and Dr Kieran Brickley, UCL UCL Brickley, Kieran andDr Brain

206, J.

Accepted Article disease. Alzheimer’s familial with associated protein mutations precursor amyloid and wild-type between acomparison interactions: and S.L. Cousins, N., Innocent, Cousins, S.L., Hoey, S.E., Stephenson, F.A. and and F.A.Perk Stephenson, S.L., Hoey,S.E., Cousins, Therapeutics Therapeutics andPak, Lee, H-K. D.T.S. Hoe, H.S., on post-synaptic composition precursor protein D.T.S. Pak, S., Vicini, M.D., Ehlers, B.T., Hyman, Y., Matsuoka, A., Pajoohesh-Ganji, L., Feng, Lu, C., J-Y., Lee, A., Makarova, Z., Fu, Hoe, H.-S., Chem.Biol. (NMD of N-methyl-D-aspartate three association co- the evidence Biochemical for (1999) F.A. P.L. andStephenson, L.M., Chazot, Hawkins, 18769-18774. subunits. depends surface onNR2A-2B NMDA receptor mobility D. L.(2006) andChoquet, Cognet, B., Lounis, F.A., Stephenson, S.L., L., M., Cousins, Groc, Heine, complex. protein precursor receptor/amyloid theNMDAwith Neto1associates (2013) F.A. Stephenson, and N. Innocent, S.L., Cousins, surfacecell oftheir enhancement delivery. the in toresult receptors NMDA NR2B-containing and NR2A- assembled with associates NMDA receptor subtypes with the PSD-95 family of MAGUK proteins. MAGUKproteins. the PSD-95 familywith of subtypes NMDA receptor of interaction Differential (2008) F.A. Stephenson, and A.R. Rutter, M., Papadakis, S.L., Cousins, 15 characterization. abiochemical receptors: glutamate heteromeric NMDAR1/NMDAR2A cloned of (1993) Optimalexpression F.A. Stephenson, and P.L. Chazot, M., Cik, This article This protectedis by Allcopyright. rights reserved. , 877-883. , 877-883.

274 18, 18, , 27211-27218. 27211-27218. , 47-56.

Stephenson, F.A. Neuroscience Letters Neuroscience (2012) The(2012) upside ofAPP at synapses. J. Neurochem. J. Neurochem. J. and activity, J. Biol. Chem. 284, 8495-8506. 8495-8506. 284, Chem. Biol. J. activity, and A) R2 subunits in recombinant NMDA receptors. receptors. NMDA recombinant in subunits R2 A)

(2012) NMDA receptor/amyloid NMDA precursor protein(2012) and Rebeck, G. W. (2009) The effects of amyloid of amyloid effects The W. (2009) G. Rebeck, and inton, M. inton, (2009) Amyloid 695 protein precursor

515,

111

126, 131-136. 131-136. , 1501-1513. 1501-1513. , 554-564. 554-564. Proc. Natl. Acad. Sci. USA.

J. Neurochem. J. CNS Neuroscience and Neuroscience CNS

104 Biochem. J. , 903-913. 903-913. ,

103 J. ,

Accepted Article APP family similarities ofproteins: and differences. Fade C.S., Frigerio, A,M., Minogue, D.M., Walsh, precursor Fe65/amyloid stimulate tyrosine c-Abl The (2004) J. C. C. andMiller, M. D. M., McLoughlin, Ward, H.L., S., Byers, Kesavapany, Standen, Lau, K.-F., C.L., M.S., Perkinton, large UK series. protein prion not (APP), but gene (PRNP) protein is Revesz, T., Lowe, J., Rossor, M., Collinge, J. and M McNaughton, D.,Knight, W., Guerreiro,R.,Ryan, Lowe, N., J., Poulter, M., Nicholl,D.J., Hardy, J., cells. intransfected subunitsNR2A and NR1a receptor N-methyl-D-aspartate human the of expression surface cell and targeting intracellular Assembly N. Tricaud LeB., Bourdelles R.A.J., McIlhinney Medicine in Persepctives Harbor aspartate NR1/NR2 receptors. receptors. NR1/NR2 aspartate homodimer formation is forefficient requisite expression cell offunctional, surface N-methyl-D- disulfide-linked NR1-NR1 Appropriate (2004) F. A. Stephenson, and L.M. Hawkins, M., Papadakis, Physiolo (2012) H. Zheng, and U.C. Muller, 40,000 molecular weight receptor-associated protein. protein. receptor-associated weight molecular 40,000 E, ligands and ofits apolipoprotein related protein the of protein family, precursor adul in and embryos mouse in Expression (1995) H. Berghe, Den Van F. and Leuven, Van B., Strooper, De D., Moechars, L., Overbergh, K., Lorent, Biol. Biol. oligomerisation. and pathogenic native aspects of functional and Structural homologues: G. Multhaup, B., Reif, L.M., Munter, D., Kaden, This article This protectedis by Allcopyright. rights reserved.

91, 91, 234-239. 234-239. Neurobiology of Aging of Neurobiology J. Biol. Chem. J. Biol. 2, a006288. 2,a006288. protein nuclear signaling. signaling. nuclear protein alpha-2-macroglobulin receptor/low density lipoprotein receptor- receptor/low alpha-2-macroglobulin

33, 426 – 434. 434. 426 –

279 gical functions of APP family of proteins. proteins. of ofAPP family gical functions , 14703-14712. , 14703-14712. eva, J.V., Wasco, W.(2007) J.V., D.J. AndSelkoe, Theeva, lipoprotein lipase, alpha-2-macroglobulin and the lipase, alpha-2-macroglobulin lipoprotein kinase phosphorylates the Fe65 adaptor protein to protein adaptor Fe65 the phosphorylates kinase , Molnar E., Streit P. and Whiting P. J. (1998) J. Whiting P. P. and Streit E., , Molnar t t mouse members brainofthree amyloid the of Biochem. Soc. Trans. Biochem. Soc. Trans. ead, S. (2012) Duplication of amyloid of precursorDuplication ead, S.(2012) Neuroscience Neuroscience (2012) The amyloid precursor protein and its and protein precursor The amyloid (2012) a significant cause of early onset dementia in a in dementia onset early cause of a significant Neuropharmacology J. Biol. Chem. J. Biol. 65, 1009-1025. 1009-1025. 35, 35,

279

37 416-420. 416-420. , 22084–22091. , 22084–22091. , 1355–1367. 1355–1367. , Cold Spring Cold Eur. J. Cell Accepted Article positions of molecular weight standards ofmolecularweight (xpositions 10 APLP2 where appropriate. The positions of molecular of positions The appropriate. where APLP2 immunoblots. the probe to used specificities antibody the are c-Myc GluN2B and GluN2A, C2, GluN1 respectively. pellet = immune lane 3 and = non-immune extract; lane 2 pellet soluble detergent where lane 1= is identical immunoblots the for layout gel lane The Procedures. as Experimental all under described by immunoblotting wereanalyzed and antibodies immune Ig pellets asshown immune anti-GluN2A/B oranti-GluN2A or at by (IP)100,000g andimmunoprecipitated centrifugation non- with collected extracts soluble solubilized, the detergent harvested, homogenates cell transfected orGluN2B, +GluN2A + GluN1 APLP2 GluN2B; or GluN2A GluN1+ APLP1+ clones:- the with co-transfected were cells 293 HEK and APLP2 GluN and GluN1/GluN2A Assembled Figure2: transfections. independent 3 = from n immunoprecipitations independent 3 = n least at of representative GluN2B and c-MycGluN2B are the specificitiesantibody used to probe the immunoblots. GluN2A, C2, GluN1 respectively. pellet = immune andlane 3 pellet non-immune lane = extract; 2 lane for Procedures. Thegel layout the immunoblots bywere analyzed im pellets immune and shown as antibody receptor anti-NMDA appropriate the or Ig non-immune with (IP) immunoprecipitated 100,000g and at centrifugation by extracts collected the soluble solubilized, detergent harvested, homogenates cell transfected GluN2B, + APLP2 and GluN2A + APLP2 GluN1; + APLP2 GluN2B; + APLP1 GluN2A; + APLP1 GluN1; + APLP1 the clones:- with co-transfected were cells 293 HEK GluN2 NMDA receptor subunits Figure 1:APLP1 and APLP2, similarly to APP, co-immunoprecipitate with GluN1 notbut FIGURE LEGENDS This article This protectedis by Allcopyright. rights reserved. GluN2A, GluN2B, APLP1 (M GluN2B, GluN2A, r = = 87 ± 1kDa) and APLP2 (M 3 → Da) are shown on the right. The immunoblots are The Da)areright. the shownon immunoblots munoblotting all asmunoblotting underExperimental described denotes GluN1, GluN2A, GluN2B, APLP1 and and APLP1 GluN2B, GluN2A, GluN1, denotes 1/GluN2B co-immunoprecipitate with APLP1 with 1/GluN2B co-immunoprecipitate weight standards (x 103 Da) are shown on the Da)are the standards(x shown on 103 weight is identical where detergent lane 1= soluble r = 141 ± 3 kDa) where appropriate. The ± = 141 appropriate. where kDa) 3 → denotes GluN1, GluN1, denotes Accepted Article A and B, HEK 293 cells were co-transfected in triplicate in parallel with the clones shown and cell and cell theshown cloneswith parallel in intriplicate co-transfected were cells 293 HEK A andB, subunits receptor no concomitant change in expression with surface receptor cell NMDA Figure APLP1 APP, andAPLP2, enhance andGluN1/GluN2B 4: similarly to GluN1/GluN2A extract preparations. detergent separate ea immunoprecipitations 3 n = at least independent on (x103 Da)areshown standards weight molecular antibodies. anti-APLP2 and anti-APLP1 anti-GluN2B, anti-GluN2A; C2; anti-GluN1 using immunoblotting bywere analysed and immune pellets Ig non-immune antibodies or C2 anti-GluN1 using carried immunoprecipitations ratbrain, out from adult prepared were (100,000g) Detergent extracts brain the in receptors FigureAPLP1 and 3: APLP2 transfections. 3 independent = n from immunoprecipitations independent 3 = n at least of representative are The immunoblots right. This article This protectedis by Allcopyright. rights reserved. meanare ± the APLP2.SEMfor at Results or APLP1 exogenousAPP; of in theabsence by divided ar The results Procedures. and all byimmunoblotting analysed ashomogenates Experimentalin collected quantitative described orGluN2B/APLP1 GluN1/GluN2A GluN2B/APP; cells HEK 293 D, and C GluN1/GluN2B/APLP2); 4 (GluN1/GluN2B; = n GluN1/GluN2A/APLP2); GluN1/GluN2A/APLP1; GluN1/GluN2A/APP; (GluN1/GluN2A; = 6 n transfections, independent following atleast ± the SEMmeans for arethe Values = 1. GluNR1/GluN2B/pCIS and GluNR1/GluN2A/pCIS i.e. control transfections of expression the where expression surface cell in increase fold the as expressed are results The appropriate. as antibodies Cys 46-60 anti-GluN2B Cys or 44-58 anti-GluN2A using 20hpost-transfection was measured expression receptor surface → denotes GluN1, GluN2A, GluN2B, GluN2B, APP, GluN2A, GluN1, denotes e expressed as the fold change in subunit expression i.e. in the presence presence the in i.e. expression subunit change in thefold as eexpressed associate with GluN1/GluN2A and GluN1/GluN2B and with GluN1/GluN2B NMDA GluN1/GluN2A associate GluN1/GluN2B/APP; GluN1/GluN2B/APLP1; GluN1/GluN2B/APP; the The right. immunoblots are of representative ch carried out in triplicate from at least three at triplicate from in chcarried out were co-transfected with GluN1/GluN2A or GluN1/GluN2A with co-transfected were or, GluN1/GluN2A or GluN2B/APLP2 cell GluN2B/APLP2 cell or or, GluN1/GluN2A APLP1 and APLP2. The positions of Thepositions APLP1 andAPLP2. Accepted Article 0.01; ***0.01; **** ***** p< p< 0.001; 0.005; p< 0.0005. 0.025**; < * for p p alltransfections. < transfections from n=independent 3 least 3 immunoblots This article This protectedis by Allcopyright. rights reserved.

Accepted Article This article This protectedis by Allcopyright. rights reserved.