Proc. Natl. Acad. Sci. USAVol. 89, pp. 10758-10762, November 1992Medical Sciences

Identification of a mouse brain cDNA that encodes a protein relatedto the Alzheimer disease-associated amyloid IB protein precursorWILMA WASCO*t4, KEITH BUPP*§, MARGARET MAGENDANTZ*, JAMES F. GUSELLAt, RUDOLPH E. TANZIt,AND FRANK SOLOMON**Department of Biology and Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139; and tMolecular NeurogeneticsLaboratory, Massachusetts General Hospital East, Charlestown, MA 02129

Communicated by Phillips W. Robbins, June 26, 1992

ABSTRACT We have isolated a cDNA from a mouse brainlibrary that encodes a protein whose predicted amino acidsequence is 42% identical and 64% similar to that of theamylold (3 protein precursor (APP). This 653-amino acidprotein, which we have termed the amyloid precursor-likeprotein (APLP), appears to be similar to APP in overallstructure as well as amino acid sequence. The amino acidhomologies are concentrated within three distinct regions ofthetwo proteins where the identities are 47%, 54%, and 56%. TheAPLP cDNA hybridizes to two messages of approximately 2.4and 1.6 kilobases that are present in mouse brain and neuro-blastoma cells. Polyclonal antibodies raised against a peptidederived from the C terminus of APLP stain the cytoplasm in apattern reminiscent of Golgi sai . In addition to APP,APLP also displays significant homology to the DrosophilaAPP-like protein APPL and a rat testes APP-like protein. Thesedata indicate that the APP gene is a member of a stronglyconserved gene family. Studies aimed at determining thefunctions of the proteins encoded by this gene family shouldprovide valuable clues to their potential role in Alzheimerdisease neuropathology.

The 39- to 43-amino acid peptide (BA4 is the major componentof the senile plaques and cerebrovascular amyloid depositsthat are characteristic of Alzheimer disease, Down syn-drome, and to a lesser extent, normal aging. (3A4 is derivedfrom the larger amyloid (3 protein precursor (APP), whichresembles an integral membrane-associated protein with asmall cytoplasmic C-terminal domain and a larger extracel-lular N-terminal domain (1-4). The APP gene produces atleast four major transcripts predicted to encode proteins of563, 695, 751, and 770 amino acids (5-8). With the exceptionofAPP 695, these transcripts contain an alternatively splicedexon encoding a Kunitz type protease inhibitor domain (6-8).Secreted forms of APP containing the protease inhibitordomain are identical to protease nexin-Il (9, 10), an inhibitorof various serine proteases. It has been shown that normallyAPP is secreted by cleavage at a site near the membrane-extracellular junction within the f3A4 domain (11-14). There-fore, the normal constitutive processing ofAPP precludes theformation of fA4.

In the course of cloning the human APP gene, othercross-hybridizing cDNAs were also obtained, leading to thesuggestion that APP is a member of a larger gene family (4).We have now identified a mouse brain cDNA that encodes aprotein whose predicted amino acid sequence is 42% identicaland 64% similar to APP.L This amyloid precursor-like protein(APLP) is clearly not the mouse homologue of human APP,which has previously been cloned and found to be 96.8%identical to human APP (15). APLP contains three particu-larly conserved regions of homology with APP and is en-

coded by a 2.4-kilobase (kb) message that is present in mousebrain and neuroblastoma cells. Antibodies raised against asynthetic peptide derived from the C terminus ofAPLP stainthe cytoplasm of neuroblastoma cells in a pattern similar tothat obtained with an antibody to a known Golgi protein.The cDNA described here, together with the gene encoding

APP and two other genes-one from Drosophila (16) and onefrom rat (17)-form a gene family producing proteins that arehighly conserved and may therefore share common functionsand, perhaps, undergo similar processing.

MATERIALS AND METHODSCells. Neuroblastoma NB2A cells were maintained as

previously described (18).Screening of Agtll Libraries. Three mouse brain Agtl1

cDNA libraries (Stratagene, Clontech) were screened withcDNA probes (19). Positive clones were sized by PCRamplification and subcloned in pBluescript (Stratagene) orM13 (New England Biolabs) vectors, and both strands weresequenced with Sequenase (United States Biochemical).Rapid Ampfication of cDNA Ends (RACE) Procedure for

Obtining 5' cDNA Extensions. The RACE procedure (20,21)was carried out with primers corresponding to nucleotides699-719 and 672-692 of the sequence presented in Fig. 2.Products were subcloned in pBluescript, screened by hybrid-ization to the 5' 120-base-pair (bp) EcoPJ-Pst I fragment of69A, and sequenced.RNA Analysis. Poly(A)+ RNA (22) was analyzed by North-

ern blotting according to standard methods (23, 24).Production ofAntisera to an APLP Peptide. A 20-mg sample

of the peptide CQQLRELQRH (Biopolymers Laboratory,Howard Hughes Medical Institute and Center for CancerResearch, Massachusetts Institute of Technology) was con-jugated to keyhole limpet hemocyanin (25) and used toimmunize four New Zealand White rabbits (26).

Protein Preparation. Protein from neuroblastoma cells wasisolated by rinsing the cells with phosphate-buffered saline(PBS) followed by lysing the cells in SDS sample buffer (gelloading) and boiling. Protein from mouse brain was isolatedby homogenizing in a modified RIPA buffer (50mM Tris-HCl,pH 7.4/150 mM NaCl/5 mM EDTA/1% Triton X-100/1%sodium deoxycholate/0.1% SDS/protease inhibitors) andcentrifuging at 10,000 x g for 30 min at 40C.

Abbreviations: APP, amyloid ,( protein precursor; APLP, amyloidprecursor-like protein; APPL, amyloid precursor protein-like pro-tein; RACE, rapid amplification ofcDNA ends; ORF, open readingframe.*To whom reprint requests should be addressed at the presentaddress: Department of Neurology, Molecular Neurogenetics Lab-oratory, Massachusetts General Hospital East, 149 13th Street,Charlestown, MA 02129.§Present address: Department of Molecular and Cellular Biochem-istry, University of Paris, 91405, Orsay, France.IThe sequence reported in this paper has been deposited in theGenBank data base (accession no. L04538).

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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APLP

IA69A

J-100 bse pairs= open reaing frame

FIG. 1. Schematic representation of the mouse APLP ORF andthe relationships of various cDNA clones. The 2361-bp ORF andnoncoding region ofthe APLPcDNA are shown. Also shown are therelative locations of two representative cDNA clones isolated fromAgtll libraries, 69A and lA, and a clone obtained through the RACEprocedure, J. Restriction enzyme sites: E, EcoRl; P, Pst I.

t3-Galactosidase fusion protein was generated from anEcoRI-EcoRI fragment of a Agtll clone containing 666nucleotides ofthe 3' coding portion and 260 nucleotides oftheuntranslated region of the APLP ligated into the pUEX5vector (23).For Western blot analysis, protein samples were subjected

to polyacrylamide gel electrophoresis, transferred to nitro-cellulose, and probed with rabbit antibodies and 125I-labeledstaphylococcal proteinA (27). Antibody R37 was provided byT. Ishii (Tokyo Psychiatric University).

Immunofluorescence. Neuroblastoma cells were platedonto glass coverslips 48 hr before fixation; 24 hr beforefixation, the concentration of fetal calf serum was changedfrom 10% to 0.1% to induce neurite extension. Twentyminutes before fixation, concanavalin A was added to 20mg/ml to promote cell adhesion to the coverslips. Cells werefixed in 3.7% (vol/vol) formaldehyde/PBS, permeabilized inacetone, and blocked for 30 nmn at 37°C in PBS containing 1%calf serum. Primary antibody (1:10,000) was added andvisualized with fluorescein isothiocyanate-conjugated goatanti-rabbit antibody (Cappel Laboratories). A rabbit anti-body against Golgi mannosidase II was provided by K.Moremen (28, 29).

RESULTSIdentification and Cloning of APLP. In an antibody-based

screen for cDNA clones encoding a microtubule-associated

Proc. Natl. Acad. Sci. USA 89 (1992) 10759

protein (MAP), a clone was isolated from a mouse braincDNA library which was found to have an open reading frame(ORF) homologous to that ofAPP. The epitope recognized bythe antibody was translated from the noncoding strand of theAPLP cDNA, which does not display homology to anyknown MAP gene.The cDNA clone in which the APP homology was origi-

nally identified contained a portion of the C-terminal codingsequence as well as aportion ofthe 3' untranslated region. Toextend the APLP ORF in the 5' direction, we screened twoClontech Agtll libraries with probes from the 5'-most regionsof our available cDNA clones. Repetitive screens usingprogressively more upstream probes resulted in the isolationof a 1.8-kb cDNA clone, 69A (Fig. 1), whose 5' terminus hasan EcoRI site that is present in the coding sequence ofAPLP.A variation of the RACE procedure (20, 21) allowed theisolation of several independent overlapping cDNA clonesthat extended the APP homology past the 5' EcoRI site. APCR product amplified with primers to the 5'-most 100 bp ofclone J was then used to screen a Stratagene mouse braincDNA library. Full-length APLP clones were obtained andsequenced (Fig. 2). The predicted initiator methionine is inagreement with the eukaryotic consensus initiation sequence(30).APLP Is a Member of a Family of APP-Like Proteins. The

alignment of the APP and APLP sequences (Fig. 3) revealsthat the two proteins are 42% identical and 64% similar at theamino acid level. The identities are particularly strong inthree distinct regions (Fig. 4), where the proteins are 47%,54%, and 56% identical and 67%, 73%, and 74% similar.These same three regions have been shown previously to beshared between APP and a APPL, and they have been termedthe extracellular I (El), extracellular II (EII), and cytoplasmic(C) domains by these investigators (16). The cytoplasmicdomain homology is also present in a partial cDNA clone thathas been isolated from a rat testis library (17). Only APPcontains the PA4 sequence that is found in amyloid plaques.In APLP the extracellular portion of,BA4 is absent; however,a small degree ofhomology is observed in the transmembraneportion of the domain. All four proteins contain a 3- or4-amino acid span ofcharged residues (arginine/lysine) at thecytoplasmic face of the membrane (Fig. 4). This motif hasbeen hypothesized to allow interaction with membrane phos-

1 COCcAGCC GACCCO X

121 GGTCGCCCTCCCCCT=CCTGTCATCTTCCG R V R P P L P L L L P L 8 L L L L

2418 A Q V A G L C C R L T L HN D L R T

361 CAGUCTOcACAClTCCC TAC:CTcGGE Y C R Q H Y P I L H I A R V F Q A A

481 CATGhTGTCCTH I V V P F HC L P C F V 8 A L L

601 A0CMATCAOGOCTCAGGAGC CCICAIVTCCR H Q I A Q I A C 8 S Q G L I L HGCS

721 A A A A A AP A T P N P 8 C N A A G D P S T R 8S

841 GCGoTTACT¶VTAGAQCCCCTCAOgCTG5AGAAGAAGA.CAAGAGG OQV D D Y P V Z P P Q A K IE B I k F F

961 AGOCC TAT_ Q_ A1G_R P T D C V D V Y F CG P G E I G R H

1081 A:CGICATWCOCATAACTGCAOCGAA4M R HW A N A D S Q SK N L PIX A D R

1201 CG0CAACGCCGgIMGACCCCOC: cCACcAGACAIC rIA&CAAChCC_R Q R L V Z T H A T R V I A L I N D Q

1321 TTTQCT% CCA GAGCAAGAGCAhGSGGCACV L X A L R i Y L R A Z Q XK Q R H T

1441 CAGcC CAA C CV Q T H L Q V I I I R H p,,,, L C L L

1561 CACTOGTCC ACH L G P S E L D A 8 V P C 8 8 8 S D X

1681 GATCAAG W1ATCC1VCTWG0GAGAGACTAACCCACTATGJD Q IS SS S C R I K L T P L I Q Y Z

1001 CcC G A c C A CI L A P S G T G V S R T

1921 TATOWACTATCAOCCh612 Y C T I S H C V V Z V D P X L T L 1 1

2041 CACCTTGACCCCTACCCA A CCCCCTR P *

2161 CCTAtAATTCACACCCTTAACCACACCAA2281 GATOTATGTCACTCCCTOGAATTACCATCCATCAACAC

I P.

A A v :__A _CNCQACCAGAACCCT~ACCTCC~r~AOCAGCT~ C~ 0C 1 _I t !CCAGG&GCTTCTCTTOCCTA

V 1..A.F

Q L R L Q R H G Y r..!.7..)T..Y.

D I Q R DrOCQAGCAGAAACCC_ R K K X P

kCc.R.F, L H I

411

451

491

531

571

611

651

XX:Cl -ACCCVCCC

QccIIvccl ACccToGTctcLLAYA&CTCCTCTITCCCICCOGC-p*IyA tail

FIG. 2. Composite nucleotide sequence of APLP cDNA and predicted amino acid sequence of APLP. The predicted membrane-spanningregion is underlined. The location of the peptide sequence used for the production of antisera is double underlined. Predicted N-glycosylationsites and a region surrounding a potential tyrosine phosphorylation site are underlined by dots. The polyadenylylation signal is indicated byboldface type and the stop codon is shown by an asterisk.

Q 11CoGG 51

L 91CACH 131PA0GR 171CCTP 211ccAp 251cCAp 291

Y 331WA 3E 371

E

ZL : Q E L L a

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APLP 21

APP 1

LLLPLSLLLLRAQLAVGNIAVG3SPSAAEAPGSAQVAGLCGRLTLHRDLRT:1 .1.1111 .:..:.::MLPGLALLLL .AAWrARALEVPrDCGLLAEPQIAMGCRLkORh4QN

71 GRWEPDPQRSRRCLLDPQRVLEYCRQtYPELHIARVB0AAQKIPR1WG1:1:.11 .: 1: ..: ::1:11:.1:.:.:1

50 GKWDSDP9GTKTCIMVr ILQYCQEVYPELQI¶NVVEANPVTIcWCK

121 GSGRCAHPHHEVVPFHCLPGEFVSEALLVPEmCRFLHQERiDQCFESST.1.. .111 1:1::11.11111:11111: 1:11111111 II.

100 RGRKQCKITHPHF.VIPYRCLVGEFVSDALLVPDKCKFLHQssNDVCETHL

171

149

RRHQEAQEhACSQGL LHGBM LPCGSDRFRGVEYVCCPPPATFN. . PS::I .. ..: II: 1111111 I:IIIII:IIII:... : .1HWHVAKCSEKSTNLHDYGMLLPOGIDKFRGVEFVCCPLAEEl-NVDS

219 GQ4AGDP9MSWPLQGR ..AEOD........ EEEVESFPQPVDMYFVEP:1:1...1 :: 1:1:11 1111..:.1 :

199 ADAEEDSDVI*WGADTDYADGSEKVVEVAEEEEVAEvEEEEADDDWD

260 PQ EERAPPPSSHTPV............ V PIPR. D.:1111.11..:.:1.: 11..1: .1 1

249 EDGDEVKEEEIPYEEATER¶TSIA TrESVEKVVRVPTTAASTPD

296 GDVYFEPGEIGEHHEGFLRAKMDLEERNRQINEVERENMRADSQSKNL:11 1:: 11: .11. 1:11 1!.::-. :11-1--1

299 AVDKYLETPGDENHAEHQKAKERLEAHF RmEMQVMRqEAEAERQAKNL

346 PKAERALNEHFQSILQTLEEQVSGERQRLVTHATRVIALINDQRALI II I: . I: : I. :: I::. .. II .II I::

349 PKADKKAVIQHFQECVESLEQEAANERQQLVETHMARVEAMLNDRRRLAL

396 EGFLAALQGDPPQEVLNLRRYLRAQKEQRHTLRHYQHVAAVDPEKA1.::.111:11.::1: 1::1:11111:..111:111 111.11

399 ENYITALQAVPPRPRHWVF4LYRAEQKDRQHTLKHFEVRMVDPKKA

446 QW4RFQVTLQVIEER)NQSLGLLDQNPHLAQELRPQIQELLLAEHLGP.1:1 11 111.11 1111111:11I. I:1:1:..:::111 1:

449 AQIRSQVMHILRVIYER QSLSLLYNVPAVAEEIQDEVDELLQKNiYS

496 SELDASVPGSS. . SEDKGSLQPPESKDDPPVTLPKGSM ......QESSS

499 DDVLA NISEPRISYGSAUPSLTLPr1JKLPVNGEFSLDDLQPHS

538 SGREKLTPLEQYE.QKVN& .. SAPRGFPFHSS .... DIQRDELA ......

549 FGADSVPANTIEVEPVDARPAADRGLTTRPGSGITNIKTEEISEVK0I

575 .PSGflVSREALSGLLIMGAGQGSLIVLSLLLL:..I.:1 I :: 1I..:.I1.:1

599 TLVML

607 RKKKPYGrISHOVVEVDPMLTLEEQQLRELQRHGYENPrYRFLEERP*11..11111111: :111:.1:111:::.

649 . KKKQYTSIHHGVVEVDAVTPEERHLSKMQStlYENPrYKFFEBlN

FIG. 3. Comparison ofthe APLP and APP amino acid sequences.The GCG BESTFIT analysis of the mouse APLP and human APP 695(2) is shown. Identities are indicated by a vertical line between thesymbols for the two amino acids. Similarities are indicated by a singleor double dot. Gaps produced by the BESTFIT alignment are shownby dots in the sequence. The /3A4 peptide is underlined in the APPsequence. The identities are concentrated in three regions: APLPamino acids 21-211, 316-488, and 608-653.

pholipids or to provide a stop-transfer signal for membrane-bound proteins (31).Northern Blot Analysis. Northern blots (Fig. 5) reveal that

in mouse brain and neuroblastoma cells there are two mes-sages of approximately 2.4 and 1.6 kb, with the largermessage present in greater abundance. The cDNA that wehave cloned corresponds to the 2.4-kb message, althoughboth messages are consistently seen in Northern blots thatare probed and washed under stringent conditions. Thepredominant message in brain is 2.4 kb, while in peripheralorgans (lung, heart, kidney, spleen, and liver; data notshown) only the 1.6-kb message was observed. The mouseAPLP cDNA does not hybridize to the 3.2- and 3.4-kb APPmessages under the conditions used.

Generation of Antibodies Against an APLP Peptide. Anti-bodies were raised against a synthetic peptide which corre-sponds to a unique sequence in the C terminus of mouseAPLP. Two of four injected rabbits (301 and 302) producedsera that strongly recognize a 65-kDa mouse brain proteinthat is not recognized by preimmune sera (Fig. 6A). A smallerprotein of approximately 33 kDa that is recognized by anti-serum 301 may be a proteolytic degradation product of thelarger protein; however, this remains to be shown. In Fig. 6A,the specificity of the interaction of the antibody with theseproteins is demonstrated by the ability to block the binding ofantibody 301 to the proteins by preabsorbing with the originalpeptide (lanes 2-5); an irrelevant peptide has no effect on the

3XXPCULLTLAR DOMAIX I*e I III* .. . .. . I..*.lI I I .. .

APLP 21 lLlpL sLLLLrAqlaVgnLaVgspsAkeaPgsaQvkgLC 0 RLtlHrdlrtAPP 1 MLpgL aLLLL AaWtarALeVptdgnkgllaePQIAmfC 0 RLnmHnnvqnAPPL 1 McaaLrrnLLLr slWvVlAigtaqvqAAssPrwPQIAvLCeaqiyqpqylsee

1.1 Joe * #10 I . . 11 II.. 10000 0 @ . 11APLP 71 ORWepDPq re rrCLlDpqrvLe TCrqmnPELhlarVeqAaQaipmerlCgAPP 50 OkWdsDPS gT kTCi DtKegilqYCqev1PELqITNVVEAnQpvtIqnWCkAPPL 55 ORWvtDlSkktTgpTCLrD KmdLd YCkkaYPnrdITNiVEsshyqkIggWCr

APLP 122 gtRsgrCA hp hleV vPPhCLpOEPVSeLLVPEGCrFLHQERMDqcEsstAPP 100 rGRk qCk th plffV iPyRCLvOEPVEDALLVPdkCkILNQERMDvCEth1APPL 108 qGaln aAkckguNrwikPF RCL ODyaZLEG iGClPdlhnasrCwpfv

APLP 172APP 148APPL 159

......III*.IRrHQeAqEACSsqG1lLHgsGKLLPCasDrIROyWVCCPhWHtvAkEtCSEkstnLHdyGNLLPCOIDkPRQVUFVCCPRWnQtgaaACqgrGmgmrtfaULIovm=

5YXRACNLLULAR DOMAIN 1I.. II*... 1 ..II... .IAPLP 316 AKmdLErrrmrqineVIRW amAdsQsKNL P&A DrQAlneH]lQsilQtLAPP 318 AKeRL~akHRErMsqNVRE ;eEAerQaKNL PKA DlkAviqHlQekVesLAPPL 413 sqkRLBEsHRgkvtrVxkdWsdlEekyQdmrLadaqsafIfKQrmtarPQtsVQaL

I** 11*0**1 *le 11 I .10001 1*0 * 110 11 * 01 I **0

APLP 365 UEqvsg3RQrLVETlatRViAl IXDqRhaALEgflaALQgdPPqAerVlmaLrrYLAPP 368 UqEaanNRQQLVETmaRVeOAm1NDRRNlALEnYitALQavPPrprHVfrunLkKYvAPPL 470 ZEEgnalkhQLaamuqqaVlAhllqRkReAmtcYtqALteqPPnAhHVekcLqKl L

I I* I*.*,. 1. 1 *j 0. 1. * LI * * ** I1 **... *.. I I .IAPLP 421 RAEQleqrNTLrHYqN VaaVDP EKAqQmRfQVqTHLqVleERMIQ8LgLLAPP 424 NAEQKDRqNTLk~fe. VrmVDP kKAAQiRsQVmTHLrVtyERMNQSLsLLAPPL 523 RAlhEDRahaLaNYrNl1nsggPgglEaAAseRprt 1erLididravnfptmL

CYTOPLASMIC DOMAIN

*.III..*.I * II . I .IIII...I.............. . 1v *APLP 609 KKKpYGTIS HGVVNVDPMLY 1 Z~qqLrelQrHGYZNPTYrFL erp*APP 649 KKKQYtSIH HAVVNVDaaV! P 3URHLsoKMqNTWPTYKPPZQQMQnAPPL 834 KwRtsrSpHaqgfiNVDqnV~thhPivrUZkivpnMQiNdYUMPYYKYPFvke*test 145 rKRQYGTIS HgiVUVDPMLT P 3NRHLnKMQnH5IIXKYLEQMQiQ

FIG. 4. Domains of homology. Regions of the amino acid se-quences of the mouse APLP, the human APP, the Drosophilaamyloid precursor protein-like protein (APPL), and the rat testiscDNA product (test) are compared. Amino acids that are identical inall of the sequences in the domain are shown as uppercase letters inboldface type and are identified by the presence of a vertical lineabove the sequences. Amino acids that are the same in more than onesequence are shown as uppercase letters and have a dot over thesequences. Amino acids that are not identical to any others areshown as lowercase letters. The conserved cysteines (see text) areidentified by the presence of a caret (A) underneath the sequence.Spans of particularly conserved amino acids are underlined. AnN-glycosylation signal is identified by a double underline. Stopcodons are indicated by an asterisk and the amino acid numbers ofthe sequences are shown at the beginning of each line.

interaction of the antibody with either the 65-kDa or the33-kDa protein (lane 6). Antiserum 301 also recognizes thesetwo proteins in neuroblastoma cell extracts (Fig. 6B).To further confirm the specificity of the 301 antiserum, we

determined whether the antiserum would recognize a ,B-ga-lactosidase fusion protein containing the 222 C-terminalamino acids encoded by the APLP cDNA. Fig. 6C shows aWestern blot of bacterially produced proteins that wereprobed with antiserum 301. As can be seen in lane 6,antiserum 301 does specifically interact with the ,B-galacto-sidase-APLP fusion protein.There are a number ofantibodies that have been generated

against the C terminus of APP. Because the identity betweenAPP and the mouse APLP in this region is particularly strong,

., - 41.6

t 2

FIG. 5. Northern blots of mouse brainand neuroblastomaRNA. Poly(A)+ RNA (10,ug) from neuroblastoma (lane 1) and mousebrain (lane 2) was probed with DNA corre-sponding to nucleotides 1791-2305 of thenucleotide sequence shown in Fig. 2. Sizesof hybridizing messages are indicated in kb.

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A11697

,, 66

-4 45

1 2 3 4 5 6

B c

_- 65

d* -433

q

a~~-A

1 2 3 4 5 6

1 2

FIG. 6. Western blots using antiserum 301. Mouse brain andneuroblastoma proteins were separated on a 7.5% polyacrylamidegel. (A) Mouse brain protein was probed with antiserum 301 orpreimmune serum at a dilution of 1:100. The binding of antiserum 301to the 65- and 33-kDa proteins is inhibited by the presence ofincreasing amounts of the peptide used to immunize the rabbit. Lane1, preimmune serum with no peptide; lane 2, immune serum with nopeptide; lane 3, immune serum preabsorbed with peptide at 5 ng/ml;lane 4, immune serum preabsorbed with peptide at S0 ng/ml; lane 5,immune serum preabsorbed with peptide at 500 ng/ml. Preabsorptionwith an irrelevant yeast P-tubulin peptide at 500 ng/ml had no effecton the binding (lane 6). (B) Neuroblastoma cell extracts probed withpreimmune serum (lane 1) and 301 antiserum (lane 2). Both sera wereused at a dilution of 1:100. (C) Anti-peptide antiserum recognizes a

P-gaIactosidase-APLP fusion protein. Western blots on bacteriallyproduced proteins. Lanes 1-3 were stained with preimmune serumfrom rabbit 301. Lanes 4-6 were stained with immune serum. Lanes1 and 4, induced cells containing a plasmid with its P-galactosidasegene fused to an APLP cDNA fragment inappropriately oriented forproduction of an APLP epitope. Lanes 2 and 5, uninduced cellscontaining a plasmid with its .-galactosidase gene fused in frame tothe APLP ORF. Lanes 3 and 6, same cells as in lanes 2 and 5 exceptinduced. Induced cells were grown at 42°C. Uninduced cells weregrown at 30°C. The arrowhead indicates a P-galactosidase-APLPfusion protein recognized by immune serum but not by preimmuneserum. That protein is approximately 24 kDa larger than P-galacto-sidase alone-as predicted due to the insertion of 222 additionalresidues of APLP ORE.

we predicted that some of these antisera would also interactwith the mouse APLP. One ofthese antisera (R37; refs. 2 and32) is directed against the C-terminal 15 amino acids ofAPP,a region where the two proteins are particularly similar (seeFig. 4). R37 recognizes the ,-galactosidase-APLP fusionprotein and the 65-kDa protein in mouse brain (data notshown). The 15-amino acid sequence used to raise theanti-APP antibody does not overlap the 9 amino acids used togenerate antiserum 301. These data suggest that the 65-kDaprotein contains at least two epitopes in common with theAPLP fusion protein.AaU-APLP Immunocytodemstry. The subcellular local-

ization of the protein recognized by antiserum 301 wasassayed by immunofluorescence. When neuroblastoma cells

are stained with 301 (or antiserum 302; data not shown), thepattern that is observed is a reticular staining near the nucleus(Fig. 7 a and b). When these cells are stained with an antibodyto a known Golgi enzyme, mannosidase II, a similar patternis observed (Fig. 7c). It remains unclear whether the cyto-plasmic protein stained corresponds to the 65-kDa or the33-kDa species observed on Western blots. Because thesecells were permeabilized with acetone it was not possible todetermine whether APLP resides in the plasma membrane.

DISCUSSIONThe APLP cDNA, whose predicted product displays 42%identity with APP, encodes a murine member ofthe APP-likegene family. Homology among APP, APLP, and APPL isstrongest in three distinct domains (Figs. 3 and 4) andincludes 11 cysteine residues, an acidic residue-rich region,a potential N-glycosylation site, a hydrophobic membrane-spanning region, a highly conserved cytoplasmic domain, andseveral specific amino acid sequence motifs with exact iden-tity. These findings indicate that the mouse APLP, theDrosophila APPL, the rat testis protein, and APP constitutea family of related proteins. The extensive conservation ofamino acid identity as well as both the overall and specificdomain structure within this family of proteins suggests thatthese proteins may share common functions and, perhaps, beprocessed similarly.Immunocytochemical analysis of APLP in neuroblastoma

cells reveals a pattern suggestive of Golgi staining. Antiserato APPL and APP have also been shown to recognize aprotein in the endoplasmic reticulum or Golgi complex (33,34). The N-terminal extracellular portion of both APPL andAPP can be secreted by means of cleavage near the mem-brane (13, 34, 35). Our results suggest that APLP, like APPand APPL, may be processed in the Golgi complex; however,it is not yet clear whether APLP is secreted.The cytoplasmic region is the most strongly conserved

domain in the APP-like proteins. A peptide containing aportion of the APP cytoplasmic domain can be phosphory-lated on serine and threonine residues in vitro (36). In APLP,a threonine residue resides in a position analogous to thepotentially phosphorylated serine in APP. Agents that areknown to regulate protein phosphorylation at these sites inAPP appear to affect the rate of proteolytic processing ofmature forms (37), raising the possibility that protein phos-phorylation may also affect processing of APLP.A seven-amino acid sequence in APLP contains a potential

tyrosine phosphorylation site residing eight or nine aminoacids from the C terminus of all four proteins (Fig. 4; ref. 38).The seven-amino acid sequence also contains the tetramericmotif NPXY required for the ligand-independent, coatedpit-mediated internalization of the low density lipoproteinreceptor (39). The NPXY sequence is present in the cyto-plasmic tails of at least 16 other cell surface receptor mole-cules, including the ,-integrin receptor and members of theepidermal growth factor receptor family (39), and appears toplay a role in binding clathrin.The existence of a family of APP-like proteins implies that

they may share common functions. The conservation of 11specifically spaced cysteines in the extracellular portion ofthese proteins is indicative of conserved higher-order struc-ture and suggests that these molecules may interact with acommon extracellular molecule(s). Moreover, the strongamino acid conservation within the intracellular C terminisuggests that the proteins in this family may be similarlyphosphorylated and undergo clathrin-mediated internaliza-tion. A distinct physiological role for the ubiquitously dis-tributed APP has yet to be determined. Clues to the functionof any of the members of this family of proteins should helpto elucidate the role of APP and APP-like genes in theneuropathogenesis of Alzheimer disease.

Medical Sciences: Wasco et al.

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Note. We have now isolated human APLPcDNAs and mapped APLPto the proximal long arm of human chromosome 19 (40).

We thank Christine Bulawa, Jasper Rees, and the members of theSolomon and Tanzi laboratories for helpful discussion and advice; T.Ishii and K. Moremen for antibodies; and A. Bernards and A.Snijders for the Stratagene mouse brain library. This work wassupported by National Institutes of Health Grants CA53395 (to F.S.)and R01 NS-30428-01 (to R.E.T.). W.W. was supported by a U.S.Public Health Service National Research Service Award and R.E.T.is the recipient of a French Foundation fellowship and an AmericanHealth Assistance Foundation Award.

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