The EMBO Journal vol.8 no. 5 pp. 1 361 - 1368, 1989

The primary structure of the 4 subunit of VLA-4:homology to other integrins and a possible cell -celladhesion function

Yoshikazu Takada, Mariano J.Elices,Carol Crouse and Martin E.HemlerDana-Farber Cancer Institute, Harvard Medical School, Boston,MA 02115, USA

Communicated by J.-P.Thiery

VLA4 is a cell surface heterodimer in the integrinsuperfamily of adhesion receptors. Anti-VLA-4antibodies inhibited cytolytic T cell activity, withinhibitory activity directed against the effector T cellsrather than their targets. Thus, whereas other VLAreceptors appear to mediate cell-matrix interactions,VLA4 may have a cell-cell adhesion function. Tofacilitate comparative studies of VLA4 and otherintegrins, cDNA clones for the human oe4 subunit ofVLA4 were selected and then sequenced. The 3805 bpsequence encoded for 999 amino acids, with an N-terninus identical to that previously obtained from directsequencing of purified a" protein. The a1 amino acidsequence was 17-24% simnilar to other integrin a chainswith known sequences. Parts of the a(4 sequence mostconserved in other a chains include (i) the positions of19/24 cysteine residues, (ii) three potential divalent cationbinding sites of the general structure DXDXDGXXD and(iii) the transmembrane region. However, ce4 standsapart from all other known integrin a subunit sequencesbecause (i) a'4 has neither an inserted I-domain, nor adisulfide-linked C-terminal fragment, (ii) its sequence isthe most unique and (ill) only ca4 has a potential proteasecleavage site, near the middle of the coding region, whichappears responsible for the characteristic 80 000 and70 000 Mr fragments of aCY.Key words: Integrin/cell adhesion/cytolytic T cell/cDNAsequence

result suggests a possible cell -cell adhesion function forVLA-4.The discovery of the integrin superfamily of cell adhesion

receptors has greatly contributed to our understanding of celladhesion mechanisms (Hynes, 1987). Within the integrinsuperfamily, there are at least 11 distinct receptorssubdivided into three families known as (i) VLA proteins(Hemler et al., 1987b, 1988), (ii) cytoadhesins (Ginsberget al., 1988), and (iii) LFA-1, Mac-I and plS50,95 proteins(Springer et al., 1987). VLA-4 is known to be a memberof the VLA/integrin family because the aC4 subunitassociates with the common VLA f (fll) subunit (Hemleret al., 1987a,b) and the a4 N-terminal amino acid sequenceshowed marked similarity to other integrin a subunitN-terminal sequences (Takada et al., 1987). However,VLA-4 also differs from other integrin heterodimers becausethe VLA-4 a(x subunit is only weakly associated with its j31subunit, and also the 150 000 Mr a'4 protein usuallyundergoes partial 'trypsin-like' cleavage to form 80 000 and70 000 Mr fragments (Hemler et al., 1987a). Among thehuman integrin a subunits, those from a fibronectin receptor(VLA-5) (Argraves et al., 1987; Fitzgerald et al., 1987a),collagen receptor (VLA-2) (Takada and Hemler, 1988),vitronectin receptor (Suzuki et al., 1987), platelet gpIlb/Ella(Fitzgerald et al., 1987a; Poncz et al., 1987), Mac-I(Amaout et al., 1988; Corbi et al., 1988; Pytela, 1988) andp150,95 (Corbi et al., 1987) have been sequenced and shownto have 20-60% conservation of amino acids. These six

L25

IntroductionThe cell surface heterodimer VLA-4 is highly expressed onthymocytes, peripheral blood lymphocytes, monocytes, Tand B cell lines and myelomonocytic cell lines (Sanchez-Madrid et al., 1986; Hemler et al., 1987a,b) but has beenfound in only low amounts on most adherent cells and celllines (Hemler et al., 1987a). Thus VLA-4 is different fromother VLA proteins (VLA-2, VLA-3, VLA-5, VLA-6)which are present in varying amounts on nearly all adherentcells and cell lines. Whereas the latter VLA proteins arereceptors for the extracellular matrix components collagen,laminin and fibronectin (Hynes, 1987; Wayner and Carter,1987; Gehlsen et al., 1988; Sonnenberg et al., 1988), aligand for VLA-4 has not been discovered.

Recently, the mouse equivalent of VLA-4 was implicatedas a lymphocyte homing receptor on Peyer's patch highendothelial venules (HEV) (Holzman et al., 1988). This

4-150 K

;-S-

a b c d

-110 K

e f 9 h i

Fig. 1. Sequential immunoprecipitations using anti-VLA-4 antibodies.Extract from "25-labeled HPB-MLT cells was immunodepleted bysuccessive incubations with control antibody, B-5G10 or L25 asindicated at the top of the figure. Then the remaining proteins wereimmunoprecipitated using B-5G10 (lanes a, d and g), B-5E2 (lanes b,e and h) or L25 (lanes c, f and i).

©IRL Press

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V.Takada et al

MAbDilutions:

1:1001:200

1:10001:2000

Specific 51Cr-Release (cpm)

100 200 300 400 500 600

Fig. 2. Inhibition of cytolytic T cell function by anti-VLA-4antibodies. The mAb L25 (A) and B-5G10 (B) were preincubated withcloned cytolytic T cells for 30 min as indicated and then specific 51Crrelease from target cells was determined in a cytolysis assay as

described in Materials and methods. The anti-HLA class I mAbW6/32 was used as a negative control antibody (D) and gave resultssimilar to no antibody (not shown). Also in some experiments L25 (C)or an anti-HLA class II mAb (E) was preincubated with target cellsrather than effector T cells. Each experiment was done at fourdifferent dilutions of antibody ascites fluid, and at each dilutionsamples were analyzed in quadruplicate.

a subunits can be subdivided into two groups which haveseveral distinguishing features (Takada and Hemler, 1988).Most prominently, there are (i) those containing an insertedI-domain and (ii) those which undergo protease cleavage,together with disulfide linkage of the cleaved C-terminalfragment.

In this paper, the role of VLA-4 in cytotoxic T lymphocytefunction is investigated, and a mAb (called L25) which was

previously known to block cytolytic function (Claybergeret al., 1987), is shown to recognize VLA-4. Also, tofacilitate detailed structural comparisons between VLA-4 andother integrins, the a4 subunit was cloned and sequenced.

Results

Recognition of VLA-4 by the mAb L25The mAb L25 was previously found to block cytolytic Tcell function and also recognized a series of proteins of150 000, 85 000 and 75 000 Mr (Clayberger et al., 1987).Because the sizes of those proteins resembled the VLA-4a!4 subunit and its cleaved fragments, the L25 mAb was

directly tested for VLA-4 recognition. As shown (Figure 1),both L25 and the anti-VLA-4 mAbs B-5G10 and B-5E2recognized proteins of similar sizes (lanes a-c). Further-more, when the mAb B-5G10 was used to immunodepleteall VLA-4 (lanes d and e) from an HPB-MLT cell extract,material precipitated by L25 was also removed (lane f).Conversely, preclearing with the mAb L25 (lanes g-i)caused the disappearance of VLA-4 as detected by B-5G10(lane g) or B-5E2 (lane i).

c

4-

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OCx0_

c_

C

0

0..

M

0-~~~50K

-80K

P:K

Fig. 3. Immunochemical crossreactivity between a4 and a fusionprotein made from a4 cDNA. Rabbit anti-fusion protein antibodieswere positively selected from crude anti a4 antisera by using fusionprotein-Sepharose as described in Materials and methods. The fusion-protein enriched sera was used to immunoblot the fusion protein itself(lane a) and purified VLA-4 protein (lane b). Also, un-enriched anti-a4 sera was used to immunoblot whole cell lysate from HPB-MLTcells (lane c), fusion protein (lane d), and purified VLA-4 protein(lane e).

Inhibition of cytotoxic T cell function by anti-VLA-4antibodiesTo investigate further the potential role of VLA-4 incytotoxic T cell function, the mAbs L25 and B-5G10 were

tested for blocking effects. As shown (Figure 2), fourdifferent concentrations of L25 effectively blocked cytolyticT cell activity down to 30-40% of control levels. Anotheranti-VLA-4 mAb (B-SGlO) was less potent, but also blockedcytolysis (down to 50-60% of control levels). When L25was pre-incubated with target cells rather than with killercells, it had no blocking effect. In another controlexperiment, an anti-HLA class II mAb (LB3. 1) effectivelyblocked killing, as expected, since these cytolytic T cellswere directed against class II targets. Similar blocking resultswere obtained using other CTL clones or uncloned CTL thatrecognize class I targets (data not shown). These resultsconfirm the previous L25 anti-cytolytic blocking results(Clayberger et al., 1987), and suggest that VLA-4 couldhave a cell-cell adhesion role during the association ofcytolytic T cells and their targets.

Identification of cDNA encoding for the a4 subunitTo allow detailed structural comparisons between VLA-4and other integrin a subunits, the a4 subunit was clonedand sequenced. Polyclonal anti-human VLA a4 antiserawas used to select multiple clones from a Xgtl 1 library made

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IA. Anti -VLA-4

(L25)

B. Anti-VLA-4(B-5G10)

C. Anti-VLA-4j_ ~ (L25), with targets

g D. Control+ Z HAb (W6/32)

E. Anti-Class IIMAb (LB3.1)

Function and primary structure of VLA-4 ce4 subunit

M P T IS AW GAK C A N PCG EPAAV

N (C) C A P G R P Y N V A T E S A S P5 C H NA

S A A H S H G A N P W AV S A P A N W L A MIA A N P A35' TACAGATS ~ASS'- TAS,A A..A5I,S A A.'SIS '-,SA.'1.A.

A (C) P C K N P G 5 A (C) S 5 G S P N G P (C) C K TA C409 GACP'A.ACG3'7.9'... ASA .ASAAS.'.A ('' .'..A'. S -----A-'--

A N Q W A S P 5 P C N G A V AT (C) GC H P W K N K NA59 SAAAATAAAGC'CI S TF-TC9'SCA, GGAGTSACISS CTGATTTA.GA.ACAGA.AITSASTIAA.G. A.A7AS'IICIS T7.IIA' AS~'-.

H N K P G G (C)YP V P P A P A S K P A A P (C)Y Q563'TTGGAGA.AAA"T'~',A'A7S' AA~~GG.IAA-A'A-SCIGTTTACACAAG-AT--A.TI''AA GG,'A'SSS-CSSIIIA'A

G ENSA S C) QAAG GASP PAYTKADSI A G AP GSS (SISWGIS CTTTTTGTCTACA A7ATAG IA ~AAATAAATACAAGSITTTTTAGAIA.AACA.A.A.A7CA.TAAA_ATTTGGAAS'TA-'7ASAT- -

SPAvy N') GITTN K A K A PG ADK S N SAVKSf GAPY G Y SAG AS.8 AT'TAITGTCGGAGCCASG'AT'A-,'SAASTASISASTCGG IIA.GACT ACATGAGCAG AISAASTS'GTAIG5ATATATATT 'A ''TTSA'S"A,AS A A A A A A G CG A 5P H G K A 5 A K N 25S

GIG AICTIACAT5G.AAAATk" S'A.'AAAA5 'IGGAT ,'A'77''~A CTICT'SIITIS'r,STG95A ''CA.A'GIA5ATG,- 71A-A N K C K K C S Y G A S A SC) A A S L N AN S G A S-0 L v 2-

AS021 SCACCCATGCAGAS' C:7AAl ASGCGAAGGAA~ASTIS"GTTIACATCAATCPSTSAS CTSGGGAGAGA.ATGAA.GAISATG'S AAIA,~'-~'-SA PNA SAGI P I CP PAPAVY NASG A GNAVNNAN A ATN LA 3l

112A AGTGACAAATA,.'S"''A -T'I'SSGAATCTATAGTTA-.7'TTAGGCGACATTGACAATSGATGS'~'TTAGAAGAGTTGCATCGGGSAADKPYA A SCFG SAGAVN L GAG1 ANADG FGHANVA) C )3

1A2S GACTTGCMAGGTGC5' TAll ATG TACAATGGCCGTGCAGATGGGATCTCGTCA.AICTTCTCACAAGAAAGAAT& GGA('-TCAGATCASC AT..l TA...-

1S27 ATGTTTGGACA.TI9ATATCASSAC G TTGATGCAGATAAIA.ATGGCTATGTAGATGTAGSC.AS7ASS(G(T'TTCGG"A'G.AT,"('-' "~-.N C Q 5 S C A A A A N N C P N A AV A C A A K A SD S A A A P A'4'

NAG ACAAGACCTGTASTAA7IT'SISIHACSC TTCTAGCCACIC(GAGTCAGTAAAATAG4ACGAAA77TGA(AGTGTTGM.M(GGATGS "-T'-T~A,A A P A A A A A A L A P P A A N- A AT K S (C) N N C NW P 5 A (C)

lA GGATCTAACACTTSA ~''''ATATA.A,SSCASGGA.AGTI'SA (AGITTSA'ATTTTTG7TATAlA.GATGAGAT7GGATAS TGAGIS.ACA~~S'. ""

A L L (C)F Y K C K A P C P A L N'MN A A A N R K A S AS-1633 ICAGAGATT CTAT"7"T'AAG'SGA.AA'TCGSACSTAT7AGA.ACGCATACAIGSTSGSCCAGCAGAGAGCTAMACTSAGSCAC515 S~"-

(73) CGGA.AAGATGTGS SS-.. .....' 'A 5TTCAT55 '~A'5~GCT1ASIACITlGS'I9CATGTCA7CAGTA.AGAAI7A.A( ASASk"-~,-AKAAPAISAPAAAH A APP) C PG NA AKAS!C 56

1A3' STTCAGCCAA'( I, T.IA.HAA,GGSAAAAAASA GACATSATAAGAAACAATA.ATTI(. A.A'SST7TGTGGGSSA GA.AA.A77A'''C Q P A S K K K A N K K N PF A P P (C) A P N'(CA 5 AS

19AS GTTACTGCAAAGAT',SG5'TT'' AASCCII'ATACAASATGAAGAACATAT TGCTATGI GGAGS'ATGAAGACATAGATGT'-AA T,TAA AVSAGAK I(GSA K PPHSN'KATPLAACGSNMKGTGLN N'AV fNA6-

ASS., 5SAGATGATGSIA'A .'.A S .'- -'T'ATIAGGA .9T 5' ST' -AT".'~A''AGS'--TAGAGSATGGA.ASASAAS GA~.S.A "'',-.C A A A Y H A K P V C P H K K Q A l) A E

A1A' SAGCAGATACCTSF'.. 5TA5 A.C'A'.GICISAGI(TTGIATATA' AGAGSA5T(A"''T''AAGGATAGAAA(TTAS'''''' ,'"-',A A N S C A A A A D5(C) 5 C Y A A A S P SA V S 7

H A A A S A AH A T (C) N N S N K H P V T V A2A34 7 GTTAA.AGIATATA H'TAAS 'GAT','" TG'S TT'T'GTA'A 'A~TAS'' T5' ,'SA'SFA'AkAA'AGA-.A.A-,.A'SAS' ,-~',

ANN ASGSA.GAAATGAS-'A'5 7-'' .'"..A.A.......A.'-A--G'''I''5'AGA'

ASS)5 SATAAGC 'ST7(AS 'A''-'...A' T9 ~ASC TA(5(5'~ A.PAA(. A' '''S AAA''GA' ~AAASASTS'SIGT,' 'A~S.S -S

Q A L K C A P 5 A A K A K P PY (C) K A D H H (C) N '

2(755 GGGA.AAATSSAAA '.SAAAGAS,AA T.S"'7A'S.' SG''SAAGS. 5(G'~GCAA r"''GCKNM ACSGK GA SAGHIQR5A5 P5)I f A

SAG'5 AGAGCA.ACAGGII5555TGAS 'CAA AGAG7AAST(TGAlAA' GS G.GASGISATGA A'.ST S7 ' ATC TAP A A G SPA SPN PA GAIF N KAHENV A A

GA5. GSA AGTTATTC _ASS A

--------

55A5ATA-T A--- ''5..5'.G.A7 SA....PK SAAI SA GPASSASH IAIGACA-T'AAA'S GGT,,TASGA "I,AACGA~ASAA,STT('SASA'SAT."A.G'.AA~A~A

AATGALAWL5555A (GAG.- A"GAA"IA'.. GAA A 15'((AGA'S'SA2p6S TAGT"' -TGA7'.AAS'SS~AA'' TAGCA'A', '9T'','Tr GASA'AGAA- AAI'A

Fig. 4. Complete nucleotide sequence of an oea subunit cDNA anddeduced amino acid sequence. The deduced N-terminal amiino acidsequence (YNVDT...) which matches the N-termlinal amino acidsequence from purified a'4 protein is underlined, and also the probabletransmembrane domain is underlined. Three potential divalent cationbinding domains are indicated by small boxes, possible N-glycosylationsites are marked with *, and cysteines are indicated by' (C).

from HPB-MLT cells. Ten clones were identified as positiveduring the initial screen of 5 X I05 recombinant Xgtl 1plaques. Following plaque purification of these clones, theywere digested with E-CoRI. applied for agarose gelelectrophoresis, transferred to nitrocellulose and examinedfor cross-hybridization of each insert. Two groups of fourwere found to cross-hybridize to each other. To providepreliminary evidence that some of the cDNA insertscontained authentic a

4 cDNA, fusion proteins synthesizedby recombinant phage were examined. Fusion proteins madefrom two representative clones were coupled to Sepharoseand then used for positive enrichment of anti-fusion proteinantibodies from the anti-a4 polyclonal antibody. Afterelution from the fusion protein -Sepharose, these antibo-dieswere tested for immunoblotting of purified VLA-4 proteinimmobilized on nitrocellulose (Figure 3). Enriched anti-fusion protein 4. 10 sera recognized itself (lane a) as wellas the 150 000 Mr a

4 subunit and its 80 000 Mr fragment(lane b). Notably, the 70 000 Mr fragment of ce was notrecognized, suggesting that clone 4.10 (- 1 .8 kb) coded forepitopes on the N-terminal half of the ct'4 subunit. Un-enriched sera gave a weaker signal, but did bind to a4 froma whole cell extract (lane c), 4. 10 fusion protein (lane d)and purified a4 (including both of its fragments, lane e).Of 10 clones originally selected, four contained at'4 cDNAinserts. Enriched antisera obtained using fusionprotein-Sepharose from other clones did not bind to a4protein. Next the cDNA clone 4.10 was subcloned into

28s

8s

Actin

Fig. 5. RNA hybridization analysis. Total RNA (5 yIg per lane) fromthe leukemic cell lines MOLT-4 and HSB and from the fibroblastcell line MRC was electrophoretically separated on 1 %agarose-formaldehyde gels. transferred to nitrocellulose filters, thenprobed with cX cDNA clone 4.43. In a control experiment (below),the same RNA samples were probed with actin cDNA.

pGEM-4 and partially sequenced. The amino acid sequenceYNVDTESALLYQGPHN ... deduced from the nucleotidesequence was identical to the N-terminal sequence frompurified ac4 peptide (Takada et al., 1987). From this result,and the fusion protein results, we concluded that clone 4.10is authentic cDNA for the VLA-4 ax4 subunit.

Nucleotide and amino acid sequence for a4 cDNABecause the cDNAs initially selected using anti-ax4 sera didnot contain the entire (x4 sequence, additional overlappingclones with an overall length of 3.8 kb were obtained byscreening a XgtlO library with radiolabeled 4. 10 insert. Atotal of four different clones were analyzed, which over-lapped as described in the Methods section. After the cDNAclones were subcloned into pGEM-4 plasmid, the completenucleotide sequence was determined in both orientations froma series of overlapping deletion clones (Figure 4). Translationof the ax4 cDNA sequence yielded 1038 amino acids(encoded by 3114 bp) between the 5' and 3' untranslatedregions of - 25 and 670 bp, respectively. Preceding the N-terminal sequence of the mature protein is a methioninefollowed by 39 amino acids which fulfill the requirementsfor a signal peptide (von Heijne, 1984). Thus the openreading frame (ORF) shown in Figure 4 encodes a matureprotein of 999 amino acids predicted to be 111 000 Mr. Theaddition of 12 potential N-glycosylation sequences (Asn-Xaa-Ser/Thr, average 2500 Mr) to the core protein would resultin an estimated size of 141 000 daltons. That value is closeto the 140 000- 150 000 Mr estimated from ae subunitmigration of SDS -polyacrylamide gels.

Analysis of the N-terminal portion of the sequencerevealed the presence of seven homologous repeatingdomains (domain I, residue 16-56; II, 92-133; III,

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YV-IMSAL YQHNT FGYS laSlinp- Po 2l~ 4T GqNYNGL;AKI- G G Q I DFSSE FG ~QQFINPKGN- SGFP RM--- D VLR CI!IQTST-AgAPAVL PGS F SEFYRPqTDGVS- LG ~TSQ-POLQ PV~A ~cPI-EFDSK-PVQLT GNGS F LD1I~KDSHN[VA GATG ---¶fQ-EE V PWRAE -PSI4OFD LRIDSP-A GEGS F

FFFVP SIS-RM

LVATTQ-PG IVEc44VL q--DWSSTRIQqPI-EFDATTENANT- NARG F VQLQGS GA- IVA ---Q LM -DYS-TG P1JI QV!MVT(gLTA-FRVDSAG F VQYANS----- VVG~KTA ----Q Ld -BS-TG CP1 2lQq1P

qG CG CL.------ERNWE GSVTC NR- KNIFYIKNEI4 PT C G PDRN

c

L-L-N-T-F- TC PL- QQCGNQYYT- D- -FI LS-RLL SSLSSSEGEEPYKS Q -AG SL PLY TEKEPL---S C LST -FTLTRNVGS.----QTLQTF V---VSWSD I PWQ LETEA LAd -SGRRAEN-RDYAE.----DDPr4OFKS sv-IO--SKQD PLY TENKQE---R FLQ- GTK--TV

v--------LSL TTSPPQ-L CPTVN--QTCSENTYV--- FLFGS 4LQPQEEA---V--------- LSL ATTSPSQ-L CPTVN--NECGRNNYL-- T FLLI4TQQRL-

PFA~ FA_Lcg . 191 aa I-domain AP

S.D--pFS14gAGQGY.C----------- E GfTG PGY

YS--PGNTLSRIYVENDFSWDKRY.S -QAGELv GAPGFEAL PQE IAFLIDGSGSIIP.---187 aa 1-domain ---SGPVSRQ PRQ Q 32FLIDGSGS30S.----187 aa I-domain --- -P[G-PV GGFS~~LITTfq"- -PM-LGY----Q-HEQ- ITOXYYT

~~1d~~1kuI 1~ST-E- A RAYT- LIQILSATQEQIAESfYPEYLINLVQGQ-1JJQIY,ASSI YD EYLYS SGD- D KGNLTYV4 LNI$M.TAQAPVAD IFS_MRPGILLWHVSSQ ISFD7SSNPEXIM GYS 41P1D WL L~~SDQVAEIVSU~DPNVYSIKYNNQ-jATRTACAIFD SYGS LqNGD-G D RAARTIINVYD

M~4~SKE.---- !SI -----IRNMTRVDSDMND LG ILRN---RVQS1 A YQHI-P1P1l ------P*-------NMSE?IDNR SYG LALWK---GVQS A YQNT-

I~~I~ENM KE ASY F LL -"O----Ii!MGRpYEINFT ILVGNAQF-IVINKGg-gITVIQANDQ GSF I LLVGAP YN---SDLK-EG FIKI LQQ-GSDIRSLYNF--- ~EQ SYF L LLVG3A LLND PGRP GR LQNP ~IEPTPTtJM-SYYQ4HN - EQ SYF SV D LLVGA LYNUJgRADRtKL GR LFLQ-P PNALGAPSLGENNS SYNF---EQ Y S DYF ALFNDRGSDGKL SLQR DQTTK---QNT-GNWESNAN TQ FGA S L A --TYK. QT CPL-P ARWQCDAQVS-RQWRMKAKV Q YF D L A YYE.----QTR CPL-P RWW-CDA

-f~¶~IqA-A DQDLN IS SRrEL-f-jI4~~~P~G1ENRFS I4~~~~~D N Q-N Y~~IYN N- TK S SDGF Q

:- F~ LG DYDAG FET VFV LKS TPDEL-FLG YGGPS VLVF ~~~s L L4RSDSPFT-~-

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S NGYVDVAVG SjV LL S HNPEV-OTIl E04~WPS-q CFK E~~LY LNG ~~D GA QVVQLWSQS FTPEKITLVNKNAQ.--LCF FPSXL G~~~~~~~~~SA SIFPAN RPEERSfSLEg--N C S N

NG LG YANQVVV EAR LVQ iPAAELPQTKTGN GGAV19 Pq 7 EVYS¶IQDNK LPLSTAL FV LKAGQSGLTN L G G--N~~~H LLSPL I EFNPREV VN D~NQVVKGEGE-R LNVQ SASGLTQ VGG--QV LRPL QFIPAEI SPJ1P4SQVVSEQTLVQS-N LYID

M-PGYIVLFjYqNL R-ES F S G7D3G(OS~A -- Iil=CGEA_hM9KQNNQVAI Y IT LDFSRVT KIPERCQKMVNVQAQS--- EY7QEPS. VN-SLD-12ADS-IGFTV-VLQ-L KQEQGGVR L QATLTQMLI4NGA(!D CENI NESE P LNFI-PQKLSLNA-ELQ-L QE--PRQG T.VLL QAGTTLNLDLGGKNSP I DE R P SLNV~LPRKLNFQV-ELL-L ELKQKGAIR IL S SKNM1¶0SRGGLMQ KL ESE FMEM9ERDRLREGQIQSVVTA LALDSGRPH~ ETNSRRdlQVLGLTQT-.- ~LLL--PNCI P RLNFSKNLLGSRDLQSSV LALDPGRL ETK F~LSR¶YRVLGLKAN-- KFN LL--PScq S LR.LNF

NV IfKRSTEEPtP PKqK~NKTI ER~-H S DTL~QVyS KIGF¶LjPHENKTY IJVGSMKIILNRDISLENPGTS- EAYSET JI.ITNPAY4ETEDG IDLRQIPAAQEQPFIVSNQNKR--LTFS

rIMQAPVDSHGLR- YQSKSR DKA.Q-LL-- GEDN DLqLEVFGEQNH-VYLGDKZ~LN---- LTFN----PPTEAGNA VLNGDTHVQEQT.--RIVLD ED PLLSVTGSP-LLVGADNVLE----LQNDDYRTAADTTGLQ QFTPANISRQA.---HILLE GEDN EPI DSDQ f-IYIGDDNPLT---- LIVKGTPI{SAFGNLR EDARLFTALF.---MKE GNDN Q I FSFN(D-CLVVG--GPRE----FNVTGKPLLAFRNLR L IRYFTASL.--FEKNL]DH QDGI1~FSFPALSLLVG--SNLE----fCgNAE

r4 5 GL~FY! I--L EQN -TDNSGVV1OD-- "!fG I-YVDHX4~RID-jMSRIF E -A DF E L ASFSLPVDG .Vg--- ----AASQKSVA- D PALEREQQVTF7M

AQ-- EGGY PE SGLVRHPGNFS----SL:t --_FAVNQSRI4OV-: D P-MKAGP4ILWGGLRAA--~~ ~ ~ ~ ALEEGf~--L Q--EEENETRVVL-HcEINP-NKKNAQIGIANLI M_

Q TLQNQ SQRSWRL CSASSTEVSGALKST CSHPIFPENSEVTFNgTITF RVEGQEQGQLRSLHL SAPVG--SQGTWST HLIFRGGAQITFLAT

E~D Aii EE: l~p~1VTVAIPKY-TVNGFj PTSPY~DEIb~CMVEK-F-NLQNQA L FQALSESQ~~N 0 VNKPLYZDclSTYE S PSIVHSFEDVGD-DTK--T FDFQIL 5~4NNS DVVVFRLSVEAQAQV LN SKPEAVLFlqSCWHPRD~~QKEEDLG

EUE--S RFQLQIRS qISNPNSKIVtLDVPVRAEAQ%qLRG- SFPASLVVAAEEGE-REQNSLDSW-G

ESLGNKLIMKANMT SENNIP-RTN --8WTFQLELPVEYAVYMV--W~TSHGV----STKYLNFTAqENTSR--

MN~~ ~ ~ ~ S Q FRIt~T- E-CF 8YQRVCALE~l9Nc~ RFPKFIFL TSVPY KNQLTVQ1D~DF

PAVNHVYE (SISG SQ-ALEGQQ VT L 4T jNPINPKGLE1DPEG- HNQQ--PEVE 8HqYEL PVNGLNL I-fLPY FQG-c4L c~P-QPVNPLKVDWGLPIPSP PNPAPVVQHTYEL S¶OSFSKANLQ -YE NNN4 I-JIID--j PMZ4S-DNEINPLRIKI DTVA-VNQNQY RSjj- LVF RLNQTVIW RPQFSEN-L RS KER-L-PSN-SDF7E~LRjA---VANNRY QRD ~VINF KLNQEAVWNDVKVSHPQN-PS-L SS-EK-IAPPA-SDF I4~I-~--

H ¶IE 5 LEDVNK NVT---TRIWqgTFASS TFQT--- EAPSRSSASSGPQ-1 PEK- F K1PLHQQE LHLN---FRVWAKTFLQ~HQPF¶~--INI SFJ4QIFLPEPEQPSRLQDPVLLV D P- V---LAFIIlILPSLYQR--PLDQFVIGQ -RDHL--ITKRDLALSEGDINH l I LYVK---SqYVMWTETFMNNR1NQNI4~Y.PV----------- S - QRI DI FI NATLKGNLSFDWYIKI1HNHNLLI---.-----------PV DVPSFSVQ LDFTLKGNLSWGWVRQILQKKVSV----V

TAAAEINTYN.--,S YV-- T NKPDHEEAE-PKRVGFIS Io IL FL LVA FFR-jQ LIjAVYKALKlflYRItJP LPQKER- A WTEAGSYG--VL I iLAG t L IL FF SLS¶QSIIAWFNVSS ISYAVSISjSLPRGEA Qn -EERA--I ~LVG L T LV CFF NRP1!SSASFNVIE gYRI -IEDIY--S LTVT GIgP~APNPPVVWIL 0 L LVLV YRFFK RPPS-TAEILFNDSVFTL -GQGAFVRSQTETKVEPFK----VP'NPLP- t -4~~ LAXAALT GFR-fS-VAEIMFDTSVYSQL -GQMAFMRAQTTTnKKYK----VHNPTP-1..LL. I4A1L5j4FKR-

VLA4 SILNF RSWSYIN KSNDDVLA22KMTE"DES;ETTELVLAS GTAM- KAQLX--PPAI 1DAIIB L----E DDEEGEVNR Q-----EE IE1EQLQPHENGEGNSETMacl IDM-M GPPGAEPQp150 PEM ANGQIAPENGTQTPSPPSEK

Fig. 6. Alignment of the a chain protein sequences of VLA-4 and other integrin Ca subunits. The a4 sequence is compared to human integrin a

subunit sequences from VLA-2 (Takada and Hemler, 1988), VLA-5 (Argraves et al., 1987; Fitzgerald et al., 1987a), gp IIb (Poncz et al., 1987),VNR (Suzuki et al., 1987), Mac-i (Arnaout et al., 1988; Corbi et al., 1988) and p150 (Corbi et al., 1987). The 191 amino acid 1-domain present in

a2 (residues 159-349), as well as corresponding 187 amino acid regions in p150 and Mac-I have been omitted since there is no corresponding

sequence in a4. Residues in other sequences identical to those in a4 are boxed.

1364

V.Takada et al

VLA4VLA2VLA5IIBVNRMaclp150

VLA4VLA2VLASIIBVNRMaclp150

VLA4VLA2VLA5IIBVNRMaclp150VLA4VLA2VLA5IIBVNRMaclp150VLA4VLA2VLASIIBVNRMaclp150

VLA4VLA2VLA5IIBVNRMaclp150VLA4VLA2VLA5IIBVNRMaclp150VLA4VLA2VLASIIBVNRMacip158

VLA4VLA2VLA5IIBVNRMaclp150VLA4VLA2VLA5IIBVNRMaclp1 50VLA4VLA2VLA5IIBVNRMacipl50VLA4VLA2VLA5IIBVNRMaclp150

VLA4VLA2VLASIIBVNRMaclp150VLA4VLA2VLA5IIBVNRMaclp150

77727573756666

145131146142137120119

188375188191179358356

252440266271257420418

324513341345330492489

398590416418405569566

475658494497485545542

548726569571561720717

627797638636630788785

694865707704698867862

771939782777773934930

8461014851852848

1002998

9011065914925920

10521048

9761134991

1000997

10221118

999115210081009101811371144

Function and primary structure of VLA-4 a4 subunit

Subunitsp150 containing

I -domai n

*V Cleavedsubunits

IIb

20 30 40 50 60 70

Percent Similarity

Fig. 7. Linkage map of relative similarities between integrin axsubunits. Based on the alignment in Figure 6, percent similarities were

calculated between pairs of ca subunits (based on amino acid identity)and then linkage trees were determined by standard procedures usingthe average linkage values (Sneath and Sokal, 1973).

Table I. Comparison of subsets of a subunits which exclusively shareresidues at many positions

Subunit No. of sites with Commoncomparison shared residues features

A 1 - a,va,IIb 70 Cleaved subunits2 - a,aM,p5l0 42 I-domain subunits3 ae c ,pl50 29 -

4 caM,IIb,pl50 21 -

5 (X4,5,av 20 -

6 a:2,a,2 v 18 -

7 x5,a',pl50 17 _8 cV aM pl50 16 -

9 o4 ('X IIb 1010 x2,ctvIIb 10 _

11 a2,a4,jIIb 8 _

12 a4 ' avM 713 a5,IIb,pl50 7 -

14 ax2,ca5,lIb 6 _15 - ca2,a4,a5 5'a -associated

B 1 - a4 ,5a (,v,IIb 29 -

2 a ,a,avj,IIb 24 -

3 c4,a,aM,p 150 22 -

4 c4'2 a5jIlb 10 -

aSets of sequences (column 2), shown in order of prevalence (column1), were derived from computer-generated lists of all amino acidsequence positions in which only three (Part A) or only four (Part B)identical amino acids are shared out of the total of seven at eachposition (from seven total sequences). The number of positions at

which each set of sequences has identical amino acids is listed incolumn 3, and structural features shared within a set of sequences are

listed in column 4. Sequence information from I-domains (residues159-349 in a2, 148-334 in caM and 146-332 in plS0) was omittedfor these determinations.

165-190; IV, 216-249; V, 271 -307; VI, 333-368; VII,395 -435) spaced 22-37 amino acids apart. These domainscontain 30-41 amino acids and are 21-57% homologousto each other. Between repeating domains II and III of a4there is no large inserted 'I-domain' of the type found inVLA-2 or in the a subunits from the O2 integrin family.

Table II. Comparison of structural features of a!4 and other integrin a

subunits

Integrin I-domain Disulfide- Divalent Residues at Residues ata subunit linked cation X4 positions C4 positions

fragment sites 119;278 161 ;462

(x4 j No16F-19 C-278 li-Z C-4621

a2 Yes No [_C-I O C467 S-350 --645

aM Yes No 3 C-97 C-446 S-335 A-633

p150 Yes No 3 C-97 C-4441 A-333 --630

aL Yes No . 3 C-94 CQ440 S-325 G-622

ct5 No Yes 4 K-125 A-289 C-164 C481

IIb No Yes 4 E-117 A-295 C-167 C-484av No Yes 4 M-112 A-280 C-155 C472PS2a No Yes 4 iT-157 A-337 C-198 C-536

Structural features of a subunits are from references listed in thelegend for Figure 6, except for features of caL (Larson et al., 1989)and PS2a (Bogaert et al., 1987). Each residue listed in the last twosets of columns exactly aligns with the others in the column based on

the alignment shown in Fig. 6.

The repeated domains V, VI and VII each containsequences of DX(D/N)X(D/N)GXXD which are similar tothe EF-hand consensus metal binding domains of a numberof calcium and magnesium binding proteins includingcalmodulin, troponin C, parvalbumin (Szebenyi et al.,1981), thrombospondin (Lawler and Hynes, 1986), myosinlight chain (Reinach et al., 1986) and galactose bindingprotein (Vyas et al., 1987). These potential divalent cation-binding sites are located in a region (between amino acids280 and 414) devoid of cysteine residues and N-glycosylationsites. The presence of divalent cation-binding sites in thea'4 subunit is perhaps consistent with the recently noteddivalent cation requirement for murine VLA-4 a -, chainassociation (Holzman et al., 1988).

Northern blotting analysisThe distribution of mRNA for the a4 subunit was studiedby probing with cDNA clone 4.10. Two bands of - 5-6kb were strongly present in Molt-4 RNA, more weakly inHSB RNA and absent from fibroblast RNA (Figure 5).These results are consistent with the known cell-surfaceexpression of VLA-4 on these cells (Hemler et al., 1987a).Hybridization of the same blot with a cDNA probe for thehuman actin gene gave comparable signals in all lanes. Sincethe RNA size (5-6 kb) was somewhat larger than the cDNAclone (3.8 kb), and since no poly(A) tail is present (Figure4), it is assumed that the 3' untranslated region (and perhapsalso the 5' end) is incomplete. While there is not yet an

explanation for the appearance of two RNA bands, there isno evidence for diversity within the coding region.

Comparison of a&4 subunit sequence with otherintegrn a chainsThe alignment of the a'4 subunit sequence with the a chainsequences of human VLA-2, VLA-5 (FNR), VNR,gpIIb/fla, Mac-i and p150,95 shows that several structuralcharacteristics are shared (Figure 6). For example, of the24 a(4 cysteine residues, 19 are conserved in at least threeof the other sequences, and 14 are conserved in all seven

ca subunit sequences. Also, there is 26-39% conservationin the 23 amino acid transmembrane region and 100%

1365

V.Takada et al

conservation of the GFFKR sequence on the cytoplasmic sideof the transmembrane domain. The most striking similaritiesare evident in the region of the a!4 homologous repeats inthe N-terminal half of the molecule. As for aX4, each of theother integrin at subunits has also been noted to have sevenhomologous repeats with three or four potential divalentcation sites within repeats IV -VII (Argraves et al., 1987;Corbi et al., 1987, 1988; Fitzgerald et al., 1987a; Suzukiet al., 1987; Poncz et al., 1987; Arnaout et al., 1988;Pytela, 1988). In these repeat regions, the similarity betweena4 and the other at subunits ranged from 25-33% (inrepeats I and II) to 39-52% (in repeats III -VII). Theoverall similarity between oa4 and the other integrin asubunits is 17-24%, or 20-24% if the I-domains for a 2,aM and p150 are not included in the calculation. This is incontrast to the higher degree of similarity ( - 45 %) betweendifferent human integrin f chains (Tamkun et al., 1986;Argraves et al., 1987; Fitzgerald et al., 1987b; Kishimotoet al., 1987; Law et al., 1987). The a(4 subunit did notcontain a potential divalent cation binding site in repeat IV,and thus resembled ca!2, aiM and p150 but differed from asubunits of VLA-5, VNR and Ilb/Illa.

Potential protease cleavage sites in the VLA-4 a4subunitFor three of the integrin a subunits (a5, av, 1Ib), theC-terminal 15% of the amino acid sequence is cleaved, butthen remains attached to the rest of the a chain by a disulfidelinkage (Argraves et al., 1987; Suzuki et al., 1987; Loftuset al., 1988). The aC4 sequence contains a Lys-Arg (atpositions 852-853) which is in the same region as cleavagesites in other integrin a subunits. However, there is littleadditional homology and no evidence yet that this site iscleaved since the size of the a4 subunit protein isappropriate for its sequence length. Also, the 150 000 M,a4 subunit does not diminish in size upon reduction(Hemler et al., 1987b), suggesting that there are no disulfide-linked cleavage fragments. However, at another site in CX4(residues 564-583) there is a 'KKEK' sequence whichsomewhat resembles the protease cleavage sites in otherintegrins (Argraves et al., 1987; Suzuki et al., 1987; Loftuset al., 1988). Variable cleavage at this site would beconsistent with the previously observed splitting of a4 into80 000 and 70 000 fragments (Hemler et al., 1987a,b).Furthermore two residues after the 'KKEK', there is ana4-specific 'MKKTI' insert, perhaps also involved ina4-specific cleavage (see also Figure 6). However, unlikefor the other cleaved integrin at subunits, cleavage of a4 isvariable and incomplete and there is no evidence for disulfidelinkage of the 80 000 and 70 000 Mr cleaved a(4 fragments.

Relative similarities between integrin a subunitsThe a subunits in the integrin superfamily have previouslybeen subdivided into (i) those with I-domains and (ii) thosewith proteolytically cleaved, disulfide-linked C-terminalfragments (Takada and Hemler, 1988). Because the ag4subunit does not have a disulfide-linked C-terminal fragment,and does not contain an I-domain, it was interesting tocompare the overall similarity of this sequence with the othersix previously established integrin a sequences (Figure 7).As shown, the ca 4 sequence was a little more similar to theprotease-cleaved subunits, than to the I-domain subunits,

perhaps consistent with the anomalous protease cleavagementioned above. Also Figure 7 shows that a 2, a4 and a 5did not group together despite being members of the samefamily and sharing the same t31 subunit. Rather, a2 wasmore similar to the two other subunits which contain an I-domain, and a5 was more similar to the other subunitswhich undergo protease cleavage and have disulfide-linkedC-terminal fragments.To affirm further the linkages shown in Figure 7, patterns

of amino acid conservation among sets of a subunits wereexamined. Thus a computer-generated list was compiled ofall individual amino acid positions in which exactly three(out of seven) sequences shared residues. From that list itwas determined that a 5, a v and Ilb most often sharedamino acids (at 70 positions) and after that, ac2, CM andp150 exclusively shared residues at 42 positions (Table I,Part A). The designation of these two groups by this methodagrees with the results shown above in Figure 7 andpreviously discussed (Takada and Hemler, 1988). Residueswere shared among other sets of three a subunits lessfrequently (at 29 positions or less). Notably, the VLA asubunits (a 2, a4, a5) shared residues at only five positions,and thus this group was only 15th-most prevalent in the listof possible groups of three a subunits. The 187-191 aminoacids in the I-domains of a 2, a!M and p150 were excludedfrom the alignments used to obtain data in Table I. Becausethere are so few 'l01-specific' amino acids it appears that theVLA a subunits do not depend on large numbers of con-served amino acids for f3, association.To address further the question of a 4 similarities to other

integrin subunits, positions were enumerated in which exactlyfour (of the seven) sequences shared amino acids (Table I,Part B). The set of four sequences which most oftenexclusively shared residues was a4, a5 a and lIb (occur-ring 29 times). The next time a4 appeared in the list (inthird place) it was grouped with the 'I-domain' subunits af2,acM and p150 (occurring 22 times). This result emphasizesthat the evidence moderately favors grouping of a4 with the'cleaved' subunits compared with grouping it with the 'I-domain' subunits, a result consistent with the grouping shownin Figure 7.

Disulfides in the a4 sequenceThe majority of cysteines in both integrin a and ,B chainsare highly conserved, consistent with a major structural rolefor intrachain disulfide bonds in determining a conservedtertiary structure characteristic of integrins in general.However, in addition to the 14 cysteines conserved in nearlyall integrin a subunits, it was previously noted that therewas a pair of cysteine residues seen only in I-domain subunits(Ca2 am and p150), and another pair seen only in cleaveda subunits (a-5, aV and IIb) (Takada and Hemler, 1988).Notably, a4 had cysteine residues at two positions (165 and462 in the a 4 sequence) which matched the two positionswhere cysteines were previously found only in cleavedsubunits. On the other hand, a4 also had a cysteine in onlyone (position 178) of the two positions previously suggestedto be I-domain specific (Table II). Thus with regard to'subset-specific' cysteines, a4 resembled both the cleavedsubunits (a5, aV and IIb) and I-domain subunits (a 2, aMand p150), but again t4 appeared to be more related to theformer than the latter.

1366

Function and primary structure of VLA-4 a4 subunit

Discussion

The ability of two separate anti-VLA4 mAb (L25 andB-5G10) to block cytolytic T cell lysis of target B cellssuggests that VLA-4 may function in cell -cell interactions.Thus VLA-4 appears to be distinct from other receptors

(VLA-2, VLA-3, VLA-5 and VLA-6) in the VLA (integrin(3) adhesion family which mediate cell-matrix adhesionfunctions. A cell-cell adhesion function for VLA-4 isconsistent with VLA-4 being present on nearly alllymphocytes and monocytes, but absent from most adherentcells (Hemler et al., 1987a). The mAb L25 had previouslybeen shown to block class I and class II directed CTLfunction (Clayberger et al., 1987), but the antigen was notidentified as VLA4. Now it is clear that the proteins of150 000, 85 000 and 75 000 M, previously immunopre-cipitated by L25 (Clayberger et al., 1987) are a4 and itscleaved products. Most likely, a 130 000 Mr subunit was

not previously seen in L25 immunoprecipitations (Claybergeret al., 1987) because it had dissociated from a!4, as oftenoccurs for VLA-4 subunits (Hemler et al., 1987a).

Because VLA-4 expression is widespread on leukocytes,and because a wide variety of specific and non-specificreceptors and ligands have been identified which assist Tcell-target cell interaction (Martz, 1987), it appears mostlikely that VLA-4 would be an accessory molecule ratherthan a highly specific receptor in this process. Furthermore,VLA-4 not only appears to mediate T-B cell interaction,but another recent study has implicated mouse VLA4 inlymphocyte -endothelial cell interaction. Specifically, anti-mouse VLA-4 mAb selectively blocked organ-specifichoming to Peyer's patch high endothelial venules (Holzmanet al., 1988). At present it is difficult to understand howVLA4 could have an organ-specific role in lymphocytehoming, considering the widespread distribution of VLA-4on nearly all lymphocytes and its role also in T-B cell inter-action.

Prior to recent studies of VLA-4, other integrin receptorsknown to have cell-cell interaction functions were foundonly in the integrin (2 family. For example, LFA-1 on

lymphocytes interacts with the ligand ICAM-1 on otherlymphocytes, endothelial cells and other cells (Dustin et al.,1988). Also LFA-1 appears to be involved as a non-organ-

specific accessory molecule in lymphocyte homing (Hamannet al., 1988; Pals et al., 1988). Thus there is precedent forboth T-B interactions and T-endothelial interactionsmediated by the same integrin receptor. Also, the (2

integrins Mac-I and pl50,95 mediate granulocyte andmonocyte interaction with unknown ligand(s) on endothelialcells (Pohlman et al., 1986; TeVelde et al., 1987). In futurestudies, it will be interesting to identify the VLA4 ligand(s)and determine if it/they have any resemblance to ICAM-1or other (2 integrin ligands.An antibody (4B4) to the VLA (1 subunit (also called

(CD29) has previously been shown to subdivide lymphocytesinto subpopulations correlating with different immunologicalfunctions (Morimoto et al., 1985; Sanders et al., 1988).However, since VLA-4 is evenly distributed on lymphocytes,with no evidence for subpopulations (Hemler et al., 1987a),it is not clear that the immunological functions of VLA-4would correlate with those previously defined subpopulationfunctions.

Although VLA-4 may functionally resemble the 02integrins, it is structurally quite distinct, and in fact, itsprimary a subunit structure is unlike any of the knownintegrin a subunits. The a4 subunit has neither an I-domain,nor a typical cleaved, disulfide-linked, C-terminal fragment.Also a4 stands apart in terms of overall sequence similaritywith the other integrins, the location of certain characteristiccysteine residues, and the presence of an anomalous proteasecleavage site near the middle of the sequence. In many ofthese aspects, a4 is a little more like the cleaved subunitsand less like the subunits with I-domains (see Tables I andII). Perhaps this is consistent with the unusual cleavage whichoccurs in ae4, resulting in partial conversion to 70 000 and80 000 Mr fragments. As the list of VLA-4 functionsbegins to expand, the availability of an ca4 clone, and theaccompanying sequence information should greatly aid futurestudies correlating VLA-4 structure and function.

Materials and methods

Monoclonal antibodiesThe mAb B-5G10 and B-5E2 were produced as previously described (Hemleret al., 1987a), and L25 (Clayberger et al., 1987) was obtained fromDr B.McIntyre, M.D. Anderson Hospital, TX.

Analysis of cytolytic T cell functionHuman cytolytic T cell clones and uncloned mixed lymphocyte cultureswere derived as previously described (Brenner et al., 1985). To assaycytolytic function, T cells were incubated with 51Cr-labeled Epstein-Barrvirus-transformed B cells for 4 h in U-bottom microtiter wells. The amountof 51Cr was determined in quadruplicate for each sample and the specifickilling was calculated using standard procedures (Brenner et al., 1985). Todetermine inhibition, varying dilutions of mAbs were preincubated for 30min with either the effector T cells or with the target cells.

Isolation of cDNA clonesPhage XgtlO and Xgtl 1 cDNA libraries made from the T leukemic line HPB-MLT, were obtained from D.Dialynas (Dialynas et al., 1986). For screeningthe Xgtl 1 library, anti-a4 antiserum was prepared by immunizing rabbitswith the a4 subunit of VLA-4 isolated from HPB-MLT cells (Hemleret al., 1987a), and non-specific reactivity was removed by passage overSDS-denatured placenta extract coupled to pharose. Positive clones selectedby antibody screening of S x 10' recombinant Xgtl 1 (Young and Davis,1983) were plaque-purified. The phage DNA was digested with EcoRIrestriction enzyme and the inserts were tested for cross-hybridization bySouthern blotting (Maniatis et al., 1982). Two representative clones wereselected to produce fusion proteins in lysogenic Y1089 Escherichia coli asdescribed (Huynh et al., 1985). The fusion proteins were purified bySDS-PAGE, electroeluted, coupled to Sepharose and then used to positivelyselect anti-fusion protein antibodies from anti-a4 rabbit serum. The initiallyselected clone (clone 4.10, bp 111 - 1827) did not contain the entire a4coding region, so additional clones were selected from the HPB-MLT XgtlOlibrary by screening with 32P-labeled 4.10 cDNA probe. These were clone4.39 (bp 1-1827), clone 4.37 (bp 88-1660) and clone 4.43 (bp1025 -3805). Clone 4.43 was isolated by partial EcoRI digestion so thatit would span the EcoRI site at bp 1827. In clone 4.10, to the 5' side ofthe N-terminal amino acid codon (bp 142) there was a 30 bp sequence (notshown) which is not found in clones 4.39 and 4.37. At present it is notclear if there is variability in the leader sequence, or if the 30 bp at the5' end of clone 4.10 are a cloning artefact.

DNA sequencingPhage DNAs were purified by the plate lysate method (Maniatis et al., 1982),digested with EcoRI, and then the cDNA inserts were separated on agarosegels, electroeluted and subcloned into pGEM-4 plasmid (Promega Co.).The DNA sequences were determined by the dideoxynucleotide chain-termination method of Sanger et al., 1977) using adenosine 5'-[a-thio]tri-phosphate labeled at the a-thio position with 35S. To facilitate the sequenc-ing, a series of overlapping deletion clones of both strands were made as

described (Henikoff, 1984).

1367

V.Takada et al

AcknowledgementsWe thank J.McLean and Dr M.Brenner for T cell clones and lines usedin cytolysis studies, and D.Faulkner and T.Smith for assistance with cDNAsequence comparisons and alignments. This work was supported by NIHgrant GM 38903 and by a fellowship from the Cancer Research Institute(to M.J.E).

ReferencesArgraves,W.S., Suzuki,S., Arai,H., Thompson,K., Pierschbacher,M.D.

and Ruoslahti,E. (1987) J. Cell Biol., 105. 1183-1190.Arnaout,M.A., Gupta,S.K., Pierce,M.W. and Tenen,D.G. (1988) J. Cell

Biol., 106, 2153-2158.Bogaert,T., Brown,N. and Wilcox,M. (1987) Cell, 51, 929-940.Brenner,M.B., McLean,J., Yang,S.Y., van der Poel,J.J., Pious,D. and

Strominger,J.L. (1985) J. Immunol., 135, 384-390.Clayberger,C., Krensky,A.M., Mclntyre,B.W., Koller,T.D., Parham,P.,

Brodsky,F., Linn,D.J. and Evans,E.L. (1987) J. Immunol., 138,1510-1514.

Corbi,A.L., Miller,L.J., O'Connor,K., Larson,R.S. and Springer,T.A.(1987) EMBO J., 6, 4023-4028.

Corbi,A.L., Kishimoto,T.K., Miller,L.J. and Springer,T.A. (1988) J. Biol.Chem., 263, 12403-12411.

Dialynas,D.P., Murre,C., Quertermous,T., Boss,J.M., Leiden,J.M.,Seidman,J.G. and Strominger,J.L. (1986) Proc. Natl. Acad. Sci. USA,83, 2619-2623.

Dustin,M.L., Staunton,D.E. and Springer,T.A. (1988) Immunol. Today,9, 213-215.

Fitzgerald,L.A., Poncz,M., Steiner,B., Rall,S.C.,Jr, Bennett,J.S. andPhillips,D.R. (1987a) Biochemistry, 26, 8158-8165.

Fitzgerald,L.A., Steiner,B., Rall,S.C.,Jr, Lo,S. and Phillips,D.R. (1987b)J. Biol. Chem., 262, 3936-3939.

Gehlsen,K.R., Dillner,L., Engvall,E. and Ruoslahti,E. (1988) Science, 24,1228-1229.

Ginsberg,M.H., Loftus,J.C. and Plow,E.F. (1988) 7hrombos. Hemostas.,59, 1.

Hamann,A., Jablonski-Westrich,D., Duijvestijn,A., Butcher,E.C.,Baisch,H., Harder,R. and Theile,H.-G. (1988) J. Immunol., 140,693-699.

Hemler,M.E., Huang,C., Takada,Y., Schwarz,L., Strominger,J.L. andClabby,M.L. (1987a) J. Biol. Chem., 262, 11478-11485.

Hemler,M.E., Huang,C. and Schwarz,L. (1987b) J. Biol. Chem., 262,3300-3309.

Hemler,M.E., Crouse,C., Takada,Y. and Sonnenberg,A. (1988) J. Biol.Chem., 263, 7660-7665.

Henikoff,S. (1984) Gene, 28, 351-359.Holzman,B., Mclntyre,B.W. and Weissman,I.L. (1989) Cell, 56, 37-46.Huynh,T.V., Young,R.A. and Davis,R.W. (1985) In Glover,D.M. (ed.),DNA Cloning: A Practical Approach. IRL Press, Oxford, Vol. 1, p.49-78.

Hynes,R.O. (1987) Cell, 48, 549-554.Kishimoto,T.K., O'Connor,K., Lee,A., Roberts,T.M. and Springer,T.A.

(1987) Cell, 48, 681-690.Larson,R.S., Corbi,A.L., Berman,L. and Springer,T.A. (1989) J. Cell Biol.,

108, 703-712.Law,S.K.A., Gagnon,J., Hildreth,J.E.K., Wells,C.E., Willis,A.C. and

Wong,A.J. (1987) EMBO J., 6, 915-919.Lawler,J. and Hynes,R.O. (1986) J. Cell Biol., 103, 1635-1648.Loftus,J.C., Plow,E.F., Jennings,L.K. and Ginsberg,M.H. (1988) J. Biol.

Chem., 263, 11025-11032.Maniatis,T., Fritsch,E.F. and Sambrook,J. (1982) Molecular Cloning: A

Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, New York.

Martz,E. (1987) Hum. Immunol., 18, 3-37.Morimoto,C., Letvin,N.L., Boyd,A.W., Hagan,M., Brown,H.M.,

Kornacki, M.M. and Schlossman,S.F. (1985) J. Immunol., 134,3762-3769.

Pals,S.T., den Otter,A., Miedema,F., Kabel,P., Keizer,G.D., Scheper,R.J.and Meijer,C.J.L.M. (1988) J. Immunol., 140, 1851-1853.

Pohlman,T.H., Stanness,K.A., Beatty,P.G., Ochs,H.D. and Harlan,J.M.(1986) J. Immunol., 136, 4548-4553.

Poncz,M., Eisman,R., Heidenreich,R., Silver,S.M., Vilaire,G., Surrey,S.,Schwartz,E. and Bennett,J.S. (1987) J. Biol. Chem., 262, 8476-8482.

Pytela,R. (1988) EMBO J., 7, 1371-1378.Reinach,F.C., Nagai,K. and Jones-Kendrick,J. (1986) Nature, 322, 80-83.

Sanchez-Madrid,F., De Landazuri,M.O., Morago,G., Cebrian,M.,Acevedo,A. and Bernabeu,C. (1986) Eur. J. Immunol., 16, 1343- 1349.

Sanders,M.E., Makgoba,M.W., Sharrow,S.O., Stephany,D., Springer,T.A., Young,H.A. and Shaw,S. (1988) J. Immunol., 140, 1401-1407.

Sanger,F., Nicklen,S. and Coulson,A.R. (1977) Proc. Natl. Acad. Sci. USA,74, 5463-5467.

Sneath,P.H.A. and Sokal,R.R. (1973) Numerical Taxonomy. W.H.Freeman, San Francisco.

Sonnenberg,A., Modderman,P.W. and Hogervorst,F. (1988) Nature, 360,487-489.

Springer,T.A., Dustin,M.L., Kishimoto,T.K. and Marlin,S.D. (1987) Ann.Rev. Immunol., 5, 223-252.

Suzuki,S., Argraves,W.S., Arai,H., Languino,L.R., Pierschbacher,M. andRuoslahti,E. (1987) J. Biol. Chem., 262, 14080-14085.

Szebenyi,D.M.E., Obendorf,S.K. and Moffat,K. (1981) Nature, 294,327-332.

Takada,Y. and Hemler,M.E. (1989) J. Cell Biol., in press.Takada,Y., Strominger,J.L. and Hemler,M.E. (1987) Proc. Natl. Acad.

Sci. USA, 84, 3239-3243.Tamkun,J.W., DeSimone,D.W., Fonda,D., Patel,R.S., Buck,C.,

Horwitz,A.F. and Hynes,R.O. (1986) Cell, 46, 271-282.TeVelde,A.A., Keizer,G.D. and Figdor,C.G. (1987) Immunol., 61,

261 -267.von Heijne,G. (1984) J. Mol. Biol., 173, 243-251.Vyas,N.K., Vyas,M.N. and Quiocho,F.A. (1987) Nature, 327, 635-640.Wayner,E.A. and Carter,W.G. (1987) J. Cell Biol., 105, 1873-1884.Young,R.A. and Davis,R.W. (1983) Science, 222, 778-782.

Received on January 13, 1989; revised on February 27, 1989

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