Completesequenceofhumanvinculin and assignment to ...mProc. Natl. Acad. Sci. USA87(1990) 5669 6 5. 500-residue no. 500 1000 humanvinculin FIG. 3. Dotmatrixcomparisonofhumanandnematodevinculin

Proc. Natl. Acad. Sci. USAVol. 87, pp. 5667-5671, August 1990Biochemistry

Complete sequence of human vinculin and assignment of the geneto chromosome 10

(cell-natrix junctions/actin cytoskeleton/talin)

P. A. WELLER*, E. P. OGRYZKOt, E. B. CORBEN*, N. I. ZHIDKOVAt, B. PATEL*, G. J. PRICE**,N. K. SPURR§, V. E. KOTELIANSKYt, AND D. R. CRITCHLEY*¶*Department of Biochemistry, University of Leicester, Leicester LEl 7RH, United Kingdom; tInstitute of Experimental Cardiology, Cardiology ResearchCentre of the USSR, Academy of Medical Sciences, 3rd Cherepkovskaya Street 15A, Moscow 121552, USSR; and §Department of Human GeneticResources, Imperial Cancer Research Fund, Clare Hall Laboratories, Potters Bar, Herts EN6 3LD, United Kingdom

Communicated by Nikolay P. Dubinin, April 13, 1990 (receivedfor review March 1, 1990)

ABSTRACT We have determined the complete sequenceof human vinculin, a cytoskeletal protein associated withcell-cell and cell-matrx junctions. Comparison ofhuman andchicken embryo vinculin sequences shows that both proteinscontain 1066 amino acids and exhibit a high level of sequenceidentity (>95%). The region of greatest divergence falls withinthree 112-amino acid repeats spanning residues 259-589. In-terestingly, nematode vinculin lacks one of these central re-peats. The regions of human vinculin that are N- and C-terminal to the repeats show 54% and 61% sequence identity,respectively, to nematode vinculin. Southern blots of humangenomic DNA hybridized with short vinculin cDNA fragmentsindicate that there is a single vinculin gene. By using a panel ofhuman-rodent somatic cell hybrids, the human vinculin genewas mapped to chromosome 10qll.2-qter.

Vinculin is a cytoskeletal protein associated with the cyto-plasmic face of both cell-cell and cell-extracellular matrixadherens-type junctions (1, 2), where it is thought to functionas one of several interacting proteins involved in anchoringF-actin to the membrane (reviewed in ref. 3). In cell-matrixjunctions, these proteins include a-actinin (4) and talin (5),although talin is absent from cell-cell junctions (6). Evidencein support of such a role for vinculin comes from in, vitrobiochemical studies that show that vinculin binds to a-actinin(7, 8), an F-actin bundling protein (reviewed in ref. 9), and totalin (10). Talin in turn has been reported to bind to thecytoplasmic domain of the A1 subunit of the fibronectinreceptor (11, 12), thus completing the link between the actincytoskeleton and the extracellular matrix. Interaction be-tween matrix proteins and their receptors is thought toprovide the trigger for assembly of these specialized cellularjunctions (13), and protein phosphorylation is likely to beinvolved in the regulation of their stability (reviewed in ref.3). To obtain a more complete understanding of the role ofvinculin in adherens junctions, the sequence of chickenembryo vinculin has been determined (14-16). The sequenceof nematode Caenorhabditis elegans vinculin also has beenpublished (17). Here we report the complete sequence ofhuman vinculin 11 together with evidence that there is a singlehuman vinculin gene, which we have localized to chromo-some 10.

MATERIALS AND METHODSA partial human vinculin cDNA (HV1) was isolated from ahuman phage AgtlO fibroblast cDNA library (provided by thelate A. R. Macleod ofLudwig Institute, Cambridge, U.K.) byusing a 2.89-kilobase (kb) chicken embryo vinculin cDNAprobe (14). The HV1 cDNA was used to screen an oligo(dT)-

Sad BclI PstI PstI EcoRI KpnI KpnII I if I I I

PstI EcoRIi

HVIHV2

HV7Vh6F

- Vh1F

Vh3E

FIG. 1. Restriction map of overlapping human vinculin cDNAs.-, Coding sequence; El, untranslated sequence. Clones HV1,HV2, Vh1F, Vh6F, Vh2E, and Vh3E were completely sequenced onboth strands. The sequence across the EcoRI site within the codingregion was confirmed by using clone HV6. There were no differencesbetween the sequences ofthese cDNAs in the regions ofoverlap. Theorigins of the cDNA clones are described in Materials and Methods.

primed human endothelial cell Agtl1 library (provided by S.Orkin of Children's Hospital, Boston) (18), and HV2, HV6,and HV7 were isolated. Vh1F was isolated from a humanfibroblast Agtll library (Clontech) by screening with anantibody to human vinculin following standard procedures.Vh2E and Vh3E were isolated from a human endothelialAgtll library (a gift from M. Chao of Cornell Medical Center,New York) by using Vh1F as a probe. Vh6F was isolatedfrom a human lung fibroblast Agtll library (Clontech) byusing the 0.27-kb 5' EcoRI-BamHI fragment from HV1 as aprobe. DNA probes were labeled with [a-32P]dCTP by therandom priming method (19). Plaque screening by DNA'DNA hybridization was by standard procedures (20), with afinal wash in 0.45 M NaCI/0.045 M sodium citrate, pH 7, at650C. cDNAs were subcloned into either M13mp18 or theplasmid vectors Bluescript (Stratagene) or pTZ19 and weresequenced on both strands by the dideoxy chain-terminationmethod (21).

All of the hybrids used in this work have been described(22). Cells were maintained in RPMI 1640 medium supple-mented with 10% fetal calf serum at 370C in 5% C02/95% air.To retain human chromosomes X or 17, media were supple-mented where appropriate with 0.1 mM hypoxanthine, 10 AuMmethotrexate, and 16 ,LM thymidine. All hybrids werechecked for the presence of human chromosomes by isoen-zyme and karyoptic analysis (22) at the same passage as thatused to prepare high molecular weight genomic DNA.

tPresent address: Roche Products, Welwyn Garden City, Herts,U.K.1To whom reprint requests should be addressed.'The sequence reported in this paper has been deposited in theGenBank data base (accession no. M33308).

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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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-44 CACTTCTCTGTCGCCCGCGGTTCGCCGCCCCGCTCGCCGCCGCG

C C C C C T C TATGCCAGTGTTTCATACGCGCACGATCGAGAGCATCCTGGAGCCGGTGGCACAGCAGATCMetProValPheHisThrArgThrIleGluSerIleLeuGluProValAlaGlnGlnIle 20

C C G A G C GTCCCACCTGGTGATAATGCACGAGGAGGGCGAGGTGGACGGCAAAGCCATTCCTGACCTCSerHisLeuValIleMetHisGluGluGlyGluValAspGlyLysAlaIleProAspLeu 40

C T G C T G G AACCGCGCCCGTGGCCGCCGTGCAGGCGGCCGTCAGCAACCTCGTCCGGGTTGGAAAAGAGThrAlaProValAlaAlaValGlnAlaAlaValSerAsnLeuValArgvalGlyLysGln 60

Ser

G G A A A C C A G C C AACTGTTCAAACCACTGAGGATCAGATTTTGAAGAGAGATATGCCACCAGCATTTATTAAGThrValGlnThrThrGluAspGlnIleLeuLysArgAspMetProProAlaPheIleLys 80

A C C T GA C G AG T TGTTGAGAATGCTTGCACCAAGCTTGTCCAGGCAGCTCAGATGCTTCAGTCAGACCCTTACValGluAsnAlaCysThrLysLeuValGlnAlaAlaGlnMetLeuGlnSerAspProTyr 100

Arg Ala

A A T C C A A TTCAGTGCCTGCTCGAGATTATCTAATTGATGGGTCAAGGGGCATCCTCTCTGGAACATCASerValProAlaArgAspTyrLeuIleAspGlySerArgGlyIleLeuSerGlyThrSer 120

T A T G A T A G C CC T CGACCTGCTCCTTACCTTCGATGAGGCTGAGGTCCGTAAAATTATTAGAGTTTGCAAAGGAAspLeuLeuLeuThrPheAspGluAlaGluValArgLysIleIleArgValCysLysGly 140

A G T A A A G G A TATTTTGGAATATCTTACAGTGGCAGAGGTGGTGGAGACTATGGAAGATTTGGTCACTTACIleLeuGluTyrLeuThrValAlaGluValValGluThrMetGluAspLeuValThrTyr 160

A A G A C T AACAAAGAATCTTGGGCCAGGAATGACTAAGATGGCCAAGATGATTGACGAGAGACAGCAGThrLysAsnLeuGlyProGlyMetThrLysletAlaLysMetIleAspGluArgGlnGln 180

AT A T A TA G T C C T T GGAGCTCACTCACCAGGAGCACCGAGTGATGTTGGTGAACTCGATGAACACCGTGAAAGAGGluLeuThrHisGlnGluHisArgValMetLeuValAsnSerMetAsnThrValLysGlu 200

C AT T A T C C A A T GTTGCTGCCAGTTCTCATTTCAGCTATGAAGATTTTTGTAACAACTAAAACTCAAAAAACLeuLeuProValLeuIleSerAlaMetLysIlePheValThrThrLysAsnSerLysAsn 220

Thr Ser

G A A G C G C A G GCAAGGCATAGAGGAAGCTTTAAAAAATCGCAATTTTACTGTAGAAAAAATGAGTGCTGAAGlnGlyI1eGlUGluAlaLeuLysAsnArgAsnPheThrValGluLysMetSerAlaGlu 240

A A C A T C r R

ATTAATGAGATAATTCGTGTGTTACAACTCACCTCTTGGGATGAAGATGCCTGGGCCAGCIleAsnGlutleIleArgValLeuGlnLeuThrSerTrpAspGluAspAlaTrpAlaSer 260

A T A CTA T A GA AAAGGACACTGAAGCCATGAAGAGAGCATTGGCCTCCATAGACTCCAAACTGAACCAGGCCLysAspThrGluAlaMetLysArgAlaLeuAlaSerIleAspSerLysLeuAsnGlnAla 280

Leu Met

C GA A T A AC AC T A A A GAAAGGTTGGCTCCGTGACCCTAGTGCCTCCCCAGGGGATGCTGGTGAGCAGGCCATCAGALysGlyTrpLeuArgAspProSerAlaSerProGlyAspAlaGlyGluGlnAlaIleArg 300

Asn Pro

C T CA A T GCAGATCTTAGATGAAGCTGGAAAAGTTGGTGAACTCTGTGCAGGCAAAGAACGCAGGGAGGlnIleLeuAspGluAlaGlyLysValGlyGluLeuCysAlaGlyLysGluArgArgGlu 320

Ala

A CTT G C A C T T A TATTCTGGGAACTTGCAAAATGCTAGGGCAGATGACTGATCAAGTGGCTGACCTCCGTGCCIleLeuGlyThrCysLysMetLeuGlyGlnMetThrAspGlnValAlaAspLeuArgAla 340

Thr Leu

G TG TA A A G A G A A CAGAGGACAAGGATCCTCACCGGTGGCCATGCAGAAAGCTCAGCAGGTATCTCAGGGTCTGArgGlyGlnGlySerSerProValAlaMetGlnLysAlaGlnGlnValSerGlnGlyLeu 360

AlaThr Met R2T T G 1. C G AT G A T

GATGTGCTCACAGCAAAAGTGGAAAATGCAGCTCGCAAGCTGGAAGCCATGACCAACTCAAspValLeuThrAlaLysValGluAsnAlaAlaArgLysLeuGluAlaMetThrAsnSer 380

Leu

GCT T G C CAAGCAGAGCATTGCAAAGAAGATCGATGCTGCTCAGAACTGGCTTGCAGATCCAAATGGTLysGlnSerIleAlaLysLysIleAspAlaAlaGlnAsnTrpLeuAlaAspProAsnGly 400

Ala

AGT A C AAT A T AA G TGGACCGGAAGGAGAAGAGCAGATTCGAGGTGCTTTGGCTGAAGCTCGGAAAATAGCAGAAGlyProGluGlyGluGluGlnIleArgGlyAlaLeuAlaGluAlaArgLysIleAlaGlu 420

Ser His IleMetSer Val

G G G T C T C T G CTTATGTGATGATCCTAAAGAAAGAGATGACATTCTACGTTCCCTTGGGGAAATATCTGCTLeuCysAspAspProLysGluArgAspAspIleLeuArgSerLeuGlyGluIleSerAla 440

GluGlu

AG GC GT G C T C C T C TCTGACTTCTAAATTAGCAGATCTACGAAGACAGGGGAAAGGAGATTCTCCAGAGGCTCGALeuThrSerLysLeuAlaAspLeuArgArgGlnGlyLysGlyAspSerProGluAlaArg 460

Ala Ser His

A G AA A T AT A T T A T A A Ar R3GCCTTGGCCAAACAGGTGGCCACGGCCCTGCAGAACCTGCAGACCAAAACCAACCGGGCTAlaLeuAlaLysGlnValAlaThrAlaLeuGlnAsnLeuGlnThrLysThrAsnArgAla 480

Ile Ser Ser

A A T CT A TT T C TT G TGTGGCCAACAGCAGACCGGCCAAAGCAGCTGTACACCTTGAGGGCAAGATTGAGCAAGCAValAlaAsnSerArgProAlaLysAlaAlaValHisLeuGluGlyLysIleGluGlnAla 500

Thr Val

A A T T T G A C A A TCAGCGGTGGATTGATAATCCCACAGTGGATGACCGTGGAGTCGGTCAGGCTGCCATCCGGGlnArgTrpIleAspAsnProThrValAspAspArgGlyValGlyGlnAlaAlaIleArg 520

TT G T A T G A C C A T G CGGGCTTGTGGCCGAAGGGCATCGTCTGGCTAATGTTATGATGGGGCCTTATCGGCAAGATGlyLeuValAlaGluGlyHisArgLeuAlaAsnValMetMetGlyProTyrArgGlnAsp 540

Arg

G T A T A G T T T T A ACTTCTCGCCAAGTGTGACCGAGTGGACCAGCTGACAGCCCAGCTGGCTGACCTGGCTGCCLeuLeuAlaLysCysAspArgValAspGlnLeuThrAlaGlnLeuAlaAspLeuAlaAla 560

Ala

A TC TA G A TG T G C Go AGAGGGGAAGGGGAGAGTCCTCAGGCACGAGCACTTGCATCTCAGCTCCAAGACTCCTTA

ArgGlyGluGlyGluSerProGlnAlaArgAlaLeuAlaSerGlnLeuGlnAspSerLeu 580Ile Ala

R3--"C A A A A C G T T T

1740 AAGGATCTAAAAGCTCGGATGCAGGAGGCCATGACTCAGGAAGTGTCAGATGTTTTCAGCLysAspLeuLysAlaArgMetGlnGluAlaMetThrGlnGluValSerAspValPheSer 600

C T T T T A A T T T A T C1800 GATACCACAACTCCCATCAAGCTGTTGGCAGTGGCAGCCACGGCGCCTCCTGATGCGCCT

AspThrThrThrProIleLysLeuLeuAlaValAlaAlaThrAlaProProAspAlaPro 620

T A G A A A A T G T CT GA G1860 AACAGGGAAGAGGTATTTGATGAGAGGGCAGCTAACTTTGAAAACCATTCAGGAAAGCTT

AsnArgGluGluValPheAspGluArgAlaAlaAsnPheGluAsnHisSerGlyLysLeu 640Glu AlaAlaArg

A A A A A A A A A A T T1920 GGTGCTACGGCCGAGAAGGCGGCTGCGGTTGGTACTGCTAATAAATCAACAGTGGAAGGC

GlyAlaThrAlaGluLysAlaAlaAlaValGlyThrAlaAsnLysSerThrValGluGly 660Thr

AA C AT T AA G G T A A A A1980 ATTCAGGCCTCAGTGAAGACGGCCCGAGAACTCACACCCCAGGTGGTCTCGGCTGCTCGT

IleGlnAlaSerValLysThrAlaArgGluLeuThrProGlnValValSerAlaAlaArg 680Thr Ser

C C G A T G A A2040 ATCTTACTTAGGAACCCTGGAAATCAAGCTGCTTATGAACATTTTGAGACCATGAAGAAC

IleLeuLeuArgAsnProGlyAsnGlnAlaAlaTyrGluHisPheGluThrMetLysAsn 700

C T A G T G C G2100 CAGTGGATCGATAATGTTGAAAAAATGACAGGGCTGGTGGACGAAGCCATTGATACCAAA

GlnTrpIleAspAsnValGluLysMetThrGlyLeuValAspGluAlaIleAspThrLys 720His

A G T G G T T T A T A T A2160 TCTCTGTTGGATGCTTCAGAAGAAGCAATTAAAAAAGACCTGGACAAGTGCAAGGTAGCT

SerLeuLeuAspAlaSerGluGluAlaIleLysLysAspLeuAspLysCysLysValAla 740

T G A A A C C A A A G2220 ATGGCCAACATTCAGCCTCAGATGCTGGTTGCTGGGGCAACCAGTATTGCTCGTCGGGCC

MetAlaAsnIleGlnProGlnMetLeuValAlaGlyAlaThrSerIleAlaArgArgAla 760Met

A T A AC T A A A C T A A G2280 AACCGGATCCTGCTGGTGGCTAAGAGGGAGGTGGAGAATTCCGAGGATCCCAAGTTCCGT

AsnArgIleLeuLeuValAlaLysArgGluValGluAsnSerGluAspProLysPheArg 780

T A T GC A A G A2340 GAGGCTGTGAAAGCTGCCTCTGATGAATTGAGCAAAACCATCTCCCCAATGGTGATGGAT

GluAlaValLysAlaAlaSerAspGluLeuSerLysThrIleSerProMetValMetAsp 800

T A A T C T T TT G T T T T2400 GCAAAAGCTGTGGCTGGAAACATTTCCGACCCTGGACTGCAAAAGAGCTTCCTGGACTCA

AlaLysAlaValAlaGlyAsnIleSerAspProGlyLeuGlnLysSerPheLeuAspSer 820

CA T A T G A2460 GGATATCGGATCCTGGGAGCTGTGGCCAAGGTCAGAGAAGCCTTCCAICCTCAGGAGCCT

GlyTyrArgIleLeuGlyAlaValAlaLysValArgGluAlaPheGl rProGlnGluPro 840

T C T T T C G T AT G T2520 GACTTCCCGCCGCCTCCACCAGACCTTGAACAACTCCGACTAACAGATGAGCTTGCTCCT

AspPheProProProProProAspLeuGluGlnrLeuArgLeuThrAspGluLeuAlaPro 860

A A T A T C C A A C T A2580 CCCAAACCACCTCTGCCTGAAGGTGAGGTCCCTCCACCTAGGCCTCCACCACCAAGGAA

ProLysProProLeuProGluGlyGluValProProProArgProProProPrluGluA G A A A A A CT T C

2640 AAGGATGAAGAGTTCCCTGAGCAGAAGGCCGGGGAGGTGATTAACCAGCCAATGATGATGLysAspGluGluPheProGluGlnLysAlaGlyGluValIleAsnGlnProMetMetMet

Ala

T G T G C G G T T C2700 GCTGCCAGACAGCTCCATGATGAAGCTCGCAAATGGTCCAGCAAGGGCAATGACATCATT

AlaAlaArgGlnLeuHisAspGluAlaArgLysTrpSerSerLysGlyAsnAspIleIle

T T T A A G C A C GC A2760 GCAGCAGCCAAGCGCATGGCTCTGCTGATGGCTGAGATGTCTCGGCTGGTAAGAGGGGGC

AlaAlaAlaLysArgMetAlaLeuLeuMetAlaGluMetSerArgLeuValArgGlyGly

C A A T C C A A T T T A G A2820 AGTGGTACCAAGCGGGCACTCATTCAGTGTGCCAAGGACATCGCCAAGGCCTCAGATGAG

SerGlyThrLysArgAlaLeuIleGlnCysAlaLysAspIleAlaLysAlaSerAspGluAsn

C A A G A T T C A2880 GTGACTCGGTTGGCCAAGGAGGTTGCCAAGCAGTGCACAGATAAACGGATTAGAACCAAC

ValThrArgLeuAlaLysGluValAlaLysGlnCysThrAspLysArgIleArgThrAsn

C C G T T2940 CTCTTACAGGTATGTGAGCGAATCCCAACCATAAGCACCCAGCTCAAAATCCTGTCCOCA

LeuLeuGlnValCysGluArgIleProThrIleSerThrGlnLeuLysIleLeuSerThr

A T A T C A A A A A T3000 GTGAAGGCCACCATGCTGGGCCGGACCAACATCAGTGATGAGGAGTCTGAGCAGGCCACA

ValLysAlaThrMetLeuGlyArgThrAsnIleSerAspGluGluSerGluGlrnAlaThr

T T C A A A3060 GAGATGCTGGTTCACAATGCCCAGAACCTCATGCAGTCTGTGAAGGAGACTGTGCGGGAA

GluMetLeuValHisAsnAlaGlnAsnLeuMetGlnSerValLysGluThrValArgGlu

3120

3 180

3240

3300

3360

3420

3480

3540

3600

3660

3720

3780

3840

390039604020

4080

4140

4200

4260

4320

4380

4440

4500

4560

4620

4680

4740

4800

4860

4920

4980

5040

A A C T G AA C CT C

GCTGAAGCTGCTTCAATCAAAATTCGAACAGATGCTGGATTTACACTGCGCTGGGTTAGAAlaGluAlaAlaSerIleLysIleArgThrAspAlaGlyPheThrLeuArgTrpValArg

C A T AAAGACTCCCTGGTACCAGTAGGCACCTGGCTGAGCCTGGCTGGCACAGAAACCTCTACTALysThrProTrpTyrGlnEnd-x...s on-- - - - - -- - - - - ---------a~- -------------x-X-x--

880

900

920

940

960

980

1000

1020

1040

1060

1066

AATTAAAAAAAA 5051

FIG. 2. Complete nucleotide sequence and derived amino acid sequence of human vinculin. Positions where the chicken embryo vinculinnucleotide and derived amino acid sequences differ from human are shown above and below the human sequence, respectively. The positionsof the boundaries of each of the three 112-residue repeats in the amino acid sequence are indicated (R1-R3), and the proline-rich region (residues837-878) is boxed. One chicken embryo vinculin cDNA (cVin5) analyzed by Price et al. (15) lacks 123 bp encoding residues 167-207. The

0

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1020

1080

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162 0

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m Proc. Natl. Acad. Sci. USA 87 (1990) 5669

6

5.

500-

residue no. 500 1000

human vinculin

FIG. 3. Dot matrix comparison of human and nematode vinculinamino acid sequences using the University of Wisconsin GeneticsComputing Group programs COMPARE and DOTPLOT, with windowand stringency values of 30 and 8, respectively.

RESULTSThe relationship between the eight cDNA clones from whichwe have determined the complete sequence ofhuman vinculinis shown in Fig. 1. Sequence analysis shows that the cDNAsspan 44 base pairs (bp) of 5' untranslated sequence, thecomplete coding sequence (3198 bp), and 1852 bp of 3'untranslated sequence (Fig. 2). Comparison of the codingsequence of human vinculin with that of chicken embryovinculin (14-16) shows a high level of sequence identity bothat the DNA (82%) and amino acid (95%) levels, although the5' and long 3' untranslated sequences are divergent. Humanvinculin contains 1066 amino acids, as does the chickenembryo protein, and has a deduced molecular mass of 116,732Da. Most of the amino acid sequence divergence between thehuman and chicken embryo vinculin sequences occurs in thecentral region of the protein, which contains three repeats ofapproximately 112 amino acids (residues 259-589) ofunknownfunction (14). There are 32 amino acid sequence differences inthis repeat region, which spans 330 residues. The regionN-terminal to the repeats, which contains a talin-binding site(23), is more highly conserved with only 5 differences in 258amino acids. Similarly, there are only 13 sequence differencesin the 476 amino acids C-terminal to the repeats. This regioncontains a proline-rich sequence (residues 837-878) thought tobe important in separating the globular head of vinculin fromits extended tail (14, 15).The sequence of nematode vinculin also has been deter-

mined (17), and similarities with the partial chicken embryovinculin sequence (14) were noted. Interestingly, there areonly two central repeats in nematode vinculin, which accountsfor its lower molecular mass estimated by SDS/PAGE. Dotmatrix analysis of the complete human and nematode se-quences (Fig. 3) confirms that the N- and C-terminal domainsofvinculin are the most highly conserved regions ofthe protein(54% and 61% sequence identity, respectively), and the align-ments of these regions are shown in Fig. 4.

Southern Blot Analysis of the Human Vinculin Gene. South-ern blot analysis of human DNA was carried out with a

Anematodehumanchick

1 50MwFNPVFHTRTIE SILEPVAQQI SHLVIMHEEG EVDGK&IPDL TAPVAAVQAAMPVFHTRTIE SILEPVAQQI SHLVIMHEEG EVDGMAIPRL TAPVO&VQAA

nematode_human LVVGR TVQITEDQIL KRDNPPAFIK VENACTKLVQ AAQMLQSDPYchick VSNLVRVGKE TVQTTEDOIL KRDNPPAFIK VENA CTKLAAQMLdPY

101 150nematode S VRK IVB_ (_q% _ Vhuman SVPAYLID GSRGILSGTS DLLLTFDEAE VRKIIRVCKG ILEYLTVAEVchick SVPARDYLID GSRGILSGTS DLLLTFDEAE VRKIIRVCKG ILEYLTVAEV

-S4)

'0.s

0acs

nematodehumanchick

nematodehumanchick

151 200

VETNEDLVTY TKNLGPGN4TK NAKNIDERQQ ELTHQEHRVM LVNSMNTVKEVETMEDLVTY TKNLGPGNTK XAKMIDERQQ ELTHQEHRVM LVNSM4TVKE:201 249

LLPVLISANK . N IEEALRNFTVEGKMSA EINEIIRVLQLLPVLISAMK . IFVTT' PGIEEALKN RNFTVEKNSA EINEIIRVLQ

250 258nematode LI-rxERDhuman LTSWDEDAWchick LTSWDEDAW

Bnematodehumanchick

820 869RASPYNPPPP SSOVIRSVNA SPPTAPgIHN KEILRLTDELAPPKiPLEGESGYRILGAVA KVREAFQPQE PDFPPPPPDL EOLRLTDELA PPKPPLPEGESGYRIDGAVA KVREAFQPQE PDFPPPPPDL ELtjDLELA PPKPPLPEGE

nematode _human VPPPRPPPPE EKDEEFPEQK AGE ......V INQPHNKAAR QLHDEARKWSchick VPPPRPPPPE EKDEEFPEQK AGE..iINQENQOAAR QLHDEARKWS

914 963nematode Rhuman SKGNDIIAAA KRMALIMAEM SRLVRGGSG KRAIQCAKD IAKASDEVTRchick SKGNDIIAAA KRNALIMAE( SRLVRGGSG#4 KkALI92CAKD IAKASDEVTR

964 1006nematode VJRP5__QtT _M~ ADVI~Ghuman LAKEVARQCT DKRIRTNLLQ VCERIPTIST QLKILSTVKA TML.chick IAKEVAKQCT DKRIRTNLLQ VCERIPTIST QLKILSTV!A TML.

1007 1054nematode E Hhuman THNAQNLMQSV VRAEAA SIKIRTDAGFchick . . GRTNISDE ESEQATErMLV HNAQNIMQSV KETVREAEAA SIKIRTDAGF

1055 1066nematode R _FWU~kSNF*human TLRWVRKTPW YQ*chick TLRWVRKTPW YQ*

FIG. 4. Alignment of the N- and C-terminal regions of human,chicken embryo, and nematode vinculin. Amino acids are numberedrelative to the human sequence. Alignments were determined byusing the University of Wisconsin Genetics Computing Group pro-gram GAP. Residues identical to the human sequence are boxed. (A)N-terminal region, residues 1-258. Arrows denote the boundaries ofthe 41-amino acid region (residues 167-207) absent from the chickenembryo cDNA cVin5 (15). (B) C-terminal region, residues 820-1066.

number ofvinculin cDNA fragments as probes (Fig. 5). Whenblots were hybridized with fragments spanning the entirecoding sequence and part of the 3' untranslated region, arelatively complex pattern of hybridization was obtained(data not shown). In contrast, short fragments encodingresidues 447-539 and 704-773 as well as a 499-bp fragmentfrom the 3' untranslated sequence gave rise to single hybrid-izing bands (Fig. 5 b, c, and e), strongly suggesting that thereis a single human vinculin gene. However, a 596-bp fragmentconsisting of the 5' untranslated region and coding up toresidue 182 and a 367-bp fragment encoding residues 943-1065 hybridized to multiple bands in most enzyme digests(Fig. 5 a and d). This result suggests that the organization ofthe 5' and 3' ends of the gene may be relatively complex.Alternatively, the probes could be detecting pseudogenes orother vinculin-related sequences, although the high strin-gency at which hybridization was carried out makes thislatter possibility unlikely.Chromosome Assignment of the Human Vinculin Gene.

Southern blots of EcoRI-digested somatic cell hybrid DNAswere hybridized with the 1.5-kb human vinculin cDNA HV1(see Fig. 1), which detects four major fragments in human

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origin of this heterogeneity remains to be established. However, both the human vinculin cDNAs that span this region (HV2 and HV7) containthis sequence. The chicken embryo vinculin sequence published by Price et al. (GenBank accession no. Y00312; refs. 14 and 15) contains anerror: residues 442-447 should now read Thr-Ala-Lys-Leu-Ser-Asp, in agreement with the sequence published by Coutu and Craig (GenBankaccession no. J04126; ref. 16).

Biochemistry: Weller et al.

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FIG. 5. Analysis ofthe human vinculin gene by Southern blotting.Human genomic DNA was cleaved with the restriction endonu-cleases EcoRI (E), BamHI (B), Pst I (P), and Kpn I (K). DNAfragments were separated in a 0.8% agarose gel and transferred toHybond N (Amersham), and the blot was hybridized at 650C in 0.45M NaCl/0.045 M sodium citrate (pH 7) with five different vinculincDNA fragments in succession. Posthybridization washes were at650C in 0.03 M NaCl/0.003 M sodium citrate (pH 7). The filters werestripped between each hybridization, and the efficiency of proberemoval was checked by autoradiography. (a) EcoRI-Sac I fragment(596 bp) from clone HV2, including the 5' untranslated sequence andthe coding sequence up to residue 182. (b) Bgl II fragment (279 bp)from HV1 encoding residues 447-539. (c) Cla I-EcoRI fragment (208bp) from HV1 encoding residues 704-773. (d) Kpn I fragment (367bp) from HV6 encoding residues 943-1065. (e) Nco I-EcoRI fragment(499 bp) from HV6 containing only the 3' untranslated sequence.Migration positions of size markers (phage A HindIII fragments) areshown in kb.

DNA of approximately 12, 6, 4, and 3 kb (Fig. 6, lanes 9 and11). In mouse DNA, this probe detects fragments of 10, 8, and3.5 kb, (Fig. 6, lanes 8 and 10) and in rat DNA of 11 kb and2 kb (Fig. 6, lane 5). Typical results obtained from screeningseven hybrids, two ofwhich were found to contain the humanvinculin gene, are shown (Fig. 6, lanes 1-7). A total of 17human-rodent cell hybrids were screened, and results fromthe analysis are shown in Table 1. The only chromosome toshow complete concordance with the presence of the humanvinculin gene is chromosome 10 (Table 1). The hybridTRAXK12 (a gift from P. J. Goodfellow, Imperial Cancer

Table 1. Assignment of the human vinculin gene to chromosome 10

FIG. 6. Chromosome assignment of the human vinculin gene.Southern blot ofEcoRI-cleaved somatic cell hybrid DNAs (lanes 1-7and 12-14) and mouse (lanes 8 and 10) and human (lanes 9 and 11)genomic DNA hybridized with a 1559-bp human vinculin cDNAprobe (HV1, see Fig. 1). Hybrids in lanes 4 and 5 are human-rat.Migration positions of size markers (phage A HindIll fragments) areshown in kb.

Research Fund, London), which contains a chromosomeX/10 translocation t(X;10)(p22.3;qll.2) was positive for thehuman vinculin gene (Fig. 6, lane 12), thus allowing furtherlocalization to chromosome 10qll.2-qter.

DISCUSSIONComparison of the human, chicken embryo, and nematodevinculin sequences shows that the regions N- and C-terminalto the central repeat region are highly conserved and pre-sumably contain domains important to the function of theprotein. In the case of the N-terminal region, we haveprovided evidence that residues 167-207 are important to thetalin-binding activity of chicken embryo vinculin (23). It isalso apparent that this region of the molecule is important forthe ability of the expressed protein to localize to cell-matrixjunctions (23, 24). Furthermore, we have shown that thedeletion of contiguous regions of as little as 7 amino acids inthe- sequence spanning residues 167-201 dramatically reducethe talin-binding activity of the N-terminal 398 amino acids ofvinculin (23). Although residues 167-207 are identical inhuman and chicken embryo vinculin, this is a region of

Chromosome

Hybrid name 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X VINC

3WCL5. . . . . + - - + + + - + + + - - + +

SIF15 - + - - - + + - - + - - + + - + +DT1.2 - - +. . . . . . .+ + - + - + + + - + + + +HORP9.5..........+ + + - +........+ +MOG34A4 + - + + + + + + - + + + + + + + + + _ + +FG10 - + - - + - - + - + - - - + + - - + +

FST9/10 - - + + - + - + - + + + - + + - + + +DT1.2.4 - - +. . . . . . .+ + - - - + + + - + + +RJDlt - - + - - - + - - +. . . . . . . .+ + + +TRAXK12t - - -. . . .- -- * + - - - + + - - + + *SIR74ii + + + +. . . . . . . .+ + + - + t - - + t +DUR4R3 - - + - + - - + - - + + + + - + - + + -

SIF4A31 - - + + + +........+ - +.......+CTP41A2 - + - - - + +.......+ - +......+CTP34B4 + + + - + + + + - - + - + - + + + - - +TWIN19-D12 + - + + - + - + - - + + - + - + + + - + -HORL9X-----------------------+

++++++++++

+, Chromosome present; -, chromosome absent; t, trace; *, hybrids containing chromosome fragments; VINC, presence (+) or absence (-)of human vinculin gene.tHuman Genetic Resources, Imperial Cancer Research Fund.*Gift from P. J. Goodfellow. For details of other hybrids, see ref. 22.

xI

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Proc. Natl. Acad. Sci. USA 87 (1990) 5671

considerable divergence in the nematode sequence (17).However, it is striking that residues 178-185 are identical inall three species. The C-terminal region of vinculin (the last170 amino acids) apparently contains a site involved intargeting the molecule to cell-matrix junctions (24). This is ahighly conserved region of the protein, which has also beensuggested to be involved in the ability of vinculin to self-associate (25). It has been calculated that the C-terminalregion of chicken embryo vinculin (residues 858-1066) has apI of 9.7, compared with a pI of 5.9 for the intact protein (16).This may account for the apparent ability of vinculin tointeract directly with phospholipids (26).

Vinculin has been reported to contain a short region ofsequence homology with actin (27). Thus, residues 300-307in the first repeat of chicken embryo vinculin (Arg-Gln-Ile-Leu-Asp-Glu-Ala-Gly) can be aligned with residues 359-366in a-actin (Lys-Gln-Glu-Tyr-Asp-Glu-Ala-Gly) (27). A num-ber of actin-binding proteins also contain a similar sequence(reviewed in ref. 27). The human and chicken embryo vin-culin sequences are identical over this region, but the nem-atode sequence is divergent, although it does contain adifferent short stretch of sequence homology with actin (17).Despite an earlier report, there is no evidence that vinculinbinds directly to actin (28). There is no significant sequencesimilarity between vinculin and any other protein in thecurrent data base. Vinculin does not contain the SH3 domainfound in a number of other proteins associated with themembrane cytoskeleton (29).There is at least one additional isoform of vinculin, termed

metavinculin, which has a higher apparent molecular weightthan vinculin and is expressed specifically in muscle (30-33).The close similarity between these isoforms suggests thatthey are generated by alternative splicing of transcripts froma single gene (34). Southern blots of chicken embryo genomicDNA cleaved with a variety of restriction endonucleases andhybridized with a short vinculin cDNA fragment containingonly the 5' untranslated sequence detected a single band ineach digest, consistent with the existence of a single vinculingene in chicken (15). The more extensive Southern blotanalysis of the human vinculin gene reported here is alsoconsistent with a single functional gene. By using the HV1cDNA as a probe, the human vinculin gene has been assignedto chromosome 10 in the region qll.2-qter. It is interestingto note that chromosome mapping of the mouse vinculinlocus with the same probe has led to assignment of the mousegene to chromosome 14, which is syntenic to human chro-mosome 10 (M. Strobel, personal communication). It shouldbe of interest to define more accurately the position of thehuman vinculin gene, as a number of loci of interest map tothe long arm of chromosome 10. These include the cell cyclecontrol gene CDC2 (22, 35) and the disease locus for multipleendocrine neoplasia types 2A and 2B (36, 37). The data onmapped loci on the long arm of chromosome 10 are summa-rized in the recent Human Gene Mapping Workshop (38).

We are grateful to Dr. V. Kushnirov for help and advice with DNAsequencing and to Ms. M. Kirillova for excellent technical assist-ance. This work was supported by a grant to D.R.C. from the MedicalResearch Council (U.K.).

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Biochemistry: Weller et al.

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