Proc. Nail. Acad. Sci. USA Vol. 88, pp. 9156-9160, October 1991 Cell Biology The uvomorulin-anchorage a is a homologue KURT HERRENKNECHT*, MASAYUKI OZAWA*t, CHRISTOPH ECKERSKORN*, FRIEDRICH LOTTSPEICHt, MARTIN LENTER*, AND ROLF KEMLER*§ *Max-Planck-Institut ffir Immunbiologie, FG Molekulare Embryologie, D-7800 Freiburg, Federal Republic of Germany; and tMax-Planck-Institut ffr Biochemie, D-8033 Martinsried, Federal Republic of Germany Communicated by Franqois Jacob, July 18, 1991 (receivedfor review June 25, 1991)

ABSTRACT The cytoplasmic region of the Ca2+- domain is well conserved in other cadherins, it is possible that dependent cell-adhesion molecule (CAM) uvomorulin associ- may also complex with other members of this gene ates with distinct cytoplasmic with molecular masses family (13, 14). Here we have produced antibodies against a of 102, 88, and 80 kDa termed a, (3, and ycatenin, respectively. catenin and show that a catenin is indeed associated with This complex formation links uvomorulin to the filament cadherins from human, mouse, and Xenopus. We have network, which seems to be of primary importance for its cloned and sequenced¶ the cDNA coding for a catenin and cell-adhesion properties. We show here that antibodies against have established the primary protein structure. Sequence a catenin also immunoprecipitate complexes that contain hu- comparison reveals homology to vinculin, a well-known man N-cadherin, mouse P-cadherin, chicken A-CAM (adhe- adherens-type and focal contact protein. rens junction-specific CAM; also called N-cadherin) or Xeno- pus U-cadherin, demonstrating that a catenin is complexed with other cadherins. In immunofluoresence tests, a catenin is MATERIALS AND METHODS colocalized with cadherins at the plasma membrane. However, Cell Lines. Mouse fibroblasts Ltk-, human HeLa, chicken in cadherin-negative Ltk- cells, a catenin is found uniformly fibroblasts CEF38, and their respective transfectants ex- distributed in the cytoplasm, suggesting some additional bio- pressing mouse uvomorulin, Li-i, H1-3, and C1-4 (10, 15) logical function(s). Expression of uvomorulin in these cells were used as well as embryonal carcinoma cells F9, PCC4, results in a concentration of a catenin at membrane areas ofcell and PAS5E. Porcine kidney LLC-PK7 and Xenopus A6 cells contacts. We also have cloned and sequenced murine a catenin. were gifts from H. Hoschutzky (Freiburg, F.R.G.) and D. The deduced sequence reveals a signfMcant homol- Wedlich (Berlin), respectively. The A6 cells were grown in ogy to vinculin. Our results suggest the possibility of a new Leibovitz L-15 medium containing 8% (vol/vol) fetal calf vinculin-related protein family involved in the cytoplasmic serum (FCS) at 240C. All other cells were cultured in Dul- anchorage of cell-cell and cell-substrate adhesion molecules. becco's modified Eagle's medium containing 10%o FCS at 370C in an atmosphere containing 10%6 Co2. For the gener- The cadherin gene family of Ca2l-dependent cell adhesion ation of F9 tumors, about 1 x 107 cells were injected molecules (CAM) was originally composed ofa rather limited subcutaneously in 129/SV mice, and solid tumors were number of transmembrane glycoproteins of which the best removed 12-15 days later and stored at -80TC. studied examples were uvomorulin/E-cadherin, liver CAM Puriication of a Catenin. Ten grams of solid F9 tumor was (L-CAM), N-cadherin, and P-cadherin (for a review, see refs. homogenized in 50 ml of Nonidet P-40 (NP-40)/TBS (2% 1 and 2). Each member was found to regulate cell adhesion NP-40/20 mM TrisHCl, pH 7.5/0.15 M NaCl/2 mM CaCl2/1 of particular cell types, and this was thought to be funda- mM phenylmethylsulfonyl fluoride) at 40C for 15 min. Insol- mental for the organization of multicellular organisms. More uble material was pelleted (48,000 X g for 30 min at 40C), and recently new members of this family have been described the supernatant was preincubated on a protein A-sepharose including M-cadherin on mouse myoblasts (3), E/P-, U-, and column (Pharmacia). Unbound material was incubated with XB-cadherin in early Xenopus development (4-6), and a new 200 mg of rabbit anti-uvomorulin IgG covalently coupled to subgroup of more distantly related desmosomal glycopro- 20 ml of protein A-sepharose and washed with 250 ml of teins (7-9). NP-40/salt buffer (0.1% NP-40/1 M NaCl/20 mM Tris-HCl, It has been shown that the cytoplasmic region ofuvomoru- pH 7.5/2 mM CaCl2). Uvomorulin-catenin complexes were lin associates with defined proteins of 102, 88, and 80 kDa eluted with 100 ml of acidic pH buffer (0.1% NP-40/0.1 M termed a, f3, and y catenin, respectively (10). The linkage of glycinehydrochloride, pH 2.5/2 mM CaCl2), and after neu- uvomorulin to actin filaments via these proteins seems to be tralization proteins were precipitated with 4 vol of 100% of crucial importance for the cell-binding function of uvo- ethanol at -20'C. morulin (11). Moreover, catenins connect uvomorulin to Protein Microsequencing. Uvomorulin-catenin complexes other integral membrane proteins, such as Na+/K+-ATPase, collected from 100 g of tumor were separated on a SDS/6% or to cytoplasmic structures, such as fodrin or ankyrin. This polyacrylamide gel and, after Coomassie-staining, bands indicates that uvomorulin is part of a cyto-cortical network containing a catenin (=20 ,ug) were excised and digested with (12). These findings support the concept that uvomorulin- 5 pug of endoproteinase LysC (Boehringer Mannheim) as mediated adhesion induces redistribution of cytoplasmic and described (16). Peptides were eluted with 75% (vol/vol) membrane proteins which, in turn, may initiate the molecular trifluoroacetic acid and separated by reversed-phase HPLC events that result in the transition of a nonpolarized to a polarized epithelial cell (2, 12). If so, catenins might play a Abbreviations: CAM, cell adhesion molecule; A-CAM, adherens central role in these processes. Since the catenin-binding junction-specific CAM (also called N-cadherin). TPresent address: Department of Biochemistry, School of Medicine, Kagoshima University, Kagoshima, Japan. The publication costs of this article were defrayed in part by page charge §To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" IThe sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank/EMBL data base (accession no. X59990).

9156 Downloaded by guest on September 29, 2021 Cell Biology: Herrenknecht et A Proc. Nati. Acad. Sci. USA 88 (1991) 9157 as described (16). Amino acid sequence analysis of the peptides was performed with a gas-phase sequenator (model 477 A; Applied Biosystems) as described (17). Antibodies. Rabbit antiserum against a catenin was pro- duced by using a synthetic peptide, His-Val-Asp-Pro-Val- Gln-Ala-Leu-Ser-Glu-Phe-Lys (localized near the carboxyl terminus, see Fig. 4), coupled to keyhole limpet hemocyanin (Sigma) by glutaraldehyde. After five subcutaneous immu- nizations in intervals of 3 weeks, specific antibodies were isolated on a peptide-e-aminohexanoyl (EAH)-Sepharose (Pharmacia) column (5 mg of peptide coupled to 1 ml of EAH-Sepharose 4B as described by Pharmacia). Rabbit antibodies against human P-cadherin and mouse N-cadherin (cross-reacting with mouse P-cadherin and human N-cad- herin, respectively) have been raised against Escherichia coli TrpE-cadherin fusion proteins, containing approximately the carboxyl-terminal half of each cadherin protein. The respec- tive cDNAs have been cloned by the polymerase chain reaction (PCR) technique (M.L. and Dietmar Vestweber, unpublished data). Monoclonal antibodies FA-5 and GC-4 against chicken A-CAM (adherens junction-specific CAM; also called N-cadherin) (18) were purchased from Sigma, and the monoclonal antibody 6D5 reacting with Xenopus U-cad- herin (5) was a generous gift of B. Angres (Tubingen, F.R.G.). Immunoblot and immunoprecipitation experiments were carried out as described (10). For indirect immunoflu- orescence tests, cells grown on cover slips were fixed with methanol (-200C), and anti-a-catenin antibodies were de- FIG. 2. Immunofluorescence staining of permeabilized PCC4 (a tected with goat fluorescein isothiocyanate-conjugated and b), HeLa (c), Ltk- cells (e), and the corresponding mouse F(ab)2 anti-rabbit immunoglobulin (Dynatech). uvomorulin-expressing transfectants H1-3 (d) and Li-i (f) with cDNA Isolation and Sequencing. An oligo(dT)-primed anti-uvomorulin (a) and anti-a-catenin (b-J). Uvomorulin and a phage Agtll cDNA library prepared from poly(A)+ RNA of catenin are colocalized at the plasma membrane (a and b). Membrane mouse embryonal carcinoma cells PCC4-aza.1 was screened staining for a catenin is observed in cells expressing N-cadherin (c), by using affinity-purified anti-a-catenin antibodies. Seven uvomorulin (f), or both (d). However, in cadherin-negative Ltk- initial clones were obtained from 500,000 phage plaques, and cells, a catenin is localized throughout the cytoplasm (e). (x200.) additional 5' sequences were cloned by using a random KS(+) U1 (Stratagene) and sequenced by using either the primed (Amersham; RPN. 1601Z) [a-32P]dCTP-labeled Klenow fragment ofDNA polymerase 1(19) or the Sequenase cDNA fragment (positions 2056-2334; see Fig. 4). All cDNA system (United States Biochemical). Sequencing was done inserts were subcloned into the EcoRI site of Bluescript on both strands of cDNA on either double-stranded or single-stranded template DNA by using the dideoxy chain- 200- termination method (19). The full-length clone is designated pBsACAT-1. Sequence Analysis. All analyses were done with programs 116 mom of the GCG package (University of Wisconsin). 97 ' RESULTS 67- "i Conventional approaches to generate antibodies against a catenin were unsuccessful, most likely because of the low 45mm .li' antigenicity of the protein. Therefore F9 tumors were gen-

45 erated to obtain large amounts of material from which to purify a catenin. Proteolytic fragments were then produced and subjected to microsequencing analysis. Several internal protein sequences were obtained (see Fig. 3), and the re- I spective peptides were synthesized for antibody production 2 3 4 5 6 7 8 9 in rabbits. One anti-peptide serum was found to recognize a FIG. 1. Immunoprecipitations (lanes 1-4) and immunoblots catenin equally well in immunoprecipitation, immunoblot, (lanes 5-10) show that a catenin associates with different cadherins. and immunofluorescence experiments. These antibodies pre- Anti-a-catenin (lane 3) and anti-uvomorulin (lane 2) (control is lane cipitated, as did anti-uvomorulin, the characteristic uvo- 1) precipitate the characteristic uvomonrlin-catenin complex from morulin-catenin complex (Fig. 1, lanes 2 and 3). The identity cell lysates of [35S]methionine-labeled HeLa (lane 4) or H1-3 cells of both complexes was confirmed by the cross-wise staining (lanes 1-3). This is confirmed by the cross-wise staining of the of the immunoprecipitates with both antibodies in immuno- immunoprecipitates in immunoblots with anti-a-catenin (lane 5) or blot experiments (Fig. 1, compare lanes 2 tnd 5 with lanes 3 anti-uvoitorulin (lane 6). Immunocomplexes collected with anti-a- and 6), as well as by peptide pattern analysis (not shown). catenin from human HeLa (lane 7), mouse PSA SE, chicken CEF38 Anti-a-catenin antibodies cross-reacted with a of (lane 9) and Xenopus A6 cells (lane 10) were stained with antibodies protein against N- and P-cadherin, A-CAM, and U-cadherin, respectively, similar size from cell lysates of different cell types and showing that a catenin associates with different cadherins from species including human, swine, rabbit, and Xenopus (not human, mouse, orXenopus. The arrowhead indicates the position of shown). In immunoblot experiments uvomorulin-negative a catenin. Molecular mass markers are given in kDa. cells had very little a catenin compared with the correspond- Downloaded by guest on September 29, 2021 9158 Cell Biology: Herrenknecht et al. Proc. Nati. Acad. Sci. USA 88 (1991) ing transfectants expressing uvomorulin. This suggests that with the respective antibodies (Fig. 2 a and b). Like uvo- uvomorulin might affect the rate of synthesis or protein morulin, a catenin was concentrated in membrane areas of stability of a catenin. Immunoprecipitates collected with cell-cell contacts. Anti-a-catenin antibodies stained equally anti-a-catenin antibody from different cell types were sub- the plasma membrane of permeabilized HeLa (positive for jected to immunoblot experiments. In each instance specific N-cadherin) and H1-3 cells (expressing uvom6rulin and staining with antibodies to human N-cadherin, mouse P-cad- N-cadherin, Fig. 2 c and d). In contrast, Ltk- cells deficient hemn, chicken A-CAM, and Xenopus U-cadherin was ob- for any cadherin, showed a catenin staining throughout the served (Fig. 1, lanes 7-10). This strongly suggests that the cytoplasm (Fig. 2e). However, a catenin became localized at complex formation with a catenin is a general feature of all the plasma membrane in LI-1 cells expressing uvomorulin cadherins. The immunoprecipitation experiments led to an (Fig. 2J). This redistribution of a catenin is most likely due to additional observation that might be ofbiological importance. complex formation with uvomorulin. Complexes collected with anti-a-catenin from HeLa cells The peptide antibodies were used to screen a Agtll cDNA contained more N-cadherin than those collected from H1-3 library from embryonal carcinoma cells PCC4. Seven initial cells, which express in addition uvomorulin (Fig. 1, lanes 3 cDNA clones were analyzed, and 5' sequences were used to and 4). This might indicate that a catenin has a stronger isolate a sequence of 3712 base pairs that hybridized to a affinity for uvomorulin than for N-cadherin, although other single 3.7-kb mRNA in Northern blot analysis (not shown). explanations are possible. An open reading frame encoding 906 amino acid residues can Immunofluorescence tests on permeabilized mouse PCC4 be defined with a calculated molecular mass of 100 kDa, cells revealed colocalization of uvomorulin and a catenin which is very close to the relative molecular mass of 102 kDa

1 CGGGhGAGCAGCGCGGCCGTCGGCTTGGCGCCTCAGGGTCGTCCCGT CCCAG 1621 AACCACATTTTAGAAGATGTGAACAAGTGTGTTATTGCTCTCCAAGAGAAAGACGTGGAT 61 TTCGCTGCAGhAATGACTGCCCCACGCAGGCAAATAAACGCCC~a N N I L E D V N K C V I A L Q E K D V D 536 N T A V H A G N I N F K N D P K 16 1681 GGGCTGGATCGCACTGCCGGTGCCATCCGAGGCCGGGCAGCCCGAGTCATTCATGTAGTC 121 AGTCTGGhGATTAGGACTCTGGCTGTTGoAwkACTIGTGGhGCCTCTTGTTACACA.GTT G L D R T A G A I R G R A A R V I N V V 556 S .L K I k T L A V K k L L X P L V T Q V 36 1741 ACCTCAGAGATGGACAACTACGAGCCAGGAGTTTACACAGAGAAGGTTCTGGAAGCCACC 181 ACAACCCTGGTAAACACCA&TAGTAAAGGGCCGTCTAATAAAAAGhGAGGCCGTTCTAAG T S E M D N Y E P G V Y T E K V L K A T 576 T T L V N T N -S K G P S N KK R G R S K 56 1801 AAGCTCCTCTCCAACACAGTCATGCCGCGCTTCACTGAGCAGGTGGAAGCAGCTGTGGAA 241 AAGGCCCATGTTTsGGCTGCATCTGTT C GC TGAGAATTTCTTGAGG K L L S N T V M P R F T E 0 V K AA V E 596 K A H V L A A S V K Q A T K N F L K K G 76 1861 GCCCTCAGCTCGGACCCGGCCCAGCCCATGGATGAGAATGAGTTCATCGACGCCTCCCGT 301 GATAAAATTGcAAAAGAGAGCCAGTTTCTCAAGGAGGAGCTTGTGGTTGCTGTAGaAGAT A L S S D P A Q P M D E N E F I D A S R 616 D K I A K K S Q F L K K K L VV A V Z D 96 1921 CTGGTGTACGACGGCATCCGGGACATCCGGAAAGCAGTGCTGATGATCAGGACCCCCGAG K 361 GTTCGaAA TGTGAAGAGCGCTGCTGGGGAGTTTGCAGATGATCCATGC L V Y D G I R D I R K A V L N I R TP 636 V R K Q G D L N K S A A G K F A DD P C 116 1981 GAGTTGGACGACTCTGACTTCGAGACTGAAGACTTTGATGTCAGAAGCAGGACCAGTGTC 421 TCTTCTGTGSAGCGAGGCAACATGGTCCGGGCAGCTCGAGCTTTGCTCTCTGCCGTCACC E L D D S D F E T E D F D V R S R T S V 656 S S V K R G N N V R A A R A LL S A V T 136 2041 CAGACAGAAGATGACCAGCTGATAGCTGGCCAGAGCGCCCGGGCGATCATGGCTCAGCTT 481 CGGCTGCTGATTCTGGCTGACATGGCAGATGTCTACAATTACTTGTTCAGCTGAAAGTT Q T E D D Q L I A G Q S A R A I M A Q L 676 R L L I L A D N A D V Y K LL V Q L K V 156 2101 CCTCAGGAGCAAAAAGCAAAGATTGCAGAACAGGTGGCCAGTTTCCAGGAAGAGAAGAGC 541 GTGGAAGaTGGTATATTGAACTGAGGAATGCTGGCAATGaACAAGACTTAGGATACAG P Q E Q K A K I A E Q V A S F Q E E K S 696 V D G I L K L R N A G N K Q D L G I Q 176 Z 2161 AAGCTGGATGCTGAAGTGTCCAAGTGGGATGACAGTGGCAATGACATCATTGTGCTGGCC 601 TACAAAGCTCTGAAACCAGAAGTAGATAAGCTGAACATCATGGCAGCAAAAAGACAAAG K L D A E V S K N D D S G N D I I V L A 716 Y K A L K P K V D K L N I N A A K R Q Q 196 2221 AAGCAGATGTGCATGATCATGATGGAGATGACCGCTTCACCC CATCCACTC 661 GAACTAAAAGAT CAGCTAG.AGGAATCCTGCAGAAA K Q M C M I M M E M T DF T R G K G P L 736 K L K D A R G I L 0 K 216 2281 AAAAACACCTCTGACGTGATCAGTGCTGCCAAGAAAATTGCGGAGGCGGGATCCAGGATG 721 AATGTTCCAATCCTCTATACTGCATCCCAGGCATGTCTACAACACCCTGATGTTGCAGCC K N T S D V I S A A K K I A K A G S R M 756 N V P I L Y T A S Q A C L Q N P D V AA 236 2341 GACAAGCTCGGCCGGACCATCGCAGACCATTGCCCAGACTCGGCATGCAAGCAGLCCTG 781 TATAGGCCAACCGGGACTTGATATACAGCAGCTGCAGCAGGCCGTCACATTTCT D K L G R T I A D H C P D S A C K Q D L 776 Y K A N R DL I Y K Q L Q Q A V T G I S 256 2401 CTGGCCTACCTTCAGCGCATCGCTCTCTACTGCCACCAGCTCAACATCTGCAGCAAAGTC 841 AATGCAGCCCAGGCTACTGCATCAGATGATGCTGCCCAGCACCAGGGTGGCAGTGGCGGA L A YL Q R I A L Y C H Q L N I C S K V 796 N A A Q A T A S D D A A Q H Q G G S G G 276 2461 AAGGCTGAAGTACAGAACCTTGGTGGAGAGCTGGTCGTCTCTGGGGTGGACAGCGCCATG 901 GAGCTGGCATACGTCTCAACAACTTTGATAAACAAATCATTGTGGACCCCTTGAGCTTC K A E V Q N L G G E L V V S G V D S A N 816 K L A Y A L N N F D K Q I I V D P L S F 296 2521 TCCCTGATCCAGGCGGCCAAGAACTTGATGAATGCTGTTGTGCAGacaGTdAAGGCGTCC 961 AGCGAGGAGCGCTTTAGG CCGTCCCTGGAGGAGCGCCTGGaAAGCATTATTAGTGGGGCT S L I Q AA K N L M N A V V Q T V K A S 836 K K F R P S L K E R L K 3 I I S G A 316 S R 2581 TACGTGGCTTCCACCAAATACCAGAAGTCACAGGGATGGCTTCCTTGAACCTTCCTGCT V A S T K Y Q K S Q 1021 GCCCTGATGGCTGACTCATCCTGCACACGTGATGACCGTCGGGAGCGCATTGTGGCAGAG Y G N A S L N L P A 856 A L N A D S S C T R D D R R K R I VA K 336 2641 GTGTCATGGAA GATGAAGGCCCCT CGTTGGTG N K N K A K K 1081 TGTAATGCTGTCCGCCAGGCTCTACAGacCTGCTCTCAGAATACATGGGCAACGCTGGa V S P K K P L V K R K Q D 876 C N A V R A L Q D L L S K Y N G N A G 356 Q 2701 GAGACGCAGACCAAGATTAAACGAGCTTCTCAAGCaCGTaaACCCAkGTcAGGCCK K A YN A 1141 CGT AAGA GAAGTGACACTCAACTCTGC AATAGATACGAC CmAGG E T Q T I R S Q KK 1K P V a b96 K K R S D A L N S A I D K N T K K T R 376 R 2761 CTGAGCGAGTTCAAAGCCATGGACAGCAI,TCTGAGCCTGCACAGCCCATGGGGTT&TGAAA A N 1201 GACTTGCGTAGACAGCTTCGCAAAGCTGTCATGGACCcAGTATTTCTTCCTGGAG L 3 * R-K1 D S I 906 L K 0 L K K A V M D H V S D S F L K 396 D R 2821 AAAACCAGTCACATGCTTCACTCAAATGI 1261 ACCAATGTCCCTCTATTAGTACTGAT TGAAGCTTCAAAGAATGGGAAGAT GTT 2881 ACAGGGAGGTAGCAGATTTCAGACTCAAC L L V L I AA K NG N K K K V 416 2941 GTTAAAGCTGGTT TAAAGCTTI T N V P K 3001 TTTATAAGTTTAATGAAAATAACAGTTCI 1321 AAcGAATATGCCCAAGTTTTTCGTGAACATGCCAACA*AC TGAGGTTGCCATCCTG 3061 TCACATTTCTGRGTAGGCTGAAGTGCCT( F V K A N K L I K V A N L 436 3121 GAGGTTGCTTTTTAGCCCTACTGTTGGGC K Y A Q F R H 3181 TGTAAATCC CCTTAACGGTCI 3241 1381 GCCTGTTCAATCTCCAACAATGAGAAGGCGTAGCTTGTCCTGTCTAAGCCAG GGAATAAACTATAGGAGACAATAaAAT41G S S N N K G V K L V R N S A S Q 456 3301 TTATG tATcG A C I K 3361 AACATTAATGGAGTTGCTTTATTCGGGcI 1441 TTAGaAGCGCTGTGTCCTCAGGTTATGCATcTGGCTTGGCAGGCsCAG 3421 TTCCCTGTTATTCCAGCTTGAATCAAI K P V I N A A L A L AA K P 476 3411 GTGTTTCCTCCTTTGG CA OAAG L A L C Q Q 3541 TACTTAGACGCACCGTGTTTGTG.AC? 1501 AGTAAACTG0CCCG CGGATCTTTTTAAAGCAG aAGTCCGT 3601 TTACTGTCTTATTTAAATTTTATG&aMCM S K L A Q K N N D L F K K Q N K K Q V R 496 3661 GTCCATTAAGAACC 1561 GGaCTTCT;CTCsAGaG V L T D A V D D I T S I D D F L A V S K 516 FIG. 3. Nucleotide and amino acid sequence of mouse a catenin. Nucleotides are numbered on the left, and amino acids (single letter notation), on the right. The cDNA sequence reveals an open reading frame of906 amino acids flanked by 72 nucleotides of5' and 922 nucleotides of 3' untranslated sequences with a typical polyadenylylation signal 14 nucleotides before the poly(A) tail. The deduced amino acid sequence contains peptide sequences (underlined) determined by direct protein sequencing analysis. The anti-a catenin antiserum was raised against a peptide near the carboxyl terminus (bold letters, boxed). Downloaded by guest on September 29, 2021 Cell Biology: Herrenknecht et al. Proc. Natl. Acad. Sci. USA 88 (1991) 9159 I II * -CATENIN 1 MTAVHAGNIN FKWDPKSLEI RTLAV RLL PLVTQVTTLV NTNSKGPSNK 50 a-CATZNIN 377 DARQLREAV IHDVDSFLE TNPLLVLIZ AKNGN. UIEVKEYAQ 421 CHICKEN 1 .. HTRTI3SILI PVAQQISHLV ItIEEGEVDG 34 CHICKEN 582 D l TQEVSOWSD TTTPIKLLAV AhTAPSDTPN RJVFEESAA 631 HUMAN 1 ...... MPVF HTRTIRSILZ PVAQQISHLV IOIEEGEVDG 34 HUMAN 582 DIA TQEVSYVVSD TTTPIKLLAV A&TAPPDAPN RVFDERAA 631 NEMATODE 1 ...... MPVF HTKTIENILS PVAQQVSRLV ILHEIAN.DG 33 NEMATODE 478 DLWDFMAL TDOVYDOVAD ITTPLKQFVE AVHADPYDPN RUQNFVDKSQ 527 CONSENSUS ------bT- -hE-hLZ Ph- -Qho-LV -----s---- CONSENSUS DL---h---h ---V-D-F-a ---Ph-----A------R- a-- -

* -CATENIN 51 KRGRSKKAHV LAASVEQATE MF..LLEKGDK IAKESQFLICE ELVVAVEDVR 98 a-CATENlh 422 VFPSEKLI EVHLACSIS .NN. EGWYL VPNSASQLEA LCPQINAAL 469 CHICKEN 35 KAIPDLTAPV SA..VQAAVS WLVRVGKETV QTTEDQILICR DMPPAFIKE 82 CHICKEN 632 NFEdmAALG ATAEKAAAVG TAN.KTTVEG IQATVIKSAK LTQVVSAAR 680 HUMAN 35 KAIPDLTAPV AA..VQAVS ULVRVGKETV QTTEDQILICR DMPPAFIKVE 82 HUMAN 632 NFENGCLG ATAEKAAAVG TAN. KSTVEG IQASVKTARE LTPQwVSAAR 680 NEMATODE 34 NAMPDLTGPV GM..VSRAVG ELIQVGYDTC DHSDDRILQQ DMPPALQRVE 81 NEMATODE 528 KLTD SQSNT TTARLVASCG PSKSKKTVEA ILDTAEKVEQ LTPQLVNIIGR 577 CONSENSUS ------v -----V--A-- U----h------a---L-- ah- --A--V- CONSENSUS ----E---h- --A------v-- h--o------L-h-An-

u-CATENIN 99 KQGDLMKSAA GEFADDPCSS VKIWNMVI8AA RALLSAVTRL LILASSIADVY 148 a-CATENIN 470 ALAkPQSKL ELFKE hKQVRVLT DAVDDITSID DFLAVIENI 519 CHICKEN 83 NACTKLVRAA QMLQADEYSV PAWDYLIDGS W2ILSGTSDL LLTFDEAEVR 132 CHICKEN 681 ILLPPI~O AYUENTEIUN ChIDNVUEE T GLYD"AIDTK SLLDASAI 730 HUMAN 83 NACTKLVQAA QMWLQSDYSV PAlDYLIDGS NGILSGTSDL LLTFDEAVR 132 681 IL10GNOA A"ENFETINU GWIDNVEIMT GLVDEAIDTK SLLDABMEAI 730 NEMATODE 82 GSSKLLEESS YSLKNDPYSV PARKtLIDGA DOILQGTSAL LLCFDESEVR 131 NEMATODE 578 VPLHNGSE. .. QINIHX QYADALHALK SHVDDAIDTG EFVAAESAN 624 CONSENSUS -h--_ DDP-S- --R--hh-s- RahL-s-o-L Lh--D--aV- CONSENSUS -----P------a--b- Qr---h--h- --VD------h--SE--h

a -CATENIN 149 ELLVQLKVVE DGILKLRNAG NEQDWIQYE ALKPEVDKLN I lAAKRRL 198 * -CATSNON 520 LEDVNIKCVIA LQKUDVDGLD RTAGAIRGRA AEVIHVVTSE MDWUGVYT 569 CHICKEN 133 EIIRVCKGIL KYLTVAEVVE T8UDL.VTYTE NLGPGMTKMA KNIDEQRL 182 CHICKEN 731 KKDLDKCGVA HANNQPQIULV AGATSIAPAA NRILLVAKRAE VEES;DPKFR 780 HUMAN 133 IIIRVCKGIL tYLTVAEVVE TM9DLVTYTE NLGPGMTENA KMIDESWL 182 HUMAN 731 KKDLDKCIMVA NANIIQPQHLV AGATSIAARA NKILLVAKRE VE3SEDPKlFR 780 NEMATODE 132 ElIIRVCRKAN DYVAVSEVIE SMADLQQFVE DISPVLHDVT NDVNLIML 181 NENATODE 625 RRYTNtOCA INGADAHGLV NKSSQIAPLG NRVLMTAQNE ADMSRPSFV 674 CONSENSUS Rhh---b--- a-h------DL----E -h-P-h--h------MQM CONSENSUS -----bC--A h------L- I---- a -Rhh-----E -aN-E-s-r-

a-CATENIN 199 KDVGNRDQMA AARGILQKNV PILYTA 224 * -CATENIN 57Q EKVLEATElLL SNTV? 585 CHICKEN 183 THQENRVHLV NSDNTVKELL PVLISA 208 CHICKEN 781 EAVEASSEL SKTISP 796 HUMAN 183 THQEHRVMLV NSllTVllELL PVLISA 208 HUmN 781 EAVKASDSL SKTISP 796 NEMATODE 182 THQVHMEILI RCtDSIKVIA PILICS 207 NEATODE 675 SRVKNAADQL HNAIPP 690 CONSENSUS -----R--h------h---- PhL--- CONSENSUS --V--A--- L ---h-P III Vinculin -ATENIN 698 U)AEVSKIWDD SGNDIIVLA QWMIM4EMT DFTHGKGPLK NTSDVISAAK -4- ':H I CKEN 905 LKDERAKUSS KGNDIIAAAK RJPLLWfEMS RLVRGGSG NKRALIQCAK S52 HUJMAN 905 LHDEAR.KWSS KGNDIIAA.A RJ6LLLMAEMS FLVHGGSG TKRA.LIQCAK 952 ... 76 . ..._...... NEMATC)DE 9'i9 LHNELKQWSS QENDIVAAAK RMAILNARLS QLVtRGGG TKKDLINCSK 886 6 208 582 796 '905 t053 ':ONSENCJ." L--9 ---V- -H-NDh--AK --hhH- -ho -- -G------hI -- -K 27% 31%zo 34%0 -CATEN'N 48 KIAEAGS1R4ID KLGRTIADHC PDSACKQDILL AYLQHRIALY HQIN;CCSKK - D C:HICKEN 9 IAKASDEVT RLAJKEVAKQC TDKRIIRTNLL QVCERLIPTIS TQLK.LSTVK - 22 224 377 585, 698 848 HJMAN 9h1 DIAKASDEVTT RLAKEVAKIQC TDKRIRTNLL QVCER.IPTIS TOQLKILSTVK 1 27 NEMATr DE 99' AIADSSEEVT RLAVQLARLC TD'KMRTALL QVSERIPTIA TQLKVLS7VyS3W F N1. X_:: -' NSENS -7 -IA ---- h- LLs --A--C -D --- t---LL ---- RIs--- -QL-h-S-VK

' - ATEN:N ; kAEV'5NLGGE .. . S: :CSAMSL IQAAK N4A VTVKASY - 4 q H '"FEN ; ATMLCRTN:. EESEQATEML VHNA1NIJKS VYETVFEAEA -:4^ a Catenin r H MiAN i , 2ATMLGRTN: SD EESEQATEML VHNAINNJQS VKETVREAEA - 4: ,EOtAT' -FE 'i -ATML. ,SAD .I G1PY;:,2FCEC7GS EE DEAM'QQL VHNAQNIJNQS VK .V-PAAEA Q e >..E; .. *:; A -F----- L V--A-PlISHUJ- V------

'I Nrw h ASTKYI1YiKS3.'G MA.:; LNNLPA:'C'- NKMKAEE K E LT.F-EF..E 'FR :K H 1.EN ;4 4 ASIRlTRTDAG F TLP VRKTFWY , -k . 44 ASTKIHTDAG F WVFXT F WY N F.KAIOMA T' F. P..ASIKTFTNSG I. PRL R WL FM''SNF .; '1' G .I,, .. AS-K------G !:

'A-l-N WHV~F '-~AL E F AM.

MA '' '.

FIG. 4. Comparison of a catenin and vinculin. Multi-alignment of homologous regions (I = amino-terminal region, II = central region, and III = carboxyl-terminal region) of mouse a catenin and vinculin from human, chicken, and C. elegans. The consensus sequence is given: upper-case letters depict absolute homology and lower-case letters depict conservative differences where h = hydrophobic, a = acidic, o = oxygen-containing side chain, r = aromatic, b = basic, and s = small side chain. (Right Lower) Schematic comparison of mouse a catenin and chicken vinculin according to dot-blot matrix analysis. Numbering of vinculin was done according to ref. 20. Homologous regions are indicated by dotted boxes; the percentage identity is indicated.

determined in SDS/polyacrylamide gels for a catenin. Sev- proline-rich sequence characteristic of vinculin. Regions of a eral peptide sequences obtained from direct protein sequenc- catenin that are nonhomologous to vinculin have no signifi- ing, including the one used for immunization, are present in cant homology to any other proteins. the deduced amino acid sequence (Fig. 3). The protein shows no internal homology, and secondary structure prediction DISCUSSION analysis indicates a high content of a-helices and overall Here we show that a catenin is a ubiquitous cellular com- strong hydrophilicity. As expected from the protein analysis, ponent since anti-peptide antibodies detect homologues in no signal peptide or hydrophobic membrane-spanning do- different cell types from Xenopus to man. This confirms main could be identified. earlier observations that the heterotypic expression of mouse Nucleic acid and protein data-base searches identified a uvomorulin in human or chicken cells results in the formation catenin as a vinculin-related protein. The homology between of a complex between uvomorulin and endogenous proteins, vinculin and a catenin is restricted to three major regions in one of which is a catenin (10). the amino-terminal, central, and carboxyl-terminal part of In addition we show that various members of the cadherin both proteins. The homologous amino acid sequences of gene family from different cell types and species all associate human, chicken, and Caenorhabditis elegans vinculin and a with a catenin. For uvomorulin the association with a catenin catenin are aligned in Fig. 4. The strongest degree of homol- is a prerequisite for its connection to the actin filament ogy was found in the respective carboxyl-terminal regions. A network (11). A similar transmembrane linkage of other schematic comparison of vinculin and a catenin is given in cadherins to cytoskeletal structures has not yet been re- Fig. 4. ported. Although the function of the association between The most significant difference between the two proteins is these cadherins and a catenin remains elusive, it is reason- the absence in a catenin of the repeated units and the able to assume that such a conserved molecular interaction Downloaded by guest on September 29, 2021 9160 Cell Biology: Herrenknecht et al. Proc. Natl. Acad. Sci. USA 88 (1991) might reflect some common functional properties one of a- or F-actin (32) on the other. In conclusion, our work which might be to mediate the cytoplasmic anchorage of all suggests the existence of a new vinculin-related family of cadherins. These complex formations might also regulate proteins involved in the cytoplasmic anchorage of cell-cell other cellular functions ofcadherins, and it will be interesting and cell-substrate adhesion molecules. to see whether, similar to the uvomorulin-catenin complex, (3 and 'y catenin can also be found in association with other cadherins. If so, it may be that catenins play a key role in the Note. Since the submission of this manuscript, Nagafuchi et al. (33) function of all cadherins. have reported the molecular cloning of the 102-kDa cadherin- It is becoming increasingly evident that a given cell can associated protein, termed CAP 102. Sequence comparison revealed express more than one cadherin at a time. This raises the that CAP 102 is identical to a catenin. question of how this cell can regulate the selectivity of adhesion by one type ofcadherin interaction. Foruvomorulin We thank A. Rolke for technical assistance, L. Lay for preparing it has been shown that complete adhesive function is possible the photographs, and R. Schneider for typing the manuscript. We only when uvomorulin complexes with catenins (10, 11). It is also thank S. Wood for discussions and critical proofreading. This tempting to speculate that a differential complex formation of work was supported by the Deutsche Forschungsgemeinschaft. catenins with one or the other cadherin could direct the selectivity of cell adhesion. Although preliminary, such a 1. Takeichi, M. (1988) Development 102, 639-655. 2. Kemler, R., Ozawa, M. & Ringwald, M. (1989) Curr. Opinion Cell Biol. view is supported by our results concerning the different 1, 892-897. amounts of N-cadherin associated with a catenin in Hela and 3. Donalies, M., Cramer, M., Ringwald, M. & Starzinski-Powitz, A. (1991) H1-3 cells. Proc. Natd. Acad. Sci. USA, in press. Our immunofluorescence studies demonstrate that a cat- 4. Ginsberg, D., DeSimone, D. & Geiger, B. (1991) Development 111, enin is localized close to the plasma membrane and is 315-335. in membrane areas of cell contacts whenever 5. Angres, B., Muller, A. H. J., Kellermann, J. & Hausen, P. (1991) concentrated Development 111, 829-844. cadherins are coexpressed in the same cell. However, a 6. Herzberg, F., Wildermuth, V. & Wedlich, D. (1991) Mech. Dev. 35, catenin can be detected in cells such as Ltk- that express no 33-42. cadherin molecule as determined by PCR analysis (unpub- 7. Koch, P. J., Walsh, M. J., Schmelz, M., Goldschmidt, M. D., Zimbel- lished observations). In these cells no association ofa catenin mann, R. & Franke, W. W. (1990) Eur. J. Cell Biol. 53, 1-12. with the plasma membrane is detected; rather, the protein 8. Wheeler, G. N., Parker, A. E., Thomas, C. L., Ataliotis, P., Poynter, in the This D., Arnemann, J., Rutman, A. J., Pidsley, S. C., Watt, F. M., Rees, seems to be uniformly distributed cytoplasm. D. A., Buxton, R. S. & Magee, A. I. (1991) Proc. Natd. Acad. Sci. USA observation may reflect some additional biological properties 88, 4796-4800. of a catenin. The a catenin detected in Ltk- cells is func- 9. Mechanic, S., Raynor, K., Hill, J. E. & Cowin, P. (1991) Proc. Natd. tional, at least with respect to its interaction with uvomorulin, Acad. Sci. USA 88, 4476-4480. since a catenin becomes concentrated at the plasma mem- 10. Ozawa, M., Baribialt, H. & Kemler, R. (1989) EMBO J. 8, 1711-1717. 11. Ozawa, M., Ringwald, M. & Kemler, R. (1990) Proc. Natl. Acad. Sci. brane after expressing uvomorulin in these cells. USA 87, 4246-4250. Nucleic acid and protein data-base searches identified a 12. McNeil, H., Ozawa, M., Kemler, R. & Nelson, W. J. (1990) Cell 62, catenin as a vinculin-related protein. Vinculin is a cytoskel- 309-316. etal protein associated with both cell-cell and cell- 13. Nagafuchi, A. & Takeichi, M. (1989) Cell Regul. 1, 37-44. extracellular matrix adherens-type junctions (21, 22). Bio- 14. Wheelock, M. J. & Knudsen, K. A. (1991) Dferentiation 476, 35-42. 15. Ringwald, M., Schuh, R., Vestweber, D., Eistetter, H., Lottspeich, F., chemical binding studies indicate an interaction of vinculin Engel, J., D6lz, R., Jahnig, F., Epplen, J., Mayer, S., Mfller, C. & with (23, 24) and a actinin (25, 26), which is thought to Kemler, R. (1987) EMBO J. 6, 3647-3653. be part of the apparent transmembrane linkage of the extra- 16. Eckerskorn, C. & Lottspeich, F. (1989) Chromatographia 28, 92-94. cellular matrix to the cytoplasmic system (22). 17. Eckerskorn, C., Mewes, W., Goretzki, F. & Lottspeich, F. (1988) Eur. vinculin and a catenin is restricted to J. Biochem. 176, 509-519. The homology between 18. Volk, T. & Geiger, B. (1984) EMBO J. 3, 2249-2260. three major regions in the amino-terminal, central, and car- 19. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natd. Acad. Sci. boxyl-terminal parts ofboth proteins, the strongest degree of USA 74, 5463-5467. homology being found in the C-terminal portion (20, 27-29). 20. Price, G. J., Jones, P., Davison, M. D., Patel, B., Bendori, R., Geiger, For vinculin this region is thought to exhibit self-association B. & Critchley, D. R. (1989) Biochem. J. 259, 453-461. a 21. Geiger, B. (1979) Cell 18, 193-205. properties (30); however, it is not yet known whether 22. Burridge, K., Fath, K., Kelly, T., Nuckolls, G. & Turner, C. (1988) catenin undergoes similar di(oligo)merization. Another ap- Annu. Rev. Cell Biol. 4, 487-525. pealing possibility is that a catenin might interact directly 23. Otto, J. J. (1983) J. Cell Biol. 97, 1283-1287. with vinculin. Vinculin is found to be associated with the 24. Buwridge, K. & Mangeat, P. (1984) Nature (London) 30, 744-746. adherens-type junctions, but its exact function in this sub- 25. Belkin, A. M. & Koteliansky, V. E. (1987) FEBS Lett. 22E@, 291-294. 26. Wachsstock, D. H., Wilkins, J. A. & Lin, S. (1987) Biochem. Biophys. cellular structure is not known. It may well be that a catenin Res. Commun. 146, 554-560. and vinculin interact with each other and are part of a 27. Weller, P. A., Ogryzko, E. P., Corben, E. B., Zhidkova, N. I., Patel, B., multiprotein complex. Price, G. J., Spurr, N. K., Koteliansky, V. E. & Critchley, D. R. (1990) Perhaps most significantly, a catenin contains a short Proc. Natl. Acad. Sci. USA 87, 5667-5671. which is conserved in all 28. Coutu, M. D. & Craig, S. W. (1988) Proc. Natl. Acad. Sci. USA 85, peptide, Arg-Gln-Gln-Glu-Leu, 8535-8539. and located in a region thought to regulate the 29. Barstead, R. J. & Waterston, R. H. (1989) J. Biol. Chem. 264, 10177- binding of vinculin to talin (31). This suggests that a catenin 10185. could also associate with talin. Although there is no exper- 30. Bendori, R., Salomon, D. & Geiger, B. (1989) J. Cell Biol. 106, 2383- imental evidence for such an association, the detection of a 2393. Ltk- such a 31. Jones, P., Jackson, P., Price, G. J., Patel, B., Ohanion, V., Lear, A. L. catenin in cadherin-negative cells suggests & Critchley, D. R. (1989) J. Cell Biol. 109, 2917-2927. possibility. This might indicate that a catenin exhibits mul- 32. Westmeyer, A., Ruhnau, K., Wegner, A. & Jockusch, B. M. (1990) tiple biological properties. For example, it may exhibit sim- EMBO J. 9, 2071-2078. ilarity to vinculin in its interaction with talin on one side and 33. Nagafuchi, A., Takeichi, M. & Tsukita, S. (1991) Cell 65, 849-857. Downloaded by guest on September 29, 2021