RESEARCH ARTICLE 3873 mARVCF cellular localisation and binding to is influenced by the cellular context but not by alternative splicing

Zoe Waibler, Annette Schäfer and Anna Starzinski-Powitz* Institut der Anthropologie und Humangenetik fuer Biologen, Johann-Wolfgang-Goethe-Universitaet Frankfurt, Siesmayerstrasse 70, D-60054 Frankfurt/Main, Germany *Author for correspondence (e-mail: [email protected])

Accepted 23 July 2001 Journal of Cell Science 114, 3873-3884 (2001) © The Company of Biologists Ltd

SUMMARY

ARVCF, a member of the family, is thought to isoforms to M-, N-, or E- is generally unaffected contribute to the morphoregulatory function of the by their altered N- and C-termini, as revealed by the MOM cadherin-catenin complex. Recently, we reported the recruitment assay. However, mARVCF isoforms isolation and characterisation of murine ARVCF reproducibly exhibit differential localisation in distinct (mARVCF), particularly its interaction with M-cadherin. cellular environments. For example, mARVCF isoforms Here, we describe the identification of novel mARVCF are unable to colocalise with N-cadherin in EJ28 isoforms that arise by alternative splicing. At the N- cells but do so in HeLa cells. Our results suggest that the terminus, alternative splicing results in the inclusion or subcellular localisation of mARVCF may be determined omission of a coiled-coil region probably important for not only by the presence or absence of an appropriate -protein interactions. At the C-terminus, four interaction partner, in this case cadherins, but also by the isoforms also differ by domains potentially important for cellular context. selective protein-protein interaction. The eight putative mARVCF isoforms were expressed as EGFP-fusion in six different cell lines that exhibit a distinct Key words: p120(ctn) subfamily, MOM recruitment assay, Armadillo pattern of cadherins. Apparently, binding of the mARVCF repeat protein

INTRODUCTION The human maps to 22q11, the so-called DiGeorge critical region (Sirotkin et al., 1997; Bonne et al., One of the most recently identified members of the p120(ctn) 1998), which is hemizygous in 80-85% of DiGeorge patients subfamily, the murine ARVCF protein (armadillo repeat gene and those with velo cardio facial syndrome (Desmaze et al., deleted in velo cardio facial syndrome) comprises an N- 1993; Kelly et al., 1993; Morrow et al., 1995). Human ARVCF terminal coiled-coil region and a central armadillo repeat appears to be more or less ubiquitously expressed, being found region. This structure closely resembles that of p120(ctn) itself in a variety of tissues including heart, skeletal muscle, lung, (Sirotkin et al., 1997; Kaufmann et al., 2000; Mariner et al., brain, liver, pancreas and kidney (Sirotkin et al., 1997). 2000; Anastasiadis and Reynolds, 2000). Not only do the Murine and human ARVCF can associate with the armadillo repeat regions of both proteins share 56% homology membrane proximal amino acids in the cytoplasmic region of but also exon-intron boundaries of the are very similar cadherins such as E-cadherin in epithelial cells and M-cadherin (Keirsebilck et al., 1998). The 35 N-terminal in muscle cells (Reynolds et al., 1994; Kaufmann et al., 2000; coiled-coil region of ARVCF (Sirotkin et al., 1997; Kaufmann Mariner et al., 2000). This was shown in detail by binding et al., 2000) is generally known as a motif mediating protein- assays using GST-fusion proteins comprising the cytoplasmic protein interactions, although such interactions have not yet domain of M-cadherin plus several deletion mutants, been demonstrated for ARVCF or any other member of this demonstrating that the 55 membrane-proximal CPD amino subfamily. ARVCF’s armadillo repeat region is characterised acids of M-cadherin are necessary and sufficient for ARVCF by 10 of these repeats and a putative nuclear localisation signal binding. Vice versa, all ten armadillo repeats of ARVCF are (NLS) within this region, in addition to a putative nuclear necessary for efficient M-cadherin binding. Deletion of repeats export signal (NES) in the C-terminus of the protein (Sirotkin 1 to 4 or 1 to 5 abolished the ability of ARVCF to colocalise et al., 1997; Kaufmann et al., 2000). The armadillo motif, with N-cadherin in rat ventricular cardiomyocytes, although originally identified in a segment polarity gene in drosophila such deletions still facilitated some interactions in vitro (Wieschaus and Rigglemann, 1987), consists of an imperfect (Kaufmann et al., 2000). However, whether ARVCF directly series of 42 amino acids that form a positively charged groove connects the cadherin complex to the or is (Riggleman et al., 1989). involved in cadherin clustering is not yet clear. 3874 JOURNAL OF CELL SCIENCE 114 (21) In human ARVCF two alternative splicing events have been show that the appearance of the isoforms varies depending on reported. One concerns the N-terminus leading to the removal the cell line or tissue examined. Cloned as EGFP-fusion of the coiled-coil domain and the use of an alternative start proteins and expressed in different cell lines we demonstrate codon. The second splice event leads to the insertion of an 18 that the localisation of mARVCF isoforms is not influenced by exon in the armadillo region that alters the putative the N- or C-terminus of the protein but depends on the cellular NLS (Sirotkin et al., 1997). It has also been shown for context. Using the MOM recruitment assay we examined the p120(ctn) and other members of the subfamily that different ability of all isoforms to associate with M-, E- or N-cadherin isoforms can arise by alternative splicing (Hatzfeld, 1999; in different cell types. Paulson et al., 2000). For p120(ctn) itself this applies to the N- terminus where alternative splicing leads to the use of different start codons. Furthermore, the armadillo repeat region and the MATERIALS AND METHODS C-terminus can be altered by using three alternative exons (Keirsebilck et al., 1998). Cells, cell culture, plasmid transfection Cadherins are a multigene family of calcium-dependent Mouse myoblasts, i28, derived from a primary satellite cell culture transmembrane cell-cell adhesion glycoproteins that mediate (Kaufmann et al., 1999b) were grown in Ham’s nutrient mixture F10 homophilic interactions and are expressed in a tissue-specific (Gibco) supplemented with 20% fetal calf serum (FCS; Sigma) in 5% CO2 at 37°C. To initiate myogenic differentiation, growth medium manner (Ringwald et al., 1987; Takeichi, 1991; Geiger and was replaced by differentiation medium consisting of Dulbecco’s Ayalon, 1992; Shapiro et al., 1995; Huber et al., 1996). Many modified Eagle’s (DMEM) medium (Gibco) with 10% horse serum of the cadherins have been classified according to the tissues (Sigma, Buchs, Switzerland). MCF7 cells, HeLa cells, CMT cells, from which they have been isolated, such as P-cadherin from EJ28 cells, COS-7 cells and RT112 cells were grown in DMEM placenta, E-cadherin first isolated from epithelial cells or M- supplemented with 10% FCS. The cells were transfected with 2 µg cadherin from muscle. The classical cadherins (and M- plasmid DNA using SuperFect Transfection Reagent, or with 1.5 cadherin) consist of an N-terminal extracellular domain, a short µg plasmid DNA using PolyFect Transfection Reagent, both from transmembrane region and a cytoplasmic domain (CPD) Qiagen (Hilden, Germany). For the MOM recruitment assay, cells were cotransfected with 1 µg pMOM-M/E/N-cadherin vector and 1 averaging 150-160 amino acids, which all exhibit a high degree µ of homology with each other (Chothia and Jones, 1997; g of either of the mARVCF EGFP-fusion plasmids (see plasmid constructions). The different cell types were subjected to Humphries and Newham, 1998; Kaufmann et al., 1999a). Most immunofluorescence microscopy 24-48 hours after transfection. Cells cadherins are known to form two distinct complexes with used in addition to i28 were: MCF7 human breast carcinoma cells via their CPD (Ozawa et al., 1989; Hirano et al., 1992; (ATCC HTB-22); COS-7 kidney cells from African green monkey Aberle et al., 1994; Butz and Kemler, 1994; Hinck et al., 1994; (ATCC CRL-1651); HeLa human cervix carcinoma cells (ATCC Näthke et al., 1994; Knudsen et al., 1995; Hertig et al., 1996; CCL-2.1); RT112 human, non-invasive bladder carcinoma cells and Kuch et al., 1997; Finnemann et al., 1997; Yap et al., 1998; EJ28 human, invasive bladder carcinoma cells (Gaetje et al., 1997). Allport et al., 2000). One complex is composed of the respective cadherin, β-catenin and α-catenin, a second Antibodies complex contains cadherin, (also called γ-catenin) The monoclonal antibody 4A6 described previously (Rüdiger et al., and α-catenin. α-catenin joins the complex by binding to β- 1997) was used to identify the birch profilin (BP) tag. Monoclonal anti-GFP antibody was obtained from Clontech (Heidelberg, catenin or plakoglobin and connects this cadherin-catenin Germany). Polyclonal antibodies against the extracellular domain of complex to components of the cytoskeleton (Hirano et al., M-cadherin were affinity-purified as described (Rose et al., 1994; 1987; Tsukita et al., 1992). β-catenin interacts with the C- Kaufmann et al., 1999b). Monoclonal pan-cadherin (clone CH-19) terminal part of cadherin’s CPD, whereas ARVCF and and monoclonal N-cadherin antibody (anti-A-CAM, clone GC-4) p120(ctn), for example, bind to the juxtamembrane region of were obtained from Sigma (Buchs, Switzerland). E-cadherin antibody the cadherin’s cytoplasmic tail, as discussed above. ARVCF was obtained from Monosan (Germany). Secondary antibody (Alexa and p120(ctn) compete for the same binding site in the CPD Fluor 568) was obtained from Molecular Probes (Leiden, The of cadherins (Mariner et al., 2000) but the different functions Netherlands). of the two molecules are as yet unknown. Immunofluorescence Many proteins of the armadillo repeat family are known to Cells grown on coverslips were rinsed in PBS and fixed in 4% enter the nucleus, although the mechanism and functional β paraformaldehyde (PFA) in PBS at room temperature for 10 minutes. consequences of this have only been described for -catenin. After fixation, cells were permeabilised by incubation with 0.2% In addition to its interaction with cadherins, β-catenin can enter Triton X-100 in PBS for 10 minutes, washed three times with PBS the nucleus alone or complexed with Tcf/Lef, a transcription and incubated with the relevant antibodies diluted in PBS/10% FCS factor of the Lef1/TCF family. Together with Tcf/Lef, β- for 1 hour (RT). After washing three times with PBS, binding of the catenin can stimulate the transcription of different target genes primary antibodies was detected by species-specific fluorochrome- (Behrens et al., 1996; Molenaar et al., 1996; van de Wetering conjugated secondary antibodies diluted in PBS/10% FCS. Controls et al., 1997). Similary, ARVCF and p120(ctn) show a dual in the absence of primary antibodies confirmed the specificity of the immunolabelling. Fluorescence was monitored with a Zeiss Axiophot localisation at cell-cell junctions and under some × × × circumstances in the nucleus (van Hengel et al., 1999; microscope. Pictures were taken with 40 , 63 or 100 objectives. Kodak Elite 400 film (400 ASA; Eastman Kodak, Rochester, NY) was Kaufmann et al., 2000; Mariner et al., 2000). Their role in the used for colour slides. nucleus, however, remains to be determined. We report here the identification and cloning of novel N- and Immunoblotting C-terminal isoforms of murine ARVCF resulting, for example, SDS-PAGE and immunoblots were performed as described for E- from the use of different start codons. By using RT-PCR, we cadherin (Butz and Kemler, 1994) and M-cadherin (Kuch et al., 1997; Differential binding of mARVCF to cadherins 3875

Kaufmann et al., 1999b). Membranes were incubated with a primary Nucleotide sequence analysis GFP-antibody for 1 hour followed by incubation with alkaline All clones were sequenced by SeqLab (Göttingen, Germany). DNA phosphatase (AP)-conjugated secondary antibody (Dianova, sequence analysis and homology searches were performed using Hamburg, Germany), which was visualised using the phosphatase HUSAR from DKFZ Heidelberg and the Blast-program-packet from substrates nitroblue-tetrazolium and 5-bromo-4 chloro-3-indolyl NCBI, USA. (NBT/BCIP) (Boehringer Mannheim, Germany). Isolation of mARVCF splice variants by RT-PCR RESULTS The different splice variants of mARVCF were obtained by RT-PCR using Pfx-polymerase (Gibco). First strand cDNA was prepared from RNA templates extracted from differentiating i28 cells 30 hours after Identification and characterisation of novel murine induction for fusion using the primers indicated in Fig. 1 (primer ARVCF splice variants sequences in 5′→3′ orientation: 5′ UTR: GCCTGTCTTGGGGG- Splice variants of murine ARVCF were obtained by RT-PCR CGGA; 6R: ACTCGGTCCAAGCTGCCC; Seq5-5: ATCGCGCTG- using primer combinations chosen in such a way that they CGCAACCTCTCA; Seq5-6: TGCAGAGGGATGGCTGGACGA; covered the entire mARVCF sequence (for primer sequences Ex19as: GGATACTGGCACACAGGTGG; 11R: TCTCCTACCA- µ see Materials and Methods). Three of the primer combinations CACAGCACC). Subsequently, 1 l Taq-polymerase (Gibco) was generated additional bands, which were presumed to arise added to produce 3′-overhanging adenine nucleotides for cloning the fragments into the vector pGEM-T Easy (Promega). The fidelity of from alternative splicing within both mARVCF’s N- and C- the amplified fragments was confirmed by DNA sequencing. terminus. The positions of these three primer sets are indicated in Fig. 1A. PCR fragment sizes generated from the known Plasmid construction for characterisation of different mARVCF mRNA sequence and the sizes of those obtained mARVCF splice variants from alternative splice variants are indicated in Table 1. To clone the C-terminal splice variants of mARVCF as full length Cloning of the novel PCR fragments into pGEM-T Easy and constructs, the pGEM-T Easy vectors containing the four different subsequent DNA sequence analysis revealed one novel N- C-terminal fragments (see previous section) and full length mARVCF terminal splice variant of mARVCF and three new variants in (Kaufmann et al., 2000) were used as templates for PCR. Two ′ ′ the C-terminus (Fig. 1B,C). The alternative 5 -end of fragments were produced with overlaps at the 3 -end of the N- mARVCF (5′alt) lacks exon 3, which contains both the terminal fragment and the 5′-end of the C-terminal fragment. These two PCR products were used in a linear amplification where the base- methionine start codon for the full-length (FL) construct and paired overlaps served as a primers. The product obtained was re- the coiled-coil domain. The first methionine in exon 4 is now amplified with the outer primers containing an EcoRI restriction site the putative translation start. in the sense primer and a SalI restriction site in the reverse primer; The previously known C-terminus of mARVCF mRNA, the resulting PCR products were inserted into the EcoRI and SalI called C11, was recently identified by a yeast two-hybrid assay restriction sites of the eukaryotic expression vector pEGFP-C2 using the cytoplasmic domain of M-cadherin as bait (Clontech, Heidelberg, Germany). The mARVCF variants with (Kaufmann et al., 2000). In the variant mARVCF 3/5, exon 19 alternative 5′-ends were similarly produced by PCR using a sense (present in C11) is spliced out (Fig. 1B,C). This changes the primer containing an EcoRI restriction site and a reverse primer reading frame between exon 18 and exon 20, generating an containing a SalI restriction site and the corresponding full length earlier stop codon and thus a truncated protein. In variant construct as a template. The resulting PCR products were also inserted into the EcoRI and SalI restriction sites of the eukaryotic mARVCF 3/7, exon 19 is replaced by the 120 nucleotide long expression vector pEGFP-C2. All PCRs used the Pfu-polymerase exon B, containing a stop codon just before exon 20. Analysis (Promega). of the cDNA-derived amino acid sequence showed that exon The vector pMOM was used for the analysis of intracellular B maintains the same reading frame as the rest of mARVCF. recruitment of ARVCF by different cadherins (for plasmid Exon B has been found in human ARVCF (hARVCF) (Sirotkin construction see Kaufmann et al.) (Kaufmann et al., 2000). Mouse M- et al., 1997) and, as in hARVCF, introduces a putative PDZ- cadherin (residues 626-784; GenBank accession no. M74541) or binding domain at the very C-terminus of the truncated mouse N-cadherin cytoplasmic domain (residues 747-906; GenBank mARVCF, which is not present in any other splice variant accession no. AB008811) were amplified either from a plasmid (M- identified so far (Fig. 1C). cadherin) or by RT-PCR from mRNA of mouse heart tissue (N- The longest open reading frame of mARVCF is encoded by cadherin) with primers containing a BamHI (sense primer) and EcoRI restriction site (reverse primer) for insertion of the PCR fragments into the third new C-terminal isoform Y, which shows an in-frame pMOM. insertion of 273 base pairs between exon 18 and 19 (Fig. GST-fusion constructs were generated by cloning the cytoplasmic domain of the respective cadherin as a PCR-product into the prokaryotic expression vector pGEX-5X-1 (Pharmacia, Freiburg, Table1. Oligonucleotide primer pairs identifying novel Germany) using BamHI and EcoRI restriction sites. mARVCF isoforms by RT-PCR Expected size (bp) Size (bp) of additionally In vitro GST binding assay Primer pair of PCR fragments amplified PCR fragments mARVCF splice variants FL-C11 and FL-3/7 were cloned into the 5′UTR/6R 1145 914 expression vector pcDNA3.1 (Invitrogen, Netherlands) and Seq 5-5/Ex19as 609 882 synthesised by in vitro transcription-translation in the presence of 35S- Seq 5-6/11R 585 493 and 613 methionine using the TNT™-coupled reticulocyte lysate (Promega, Mannheim, Germany). GST-fused cytoplasmic domain of the The expected sizes of the PCR-products from the known cDNA sequence respective cadherin was expressed in and purified from E. coli strain (GenBank accession no. AJ243418; Z.W., A.S. and A.S.-P., unpublished) are BL21pLys.S. The in vitro GST binding assay was performed as given in the left column, the right column indicates the additional RT-PCR described (Kaufmann et al., 2000). fragment sizes generated by the primer pairs. bp, base pairs. 3876 JOURNAL OF CELL SCIENCE 114 (21) 1B,C). Analysis of the extended peptide sequence has not revealed any homology to known protein A 5`UTR coiled-coil 3`UTR domain motifs. armadillo repeats Finally, primer pairs were chosen to discover possible splice variants within the armadillo 5`UTR 6R Seq5-5 Ex19as repeat region. Alternative splicing has been reported in this region for human p120(ctn), as Seq5-6 11R well as human and xenopus ARVCF (Sirotkin et al., 1997; Keirsebilck et al., 1998; Paulson et al., 2000) resulting in an 18 base pair insertion in B N-terminal splice variants: armadillo repeat six that converts the putative mARVCF full length (FL) nuclear localisation motif present at this position E3 E4 into a shorter NLS. This alternative exon, or any E4 other alternative exons within the armadillo repeat mARVCF 5`alt region, could not be detected within murine ARVCF using RT-PCR (data not shown). C-terminal splice variants: Altogether, eight different mARVCF splice variants are possible by combining one out of four E18 E19 E20 mARVCF C11 C-termini with either full length or the 5′ alternative N-terminus. Fig. 1B schematically E18 E20 mARVCF 3/5 summarises the N- and C-terminal variants and

Fig. 1C indicates the amino acid sequences E18 B E20 derived from the different putative mARVCF mARVCF 3/7 isoforms. E18 Y E19 E20 mARVCF Y Murine ARVCF mRNA expression The next question was whether one splice variant is preferentially expressed in different cell lines or tissue types. In order to determine the relative C ′ ′ quantity of the 5 - and the 3 - variants we Full length MEDCNVHSAA SILASVKEQE ARFERLTRAL EQERRHVALQ LERAQQPGMS performed RT-PCR with two pairs of 5`alt ------oligonucleotide primers that either amplify both N- Full length SGGMVGSGQP LPMAWQQLVL QGQSPGSQAS LATMPEAPEV LEETVTVEED// terminal variants or, alternatively, all four C- 5`alt ------MPEAPEV LEETVTVEED// terminal variants simultaneously. Templates were cDNAs prepared from mRNA of differentiating i28 cells, CMT cells (derived from mouse colon Y //GDTSEKELLR VSNSKQVSRE ACQENSQIVL SLIRIGGHGW SGTHLQGPGS carcinoma) and total mouse heart. As shown in Fig. C11 //GDTSEKELLR VYGQGVYCGP LEKAASTTCV PVSWLHVPAS GALAQLFVLR 3/7 //GDTSEKELLR PDPGRKAPPP GPSRPSVRLV DAVGDTKPQP V DSWV * 2A, the N-terminal full-length mARVCF mRNA is 3/5 //GDTSEKELLR GPGPAVCS* much more abundant than that encoding the 5′- Y SLYIHHQSDF STEGVWCTHL PQRSRQGQLC LATGCVGPES PSKRICTFLL alternative end missing the coiled-coil domain. C11 ------However, at the 3′-end it is evident that the relative Y HDRIEVAGRG PEGPPS* amounts of the four C-terminal isoforms are very C11 -DRIEVAGRG PEGPPS* similar in heart and CMT cells. Isoforms 3/5 and Y are expressed at low levels, whereas variants C11 and 3/7 are predominant. In i28 cells the mRNA of Fig. 1. (A) Schematic overview of murine ARVCF with the N-terminal coiled-coil mARVCF splice variant Y is even less abundant, domain and the central armadillo repeat region (GenBank accession no. AJ243418). The positions of the three gene-specific primer sets generating novel but can easily be amplified using exon Y specific isoforms when used in RT-PCR are marked with arrows. (UTR, untranslated primers (data not shown). Variant 3/5 mRNA is region). (B) Schematic drawing of the two N-terminal and four C-terminal splice more abundant in this cell line than in CMT cells variants of murine ARVCF obtained by RT-PCR. Exons (E) were postulated by or heart tissue. Using this primer pair it was comparing the known mouse and human ARVCF cDNA sequences with the human difficult to discriminate between isoform C11 and genomic ARVCF clone (AC005663). Numbers of the exons correspond to the 3/7 because they differ in length by only 28 human sequence. (C) Amino acid sequence comparison of the N- and C-terminal nucleotides. Therefore, one sense primer in exon 15 isoforms of mARVCF. The asterisks mark the stop codons; the putative PDZ- was used together with two antisense primers: one binding domain in isoform 3/7 is underlined. specific for the 3′-end of exon B and an other specific for the 5′-end of exon 19 generating bands that differ in In order to show that all cell lines (i28, MCF7, RT112, EJ28, length by 47 nucleotides. The exon 19-specific primer is also HeLa and COS-7) that were used for further experiments able to amplify isoform Y but this was not relevant owing to the express endogenous ARVCF, we performed RT-PCR analyses. very rare appearance of this variant (Fig. 2B). Fig. 2C shows that One pair of oligonucleotide primers amplified ARVCF in the variant 3/7 is more abundant than variant C11 in i28 cells, heart region encoding the armadillo repeats that is not affected by tissue and CMT cells. alternative splicing. As an internal standard the housekeeping Differential binding of mARVCF to cadherins 3877

Fig. 3. (A) Western blot analysis of the eight potential isoforms cloned as EGFP-fusion proteins. COS-7 cells were transfected with pEGFP-splice variant expression plasmids indicated above. 48 hours after transfection extracts of COS-7 cells were prepared and separated by SDS-PAGE; each lane contains 20 µg of total protein. (B) Western blot analysis of splice variant FL-3/7 as EGFP-fusion protein. i28, MCF7, RT112, EJ28, HeLa and COS-7 cells (indicated above) were transfected with pEGFP-splice variant FL-3/7 expression plasmid. 48 hours after transfection cell extracts were prepared and separated by SDS-PAGE; each lane contains 20 µg of total protein. The western blots were probed with GFP-antibodies followed by incubation with alkaline phosphatase conjugated Fig. 2. RT-PCR analysis of alternative splicing events in (A) the N- secondary antibody, and visualised by NBT/BCIP. (FL, full length). and (B,C) C-terminal region of mARVCF mRNA. The mRNA used for RT-PCR was isolated from differentiating mouse myoblasts (i28), total mouse heart (heart) and CMT-cells derived from mouse colon results indicated that all eight constructs can be expressed and carcinoma (CMT). (A) Agarose gel electrophoresis of RT-PCR that the corresponding proteins appeared at the expected products amplified with a sense primer in the 5′UTR and an position in the blot. Furthermore, we expressed the most antisense primer within exon 6 amplifying both N-terminal isoforms abundant splice variant full length 3/7 (FL-3/7) as a EGFP- at once. (B) Agarose gel electrophoresis of RT-PCR products fusion protein in each cell line used for further investigations. amplified with a sense primer in exon 15 and an antisense primer in Western blot analysis of the protein extracts from the exon 20 amplifying all four C-terminal isoforms at once. transfected cells lines indicates that EGFP-FL-3/7 protein is (C) Agarose gel electrophoresis of RT-PCR products amplified with expressed at the correct size and in comparable amounts in all one sense primer in exon 15 and two antisense primer in exon B and six cell lines (Fig. 3B). exon 19. (D) Agarose gel electrophoresis of RT-PCR products amplified with (1) oligonucleotide primers specific for the armadillo The cellular context influences the localisation of repeat region of ARVCF which is not affected by alternative splicing (upper band) and (2) oligonucleotide primers specific for the mARVCF housekeeping gene BIP (binding protein) as a standard (lower band). As shown by Kaufmann et al., EGFP-ARVCF-C11 is able The mRNA used for RT-PCR was isolated from the cell lines to interact with M-cadherin and E-cadherin in the MOM indicated above. (FL, full length). recruitment assay and colocalises with N-cadherin in rat ventricular cadiomyocytes, or E-cadherin in epithelial cells gene BIP (binding protein) was amplified in the same RT-PCR. (Kaufmann et al., 2000). Furthermore, hARVCF can associate As shown in Fig. 2D, ARVCF is expressed in each cell line with the E-cadherin-catenin complex as shown by examined and the endogenous level of ARVCF expression is immonoprecipitation (Mariner et al., 2000). With regards to comparable in the studied cell lines. mARVCF, the armadillo repeat region is required for the interaction with cadherins (Kaufmann et al., 2000). To Cloning and expression of alternative splice variants investigate whether the C- or N-termini of the novel mARVCF The eight possible splice variants of mARVCF were cloned as isoforms influence the cadherin-binding and/or cellular EGFP-fusion proteins and expressed in COS-7 cells. Protein localisation of mARVCF, six different cell lines were extracts from the transfected cells were analysed by western transfected with each of the isoforms as an EGFP-fusion blots using the monoclonal anti-GFP antibody (Fig. 3A). The protein. Twenty-four to 48 hours after transfection the cells 3878 JOURNAL OF CELL SCIENCE 114 (21) were fixed and analysed for EGFP-mARVCF expression. In whereas the sites of cell-cell contacts were clearly stained. addition to the mouse muscle myoblast cell line i28, these cells Here the EGFP-fusion proteins colocalise with M-cadherin, as were human epithelial cancer cell lines MCF7 (mammary exemplified by the merged image (Fig. 4Am) with variant FL- carcinoma), RT112 (non-invasive bladder carcinoma), EJ28 3/7. (invasive bladder carcinoma), HeLa cells (cervix carcinoma) To investigate the cellular localisation of mARVCF splice and monkey COS-7 kidney cells, all of which express variants in cells expressing endogenous E-cadherin, MCF7 endogenous ARVCF as shown by RT-PCR (Fig. 2D). cells (mammary carcinoma) and RT112 cells (bladder i28 cells, the original source of the novel mARVCF splice carcinoma), both of which are human non-invasive cell lines, variants, express M-cadherin, which localises to the cell were transfected with the cDNAs encoding the splice variants membrane predominantly at cell-cell contacts (Fig. 4Ai). As as EGFP-fusion proteins and fixed 24-48 hours after described recently, mARVCF binds to the cytoplasmic domain transfection. In both MCF7 and RT112 cells each of the of M-cadherin, which correlates with the fact that the ectopic isoforms clearly localised at the cell membrane together with fragment EGFP-ARVCF-C11 is localised at the membrane in E-cadherin and was never detected in the nucleus (Fig. 4B,C; i28 cells, although it is, to a certain extent, also found in the and Fig. 4Bm,Cm). This indicated that not only mARVCF cytoplasm (Kaufmann et al., 2000). The membrane localisation fragment C11 (Kaufmann et al., 2000) but also all of the splice is reproduced when the different mARVCF isoforms are products can associate with E-cadherin. expressed in i28 cells. The nuclei of the muscle cells were In contrast to MCF7 and RT112 cells, the human invasive found to be free of the ectopically expressed mARVF isoforms bladder carcinoma cell line EJ28 does not express E-cadherin

Fig. 4. Cellular localisation of mARVCF splice variants. Mouse myoblasts i28 (A), MCF7 cells (B), RT112 (C), EJ28 (D), HeLa cells (E) and COS-7 cells (F) were transfected with the eight potential isoforms as EGFP-fusion constructs and expression visualised by fluorescence microscopy. The localisation of the eight potential isoforms was seen at the cell membrane in all cell lines tested with the exception of EJ28 cells. Here, as a representative result for the different isoforms, the localisation of EGFP-FL-3/7 is shown. As controls, each cell line was transfected with the empty EGFP-plasmid (A-co to F-co). In i28 cells, the endogenous M-cadherin was detected with M-cadherin antibodies (Ai), in MCF7 (Bi) and RT112 (Ci) cells endogenous E-cadherin was detected with the E-cadherin antibody and N-cadherin was detected with the anti A-CAM antibody in EJ28 (Di) and HeLa cells (Ei). In COS-7 cells the anti-pan-cadherin antibody was used to detect endogenous cadherin(s) (Fi). (Am-Fm) merged images of (A-F) and (Ai-Fi). Bars, 10 µm. Differential binding of mARVCF to cadherins 3879 but is positive for N-cadherin (Fig. 4Di) (A. Zeitvogel, the mitochondrial-anchored cytoplasmic domains of E- or M- unpublished). When transfected into EJ28 cells none of the cadherin. mARVCF isoforms appeared to colocalise with N-cadherin Mouse EGFP-ARVCF-C11 colocalises with N-cadherin in (Fig. 4D,Dm), although the adhesion molecule itself was found cardiomyocytes suggesting an association of these molecules correctly at the plasma membrane (Fig. 4Di). All mARVCF (Kaufmann et al., 2000). In the experiments described here, the isoforms were distributed equally in the EJ28 cells and no localisation of mARVCF isoforms was found to be distinct clear membrane-staining could be detected. However, after when EJ28 and HeLa cells were compared, although both cell transfection of HeLa cells, which are also E-cadherin-negative types express N-cadherin but not E-cadherin (Fig. 4Di,Ei) (A. but N-cadherin-positive (Fig. 4Ei), all of the EGFP-mARVCF Zeitvogel, unpublished). Thus, it was of interest to similarly isoforms clearly colocalised with N-cadherin at the sites of test the interaction of N-cadherin’s CPD with mARVCF cell-cell contact (Fig. 4E,Em). isoforms in various cellular environments, including EJ28 Finally, in monkey kidney COS-7 cells, which express cells. Co-transfection of the pMOM-N-cadherin and each endogenous cadherin(s) detectable with a pan-cadherin EGFP-mARVCF isoform revealed that the interaction between antibody (Fig. 4Fi), the mARVCF isoforms localised at cell- each of the mARVCF splice variants and N-cadherin is cell contacts (Fig. 4F). Thus, our results support the idea possible, as exemplified by FL-3/7 in Fig. 7A-C. Also in EJ28 that the subcellular localisation of mARVCF splice variants cells, such an interaction takes place between the transfected depends not only on the presence or absence of an appropriate cytoplasmic N-cadherin domain and mARVCF. This finding interaction partner, in this case the cadherins, but also on suggests that there are no or few factors preventing this protein- additional factors. protein interaction. But in contrast to the assays done with M- or E-cadherin, this interaction did not take place in every single Unequal interaction of mARVCF with E-, M- and N- cell, although both constructs were present and expressed cadherin (Fig. 7). Neighbouring cells, all of which expressed the MOM- The results above merited closer investigation into the binding N-cadherin construct and mARVCF, exhibited distinct potential of E-, M- and N-cadherin for the eight mARVCF interactions. For example, one cell showed interaction while splice variants. The general ability of M-, E- and N-cadherin the neighbour did not (Fig. 7). This inhomogeneous pattern of to interact with mARVCF was demonstrated by in vitro GST association between the cytoplasmic domain of N-cadherin and binding assays. mARVCF splice variants FL-C11 and FL-3/7 EGFP-mARVCF could be detected in every cell line examined were cloned into the expression vector pcDNA3.1 and used as with all the mARVCF isoforms. By counting 1.5×103 a template for in vitro transcription and translation in the cotransfected MCF7 cells we could show that 36.7% of these presence of 35S-methionine. The cytoplasmic domains of M-, cells were positive for FL-3/7-MOM-N-cadherin interaction, E- and N-cadherin were expressed as GST-fusion proteins in whereas 63.3% of these cells showed no interaction of EGFP- bacteria. The results revealed that in vitro translated mARVCF FL-3/7 with MOM-N-cadherin, although both constructs were splice variants FL-C11 and FL-3/7 can bind directly to the present (Fig. 7D). The results of the subcellular localisation cytoplasmic domain of all three cadherins (Fig. 5). and the MOM recruitment assays are summarised in Table 2. Furthermore, we used the MOM recruitment assay in the ARVCF belongs to the p120(ctn) subfamily of armadillo different cell lines. This assay provides a means of testing the repeat proteins and shows a high homology to the name-giving interaction between two proteins directly in mammalian cells molecule. Both relatives can bind to the juxtamembrane region (Kaufmann et al., 2000) (see also Materials and Methods). The of cadherins (Kaufmann et al., 2000; Mariner et al., 2000) and cytoplasmic domains (CPD) of the cadherins were cloned into the MOM vector to produce a birch profilin (BP)- tagged fusion protein that is anchored to the mitochondrial outer membrane via the TOM70 protein anchor, and that shows a mitochondrial staining pattern in immuno- fluorescence assays (Kaufmann et al., 2000). Following co- transfection of the MOM-construct with each of the EGFP- mARVCF splice variants, these proteins also exhibit a mitochondrial fluorescence pattern if there is protein- protein interaction. As a control, the empty EGFP-vector is co-transfected with each of the MOM-constructs. No interaction between these two components means that the EGFP shows no mitochondrial localisation pattern (Fig. 6co, coi). As exemplified in Fig. 6 for FL-C11 and MOM-M- cadherin, all mARVCF isoforms were able to interact comparably in the MOM recruitment assay with E- or M- 35 cadherin in i28 myoblasts (Fig. 6A) and in the different Fig. 5. GST binding assay. In vitro translated S-methionine-labeled mARVCF splice variants FL-C11 (lane 1) and FL-3/7 (lane 2) were carcinoma cell lines (Fig. 6C-F). In MCF7 cells, the incubated with the cytoplasmic domains of M- (lanes 4,8), E- (lanes 5,9) membrane localisation of mARVCF did not disappear or N-cadherin (lanes 6,10) as GST-fusion proteins. As controls, in vitro completely following co-transfection of either MOM-M- translated 35S-methionine-labeled mARVCF splice variants FL-C11 cadherin or MOM-E-cadherin (Fig. 6B). This suggests a (lane 3) and FL-3/7 (lane 7) were incubated with GST alone. (GST, competition between endogenous (membrane-located) and glutathione-S-transferase). 3880 JOURNAL OF CELL SCIENCE 114 (21)

Fig. 6. MOM recruitment assay. Mouse myoblast i28 cells (A, Ai), MCF7 (B, Bi), RT112 (C, Ci), EJ28 (D, Di), HeLa (E, Ei) and COS-7 cells (F, Fi) were co-transfected with MOM-M-cadherin or MOM-E-cadherin plasmids and each mARVCF isoform as an EGFP-fusion expression plasmid. The MOM-construct, detected with a BP-antibody recognising the C-terminal birch profilin (BP) tag, is directed to the mitochondrial outer membrane due to the MOM-anchor and gives rise to a mitochondrial staining pattern of the fusion protein (A-F). The co-transfected mARVCF constructs showed the same mitochondrial staining pattern (Ai-Fi) indicating an interaction with the cytoplasmic domain of the respective cadherin. In MCF7 cells the membrane staining observed when mARVCF is transfected alone does not disappear as in the other cell lines (compare Fig. 4). The example shown here represents the MOM recruitment assay with MOM-M-cadherin and EGFP-FL-C11. Control, the empty EGFP-vector was co-transfected with MOM-M-cadherin (co and coi). Bars, 10 µm. are mutually exclusive for one another in E-cadherin anti-Xpress antibodies to detect Xpress-tagged FL-3/7. complexes (Mariner et al., 2000). To investigate whether the Pictures were taken where both, FL-3/7 (Fig. 8Aa) and inability of mARVCF to associate with MOM-N-cadherin in p120(ctn) (Fig. 8Ab) were located at the plasma membrane, 63.3% of cells (Fig. 7D) is due to a competition between both hence did not interact with MOM-N-cadherin. To confirm that armadillo repeat proteins for MOM-N-cadherin binding, we these cells were indeed positive for MOM-N-cadherin, the used p120(ctn) isoform 1A, which is the most homologous to same coverslips were washed again several times with PBS and mARVCF FL-3/7, in MOM recruitment assays. EGFP- then stained with the BP antibody to detect MOM-N-cadherin. p120(ctn) was able to interact with MOM-M- and-E-cadherin The positive MOM-N-cadherin staining is given in Fig. 8Ad, in all cell lines tested (data not shown). Interestingly, cells indicating that the binding of both, mARVCF and p120(ctn) to transfected with EGFP-p120(ctn) and MOM-N-cadherin endogenous cadherins can occur in the presence of showed the same inhomogenous pattern of association between overexpressed MOM-N-cadherin. Taking into account the both partners (Fig. 8) as it was observed in cells cotransfected inhomogenous interaction pattern of the armadillo repeat with mARVCF and MOM-N-cadherin (Fig. 7). To study the proteins with MOM-N-cadherin, we also analysed cells where idea of competition for MOM-N-cadherin binding, we co- both FL-3/7 (Fig. 8Ba) and p120(ctn) (Fig. 8Bb) were able to transfected MCF7 cells with pMOM-N-cadherin, FL-3/7 fused bind to mitochondrial located MOM-N-cadherin. This showed to an Xpress-tag and EGFP-p120(ctn). Cells were stained with that these two members of the p120(ctn) subfamily can bind to Differential binding of mARVCF to cadherins 3881

Fig. 7. MOM recruitment assay with MOM-N-cadherin. Cells were co- transfected with MOM-N-cadherin and each mARVCF isoform as an EGFP- fusion plasmid. As an example, EGFP- FL-3/7 is shown in MCF7 cells, demonstrating the inhomogeneous interaction pattern between all of the mARVCF isoforms and the cytoplasmic domain of N-cadherin obtained in every cell line. (A) Indirect immunofluorescence with monoclonal birch profilin (BP) antibodies visualising the MOM-N-cadherin construct. (B) Localisation of EGFP-FL-3/7. (C) Merged image of (A) and (B). (D) 1.5×103 MCF7 cells expressing MOM-N-cadherin and EGFP-FL-3/7 were counted. In 36.7% of these cells a mitochondrial located interaction of both constructs was detected. In 63.3% of cells expressing MOM-N-cadherin and EGFP-FL-3/7 such an interaction did not take place. Error bars indicate standard deviation. Bar, 10 µm. the overexpressed MOM-N-cadherin constructs in the same DISCUSSION cell. These data clearly show that the inhomogenous pattern of In this paper we report the isolation and characterisation of association of mARVCF and the cytoplasmic domain of N- novel mARVCF splice variants. These isoforms were tested for cadherin, observed in the MOM recruitment assay, is not due their binding capacity to different cadherins in various cell to a competition between p120(ctn) and ARVCF. Both contexts. In fact, all eight derivatives showed identical armadillo proteins can bind to endogenous cadherins at the behaviour to each other in all assays performed (discussed plasma membrane even though MOM-N-cadherin is present. later). However, the distinct binding pattern of the mARVCF Alternatively, both proteins can bind to MOM-N-cadherin isoforms to cadherins in the MOM recruitment assay suggests constructs in the same cell (Fig. 8). that the binding capacity of mARVCF to E-, M- and N- In addition, the percentage of cells interacting with the cadherin varies in vivo, although all three cadherins appear to cytoplasmic domain of N-cadherin in the MOM-recruitment bind equally well in vitro to mARVCF splice variants FL-C11 assay was not altered significally by co-transfecting EGFP- and FL-3/7. Although mARVCF isoforms reacted with MOM- p120(ctn). In the absence of p120(ctn), 36.7% of co-transfected M-cadherin and MOM-E-cadherin in all cell lines tested and cells showed an interaction of mARVCF and MOM-N-cadherin in each individual co-transfected cell, their interaction with (Fig. 7D). In cells co-transfected with MOM-N-cadherin, FL- MOM-N-cadherin was rather inhomogeneous in a given cell 3/7 and p120(ctn), 32.9% showed an interaction of mARVCF population. The observation that transfected MOM-E-cadherin with MOM-N-cadherin, and 35.2% of them exhibited an (AND/OR M-cadherin) protein did not completely abolish interaction of p120(ctn) with the CPD of N-cadherin (Fig. 8Af). binding of EGFP-mARVCF isoforms to endogenous (junction- Thus an interaction between mARVCF and MOM-N-cadherin localised) E-cadherin in MCF7 cells implies a competition for is not affected significantly by the co-transfection of p120(ctn). mARVCF binding between the endogenous E-cadherin and the

Table 2. Summary of the expression data of mARVCF isoforms and MOM recruitment assay MOM-cadherin interaction Cellular localization M E N i28 MCF7 RT112 EJ28 HeLa COS-7 FL C11 + + +/− mm m cmm FL 3/5 + + +/− mm m cmm FL 3/7 + + +/− mm m cmm FL Y + + +/− mm m cmm 5′ C11 + + +/− mm m cmm 5′ 3/5 + + +/− mm m cmm 5′ 3/7 + + +/− mm m cmm 5′ Y+++/− mm m cmm

Cell lines tested were i28, MCF7, RT112, EJ28, HeLa and COS-7. (+, interaction; +/−, interaction only in some cells; c, cytoplasm; m, membrane; M, M- cadherin; E, E-cadherin; N, N-cadherin; FL, full length; 5′, 5′ alternative end.) 3882 JOURNAL OF CELL SCIENCE 114 (21)

Fig. 8. MOM recruitment assay with FL-3/7, p120(ctn) and MOM-N-cadherin. MCF7 cells were co-transfected with pMOM- N-cadherin, mARVCF splice variant FL-3/7 fused to an Xpress- tag and p120(ctn) as a EGFP- fusion protein. Cells were stained with a Xpress antibody to detect FL-3/7. As shown in B, FL-3/7- Xpress (a) and EGFP-p120(ctn) (b) can bind to MOM-N-cadherin (not stained) in the same cell. In c, the merged image of a and b is shown. (A) Cells were treated as in B and pictures were taken where both FL-3/7-Xpress (a) and EGFP-p120(ctn) (b) were detected at the plasma membrane. (c) The merged image of (a) and (b). To confirm that these cells were also positive for MOM-N- cadherin, the same coverslips were washed again and stained for MOM-N-cadherin with the birch profilin antibody (BP); the positive BP staining is given in d. (e) The merged image of a, b and d where the MOM-N-cadherin staining is coloured artificially blue. In f, 500 cells expressing FL-3/7-Xpress, EGFP-p120(ctn) and MOM-N-cadherin were counted. In 32.9% (mARVCF) or 35.2% (p120(ctn)) of these cells a mitochondrial located interaction of both constructs was detected. In 67.1% (mARVCF) or 64.8% (p120(ctn)) of cells expressing MOM-N-cadherin, EGFP-FL-3/7 and EGFP-p120(ctn) such an interaction did not take place. Error bars indicate s.d. Bar, 10 µm.

E- or M-cadherin CPD-MOM fusion protein. However, this is constructs in the same cell. Taking into account the results of most likely influenced by additional cellular factors since this Mariner et al. (Mariner et al., 2000), it is obvious that these type of competition does not occur in RT112 cells that also two members of the p120(ctn) subfamily do not bind express E-cadherin. simultaneously to the same MOM-N-cadherin molecule. The In contrast to M- and -E-cadherin, N-cadherin exhibits an inhomogeneous pattern of interaction might be the result of inhomogenous pattern of association with the mARVCF heterogeneity in the cellular context (e.g. expression profile of isoforms, although it is generally able to interact with them. regulatory proteins) leading to differential modifications of the Not every cell expressing the MOM-N-cadherin fusion protein interaction partners in individual cells. and a mARVCF isoform permits the protein interaction. It is also interesting to note that the mARVCF isoforms ARVCF belongs to the p120(ctn) subfamily of armadillo cannot colocalise with N-cadherin in EJ28 carcinoma cells but proteins and shows a high homology to the namegiving do so in HeLa cells. Both cell types express N-cadherin and molecule. Both relatives can bind to the juxtamembrane region not E-cadherin (A. Zeitvogel, unpublished; and this paper). of cadherins (Kaufmann et al., 2000; Mariner et al., 2000) Furthermore, it could be shown by Kaufmann et al. (Kaufmann and are mutually exclusive for one another in E-cadherin et al., 2000) that EGFP-ARVCF-C11 colocalises with N- complexes (Mariner et al., 2000). Our results clearly cadherin in rat ventricular cardiomyocytes. This indicates that demonstrate that the inhomogeneous pattern of interaction of a given cellular background may determine the capacity of mARVCF and the CPD of N-cadherin observed in the MOM mARVCF to interact with its partner, in this case N-cadherin. recruitment assay, is not due to a competition between In line with this, a previous report described the regulation of mARVCF and p120(ctn) for N-cadherin binding. The two p120(ctn) binding to the cytoplasmic domain of cadherins by members of the p120(ctn) subfamily can interact with phosphorylation, thereby modulating cadherin-mediated endogenous, membrane-located cadherins in the presence adhesion in either a positive or negative manner. This process of overexpressed MOM-N-cadherin. Alternatively, both appears to depend on the cell context (Yap et al., 1998; Aono mARVCF and p120(ctn) can be recruited to MOM-N-cadherin et al., 1999; Anastasiadis and Reynolds, 2000). Differential binding of mARVCF to cadherins 3883

The evidence for the inhomogenous association of mARVCF We thank Jean-Claude Perriard for critical reading of the isoforms with N-cadherin might also be relevant in the context manuscript, Beata Krebs for technical assistance, Heike Handrow- of results that imply that expression of N-cadherin is associated Metzmacher for help with the genomic ARVCF analysis and Frank with metastasis and cell migration (Nieman et al., 1999; Hazan Nonnenmacher for providing EGFP-p120(ctn). This work was et al., 2000). In particular, over-expression of N-cadherin in supported by the Deutsche Forschungsgemeinschaft through SFB 474 MCF7 cells leads to abnormal invasive behaviour of these cells (B2) to A.S.-P. in cell culture (Hazan et al., 2000). In turn, expression of E- cadherin in invasive cells abolishes invasion (Frixen et al., REFERENCES 1991). Furthermore, N-cadherin has been reported to be a path- finding molecule for migrating dermomyotomal cells during Allport, J. R., Muller, W. A. and Luscinskas, F. W. (2000). Monocytes chicken embryogenesis (Brand-Saberi et al., 1996). induce reversible focal changes in vascular endothelial cadherin Considering the concept that cell invasion and migration is complex during transendothelial migration under flow. J. Cell Biol. 148, incompatible with strong adhesion (as provided by E-cadherin) 203-216. Anastasiadis, P. Z. and Reynolds, A. B. (2000). The p120 catenin family: but still requires guidance mediated by low affinity cadherins complex roles in adhesion, signaling and cancer. J. Cell Sci. 113, 1319- (such as N-cadherin) that allow movement, mARVCF might 1334. play a modulatory role in these processes. Aono, S., Nakagawa, S., Reynolds, A. B. and Takeichi, M. (1999). p120(ctn) Apparently, the binding of the identified mARVCF isoforms acts as an inhibitory regulator of cadherin function in colon carcinoma cells. to cadherins is generally unaffected by the altered N- and C- J. Cell Biol. 145, 551-562. Aberle, H., Butz, S., Stappert, J., Weissig, H., Kemler, R. and Hoschuetzky, termini of the mARVCF proteins arising by differential splicing. H. (1994). Assembly of the cadherin-catenin complex in vitro with This is in line with results described by Kaufmann et al. recombinant proteins. J. Cell Sci. 107, 3655-3663. showing that the armadillo repeat region of mARVCF is Behrens, J., von Kries, J. P., Kühl, M., Bruhn, L., Wedlich, D., Grosschedl, required and sufficient for cadherin binding (Kaufmann et al., R. and Birchmeier, W. (1996). Functional interaction of β-catenin with the transcription factor LEF-1. Nature 382, 638-642. 2000). Alternative splicing events have been reported for most Bonne, S., van Hengel, J. and van Roy, F. (1998). Chromosomal mapping of the p120(ctn) subfamily members (Paffenholz and Franke, of human armadillo genes belonging to the p120(ctn)/plakoglobin 1997; Keirsebilck et al., 1998; Hatzfeld, 1999) suggesting that subfamily. Genomics 51, 452-454. the isoforms may be important modulators of the function of Brand-Saberi, B., Gamel, A. J., Krenn, V., Mueller, T. S., Wilting, J. and these proteins. Modulation might, for example, occur due to the Christ, B. (1996). N-cadherin is involved in myoblast migration and muscle ′ differentiation in the avian limb bud. Dev. Biol. 178, 160-173. presence (as in full length mARVCF) or absence (such as in 5 - Butz, S. and Kemler, R. (1994). Distinct cadherin-catenin complexes in alt mARVCF) of the N-terminal coiled-coil domain, which may Ca(2+)-dependent cell-cell adhesion. FEBS Lett. 355, 195-200. allow the recruitment of different partners into the junctional Chothia, C. and Jones, E. Y. (1997). The molecular structure of cell adhesion cadherin-catenin complexes. Similarly, the expression of a molecules. Annu. Rev. Biochem. 66, 823-862. Deguchi, M., Iizuka, T., Hata, Y., Nishimura, W., Hirao, K., Yao, I., putative PDZ-binding domain in mARVCF is regulated through Kawabe, H. and Takai, Y. (2000). PAPIN. A novel multiple PSD-95/ alternative splicing and only occurs in variant 3/7. Dig-A/ZO-1 protein inetracting with neural plakophilin-related The PDZ domain has been generally shown to mediate armadillo repeat protein/delta-catenin and p0071. J. Biol. Chem. 275, protein-protein interactions (Fanning and Anderson, 1996; 29875-29880. Fanning and Anderson, 1998). For example, it has been Desmaze, C., Prieur, M., Amblard, F., Aikem, M., LeDeist, F., Demczuk, S., Zucman, J., Plougastel, B., Delattre, O., Croquette, M. F. et al. (1993). demonstrated that one PDZ domain of the junction protein Phisical mapping by FISH of the DiGeorge critical region (DGCR): PAPIN is responsible for the interaction of this molecule with involvement of the region in familial cases. Am. J. Hum. Genet. 53, 1239- the armadillo repeat proteins δ-catenin or p0071 (Deguchi et 1249. al., 2000). Furthermore, β-catenin can associate with LIN-7 via Fanning, A. S. and Anderson, J. M. (1996). Protein-protein interactions: PDZ domain networks. Curr. Biol. 6, 1385-1388. its PDZ domain at cell junctions (Perego et al., 2000). Fanning, A. S. and Anderson, J. M. (1998). PDZ domains and the formation Postulating a similar function of the potential PDZ-binding of protein networks at the plasma membrane. Curr. Top. Microbiol. domain in mARVCF, it might be that this domain can modulate Immunol. 228, 209-233. the interaction of the cadherin-catenin complex by recruiting Finnemann, S., Mitrik, I., Hess, M., Otto, G. and Wedlich, D. (1997). additional proteins to the complex. Uncoupling of XB/U-cadherin-catenin complex formation from its function in cell cell adhesion. J. Biol. Chem. 272, 11856-11862. With human p120(ctn) alternative splicing leads to at least Frixen, U. H., Behrens, J., Sachs, M., Eberle, G., Voss, B., Warda, A., 32 potential isoforms. In the N-terminal region four different Lochner, D. and Birchmeier, W. (1991). E-cadherin-mediated cell-cell variants have been described that use four different start adhesion prevents invasiveness of human carcinoma cells. J. Cell. Biol. 113, methionines (isoforms one to four, of which variants two to 173-185. Gaetje, R., Kotzian, S., Herrmann, G., Baumann, R. and Starzinski- four lack the coiled-coil region). These can be combined with Powitz, A. (1997). Nonmalignant epithelial cells, potentially invasive in three alternative exons A, B, and C, in the armadillo repeat human endometriosis, lack the tumor suppressor molecule E-cadherin. Am. region or the C-terminus (Keirsebilck et al., 1998). So far, J. Pathol. 150, 461-467. however, only the differential expression of exon B could be Geiger, B. and Ayalon, O. (1992). Cadherins. Annu. Rev. Cell Biol. 8, 307- assigned to a defined mechanism, namely the insertion of a 332. Hatzfeld, M. (1999). The armadillo family of structural proteins. Int. Rev. functional NES (nuclear export signal) directing the molecule Cytol. 186, 179-224. out of the nucleus (van Hengel et al., 1999). Thus, the major Hazan, R. B., Phillips, G. R., Qiao, R. F., Norton, L. and Aaroson, S. A. roles for the isoforms of both ARVCF and p120(ctn) remain to (2000). Exogenous expression of N-cadherin in breast cancer cells induces be elucidated. cell migration, invasion, and metastasis. J. Cell Biol. 148, 779-790. Hertig, C. M., Butz, S., Koch, S., Eppenberger-Ebehardt, M., Kemler, R. In summary, our data imply that the function of mARVCF and Eppenberger, H. M. (1996). N-cadherin in adult rat cardiomyocytes may be regulated by multiple mechanisms including alternative in culture. J. Cell Sci. 109, 11-20. splicing, cellular context and the cadherins themselves. Hinck, L., Näthke, I. S., Papkoff, J. and Nelson, W. J. (1994). Dynamics of 3884 JOURNAL OF CELL SCIENCE 114 (21)

cadherin-catenin complex formation: novel protein interactions and independent proteins structurally related in different species. EMBO J. 8, pathways of complex assembly. J. Cell Biol. 125, 1327-1340. 1711-1717. Hirano, S., Nose, A., Hatta, K., Kawakami, A. and Takeichi, M. (1987). Paffenholz, R. and Franke, W. (1997). Identification and loclization of a Calcium-dependent cell-cell adhesion molecules (cadherins): subclass neurally expressed member of the plakoglobin/armadillo multigene family. specifities and possible involvement of bundles. J. Cell Biol. 105, 2501- Differentiation 61, 293-304. 2510. Paulson, A. J., Mooney, E., Fang, X., Ji, H. and McCrea, P. D. (2000). Hirano, S., Kimoto, N., Shimoyama, Y., Hirohashi, S. and Takeichi, M. XARVCF, xenopus member of the p120 catenin subfamily associates with (1992). Identification of a neural alpha-catenin as a key regulator of cadherin cadherin juxtamembrane region. J. Biol. Chem. 275, 30124-31031. function and multicellular organization. Cell. 70, 293-301. Perego, C., Vanoni, C., Massari, S., Longhi, R. and Pietrini, G. (2000). Huber, O., Bierkamp, C. and Kemler, R. (1996). Cadherins and catenins in Mammalian LIN-7 PDZ proteins associate with beta-catenin at the cell-cell development. Curr. Opin. Cell Biol. 8, 685-691. junctions of epithelia and neurons. EMBO 19, 3978-3989. Humphries, M. J. and Newham, P. (1998). The structure of cell adhesion Reynolds, A. B., Daniel, J., McCrea, P. D., Wheelock, M. J., Wu, J. and molecules. Trends Cell Biol. 8, 78-83. Zhang, Z. (1994). Identification of a new catenin: the tyrosine kinase Kaufmann, U., Martin, B., Link, D., Witt, K., Zeitler, R., Reinhard, S. and substrate p120cas associates with E-cadherin complexes. Mol. Cell. Biol. Starzinski-Powitz, A. (1999a). M-cadherin and its sisters in development 14, 8333-8342. of straited muscle. Cell. Tissue Res. 296, 191-198. Riggleman, B., Wieschaus, E. and Schedl, P. (1989). Molecular analysis of Kaufmann, U., Kirsch, J., Irintchev, A., Wernig, A. and Starzinski-Powitz, the armadillo : uniformly distributed transcripts and a protein with A. (1999b). The M-cadherin catenin complex interacts with microtubules in novel internal repeats are associated with a Drosophila segment polarity skeletal muscle cells: implications for the fusion of myoblasts. J. Cell Sci. gene. Genes Dev. 3, 96-113. 112, 55-67. Ringwald, M., Schuh, R., Vestweber, D., Eistetter, H., Lottspeich, F., Engel, Kaufmann, U., Zuppinger, C., Waibler, Z., Ruediger, M., Urbich, C., J., Dölz, R., Jähnig, F., Epplen, J., Mayer, S., Müller, C. and Kemler, R. Martin, B., Jockusch, B., Eppenberger, H. and Starzinski-Powitz, A. (1987). The structure of the cell adhesion molecule uvomorulin. Insights (2000). The armadillo repeat region targets ARVCF to cadherin-based into the molecular mechanism of Ca2+ dependent cell adhesion. EMBO J. cellular junctions. J Cell Sci. 113, 4121-4135. 6, 3647-3653. Keirsebilck, A., Bonne, S., Staes, K., van Hengel, J., Nollet, F., Reynolds, Rose, O., Rohwedel, J., Reinhardt, S., Bachmann, S., Cramer, M., Rotter, A. and van Roy, F. (1998). Molecular cloning of the human p120ctn catenin M., Wobus, M. and Starzinski-Powitz, A. (1994). Expression of M- gene (CTNND1): Expression of multiple alternative spliced isoforms. cadherin protein in myogenic cells during prenatal mouse development Genomics 50, 129-146. and differentiation of embryonic stem cells in culture. Dev. Dyn. 201, 245- Kelly, D., Goldberg, R., Wilson, D., Lindsay, E., Carey, A., Goodship, J., 259. Burn, J., Cross, I., Shprintzen, R. J. and Scambler, P. J. (1993). Rüdiger, M., Jockusch, B. M. and Rothkegel, M. (1997). A novel epitope- Confirmation that the velo-cardio-facial syndrome is associated with haplo- antibody combination for the detection of protein expression in prokaryotic insufficiency of genes at chomosome 22q11. Am. J. Med. Genet. 45, 308- and eukaryotic cells. Biotechniques 23, 96-97. 312. Shapiro, L., Fannon, A. M., Kwong, P. D., Thompson, A., Lehmann, M. Knudsen, K. A., Soler, A. P., Johnson, K. R. and Wheelock, M. J. (1995). S., Grübel, G., Legrand, I., Als-Nielsen, J., Colman, D. R. and Interaction of alpha-actinin with the cadherin/catenin cell-cell adhesion Hendrickson, W. A. (1995). Structural basis of cell-cell adhesion by complex via alpha-catenin. J. Cell Biol. 130, 67-77. cadherins. Nature 374, 327-336. Kuch, C., Winnekendonk, D., Butz, S., Unvericht, U., Kemler, R. and Sirotkin, H., O’Donnell, H., DasGupta, R., Halford, S., St Jore, B., Puech, Starzinski-Powitz, A. (1997). M-cadherin-mediated cell adhesion and A., Parimoo, S., Morrow, B., Skoultchi, A., Weissman, S. M. et al. (1997). complex formation with the catenins in myogenic mouse cells. Exp. Cell Identification of a new human catenin gene family member (ARVCF) from Res. 232, 331-338. the region deleted in velo-cardio-facial syndrome. Genomics 41, 75-83. Mariner, D. J., Wang, J. and Reynolds, A. B. (2000). ARVCF localizes to Takeichi, M. (1991). Cadherin cell adhesion receptors as a morphogenic the nucleus and adherens junction and is mutually exclusive with p120ctn regulator. Science 251, 1451-1455. in E-cadherin complexes. J. Cell Sci. 113, 1481-1490. Tsukita, S., Tsukita, S., Nagafuchi, A. and Yonemura, S. (1992). Molecular Molenaar, M., van de Wetering, M., Oosterwegel, M., Peterson-Maduro, linkage between cadherins and actin filaments in cell-cell adhesion J., Godsave, S., Korinek, V., Roose, J., Destrée, O. and Clevers, H. junctions. Curr. Opin. Cell Biol. 4, 834-839. (1996). XTcf-3 transcription factor mediates β-catenin - induced axis van de Wetering, M., Cavallo, R., Dooijes, D., van Beest, M., van Es, J., formation in Xenopus embryos. Cell 86, 391-399. Loureiro, J., Ypma, A., Hursh, D., Jones, T., Bejsovec, A. et al. (1997). Morrow, B., Goldberg, R., Carlson, C., DasGupta, R., Sirotkin, H., Armadillo coactivates transcription driven by the product of the Drosophila Collins, J., Dunham, I., O’Donnell, H., Scambler, P., Shprintzen, R. and segment polarity gene dTCF. Cell 88, 789-799. Kucherlapati, R. (1995). Molecular definition of the 22q11 deletions in van Hengel, J., Vanhoenacker, P., Staes, K. and van Roy, F. (1999). Nuclear velo-cardio-facial syndrome. Am. J. Hum. Genet. 56, 1391-1403. localisation of the p120 ctn Armadillo-like catenin is counteracted by a Näthke, I., Hinck, S., Swedlow, J. R., Papkoff, J. and Nelson, J. W. (1994). nuclear export signal and by E-cadherin expression. Proc. Natl. Acad. Sci Defining interactions and distributions of cadherin and catenin complexes USA 96, 7980-7985. in polarised epithelial cells. J. Cell Biol. 125, 1341-1352. Wieschaus, E. and Riggleman, R. (1987). Autonomous requirements for the Nieman, M. T., Prudoff, R. S., Johnson, K. R. and Wheelock, M. J. (1999). segment polarity gene armadillo during Drosophila embryogenesis. Cell 49, N-cadherin promotes motility in human breast cancer cells regardless of 177-184. their E-cadherin expression. J. Cell Biol. 147, 631-644. Yap, A. S., Niessen, C. M. and Gumbiner, B. M. (1998). The juxtamembrane Ozawa, M., Baribault, H. and Kemler, R. (1989). The cytoplasmatic region of cadherin cytoplasmic tail supports lateral clustering, adhesive domain of the cell adhesion molecule uvomorulin associates with three strengthening, and interaction with p120(ctn). J. Cell Biol. 4, 779-789.