RESEARCH ARTICLE 3873 mARVCF cellular localisation and binding to cadherins 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 catenin family, is thought to isoforms to M-, N-, or E-cadherin 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 carcinoma 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-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 proteins 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 gene maps to chromosome 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 genes are very similar cadherins such as E-cadherin in epithelial cells and M-cadherin (Keirsebilck et al., 1998). The 35 amino acid 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 cytoskeleton 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 base pair 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 catenins 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, plakoglobin (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.