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Calcium-Sensitive Intercadherin Interaction 4381 Journal of Cell Science 112, 4379-4387 (1999) 4379 Printed in Great Britain © The Company of Biologists Limited 1999 JCS0706 Removal of calcium ions triggers a novel type of intercadherin interaction Regina B. Troyanovsky, Jörg Klingelhöfer and Sergey Troyanovsky* Division of Dermatology, Washington University Medical School, St Louis, MO 63110, USA *Author for correspondence (e-mail: [email protected]) Accepted 20 September; published on WWW 17 November 1999 SUMMARY Depletion of Ca2+ ions from epithelial cell cultures has been ‘calcium-sensitive’ complexes. Furthermore, experiments shown to result in the rapid destruction of intercellular with this mutant revealed that EGTA induced lateral junctions. To understand the mechanism of this effect we Trp156/Val157-independent homodimerization of E- have examined how removal of calcium ions from the cadherin. Deletion mutagenesis of E-cadherin showed that culture medium of A-431 epithelial cells affects complexes these complexes are mediated by at least two extracellular incorporating the cell-cell adhesive receptors, E-cadherin, cadherin domains, EC3 and EC4. Notably, protein kinase desmoglein or desmocollin. Sedimentation and biochemical inhibitor H-7 which confers EGTA-independence of the analysis demonstrated that calcium removal triggers a adhesive E-cadherin complexes does not block this rapid formation of a novel type of complex formed via association. We propose that this novel type of direct lateral E-cadherin-desmoglein, E-cadherin- intercadherin interaction is involved in the assembly of desmocollin and desmoglein-desmocollin dimerization of adherens junctions and their disassembly in low-calcium the extracellular cadherin regions. Replacement of Trp156 medium. and Val157 of E-cadherin, that has been shown to abolish lateral and adhesive E-cadherin homodimerization in standard cultures, did not influence the formation of these Key words: Cadherin, Catenin, Intercellular adhesion INTRODUCTION of this process are not yet understood, though some critical clues for its comprehension were evolved in recent years. Classic and desmosomal cadherins are two subfamilies of Cadherins of both groups are single-pass transmembrane cadherins serving as structural transmembrane elements in proteins containing four homologous extracellular cadherin specialized, morphologically distinct intercellular junctions domains (EC1-4, numbered from the N terminus). The crystal termed adhering junctions (Schäfer et al., 1993; Schmidt et al., structure of the EC1 domain of N-cadherin revealed that it is 1994, and references therein). These junctions are able to form lateral dimers. This interaction is mediated by a characterized by a dense cytoplasmic plaque which joins the Trp residue (Trp156 in E-cadherin; here and below the E- adhesive transmembrane junctional core with the intracellular cadherin sequence is numbered according to GenBank cytoskeleton. The group of adhering junctions referred to as an accession # Z13009, cf. Bussemakers et al., 1993) that is ‘adherens junction’, incorporates classic cadherins (e.g. E- conserved among classic and desmosomal cadherins. In cadherin) and anchors bundles of microfilaments (reviewed by addition, the EC1 domain can form antiparallel dimers which Geiger and Ayalon, 1992; Kemler, 1992; Takeichi, 1995; Yap are likely to establish a direct link between opposing cells et al., 1997). Desmosomes are another type of adhering (Shapiro et al., 1995). In our recent work (Chitaev and junctions present in epithelial cells (Schwarz et al., 1990; Troyanovsky, 1998), we presented substantial evidence Garrod et al., 1996; Troyanovsky and Leube, 1998). They demonstrating that E-cadherin forms both lateral and adhesive contain desmosomal cadherins (e.g. desmoglein, Dsg and dimers in vivo. Furthermore, some features of the identified desmocollin, Dsc) and are coupled with intermediate filaments. lateral complex are consistent with the model proposed by The intracellular domains of classic and desmosomal cadherins Shapiro et al. (1995). It was nearly abolished by the double contain a characteristic segment that is highly conserved substitution Trp156Ala/Val157Gly. In addition, formation of between members of the family and mediates binding of this complex was not dependent on the presence of the Ca2+- cadherins to cytoplasmic proteins collectively termed catenins binding sites between the EC1 and EC2 domains. Little is (α- and β-catenins, and plakoglobin). The binding to catenins known about how the lateral and adhesive E-cadherin has a critical function in cell-cell adhesion (Nagafuchi and complexes, which are likely to represent two consecutive steps Takeichi, 1988; Ozawa et al., 1989, 1990b; Ozawa and Kemler, in adherens junction assembly (Shapiro et al., 1995; Brieher et 1998; Knudsen et al., 1995; Rimm et al., 1995; Angres et al., al., 1996; Tomschy et al., 1996; Chitaev and Troyanovsky, 1996; Chitaev and Troyanovsky, 1998). Most molecular details 1998), are incorporated into mature adherens junctions. During 4380 R. B. Troyanovsky, J. Klingelhöfer and S. Troyanovsky this process the E-cadherin-catenin complex is recruited into provided by Dr W. W. Franke); anti-myc (clone 9E10, provided by Dr a Triton X-100 insoluble pool and associates with R. Kopan, Washington University, St Louis, MO); rabbit anti-myc microfilaments (McNeill et al., 1993; Adams et al., 1996; antibody (Santa Cruz Biotechnology, Santa Cruz, CA); anti-flag M2 Gloushankova et al., 1998). (Sigma, St Louis, MO); anti-α-catenin, anti-E-cadherin (Mab β One of the fundamental features of the cadherin-based C20820), anti- -catenin and anti-EGF receptor (Transduction adhesion is its sensitivity to the extracellular Ca2+ Laboratories, Lexington, KY); and anti CD44 (Zymed Laboratories Inc., San Francisco, CA). concentrations (Kartenbeck et al., 1982; Mattey and Garrod, To remove extracellular calcium, EGTA (Sigma) was added to a 1986; Volberg et al., 1986; Green et al., 1987). Importantly, the final concentration of 10 mM. Experiments performed with a lower current understanding of cell-cell adhesion is based on concentration of EGTA (5 µM) gave identical results. Treatment with experiments with cells dissociated by calcium chelators. The H-7 inhibitor (Sigma) was carried out as described by Citi (1992). mechanism of such dissociation, however, is still poorly To determine the localization of E-cadherin complexes, cells were understood. Our experiments (Chitaev and Troyanovsky, 1998) digested with 0.05% trypsin in PBS containing 0.02% EDTA for 1 showed that the adhesive E-cadherin complex was stable in the minute at 37°C. After the addition of soybean trypsin inhibitor (final absence of calcium ions in vitro or in cultured cells at 4°C, but concentration 2 mg/ml), cells were subjected to immunoprecipitation immediately disappeared at 37°C. Such a temperature- analysis. sensitive, immediate response to changes in the extracellular Immunoprecipitation and sedimentation analysis calcium concentration suggests involvement of specific signal For most immunoprecipitation experiments, 2×106 cells were cultured transduction pathways. This point of view is consistent with in a 10-cm tissue culture dish at 37°C for about 72 hours. In co-culture the observation that the protein kinase inhibitor H-7 prevents experiments, 6×106 cells producing myc- and flag-tagged forms of E- dissociation of adherens junctions and desmosomes in low cadherin were mixed in a 1:1 ratio and were cultured in a 10-cm dish calcium concentration (Citi, 1992; Pasdar et al., 1995). for 24 hours. Immunoprecipitation assay and sucrose gradient Examination of this effect led authors to propose that centrifugation were described previously (Troyanovsky et al., 1994; dissociation of the adherens junctions upon removal of Chitaev and Troyanovsky, 1998). In brief, the confluent monolayer extracellular calcium ions is caused by contraction of the (approximately 107 cells) was washed and extracted in 1.5 ml of immunoprecipitation lysis buffer (IP-buffer; 50 mM Tris-HCl, pH 7.4, cortical microfilament cytoskeleton (Citi et al., 1994; µ Denisenko et al., 1994; Volberg et al., 1994). However, how 150 mM NaCl, 1 mM DTT, 20 M p-APMSF, 2 mM EDTA, and 1% 2+ NP-40). The lysates were subjected to immunoprecipitation by the decrease in extracellular concentration of Ca ions subsequent incubations with specific antibody and Protein A- generates signaling across the plasma membrane and initiates Sepharose. For sucrose gradient centrifugation, confluent monolayer actomyosin-driven contraction is not known. cells from three 10 cm dishes were lysed with 2 ml of IP-buffer. In an attempt to understand the molecular mechanisms Lysates (1 ml) were precleaned by centrifugation at 100,000 g for 1 regulating the assembly of desmosomes and adherens hour, and then loaded on top of a 12 ml linear 5-20% (wt/wt) sucrose junctions, we studied the behavior of the adhesive and lateral gradient prepared in IP-buffer. Gradients were centrifuged at 200,000 E-cadherin complexes in response to shifts in the extracellular g for 17 hours in a SW40Ti rotor (Beckman Instruments) at 4°C, calcium level. Surprisingly, we found that removal of calcium fractionated from bottom to top into 12 fractions (1 ml each), and ions from the growth medium causes immediate assembly of analyzed by co-immunoprecipitation. The following protein standards novel intercadherin complexes in A-431 epithelial cells of known S values were centrifuged on replicate gradients: BSA, 4.5S; IgG, 7.5S; catalase, 11.35S; apoferritin,
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