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 . 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 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 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). 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 ). The binding to catenins known about how the lateral and adhesive E-cadherin has a critical function in cell- (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; (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, 17S. incorporating either several (two or more) E-cadherin molecules or both E-cadherin and desmosomal cadherins. These complexes are independent of Trp156 of E-cadherin and required the integrity of the EC3 and EC4 domains. This novel RESULTS type of intercadherin interaction could play an important role in normal junction assembly and in dissociation of the adherens Removal of calcium ions triggers lateral junctions in low-calcium medium. intercadherin association We have studied whether E-cadherin may form complexes with desmosomal cadherins in A-431 cells. Total lysates of standard MATERIALS AND METHODS confluent cultures of these cells were immunoprecipitated either with anti-Dsg or anti-E-cadherin (Fig. 1A,B). DNA constructs, cell culture, DNA transfection and Obtained immunoprecipitates were analyzed by western blot immunofluorescence microscopy for the presence of different cadherins or catenins. The construction of the expression plasmids coding for E-cadherin Surprisingly, long exposure of these blots demonstrated that with an internal deletion His773-Leu791 and tagged C-terminally either two different cadherins, E-cadherin and Dsg, may form a by myc (Ec1M) or by flag (Ec1F) epitopes, and for mutants of Ec1M heteromeric complex. Another desmosomal cadherin, Dsc was such as Ec1WVM, Ec1QNM, Ec1∆(748-882)M, and Ec1∆(159- not found in the anti-E-cadherin or anti-Dsg 536)M was described recently (Chitaev and Troyanovsky, 1998). immunoprecipitates (Fig. 1). Dsc-Dsg and Dsc-E-cadherin PCR-mediated mutagenesis was used to construct plasmids encoding complexes also were not detected in the reciprocal experiments internal deletion mutants of Ec1M (see Fig. 5A for details). in which immunoprecipitates obtained with anti-Dsc antibody Transfection of human epidermoid carcinoma A-431 cells (ATCC, CRL1555) and selection, growth, and immunofluorescence were analyzed by anti-E-cadherin or anti-Dsg staining (not microscopy were done as described (Chitaev et al., 1996). The shown). These experiments also showed that a minor pool of following mouse monoclonal antibodies were used: anti-plakoglobin Dsg associated with β-catenin (Fig. 1B). (clone 11E4, Zymed laboratories, San Francisco, CA); anti- To define whether E-cadherin and Dsg molecules in the desmoglein and anti-desmocollin (clones 3.10 and U114, respectively, complex have parallel or antiparallel alignment, we performed Calcium-sensitive intercadherin interaction 4381

Fig. 1. Formation of the calcium-sensitive heterocadherin complexes. (A-C) Total lysates of A-431 cells before (−) or after incubation with EGTA (+) were immunoprecipitated under standard conditions or in the presence of 0.25% SDS (C, + SDS) with anti-E-cadherin (IP-Ec; A and C) or anti-Dsg (IP-Dsg, B) antibodies. Immunoprecipitates were analyzed by western blot for the presence of E-cadherin (Ec); Dsg (Dsg); Dsc (Dsc) and β-catenin (β-cat). D, wild-type HT-1080 cells (lanes 1 and 2) and HT-1080 cells producing recombinant Ec1M (lanes 3 and 4) were lysed without (−) or after (+) EGTA treatment, immunoprecipitated with anti-Dsg antibody, and immunoprecipitates obtained were analyzed by western blot with Fig. 2. Sedimentation analysis (A) and dynamics (B and C) of the β β antibodies against Dsg (Dsg), myc epitope (Myc), or -catenin ( - calcium-sensitive heterocadherin complexes. (A) Total lysate of cat). Note that only E-cadherin-producing HT-1080 cells exhibit wild-type A-431 cells treated before lysis for 10 minutes with 10 strong calcium-sensitive Dsg-β-catenin association. mM EGTA was subjected to sucrose gradient centrifugation. The collected fractions (numbered 1 to 12, with 1 at the bottom of the gradient) were immunoprecipitated with anti-Dsg mAb, separated by SDS-PAGE and analyzed by immunoblotting with antibodies against co-immunoprecipitation experiments with A-431 cells that Dsg (Dsg), β-catenin (β-cat) or E-cadherin (Ec). The filter stained were dissociated by EGTA treatment prior to lysis. with anti-Dsg mAb was overexposed to show presence of minor pool Surprisingly, this treatment strongly increased the amount of of Dsg in fractions 3-6. Arrowhead indicates the position of the the Dsg-E-cadherin complexes and resulted in formation of lateral and the adhesive E-cadherin complexes described by Chitaev Dsg-Dsc and E-cadherin-Dsc complexes (Fig. 1). The same and Troyanovsky (1998). The peak distribution of protein standards effect, an increase of the E-cadherin-Dsg complex and an of known S values (bovine serum albumin, 4S; rabbit IgG, 7.5S; appearance of the E-cadherin-Dsc and the Dsg-Dsc complexes, catalase, 11.4S; apoferritin, 17S) was determined in a parallel was also caused by replacement of the standard culture gradient and is shown at the bottom. (B) Wild-type A-431 cells growing under standard conditions (0) or after incubation with 10 medium for 10 minutes with low calcium medium (not mM EGTA for 30 seconds (30′′), 1, 2, 5, or 10 minutes (1′, 2′, 5′, 10′ shown). Both treatments did not change amounts of E- correspondingly) were immunoprecipitated using anti-E-cadherin cadherin or desmosomal cadherins in the NP-40-soluble or antibody. The immunoprecipitates obtained were analyzed by insoluble fractions (not shown). This calcium-sensitive western blotting with E-cadherin (Ec), Dsg (Dsg) or Dsc (Dsc) intercadherin association was unaltered upon addition of antibodies. (C) A-431 cells were treated for 10 minutes with EGTA, 0.25% SDS into the IP-lysis buffer and washing solution (Fig. then washed in PBS and further cultivated in standard growth ′ ′ 1C). These conditions, however, completely released β- medium for 10 (10 ), 20 (20 ) minutes, or 1, 2 or 4 hours (1, 2, 4, catenin/plakoglobin from E-cadherin immunoprecipitates respectively) and then analyzed as indicated in B. 4382 R. B. Troyanovsky, J. Klingelhöfer and S. Troyanovsky suggesting that catenins do not mediate association between E- cadherin and desmosomal cadherins. Anti-Dsg staining of the E-cadherin immunoprecipitate of the EGTA-treated cells analyzed under nonreducing conditions yielded only a band of monomeric Dsg (not shown) indicating that the detected calcium-sensitive intercadherin association was not caused by disulfide crosslinking. We next studied whether β-catenin associates with Dsg via E-cadherin or whether these two proteins form a separate complex. To clarify this, we examined the Dsg-β-catenin interaction in E-cadherin-negative HT-1080 cells. Fig. 1D shows that β-catenin was not co-immunoprecipitated with Dsg in the HT-1080 cells growing under normal conditions and only very weak binding was detected in EGTA-treated HT-1080 cells. In contrast, the Dsg-β-catenin complex was efficiently formed in HT-1080 cells transfected to produce myc tagged E- cadherin (Ec1M) upon addition of EGTA. These experiments strongly support the idea that the Dsg-β-catenin interaction is due to association of the conventional E-cadherin-catenin Fig. 3. Western blot analysis of total lysates (A, Tot. Lysates) or anti- complex with Dsg. A weak Dsg-β-catenin binding found in the E-cadherin immunoprecipitates (B, IP-Ec) of A-431 cells either wild-type HT-1080 cells under low calcium conditions (Fig. 1, untreated (−) or treated with EGTA (+) for 1, 10 or 20 minutes (1, 10 lane 2) is probably caused by an interaction between Dsg and or 20, respectively) before lysis. Where indicated (trypsin), cells after N-cadherin, an endogenous classic cadherin of HT-1080 cells EGTA treatment were incubated with trypsin/EGTA mixture for an (Sacco et al., 1995). The lack of an high affinity anti-N- additional 1 minute. Blots were stained with E-cadherin (Ec) or Dsg (Dsg) antibodies. Note that Dsg in the Dsg-E-cadherin complex is cadherin antibody prevented us from testing this hypothesis. sensitive to trypsin treatment. Low molecular mass bands represent To estimate the overall size of the E-cadherin-Dsg complex, proteolytic intracellular fragments of E-cadherin. the total lysate of the EGTA-treated A-431 cells was subjected to sucrose gradient centrifugation. Examination of the Dsg distribution along the gradient showed that the major pool of this protein sedimented at 8S and is consistent with our complex found in cells cultured in normal medium was previous observation (Chitaev et al., 1998). In addition to this insensitive to trypsin treatment, suggesting that in normal cells form of Dsg, overexposure of the same filters demonstrated this complex either has an intracellular localization or has a that Dsg has minor forms sedimenting up to 16S (Fig. 2A). trypsin-resistant conformation. Staining of total cell lysates of Anti-Dsg immunoprecipitation of each gradient fraction the trypsin-treated cells by antibodies against intracellular revealed that these heavy forms of Dsg associate with E- epitopes of Dsg and E-cadherin demonstrated generation of the cadherin and β-catenin (Fig. 2A). Interestingly, in contrast to intracellular fragments of these proteins. These fragments were the 13S lateral or adhesive E-cadherin complexes that we not co-immunoprecipitated (Fig. 3B) suggesting that E- described recently (Chitaev and Troyanovsky, 1998), this cadherin interacts with Dsg via extracellular segments. complex has a much broader distribution sedimenting between 11 and 16S. This broad distribution suggests that this complex Calcium removal induces a novel type of the E- has either a variable composition and/or a polymorphic cadherin dimers conformation. The data presented above show that the removal of In a special set of experiments we studied the dynamics of extracellular Ca2+ ions triggers rapid formation of the E- assembly and disassembly of the intercadherin complexes. The cadherin-Dsg and E-cadherin-Dsc complexes. To delineate the results of these experiments (Fig. 2B,C) show that E-cadherin- portions of E-cadherin required for formation of such Dsg or E-cadherin-Dsc complexes appeared very rapidly after complexes, we studied A-431 cells producing recombinant removal of calcium ions from the culture medium. Their forms of human E-cadherin, Ec1M or its mutants. Ec1M amount reached a plateau approximately five minutes after protein was tagged by myc epitope and contained a 19 amino cultivation of the cells in medium with EGTA. Addition of acid long internal deletion (His773-Leu791) within the normal medium to the EGTA-treated cells completely intracellular region. This deletion completely abolished normalized the amount of these complexes during a few hours. binding of this mutant to the anti-E-cadherin C2080 antibody These data suggest that normalization of the extracellular (Chitaev and Troyanovsky, 1998). Thus, endogenous E- calcium level leads to gradual dissociation of the Dsg-E- cadherin and Ec1M can easily be distinguished by specific cadherin complex and/or incorporation of this complex into the antibodies thereby allowing the detection of E-cadherin insoluble pool. homoassociation in co-immunoprecipitation experiments. In To show that E-cadherin-Dsg association takes place on the our recent paper (Chitaev and Troyanovsky, 1998) we showed cell surface, the cells were treated with trypsin after addition that Ec1M protein behaves similarly to wild-type E-cadherin. of EGTA (Fig. 3). This experiment showed that practically all In the present work, using A-431 cells stably expressing Ec1M, Dsg-E-cadherin complexes are localized on the cell surface at we showed that this protein, similar to endogenous E-cadherin, least up to 30 minutes after reduction of the calcium associates with Dsg upon removal of Ca2+ ions (Fig. 4A, lanes concentration. Interestingly, the level of the Dsg-E-cadherin 1 Ð and +). Calcium-sensitive intercadherin interaction 4383

The Ec1∆(744-879)M mutant of the Ec1M protein lacking the intracellular region responsible for binding to catenins and p120 had the same ability as Ec1M to form a complex with Dsg (Fig. 4, lanes 2 Ð and +). This observation supports the conclusion made above that Ca2+-sensitive interactions are not mediated by catenins. Inactivation of two of the three Ca2+- binding sites in the E-cadherin EC1/EC2 domains that had been defined by crystallographic and mutational analysis (Ozawa et al., 1990a; Nagar et al., 1996) was done by double amino acid substitution Gln225Ala/Asn256Ala (mutant Ec1QNM, Fig. 4A, lanes 3 Ð and +). This alteration did also not abolish the association with Dsg. Only deletion of the entire extracellular cadherin-like repeats 1-3 of E-cadherin in the Ec1∆(155-532)M mutant completely abolished this binding. This suggests that extracellular regions are involved in these interactions (Fig. 4A, lanes 5 Ð and +). Notably, double mutation of Trp156Ala/Val157Gly (Ec1WVM mutant) did not inhibit the Ca2+-sensitive association with Dsg (Fig. 4A, lanes 4 Ð and +). Furthermore, since mutant Ec1WVM did not form lateral or adhesive dimers with endogenous E-cadherin under standard culture conditions (see Chitaev and Troyanovsky, 1998), anti-E-cadherin analysis of the anti-myc immunoprecipitates revealed clearly the strong Ca2+-sensitive interaction of this mutant with endogenous E- cadherin (Fig. 4A, lanes 4 + and −). Western blot analysis of the anti-myc immunoprecipitates obtained after sucrose gradient centrifugation of the total lysates of Ec1WVM-producing cells showed that addition of EGTA in the culture medium significantly changed the sedimentation characteristics of the Ec1WVM mutant (Fig. 4B). Upon normal culture conditions mutant Ec1WVM was recovered from fractions distributed below of 13.5S value (corresponding to fraction 5), while a significant pool of the mutant obtained from EGTA-treated cells sedimented far above this value. Analysis of the same immunoprecipitates with anti- Dsg and anti-E-cadherin antibodies revealed broad peaks of the Ec1WVM-Dsg and Ec1WVM-E-cadherin complexes, similar to the peak of E-cadherin-Dsg complexes in wild-type A-431 cells. Thus, removal of extracellular calcium ions induced E- cadherin homoassociation. This homoassociation was not seen 156 Fig. 4. Association of Ec1M and its mutants with endogenous E- in cells producing Ec1M since the Trp -mediated lateral E- cadherin and Dsg (A) and sedimentation properties of the calcium- cadherin 13S complex prevented us from noticing the sensitive Ec1WVM/Dsg and Ec1WVM/E-cadherin complexes (B). formation of the calcium-sensitive association. We noted, (A) Untreated (−) or EGTA treated (+), A-431 cells stably producing however (Chitaev and Troyanovsky, 1998) that the combined Ec1M (lanes 1); Ec1∆(748-882)M (lanes 2); Ec1QNM (lanes 3); amount of the E-cadherin 13S complexes was surprisingly Ec1WVM (lanes 4); Ec1∆(159-536)M (lanes 5) were unchanged upon EGTA treatment, despite the dissociation of immunoprecipitated with anti-myc antibody. Immunoblot analysis of adhesive complexes. Therefore, the constant amount of 13S these immunoprecipitates with anti-myc (MYC); anti-E-cadherin complexes suggests that the decrease of adhesive complexes in (Ec); and anti-Dsg (Dsg) antibodies show that all mutants except low calcium is counterbalanced by an increased production of Ec1D(159-536)M establish complexes with Dsg upon removal of lateral Ca2+-sensitive complexes. extracellular calcium. This condition induces association of the mutant Ec1WVM with endogenous E-cadherin. Molecular weight In an attempt to determine more precisely the region of E- markers are shown in kDa. (B) A-431 cells stably producing cadherin responsible for the formation of the calcium-sensitive Ec1WVM mutant either untreated (−) or after EGTA treatment (+) complexes, we transfected A-431 cells with Ec1M mutants were subjected to sucrose gradient centrifugation. Anti-myc lacking either one of EC domains. Fig. 5 shows that deletion immunoprecipitates obtained from each fraction were separated by of EC3 or EC4 domains significantly decreased the amount of SDS-PAGE, transferred to nitrocellulose and stained with anti-myc Dsg in the anti-myc co-immunoprecipitates. Formation of the (Ec1WVM), anti-Dsg (Dsg) or anti-E-cadherin (Ec) antibodies. Note complexes was nearly abolished by deletion of both these that Ec1WVM-Dsg and Ec1WVM-E-cadherin complexes are domains in the mutant Ec1∆(3+4)M. These data showed distributed similarly along the gradient and that their peaks roughly for the first time that EC domains, other than amino- correspond to the peak of the 13S E-cadherin complexes terminal EC1 domain, may participate in E-cadherin hetero- (arrowhead). or homodimerization. Interestingly, under normal culture 4384 R. B. Troyanovsky, J. Klingelhöfer and S. Troyanovsky conditions, all deletion mutants except Ec1∆1M lacking EC1 maintenance of the adhesive dimers. In our attempt to domain co-immunoprecipitated relatively normal amount of understand the mechanism of formation of the calcium- the endogenous E-cadherin. The data suggest that these sensitive Trp156-independent lateral E-cadherin complexes, deletions did not affect the formation of the lateral and/or we studied the association between Ec1WVM protein and adhesive cadherin dimers (Fig. 5). endogenous E-cadherin and Dsg under these conditions. These experiments demonstrated that the presence of EGTA in the Extracellular calcium depletion but not concomitant lysis buffer or in the culture medium at 4¡C is not sufficient to dissociation of the adhesive E-cadherin dimers induce formation of the calcium-sensitive complexes (Fig. 5A). induces formation of the calcium-sensitive This suggests that the calcium-sensitive inter-cadherin complexes association might be connected in some way to dissociation of We have shown (Chitaev, Troyanovsky, 1998) that cultivation the adhesive E-cadherin complexes. To test this hypothesis, we of A-431 cells with 10 mM EGTA at 4¡C or addition of 10 mM studied whether the dynamics of the calcium-sensitive EGTA directly into the lysis buffer (but not in the culture association correlates with the rate of disassembly of the medium) did not dissociate adhesive E-cadherin complexes. adhesive complexes. In order to reveal the dynamics of the These data indicate that calcium ions do not participate in the adhesive E-cadherin complexes, two A-431 sublines producing either myc-tagged (Ec1M) or flag-tagged (Ec1F) forms of E- cadherin were co-cultivated overnight, treated by EGTA for different time intervals, lysed and then immunoprecipitated with anti-myc antibody. Staining of these immunoprecipitates with anti-flag showed the amount of adhesive dimers (Chitaev and Troyanovsky, 1998) and with anti-Dsg showed calcium- sensitive E-cadherin/Dsg complexes. This experiment demonstrated a striking inverse correlation between these complexes (Fig. 5B). Dissociation of adherens junctions can be blocked by preincubation of the cells with the protein kinase inhibitor H- 7 (Citi, 1992; Citi et al., 1994). While the exact mechanism of this effect remains to be elucidated, the data suggest that the primary effect of H-7 is an inhibition of actomyosin-driven contractility of the cortical cytoskeleton (Volberg et al., 1994). It was suggested that the depletion of calcium ions triggers contraction of the adherens junction-associated microfilaments that, in turn, breaks the adherens junctions. In our attempt to understand the relationship between degradation of the adhesive complexes and formation of the calcium-sensitive E- cadherin complexes we studied the effect of the H-7 inhibitor on the amount of both complexes. For this, co-cultures of the Ec1M- and Ec1F-producing A-431 cells were exposed to EGTA with or without pretreatment with H-7 for 30 minutes. In agreement with published observations (Citi et al., 1994) pretreatment of the cells for 30 minutes with 100 µM H-7 inhibited cell dissociation (not shown). Furthermore, the data presented on Fig. 5C show that H-7 pretreatment completely blocked EGTA-induced dissociation of adhesive E-cadherin complexes. These results confirm on the molecular level the conclusion made by City et al. (1994) that chelator-induced dissociation of adherens junctions is not the result of a decrease in homophilic intercadherin affinity. Finally, we compared the amount of the calcium-sensitive E-cadherin complexes in cells treated with EGTA alone and in combination with H-7 (Fig. 5C). These data showed that H-7, completely blocked EGTA-induced dissociation of the adhesive E-cadherin complexes but was unable to prevent formation of the calcium-sensitive lateral E-cadherin complexes. Similar experiments performed with A-431 cells Fig. 5. Western blots of co-immunoprecipitates obtained using anti- producing the Ec1WVM mutant demonstrated that H-7 myc 9E10 antibody from untreated (−) or EGTA treated (+) A-431 cells stably producing Ec1M, Ec1∆1M, Ec1∆2M, Ec1∆3M, pretreatment also did not block formation of calcium-sensitive Ec1∆4M, Ec1∆5M, Ec1∆(2+3)M, and Ec1∆(3+4)M. Blots were inter-E-cadherin complexes (data not shown). Thus, our data developed with anti-myc (Myc), Anti-Dsg (Dsg), or anti-E-cadherin indicate that the dissociation of adhesive complexes is not a (Ec) antibodies. Note that deletion of the EC domains 3 and 4 necessary prerequisite for the formation of the calcium- completely abolished Dsg co-immunoprecipitation. sensitive E-cadherin complexes. Calcium-sensitive intercadherin interaction 4385

proposed by Shapiro et al. (1995). In the present work we show that transferring the cells to low calcium medium induces a rapid assembly of a novel type of lateral E-cadherin complexes. The major structural feature of the new lateral complexes is their independence of a Trp156 residue. Notably, this Trp156- independent, calcium-sensitive lateral association occurs not only between two E-cadherin molecules, but also between E- cadherin and both desmosomal cadherins, Dsg and Dsc. On the other hand, this association is cadherin-specific, since, using co-immunoprecipitation assays, we were not able to detect a calcium-sensitive association between E-cadherin and other transmembrane molecules of A-431 cells, such as EGF receptor or CD44 (data not shown). The detailed composition of this Trp156-independent lateral complex is not completely clear. It sediments in a much broader peak than Trp156- dependent lateral or adhesive E-cadherin complexes, indicating that it is polymorphic, e.g. contains two or more of the conventional cadherin/catenin complexes aligned in parallel fashion. Our mutagenesis experiments indicate that the calcium-sensitive E-cadherin association requires EC3 and EC4 extracellular domains, but not the intracellular, catenin- binding region of E-cadherin and extracellular EC1 domain mediating adhesive and lateral E-cadherin dimerization under Fig. 6. Degradation of the adhesive E-cadherin complexes is not standard culture conditions. Since conformation of the EC essential for assembly of the calcium-sensitive intercadherin domains is calcium-dependent (Takeichi et al., 1981; Volk et complexes. (A) A-431 cells producing Ec1WVM mutant were al., 1990; Pokutta et al., 1994), it appears reasonable to propose immunoprecipitated with anti-myc and immunoprecipitates were that this interaction is induced by conformational changes in analyzed for myc (Myc); E-cadherin (Ec), or Dsg (Dsg) by cadherin structure after dissociation of the calcium ions. The immunoblotting. Before lysis cells were either untreated (1 and 4), or treated at 370 C (2) or at 4¡C (3) with 10 mM EGTA for 10 minutes. fact that assembly of the calcium-sensitive E-cadherin In lane 4 10 mM EDTA was added to the lysis buffer. (B) To analyze complexes is strongly temperature-dependent, suggests, dynamics of the adhesive and calcium-sensitive E-cadherin however, that some additional factors could be involved in this complexes, A-431 cells stably expressing EC1F were co-cultivated process. overnight with cells expressing Ec1M. Before lysis EGTA was added It has been shown that reduction in extracellular calcium in the growth medium for 1, 2, 5 or 10 minutes (indicated above, 0 is concentration causes an immediate splitting of cell-cell untreated control). Then cells were co-immunoprecipitated with anti- junctional structures (Kartenbeck et al., 1982; Mattey and myc and assayed for the presence of the myc-tagged (Myc) and Flag- Garrod, 1986; Volberg et al., 1986; Green et al., 1987) and tagged (Flag) forms of E-cadherin, or Dsg (Dsg). Disappearance of dissociation of the E-cadherin adhesive complex (Chitaev and the adhesive complexes as assayed by anti-flag staining correlates Troyanovsky, 1998). Notably, pretreatment of epithelial cells with assembly of the calcium-sensitive lateral intercadherin 2+ complexes. (C) Cells expressing Ec1M were co-cultured as indicated with protein kinase inhibitor H-7 confers Ca independence in B with Ec1F-producing cells. In lanes 1 and 3 cells were not on cell-cell junctions (Citi, 1992). Experiments presented here treated with EGTA; in lanes 2 and 4 cells were incubated with 10 revealed that, although H-7 completely prevents EGTA- mM EGTA for 10 minutes at 37¡C (+); in lanes 3 and 4 cultures were induced dissociation of the adhesive E-cadherin complexes, it preincubated with 300 mM of H-7 (H-7). Cellular lysates were does not abolish the induction of the calcium-sensitive immunoprecipitated with anti-myc and analyzed by immunoblotting complexes. Thus, the calcium-sensitive E-cadherin association with anti-myc (Myc), anti-cadherin (Ec); anti-flag (Flag); anti-Dsg is not caused by splitting of the intercellular junctions and (Dsg); and anti-Dsc (Dsc) antibodies. Note that pretreatment with H- dissociation of the adhesive complexes, in particular. It 7 prevents degradation of the adhesive complexes but does not inhibit strongly supports the idea that loss of calcium from E-cadherin assembly of the intercadherin complexes. results albeit through some intermediate steps in activation of the new intercadherin-binding activity. The present study also shows strong similarities in kinetics DISCUSSION of the assembly of the calcium-sensitive and disassembly of the adhesive E-cadherin complexes upon removal of Ca2+ ions. Lateral oligomerization of cell-surface receptors is a widely It suggests that both processes might be directly related to each distributed mechanism regulating receptor activity. Thus, it was other. How could the intercadherin linkage induced by suggested that adhesion potential of E-cadherin depends on its depletion of the extracellular Ca2+ ions lead to the splitting of lateral homodimerization (Brieher et al., 1996; Tomschy et al., cell-cell junctions? Experiments reported by Citi et al. (1994), 1996). Little is known, however, about how many types of Denisenko et al. (1994), and Volberg et al. (1994) demonstrated lateral E-cadherin-containing complexes can be assembled. that the H-7 inhibitor affects the contractility of the adherens Recently we have shown that the homodimerization of E- junction-associated microfilaments. It was suggested that cadherin depends on its Trp156/Val157 residues (Chitaev and these microfilaments provide pulling forces required for Troyanovsky, 1998), which is consistent with the model disintegration of the adhesion structures. Experiments reported 4386 R. B. Troyanovsky, J. Klingelhöfer and S. Troyanovsky in the present work support this point of view. They complexes to the cortical cytoskeleton. In theory Trp156- unambiguously demonstrate that H-7 protein kinase inhibitor independent association could be induced by changes in renders adhesive E-cadherin complexes insensitive to Ca2+ occupancy of the calcium-binding sites of E-cadherin. The ions. Furthermore, additional experiments (not shown) showed significance of the divalent cation occupancy is widely that cytochalasin D, a potent inhibitor of actin polymerization, accepted in -mediated transmembrane signal also prevents EGTA-induced dissociation of the adhesive E- transduction (see for recent review, Humphries, 1996). cadherin dimers. One interpretation of the present results is that In summary, our data show that E-cadherin has at least two formation of the Trp156-independent lateral complexes triggers alternative pathways to form lateral complexes. Although the contraction of the cortical cytoskeleton that is mediated much more needs to be done to gain further insight into the either by cadherin-dependent signalling or by direct anchorage molecular mechanisms of E-cadherin mediated adhesion, the of this type of complexes to the cytoskeleton. results of this study suggest a complex order of transitional Experiments presented also show that the reduction of the stages in the organization of the E-cadherin/catenin complex extracellular Ca2+ concentration leads to immediate formation during its integration into the mature adherens junctions. We of complexes incorporating both desmosomal cadherins, Dsg also show how depletion of calcium ions may activate one of and Dsc. Complexes with similar compositions were not these stages. detected in A-431 cells growing at a normal Ca2+ concentration. This oligomerization of desmosomal cadherins We thank Drs A. Eisen (Washington University, St Louis, MO) and could explain the assembly of half-desmosomal structures in R. Leube (Johannes Gutenberg-University, Mainz, Germany) for epithelial cells cultured in low calcium medium (Demlehner et valuable discussion and Dr W. W. Franke for providing us with anti- al., 1995). This possibility is supported by our previous desmoglein and anti-desmocollin antibodies. The work has been supported in part by National Institutes of Health grants AR44016-01 observation that assembly of the -like plaques can and AR45254-01. be promoted by clustering of the intracellular Dsc region (Troyanovsky et al., 1993). Another open question is whether the Trp156-independent, calcium-sensitive intercadherin interaction is involved in REFERENCES normal adherens junction assembly or its formation is only a consequence of the experimentally induced decrease of Adams, C. L., Nelson, W. J. and Smith, S. J. (1996). Quantitative analysis of cadherin-catenin-actin reorganization during development of cell-cell calcium concentration. Our data do not provide direct and adhesion. J. Cell Biol. 135, 1899-1911. unequivocal evidence for the existence of such an interaction Amagai, M., Fujimori, T., Masunaga, T., Shimizu, H., Nishikawa, T., in cells under normal Ca2+ conditions. It is possible, however, Shimuzu, N., Takeichi, M. and Hashimoto, T. (1995). Delayed assembly that the Trp156-independent lateral intercadherin interaction is of desmosomes in with disrupted classic-cadherin-mediated cell adhesion by a dominant negative mutant. J. Invest. Dermatol. 104, 27- responsible for the formation of the small, but detectable 32. amount of Dsg-E-cadherin dimers revealed in standard cultures Angres, B., Barth, A. and Nelson, W. J. (1996). Mechanism for transition of A-431 cells. Such a complex could be responsible for the from initial to stable cell-cell adhesion: kinetic analysis of E-cadherin- Dsg-β-catenin co-immunoprecipitation reported by Norvell mediated adhesion using a quantitative adhesion assay. J. Cell Biol. 134, and Green (1998). The exact role of this Dsg-E-cadherin 549-557. Brieher, W. M., Yap, A. S. and Gumbiner, B. M. (1996). Lateral dimerization complex, which could be a structural element in the proposed is required for the homophilic binding activity of C-cadherin. J. Cell Biol. cross-talk between desmosomes and adherens junctions 135, 487-496. (Wheelock and Jensen, 1992; Lewis et al., 1994, 1997; Amagai Bussemakers, M. J., van Bokhoven, A., Mees, S. G., Kemler, R. and et al., 1995), remains to be elucidated. Schalken, J. A. (1993). Molecular cloning and characterization of the human E-cadherin cDNA. Mol. Biol. Rep. 17, 123-128. Identification of the calcium-sensitive type of inter-E- Chitaev, N. A. and Troyanovsky, S. M. (1998). Adhesive but not lateral E- cadherin lateral dimerization in normal cells is complicated by cadherin complexes require calcium and catenins for their formation. J. Cell the presence of other forms of E-cadherin complexes with Biol. 142, 837-846. similar protein composition. Additionally, complexes formed Chitaev, N. A., Averbakh, A. Z., Troyanovsky, R. B. and Troyanovsky, S. by the Trp156-independent mechanism during normal adhesion M. (1998). Molecular organization of the desmoglein-plakoglobin complex. J. Cell Sci. 111, 1941-1949. could be tightly anchored to the cytoskeleton and would Citi, S. (1992). Protein kinase inhibitors prevent junction dissociation induced therefore be insoluble. Experiments that will define the precise by low extracellular calcium in MDCK epithelial cells. J. Cell Biol. 117, determinants involved in such interactions are essential for 169-178. understanding their functioning in normal cells. It is attractive Citi, S., Volberg, T., Bershadsky, A. D., Denisenko, N. and Geiger, B. 156 (1994). Cytoskeletal involvement in the modulation of cell-cell junctions by to speculate that lateral Trp -independent self-association of protein kinase inhibitor H-7. J. Cell Sci. 107, 683-692. E-cadherin molecules has a critical role in the biogenesis of Denisenko, N., Burighel, P. and Citi, S. (1994). Different effects of protein adherens junctions. It was shown that E-cadherin is recruited kinase inhibitors on the localization of junctional proteins at cell-cell contact into a Triton X-100 insoluble pool approximately 10 minutes sites. J. Cell Sci. 107, 969-981. after the formation of the stable intercellular contact (McNeill Demlehner, M. P., Schäfer, S., Grund, C. and Franke, W. W. (1995). Continual assembly of half-desmosomal structures in the absence of cell et al., 1993). Since the adhesive E-cadherin complexes are contacts and their frustrated endocytosis: a coordinated sisyphus cycle. J. soluble in Triton X-100 (Chitaev and Troyanovsky, 1998), one Cell Biol. 131, 745-760. may propose that in the next step of assembly they enter a Garrod, D., Chidgey, M. and North, A. (1996). Desmosomes: differentiation, Triton X-100 insoluble pool that is accompanied by critical development, dynamics and disease. Curr. Opin. Cell Biol. 8, 670-678. 156 Geiger, B. and Ayalon, O. (1992). Cadherins. Annu. Rev. Cell Biol. 8, 307- changes in their structure. Such a step could be the Trp - 332. independent lateral association of the adhesive dimers. In turn, Gloushankova, N. A., Krendel, M. F., Alieva, N. O., Bonder, E. M., Feder, this association may trigger anchorage of the adhesive H. H., Vasiliev, J. M. and Gelfand, I. M. (1998). Dynamics of contacts Calcium-sensitive intercadherin interaction 4387

between lamellae of fibroblasts: essential role of the actin cytoskeleton. Rimm, D. L., Koslov, E. P., Kebriaei, P., Cianci, C. D. and Morrow, J. S. Proc. Nat. Acad. Sci. USA 95, 4362-4367. (1995). Alpfa 1(E)-catenin is an actin-binding and -bundling protein Green, K. J., Geiger, B., Jones, J. C. R., Talian, J. C. and Goldman, R. D. mediating the attachment of F-actin to the membrane adhesion complex. (1987). The relationship between intermediate filaments and microfilaments Proc. Nat. Acad. Sci. USA 92, 8813-8817. before and during the formation of desmosomes and adherens-type junctions Sacco, P. A., McGranahan, T. M., Wheelock, M. J. and Johnson, K. R. in mouse epidermal keratinocytes. J. Cell Biol. 104, 1389-1402. (1995). Identification of plakoglobin domains required for association with Humphries, M. J. (1996). Integrin activation: the link between binding and N-cadherin and α-catenin. J. Biol. Chem. 270, 20201-20206. signal transduction. Curr. Opin. Cell Biol. 8, 632-640. Schäfer, S., Troyanovsky, S. M., Heid, H. W., Eshkind, L. G., Koch, P. J. Kartenbeck, J., Schmid, E., Franke, W. W. and Geiger, B. (1982). Different and Franke, W. W. (1993). Cytoskeletal architecture and epithelial modes of internalization of proteins associated with adherens junctions and differentiation: molecular determinants of sell interaction and cytoskeletal desmosomes: experimental separation of lateral contacts induced filament anchorage. C. R. Acad. Sci. Paris, Sciences de la vie/Life Sci. 316, endocytosis of desmosomal plaque material. EMBO J. 1, 725-732. 1316-1323. Kemler, R. (1992). Classical cadherins. Semin. Cell Biol. 3, 149-155. Schmidt, A., Heid, M. W., Schafer, S., Nuber, U. A., Zimbelmann, R. and Klymkowsky, M. and Parr, B. (1995). The body language of cells: the Franke, W. W. (1994). Desmosomes and cytoskeletal architecture in intimate connection between cell adhesion and behavior. Cell 83, 5-8. epithelial differentiation: cell type-specific plaque components and Knudsen, K. A., Peralta Soler, A., Johnson, K. R. and Wheelock, M. J. intermediate filament anchorage. Eur. J. Cell Biol. 65, 229-245. (1995). Interaction of α-actinin with the cadherin/catenin cell-cell adhesion Schwarz, M. A., Owaribe, K., Kartenbeck, J. and Franke W. W. (1990). complex via α-catenin. J. Cell Biol. 130, 67-77 Desmosomes and ; Constitutive molecular components. Lewis, J. E., Jensen, P. J. and Wheelock, M. J. (1994). Cadherin function is Annu. Rev. Cell Biol. 6, 461-491. required for human keratinocytes to assemble desmosomes and stratify in Shapiro, L., Fannon, A. M., Kwong, P. D., Thompson, A., Lehmann, M. response to calcium. J. Invest. Dermatol. 102, 870-877. S., Grubel, G., Legrand, J.-F., Als-Neilsen, J., Colman, D. R. and Lewis, J. E., Wahl, J. K., Sass, K. M., Jensen P. J., Johnson, K. R. and Hendrickson, W. A. (1995). Structural basis of cell-cell adhesion by Wheelock, M. J. (1997). Cross-talk between adherens junctions and cadherins. Nature 374, 327-337. desmosomes depends on plakoglobin. J. Cell Biol. 136, 919-934. Takeichi, M. (1995). Morphogenetic roles of classic cadherins. Curr. Opin. Mattey, D. L. and Garrod, D. R. (1986). Splitting and internalization of the Cell Biol. 7, 619-627. desmosomes of cultured kidney epithelial cells by reduction in calcium Takeichi, M., Atsumi, T., Yoshida, C., Uno, K. and Okada, T. S. (1981). concentration. J. Cell Sci. 85, 113-124. Selective adhesion of embryonal carcinoma cells and differentiated cells by McNeill, H., Ryan, T. A., Smith, S. J. and Nelson, W. J. (1993). Spatial and Ca2+-dependent sites. Dev. Biol. 87, 340-350. temporal dissection of intermediate and early events following cadherin- Tomschy, A., Fauser, C., Landwehr, R. and Engel, J. (1996). Homophilic mediated epithelial cell adhesion. J. Cell Biol. 120, 1217-1226. adhesion of E-cadherin occurs by a co-operative two-step interaction of N- Nagafuchi, A. and Takeichi, M. (1988). Cell binding function of E-cadherin terminal domains. EMBO J. 15, 3507-3514. is regulated by the cytoplasmic domain. EMBO J. 7, 3679-3684. Troyanovsky, S. M., Eshkind, L. G., Troyanovsky, R. B., Leube, R. E. and Nagar, B., Overduin, M., Ikura, M. and Rini, J. M. (1996). Structural basis Franke, W. W. (1993). Contributions of cytoplasmic domains of of calcium-induced E-cadherin rigidification and dimerization. Nature 380, desmosomal cadherins to desmosome assembly and intermediate filament 360-364. anchorage. Cell 72, 561-574. Norvell, S. M. and Green, K. J. (1998). Contributions of extracellular and Troyanovsky, S. M., Troyanovsky, R. B., Eshkind, L. G., Krutovskikh, V. intracellular domains of full length and chimeric cadherin molecules to A., Leube, R. L. and Franke, W. W. (1994). Identification of the junction assembly in epithelial cells. J. Cell Sci. 111, 1305-1318. plakoglobin-binding domain in desmoglein and its role in plaque assembly Ozawa, M., Baribault, H. and Kemler, R. (1989). The cytoplasmic domain and intermediate filament anchorage. J. Cell Biol. 127, 151-160. of the uvomorulin associates with three independent Troyanovsky, S. M. and Leube, R. E. (1998). Molecular dissection of proteins structurally related in different species. EMBO J. 8, 1711-1717. desmosomal assembly and intermediate filament anchorage. Subcell. Ozawa, M., Engel, J. and Kemler, R. (1990a). Single amino acid Biochem. 31, 263-290. substitutions in one Ca2+-binding site of uvomorulin abolish the adhesive Volberg, T., Geiger, B., Kartenbeck, J. and Franke, W. W. (1986). Changes function. Cell 63, 1033-1038. in membrane-microfilament interaction in intercellular adherens junctions Ozawa, M., Ringwald, M. and Kemler, R. (1990b). Uvomorulin-catenin upon removal of extracellular Ca2+ ions. J. Cell Biol. 102, 1832-1842. complex formation is regulated by a specific domain in the cytoplasmic Volberg, T., Geiger, B., Citi, S. and Bershadsky, A. (1994). Effect of protein region of the cell adhesion molecule. Proc. Nat. Acad. Sci. USA 87, 4246- kinase inhibitor H-7 on the contractility, integrity and membrane anchorage 4250. of the microfilament system. Cell Motil. Cytoskel. 29, 321-338. Ozawa, M. and Kemler, R. (1997). The membrane-proximal region of the E- Volk, T., Volberg, T., Sabanay, I. and Geiger, B. (1990). Cleavage of A-CAM cadherin cytoplasmic domain prevents dimerization and negatively regulates by endogenous proteinases in cultured lens cells and in developing chick adhesion activity. J. Cell Biol. 142, 1605-1613. embryos. Dev. Biol. 193, 314-323. Pasdar, M., Li, Z. and Chan, H. (1995). Desmosome assembly and Wheelock, M. J. and Jensen, P. J. (1992). Regulation of disassembly are regulated by reversible protein phosphorylation in cultured intercellular junction organization and epidermal morphogenesis by E- epithelial cells. Cell Motil. Cytoskel. 30, 108-121. cadherin. J. Cell Biol. 117, 415-425. Pokutta, S., Herrenknecht, K., Kemler, R. and Engel, J. (1994). Yap, A. S., Brieher, W. M. and Gumbiner, B. M. (1997). Molecular and Conformational changes of the recombinant extracellular domain of E- functional analysis of cadherin-based adherens junctions. Annu. Rev. Cell cadherin upon calcium binding. Eur. J. Biochem. 223, 1019-1026. Dev. Biol. 13, 119-146.