The cell-adhesion protein E-cadheriduvomorulin exhibits a calcium-dependent homoassociation. The effect of Ca2+ on the extracellular fragment of E-cadherin was studied using the recombinant protein expressed in the baculovirus expression system. The recombinant and native fragment of E-cadherin were found to be similar by many biochemical criteria [Herrenknecht, K. & Kemler, R. (1993) J. Cell Sci. 17, 147-1541. A large and reversible conformational transition was observed upon Ca2+ depletion. A change from a rod-like structure, 22 nm in length, to a more globular assembly of the five subdomains became evident by electron-microscopical analysis. In the presence of Ca", the circular dichroic spectra indicated predominantly p-structure but a more negative ellipticity was observed in the absence of Ca2+. The intrinsic tryptophan fluorescence decreased by 12% upon Ca2+ depletion. Both effects were used for calcium titrations which indicated calcium binding to several sites with average Kd values of 45-150 pM. Cleavage of the protein fragment by trypsin occurred only at low Caz+ concentrations and from the calcium-dependence of cleavage rates, a Kd value of 24 pM was derived. The major site of cleavage was identified by partial sequencing to be located between the two putative calcium-binding sites in the third subdomain from the N-terminus. In agreement with earlier results with the native fragment, the recombinant protein did not associate in the presence or absence of Ca2+. We suggest the calcium-dependent homoassociation therefore depends on additional effects connected with the cell surface association of E-cadherin.

The cytoplasmic region of the Ca2+-dependent cell-adhesion molecule (CAM) uvomorulin associates with distinct cytoplasmic proteins with molecular masses of 102, 88, and 80 kDa termed a, (3, and ycatenin, respectively. This complex formation links uvomorulin to the actin filament network, which seems to be of primary importance for its cell-adhesion properties. We show here that antibodies against a catenin also immunoprecipitate complexes that contain human N-cadherin, mouse P-cadherin, chicken A-CAM (adherens junction-specific CAM; also called N-cadherin) or Xenopus U-cadherin, demonstrating that a catenin is complexed with other cadherins. In immunofluoresence tests, a catenin is colocalized with cadherins at the plasma membrane. However, in cadherin-negative Ltk-cells, a catenin is found uniformly distributed in the cytoplasm, suggesting some additional biological function(s). Expression of uvomorulin in these cells results in a concentration of a catenin at membrane areas ofcell contacts. We also have cloned and sequenced murine a catenin.The deduced amino acid sequence reveals a signfMcant homology to vinculin. Our results suggest the possibility of a new vinculin-related protein family involved in the cytoplasmic anchorage of cell-cell and cell-substrate adhesion molecules.The cadherin gene family of Ca2l-dependent cell adhesion molecules (CAM) was originally composed of a rather limited number of transmembrane glycoproteins of which the best studied examples were uvomorulin/E-cadherin, liver CAM (L-CAM), N-cadherin, and P-cadherin (for a review, see refs. 1 and 2). Each member was found to regulate cell adhesion of particular cell types, and this was thought to be fundamental for the organization of multicellular organisms. More recently new members of this family have been described including M-cadherin on mouse myoblasts (3), E/P-, U-, and XB-cadherin in early Xenopus development (4-6), and a new subgroup of more distantly related desmosomal glycoproteins (7-9).It has been shown that the cytoplasmic region of uvomorulin associates with defined proteins of 102, 88, and 80 kDa termed a, f3, and y catenin, respectively (10). The MATERIALS AND METHODSCell Lines. Mouse fibroblasts Ltk-, human HeLa, chicken fibroblasts CEF38, and their respective transfectants expressing mouse uvomorulin, Li-i, H1-3, and C1-4 (10, 15) were used as well as embryonal carcinoma cells F9, PCC4, and PAS5E. Porcine kidney LLC-PK7 and Xenopus A6 cells were gifts from H. Hoschutzky (Freiburg, F.R.G.) and D. Wedlich (Berlin), respectively. The A6 cells were grown in Leibovitz L-15 medium containing 8% (vol/vol) fetal calf serum (FCS) at 240C. All other cells were cultured in Dulbecco's modified Eagle's medium containing 10%o FCS at 370C in an atmosphere containing 10%6 Co2. For the generation of F9 tumors, about 1 x 107 cells were injected subcutaneously in 129/SV mice, and solid tumors were removed 12-15 days later and stored at -80TC.Puriication of a Catenin. Ten grams of solid F9 tumor was homogenized in 50 ml of No...

The calcium-dependent class of cell adhesion molecules known as cadherins mediate homotypic cell interactions in most epithelia. We have now investigated the expression and distribution of cadherins and cadherin-associated molecules in the developing and maturing rat testis. E-Cadherin was not detected in the seminiferous tubule at any time in development or in the adult. In contrast, Leydig cells expressed E-cadherin between day 15 of gestation and postnatal day 3. alpha- and beta-catenins were expressed throughout the developing testis, but were particularly prominent in Leydig cells. In the maturing testis, alpha-catenin and plakoglobin became progressively more restricted to the basal part of the seminiferous epithelium and by 23 days exhibited a pattern characteristic of the Sertoli cell junctional complex. beta-Catenin recruitment to the Sertoli cell junctional complex was not complete until 60 days. alpha-Catenin and plakoglobin were not present at sites of Sertoli cell-germ cell contacts. Northern blot analysis of testicular RNA showed three mRNA species hybridizing with N-cadherin cDNA. A pan-cadherin antibody specific for a region of the highly conserved C-terminal of all cadherins stained sites of Sertoli-spermatocyte and Sertoli-round spermatid contact in the adult rat seminiferous epithelium, but did not stain the Sertoli cell tight junctional complex. Western blots of testicular extracts indicated that the molecule(s) recognized by these antibodies had an approximate molecular mass of 120 kilodalton, typical of members of the cadherin family. Therefore, although Sertoli cells do not express E-cadherin, another member(s) of the cadherin family is present in the testis, but may not be directly involved in tight junction dynamics as in other cells. Instead, cadherin-mediated adhesion is likely to be involved in Sertoli cell-germ cell interactions. As catenins are not present at these sites, our results suggest a catenin-independent role of cadherins in germ cell adhesion to Sertoli cells.

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Molecular analysis of the cadherin-catenin complex elucidated the central role of beta-catenin in this adhesion complex, as it binds to the cytoplasmic domain of E-cadherin and to alpha-catenin. beta-Catenin may also function in signalling pathways, given its homology to the gene product of the Drosophila segment polarity gene armadillo, which is known to be involved in the wingless signalling cascade. To study the function of beta-catenin during mouse development, gene knock-out experiments were performed in embryonic stem cells and transgenic mice were generated. beta-Catenin null-mutant embryos formed blastocysts, implanted and developed into egg-cylinder-stage embryos. At day 7 post coitum, the development of the embryonic ectoderm was affected in mutant embryos. Cells detached from the ectodermal cell layer and were dispersed into the proamniotic cavity. No mesoderm formation was observed in mutant embryos. The development of extraembryonic structures appeared less dramatically or not at all affected. Our results demonstrate that, although beta-catenin is expressed rather ubiquitously, it is specifically required in the ectodermal cell layer.