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© The Rockefeller University Press,
0021-9525/1997//1109 $5.00
The Journal of Cell Biology, Volume 136, Number 5,
, 1997 1109-1121
Article |
LI-Cadherin–mediated Cell–Cell Adhesion Does Not Require Cytoplasmic Interactions
Department of Biochemistry, University of Ulm, Germany, Department of Anatomy and Cell Biology, University of Michigan, Ann Arbor, Michigan 48109
The adhesive function of classical cadherins depends on the association with cytoplasmic proteins, termed catenins, which serve as a link between cadherins and the actin cytoskeleton. LI-cadherin, a structurally different member of the cadherin family, mediates Ca2+-dependent cell–cell adhesion, although its markedly short cytoplasmic domain exhibits no homology to this highly conserved region of classical cadherins. We now examined whether the adhesive function of LI-cadherin depends on the interaction with catenins, the actin cytoskeleton or other cytoplasmic components. In contrast to classical cadherins, LI-cadherin, when expressed in mouse L cells, was neither associated with catenins nor did it induce an upregulation of β-catenin. Consistent with these findings, LI-cadherin was not resistant to detergent extraction and did not induce a reorganization of the actin cytoskeleton. However, LI-cadherin was still able to mediate Ca2+dependent cell–cell adhesion.
To analyze whether this function requires any interaction with proteins other than catenins, a glycosyl phosphatidylinositol–anchored form of LI-cadherin (LI-cadherinGPI) was constructed and expressed in Drosophila S2 cells. The mutant protein was able to induce Ca2+-dependent, homophilic cell–cell adhesion, and its adhesive properties were indistinguishable from those of wild type LI-cadherin. These findings indicate that the adhesive function of LI-cadherin is independent of any interaction with cytoplasmic components, and consequently should not be sensitive to regulatory mechanisms affecting the binding of classical cadherins to catenins and to the cytoskeleton. Thus, we postulate that the adhesive function of LI-cadherin is complementary to that of coexpressed classical cadherins ensuring cell–cell contacts even under conditions that downregulate the function of classical cadherins.
Abbreviations used in this paper: CRD, cross-reacting determinant; GPI, glycosyl phosphatidylinositol; PCMBS, p-chloromercuriphenylsulfonic acid; PI-PLC, phosphatidylinositol-specific phospholipase C.
The current address of D. Berndorff is Research Laboratories of Schering AG, Müllerstr. 178, 13342 Berlin, Germany.
Cadherins are a multifunctional family of transmembrane glycoproteins mediating Ca2+-dependent adhesion of adjacent cells in a homophilic manner (Takeichi, 1988, 1991; Geiger and Ayalon, 1992; Kemler, 1993). Members of this family have been reported to be involved in morphogenesis (Takeichi, 1995), the development of junctional complexes and cell polarity (Nelson, 1992), invasiveness and metastasis (Birchmeier and Behrens, 1994), and most recently, transmembrane transport (Dantzig et al., 1994; Thomson et al., 1995).
Classical cadherins are composed of a highly conserved cytoplasmic domain of 
160 amino acids, a single transmembrane domain, and a large extracellular portion that is organized in a series of five structurally related tandem repeats (Ranscht, 1994). The conserved intracellular domain of classical cadherins is known to associate with a group of cytoplasmic proteins, termed catenins (Ozawa et al., 1989), which serve as a link between cadherins and the cortical cytoskeleton (Hirano et al., 1987). As demonstrated by several experiments, the formation of complexes with catenins is essential for cadherins to function as adhesion molecules. First evidence for the crucial role of this association came from studies, in which cadherins were rendered nonfunctional by COOH-terminal truncations affecting the catenin-binding site (Nagafuchi and Takeichi, 1988, 1989; Ozawa et al., 1989, 1990). Furthermore, in nonadhesive PC9 cells lacking 
-catenin, strong cell–cell adhesion could be restored by transfection with -catenin cDNA indicating that the expression of -catenin is required for the adhesive function of cadherins (Hirano et al., 1992). -Catenin is homologous to vinculin (Herrenknecht et al., 1991; Nagafuchi et al., 1991) and is a candidate for linking the cadherin /catenin complex to the actin-based cytoskeleton (Ozawa et al., 1990; Nagafuchi et al., 1994). β-Catenin exhibits homology to plakoglobin, a component of desmosomal plaques and adherens junctions (Cowin et al., 1986), and to the product of the Drosophila segment polarity gene armadillo (McCrea et al., 1991; Butz et al., 1992; Peifer et al., 1992). The primary structure of 
-catenin has not yet been established, but there is growing evidence that it might be identical to plakoglobin (Knudsen and Wheelock, 1992; Peifer et al., 1992; Piepenhagen and Nelson, 1993). Like the armadillo protein, β-catenin is thought to be involved in signal transduction and developmental patterning (reviewed by Gumbiner, 1995; Kühl and Wedlich, 1996). Recent studies suggested that β-catenin might be a target molecule for the regulation of cadherin function, since epithelial cells transformed with the v-Src tyrosine kinase acquired a more mesenchymal morphology, that was correlated with a strong phosphorylation of β-catenin and the perturbation of cadherin activity (Matsuyoshi et al., 1992; Behrens et al., 1993; Hamaguchi et al., 1993). A similar change in morphology could be induced by treatment with EGF or hepatocyte growth factor/scatter factor, which caused tyrosine phosphorylation of β-catenin as well as of plakoglobin (Weidner et al., 1990; Shibamoto et al., 1994). These observations suggest that tyrosine phosphorylation of catenins affects cadherin- mediated cell–cell adhesion.
Recently, LI-cadherin was characterized as a novel member of the cadherin family specifically expressed in polarized epithelia of liver and intestine (Berndorff et al., 1994). In intestinal epithelial cells, LI-cadherin is evenly distributed over the lateral contact zones but is excluded from adherens junctions, whereas coexpressed E-cadherin is concentrated in this specialized membrane region. LIcadherin exhibits an unusual structure, since its extracellular domain is composed of seven cadherin-type repeats instead of five typical for classical cadherins. In addition, its short cytoplasmic domain consists of only 20 amino acids exhibiting no homology to this highly conserved region of classical cadherins. Nevertheless, LI-cadherin was shown to act as a functional Ca2+-dependent cell adhesion molecule when expressed in Drosophila S2 cells (Berndorff et al., 1994).
The strikingly divergent structure of the cytoplasmic domain of LI-cadherin prompted us to investigate whether this region is of similar importance for the adhesive function of LI-cadherin as it is for classical cadherins. The general relevance of this question is emphasized by the recent discovery of two cadherins, HPT-1 (Dantzig et al., 1994) and Ksp-cadherin (Thomson et al., 1995), which are homologous to LI-cadherin, and may thus together constitute a new subfamily of cadherins. Our results show that LI-cadherin is neither associated with catenins, nor is it tightly connected to the actin-based cytoskeleton. Nevertheless, LI-cadherin is able to mediate Ca2+-dependent cell–cell adhesion of transfected L cells even after disruption of the actin cytoskeleton. We were able to demonstrate that the adhesive properties of LI-cadherin are fully retained in a construct, in which the transmembrane and the cytoplasmic domain have been exchanged for a glycosyl phosphatidylinositol (GPI)1 anchor. Apparently, the cell–cell adhesion mediated by LI-cadherin is independent of any direct interactions with cytoplasmic components. Since it cannot be affected by the same mechanisms and interactions controlling the function of classical cadherins, we assume that the adhesive function of LI-cadherin is complementary to that of coexpressed classical cadherins.
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Materials and Methods |
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Cell Culture and Transfection
Parental mouse L cells (obtained from Amer. Type Culture Collection, Rockville, MD, No. CCL-1.3) were grown in DMEM supplemented with 10% FCS. Transfected cells were grown in the same medium in the presence of 0.2 mg/ml of G418 (Gibco BRL, Eggenstein, FRG). L cells were transfected with pRc/LIC by a modified calcium phosphate method. Briefly, 1 µg of the expression vector was precipitated and added to 0.5 x 106 cells grown on a 60-mm dish. After incubation for 5 h, cells were washed and were allowed to recover for 48 h in fresh medium. Transfected cells were selected in the presence of 1 mg/ml G418, and clones were established using cloning rings. Several LI-cadherin–expressing clones were isolated, and three clones, 12.1.10, 14.3.4, and 17.11.7, expressing approximately the same amount of LI-cadherin as assessed by Western blot analysis were used for subsequent experiments. For each of these clones identical results were obtained. Although the cells were truly clonal, expression of LI-cadherin in all isolated clones was unstable and LI-cadherin–negative cells appeared after several passages. To obtain a large number of cells expressing LI-cadherin at the same level, fluorescence activated cell sorting was used. For each separation, 1.0 x 107 cells were washed with PBS, detached with 2 mM EDTA in PBS containing 2% chicken serum, harvested by centrifugation, and resuspended in 1 ml of a 1:2-dilution of DMEM, 8% FCS in PBS (DMEM/PBS). Cells were incubated with 40 µg/ml anti–LI-cadherin pAb120 for 60 min at 4°C. After washing in DMEM/PBS, cells were resuspended in 1 ml of the same buffer supplemented with FITC-conjugated goat anti–rabbit antibodies (Sigma) and incubated for 45 min at 4°C in dark. Cells were then washed three times in PBS, resuspended in 1 ml FCS-free DMEM, and kept on ice until being separated on a FACS VantageTM System (Becton Dickinson). As a control, cells were incubated with DMEM/PBS followed by an incubation with the same FITC-labeled secondary antibodies. Cells were gated using forward versus side scatter to exclude dead cells and debris. Only those cells showing the highest expression levels of LI-cadherin (10% of the total population) were isolated and plated directly on glass coverslips in 24-well plates. L cells expressing Xenopus XB/U-cadherin were generated as described elsewhere (Kühl et al., 1996).
Drosophila (S2) cells (Schneider, 1972) were grown in revised Schneider's medium (Gibco BRL) supplemented with 12.5% FCS (Sigma). Cells were maintained at 25°C with air as the gas phase. For transfection, the expression vectors pRmHa-LI or pRmLIGPI were mixed at a molecular ratio of 10:1 with pPC4, a plasmid conferring -amanitin resistance as the selectable marker (Jokerst et al., 1989), and coprecipitated with calcium phosphate according to Sambrook et al. (1989). Cells (107 in a 60-mm-dish) were incubated overnight with the precipitate, washed, and were allowed to recover for 72 h in fresh medium. After 3 wk of selection in medium containing 5 µg/ml -amanitin (Sigma), transfected cells were cloned in 0.3% soft agar as described previously (Berndorff et al., 1994). Individual clones were induced with 0.7 mM CuSO4 for 2–3 d and were assayed by Western blotting for high protein expression. The clones with highest expression of LI-cadherin or LI-cadherinGPI were designated S2/LI-cad and S2/LI-cadGPI, respectively, and were used for all subsequent experiments.
Construction of cDNA Expression Vectors
Full-length cDNA of rat LI-cadherin was excised from plasmid pTB2 (Berndorff et al., 1994) by digestion with NotI and partial digestion with ApaI, and was inserted into NotI/ApaI-restricted pRc/CMV (Invitrogen, NV Leek, NL). The resultant plasmid was designated pRc/LIC.
For the construction of a GPI-anchored form of LI-cadherin, a 2.5-kb cDNA fragment encoding the first 789 amino acids of LI-cadherin was isolated from pRmHa-LI (Berndorff et al., 1994) by digestion with KpnI and partial digestion with AccI. A DNA fragment encoding the Drosophila fasciclin I GPI anchor signal (Zinn et al., 1988) was adapted by PCR from a fasciclin I cDNA in pBluescript SK/+ using primer I (5'-CAACGTATACGGCCCGATGTTG-3') and primer II (5'-GCGGATCCGGATTTGTTTTTACATATCGG-3'). Primer I is identical to the coding strand of the fasciclin I cDNA (nucleotides 1967–89) but causes the deletion of one nucleotide to adopt the correct reading frame. Primer II introduces the underlined BamHI restriction site at the 3' end of the PCR product. The PCR product was digested with AccI and BamHI and was ligated in tandem with the 2.5-kb KpnI/Acc I-fragment of pRmHa-LI into KpnI/BamHI-restricted pRmHa-3. The correct ligation product, the plasmid pRmLIGPI, was verified by DNA sequencing of both strands using the dideoxy method (Sanger et al., 1977).
SDS-PAGE and Western Blotting
SDS-PAGE was performed according to Laemmli (1970) and proteins were electrophoretically transferred to HybondTM-C membranes (Amersham Buchler GmbH, Braunschweig, FRG). Membranes were blocked for 1 h in TBST (25 mM Tris/HCl, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.1% Tween 20) containing 5% nonfat dry milk, incubated for 1 h each with primary antibody and the appropriate peroxidase-conjugated secondary antibody (both in TBST, 5% nonfat dry milk), and were developed with the ECL detection system (Amersham). For reprobing with another antibody, membranes were stripped overnight at 42°C in 65 mM Tris/HCl, pH 6.6, containing 2% SDS and 100 mM β-mercaptoethanol, washed thoroughly with TBST, blocked, and processed as described above.
Immunocytochemistry
L cells were grown to confluency on glass coverslips, fixed in a fresh solution of PLP (26 mM Na-phosphate, pH 7.4, 10 mM NaIO4, 94 mM lysine, 2% paraformaldehyde) for 20 min at room temperature and rinsed in PBS containing 0.1 M glycine. For the staining of cytoplasmic proteins, cells were permeabilized for 5 min with 0.2% Triton X-100 in PBS. After washing with PBS, the cells were incubated for 30 min in blocking buffer (PBS, 1% FCS, 1% BSA). Incubation with primary antibody was in blocking buffer for 1 h, followed by washing and incubation with fluorophore-conjugated secondary antibody (in blocking buffer) for 1 h. After washing, cells were mounted in Elvanol and examined using a Zeiss Axiophot fluorescence microscope. For detergent extraction, cells were preincubated for 5 min at 4°C in PBS containing 5% NP-40, washed in PBS, and processed as described above.
Immunofluorescence microscopy of S2 cells was performed as described previously (Berndorff et al., 1994). Briefly, cells were harvested after aggregation, washed twice with TBS/C (25 mM Tris/HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 2 mM CaCl2) and fixed at room temperature for 15 min in TBS/C containing 3.5% formaldehyde. Fixed cells were washed and stained with FITC-labeled anti–LI-cadherin pAb120 for 1 h. The cells were finally resuspended in fluorescence buffer (885 mM Tris/HCl, pH 8.0, 0.5% n-propyl gallate, 10% glycerol) and mounted on slides.
Cell Adhesion Assays
Aggregation assays with L cells were performed as described previously (Ozawa et al., 1990). Briefly, cells were washed and treated with 0.01% trypsin in HBS (10 mM Hepes, pH 7.4, 37 mM NaCl, 5.4 mM KCl, 0.34 mM Na2HPO4, 5.6 mM glucose) containing 2 mM CaCl2 for 10 min at 37°C. After washing in a 1:2 dilution of DMEM (containing 4% FCS) in HBS, cells were resuspended in the same buffer supplemented with 5 µg/ml DNase I. The single cell suspension (5.0 x 105 cells in 500 µl) was allowed to aggregate for 30 min at room temperature in 24-well plates on a rotary shaker (80 rpm). Aggregation was either performed in buffer without additive, or in buffer supplemented with 2 mM EDTA or with anti–LI-cadherin pAb120. To disrupt the cytoskeleton before the aggregation assay, cells were preincubated with 1 µM cytochalasin D for 30 min at 37°C.
Transfected S2 cells were induced with 0.7 mM CuSO4 for 2 d at 25°C, collected by centrifugation and resuspended in Schneider's medium to a density of 1.0 x 106 cell/ml. Cells were gently dissociated by pipetting and 500 µl of the single cell suspension were agitated at room temperature for 1 h in 24-well plates on a rotary shaker (80 rpm). Aggregation assays were performed in Schneider's medium (containing 5 mM CaCl2) or in the same medium supplemented with either 30 mM EDTA or with anti–LIcadherin pAb120. For pretreatment with PI-PLC, the cell suspension (5.0 x 105 cells in 250 µl) was incubated with 1 U/ml PI-PLC from B. thuringiensis for 2 h at 37°C. Subsequently the cell suspension was diluted to 1.0 x 106 cells/ml with medium and the extent of aggregation was measured as described previously (Berndorff et al., 1994).
In cell mixing experiments, one cell line was labeled in vivo by adding 1% (vol/vol) of the fluorescent membrane dye DiI (0.5 mM stock solution in ethanol) to the cell suspension. After incubation for 15 min at 37°C, excess dye was removed by washing the cells twice in PBS. Cells were resuspended in medium to a density of 1.0 x 106 cells/ml, mixed with unlabeled cells, induced, and agitated on a rotary shaker (80 rpm) for 16 h at room temperature.
Isolation of Membrane Proteins
S2 cells were induced as described and harvested by centrifugation. About 2 x 107 cells were resuspended in 1 ml TBS/C containing 2% protease inhibitor mix (1 mg/ml leupeptin, pepstatin A, and chymostatin, each), as well as 5 mM p-chloromercuriphenylsulfonic acid (PCMBS) to inhibit any endogenously expressed PI-PLC activity in S2 cells. Cellular membranes were prepared as described (Hortsch, 1994) and their protein content was determined using the BCA protein assay (Pierce, Rockford, IL).
Treatment with PI-specific Phospholipase C
To remove trace amounts of PCMBS before PI-PLC digestion, cellular membranes containing 100 µg protein were washed twice with TBS, pH 7.4, and resuspended in 49 µl TBS, pH 7.4 containing 2 mM DTT, 2.5 mM EDTA, 0.2% Triton X-100 and 2% protease inhibitor mix. After addition of 1 µl of PI-PLC from T. brucei (generously provided by Dr. Peter Overath, Max-Planck-Institute of Biology, Tübingen, Germany), samples were incubated for 4 h at room temperature, mixed with 50 µl 2 x SDS sample buffer and boiled for 5 min. Proteins were separated by SDS-PAGE and subjected to Western blot analysis as described.
Metabolic Labeling and Immunoprecipitation
To collect radiolabeled immunoprecipitates, L cells were incubated with 5 MBq TRAN35S-label (ICN Biomedicals GmbH, Eschwege, FRG) in methionine-free MEM (Gibco BRL) for 16 h. Cells were washed and lysed in 500 µl extraction buffer (0.5% NP-40, 0.5% Triton X-100, 2 mM PMSF, 2 mM CaCl2, 2% protease inhibitor mix in TBS, pH 8.0) for 2 h at 4°C. Lysates were cleared by centrifugation and incubated with primary antibody (50 µg pAb120 or 5 µg 6D5) for 1 h. Immune complexes were incubated for 1 h with 50 µl of a 10% protein A–Sepharose suspension. Beads were washed three times in washing buffer (50 mM Tris/HCl, pH 8.5, 500 mM NaCl, 1 mM MgCl2, 2 mM CaCl2, 0.05% NP-40 containing 1 mg/ml ovalbumin), and finally boiled in SDS sample buffer. The dissociated proteins were separated by SDS-PAGE and analyzed by fluorography using the Entensify Universal Autoradiography Enhancer (DuPont New England Nuclear®, Bad Homburg, FRG).
For metabolic labeling of S2 cells, 107 cells were washed twice with TBS, pH 7.4, resuspended in 5 ml medium containing 100 µCi [1-3H]ethanolamine hydrochloride and induced with 0.7 mM CuSO4. After an overnight incubation, the cells were lysed and labeled proteins were analyzed by immunoprecipitation and fluorography as described above.
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Results |
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120 kD (Fig. 1 A) representing N-glycosylation variants of LI-cadherin as could be shown by PNGase F-digestion (not shown). No proteins were stained in nontransfected L cells (Fig. 1 A). To determine the distribution of LI-cadherin in transfected L cells, immunofluorescence staining using anti–LI-cadherin mAb 47.2 was performed. Although LI-cadherin was expressed on the cell surface and appeared concentrated at sites of cell–cell contact (Fig. 1 B, b), the cells did not acquire the cobblestonelike appearance of L cells expressing classical XB/U-cadherin (Fig. 1 B, c). While nontransfected L cells showed the typical spindle-shaped morphology of fibroblasts (Fig. 1 B, d), expression of LI-cadherin induced a small change of this phenotype, resulting in extended regions of cell–cell contact (Fig. 1 B, e). L cells expressing XB/U-cadherin exhibited a rather epithelial phenotype and appeared tightly connected with cell–cell contacts being barely visible in phase contrast views (Fig. 1 B, f). In contrast, LI-cadherin– transfected cells never formed an entirely closed monolayer even when grown to confluency (Fig. 1 B, e).
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- and β-catenin could be coprecipitated with XB/U-cadherin (Fig. 2, lane 4). In contrast, no proteins were coprecipitated under the same conditions with LI-cadherin using anti–LI-cadherin pAb120 (Fig. 2, lane 2). It has been suggested that the introduction of cateninbinding sites into L cells, due to the transfection with classical cadherins, either induces the upregulation of expression or leads to a reduced degradation of catenins (Nagafuchi et al., 1991, 1994; Shibamoto et al., 1995). Therefore, we determined whether the cellular concentration of β-catenin is influenced by the expression of LI-cadherin in transfected L cells. As shown in Fig. 3, the expression level of β-catenin in L cells remained unchanged after transfection with LI-cadherin cDNA (Fig. 3, lanes 4 and 5) while it was significantly elevated in cells expressing XB/U-cadherin (Fig. 3, lane 6). The combined results of both experiments indicate that LI-cadherin is not able to interact with catenins when expressed in L cells.
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50–100 cells (Fig. 6 a). Aggregation could be completely inhibited by the removal of Ca2+ with EDTA or by incubation with anti–LIcadherin pAb120 (Fig. 6, b and c). However, disruption of the actin-based cytoskeleton by preincubation with cytochalasin D had no effect on LI-cadherin–mediated cell aggregation (Fig. 6 d), whereas XB/U-cadherin–expressing cells remained disperse under these conditions (not shown). These results demonstrate that LI-cadherin is a functional Ca2+-dependent cell adhesion molecule when expressed in L cells.
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Construction of GPI-anchored LI-CadherinGPI
An artificial GPI-anchored form of LI-cadherin (LI-cadherinGPI) was generated, thus excluding any direct interaction of the mutant protein with cytoplasmic components (Fig. 7). In the fusion protein the extracellular domain of LI-cadherin is linked directly to the GPI anchor signal sequence of fasciclin I, a homophilic neural cell adhesion molecule expressed on a subset of fasciculating axons in both, the grasshopper and the Drosophila embryo (Zinn et al., 1988; Elkins et al., 1990; Hortsch and Goodman, 1990). When processed correctly, LI-cadherinGPI should contain the complete extracellular domain of LI-cadherin, followed by the last 28 amino acids of mature fasciclin I and the carboxyterminally linked GPI-anchor. Since the domains responsible for the adhesive function of fasciclin I are located near the amino terminus (Seeger, M., personal communication), it can be ruled out that the small carboxy-terminal fasciclin I–derived portion does contribute to the adhesive properties of the fusion protein.
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LI-CadherinGPI Is Expressed as a GPI-anchored Integral Membrane Protein in S2 Cells
To examine whether LI-cadherinGPI is correctly processed and bound to the plasma membrane via a GPI anchor, detergent-treated membrane fractions from parental and transfected S2 cells were incubated with PI-specific phospholipase C (PI-PLC) from T. Brucei, separated by SDSPAGE, and analyzed by immunoblotting. Staining with anti–LI-cadherin pAb120 showed that LI-cadherin and LI-cadherinGPI were expressed in similar amounts by the clonal cell lines S2/LI-cad and S2/LI-cadGPI (Fig. 8 A, lanes 3 and 5). Both proteins have an apparent molecular mass of 110 kD which was not significantly changed upon PIPLC treatment (Fig. 8 A, lanes 3–6). No immunoreactive proteins were found in membranes of untransfected S2 cells (Fig. 8 A, lanes 1 and 2). The blot was stripped and reprobed with a polyclonal antibody (anti-CRD pAb) against the cross-reacting determinant of GPI anchors, an epitope which is exposed in GPI-anchored molecules solely after digestion with PI-PLC (Zamze et al., 1988). CRD-specific antibodies were unable to detect any membrane proteins produced by either untransfected or S2/LIcad cells, irrespective of PI-PLC treatment (Fig. 8 A, lanes 7–10). Likewise, undigested membranes from S2/LI-cadGPI cells did not contain any immunoreactive proteins (Fig. 8 A, lane 11). However, after incubation with PI-PLC, a single protein band was detected in these membranes at 110 kD (Fig. 8 A, lane 12), indicating that LI-cadherinGPI is correctly processed in Drosophila S2 cells and is recognized as a substrate by PI-specific PLC.
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Taken together these experiments demonstrate that LIcadherinGPI is correctly processed and expressed in S2 cells. It is attached to the plasma membrane via an intact GPI anchor that is susceptible to cleavage by PI-PLC.
The Adhesive Function Is Preserved in LI-CadherinGPI
To quantitatively compare the cell adhesion activity of native and GPI-anchored LI-cadherin, a cell adhesion assay was performed, and aggregation was calculated as percent reduction in particle number over an incubation period of 60 min. In the presence of Ca2+, LI-cadherinGPI mediated cell–cell adhesion to the same extent as wild-type LI-cadherin (Fig. 9, Ca2+). Under these conditions, no significant aggregation of untransfected S2 cells was observed (Fig. 9, control). Addition of EDTA or anti–LI-cadherin pAb120 entirely inhibited the aggregation of both S2/LI-cad and S2/LI-cadGPI cells. The complete inhibition of LI-cadherinGPI-mediated cell–cell adhesion by anti–LI-cadherin antibodies and its strict Ca2+ dependence rules out that the fasciclin I–derived portion of the fusion protein is contributing to its adhesive function.
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Discussion |
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To examine whether the cytoplasmic domain is of similar importance for the adhesive function of LI-cadherin as it is for classical cadherins, we analyzed the interaction of LI-cadherin with cytoplasmic components in transfected L cells. In contrast to classical cadherins (Nagafuchi and Takeichi, 1989; Ozawa et al., 1989), no catenins or other copurified proteins were found in LI-cadherin immunoprecipitates from metabolically labeled cells. Furthermore, expression of LI-cadherin did not induce the upregulation of β-catenin expression observed for classical cadherins (Nagafuchi et al., 1991, 1994; Shibamoto et al., 1995). These observations demonstrate that the cytoplasmic domain of LI-cadherin is not associated with catenins. This can be explained by the lack of homology of this domain to the recently identified region of E-cadherin, which is essential for the interaction with catenins (Stappert and Kemler, 1994). It has been reported that nonfunctional cadherin molecules without catenin-binding activity can be easily extracted with nonionic detergents, while intact cadherins are resistant to this treatment due to their ability to interact with the cytoskeleton (Nagafuchi and Takeichi, 1988; Ozawa et al., 1989). We have found that LI-cadherin can be completely extracted with NP-40 under conditions where significant amounts of the classical XB/U-cadherin remain attached to the cytoskeleton. In addition, while classical cadherins colocalize with actin (Hirano et al., 1987; see Fig. 5) and induce a redistribution of cytoskeletal proteins to the plasma membrane (McNeill et al., 1990), expression of LI-cadherin in transfected L cells did not result in a reorganization of the actin cytoskeleton. These results clearly demonstrate that LI-cadherin is not firmly attached to the actin cytoskeleton. This is consistent with the finding that the morphology of transfected L cells was only slightly changed due to the expression of LI-cadherin. Although sites of cell–cell contact were enlarged, LI-cadherin did not induce the epithelial phenotype adopted by L cells expressing classical cadherins. Despite these obvious differences, LI-cadherin was capable of mediating Ca2+-dependent cell–cell adhesion. In contrast to classical cadherins, however, adhesion by LI-cadherin was independent from an intact actin cytoskeleton.
There are two possible explanations for the ability of LIcadherin to mediate cell–cell adhesion without binding to the cytoskeleton via catenins: One is based on a recently proposed model, in which the adhesive forces of individual cadherin molecules are bundled in a so-called "adhesion zipper" (Shapiro et al., 1995). Each element of the zipper is believed to consist of a cadherin dimer stabilized by hydrophobic interactions between adjacent cadherin molecules of one cell. In this respect it is conceivable that the lateral association of the aligned molecules is strengthened by the two additional cadherin-type repeats present in the extracellular domain of LI-cadherin. This stabilization may compensate for the missing intracellular linkage to the cytoskeleton, which induces the clustering of classical cadherins in adherens junctions. This hypothesis is subject of current investigations.
The second possibility is that the clustering of LI-cadherin molecules is promoted by the interaction with auxiliary proteins, which bind to the short cytoplasmic domain of LI-cadherin but do not coprecipitate under standard conditions. This is conceivable, since other adhesion molecules containing only short intracellular domains have been reported to associate with cytoplasmic proteins, and thus become linked to the cytoskeleton. For example, the cytoplasmic 47 amino acids of β1 integrin are able to bind -actinin (Otey et al., 1990), as well as paxillin and pp125FAK (Schaller et al., 1995). Furthermore, L-selectin has recently been shown to interact directly with -actinin although its predicted cytoplasmic domain contains only 17 amino acids (Pavalko et al., 1995).
To test the second hypothesis, a GPI-anchored form of LI-cadherin (LI-cadherinGPI) was constructed, thus excluding any interaction with cytoplasmic components. A similar approach has been used to demonstrate that the homophilic adhesive activity of Drosophila neuroglian is independent of its intracellular interaction with ankyrin (Hortsch et al., 1995). No interaction of a GPI-anchored neuroglian with the membrane cytoskeleton has been observed (Dubreuil et al., 1996). In the present report the transmembrane and cytoplasmic domains of LI-cadherin were exchanged for the GPI anchor signal sequence of fasciclin I (Zinn et al., 1988). When expressed in Drosophila S2 cells, which have been used successfully for the functional analysis of both LI-cadherin (Berndorff et al., 1994) and fasciclin I (Elkins et al., 1990), LI-cadherinGPI was correctly processed and linked to a GPI anchor. Despite the obvious lack of cytoplasmic interactions, LI-cadherinGPI mediated Ca2+-dependent adhesion of transfected S2 cells to the same extent as wild-type LI-cadherin. Aggregation could be suppressed by calcium withdrawal, addition of LI-cadherin–specific pAb120 or preincubation with PIPLC. Using cell mixing experiments we were able to show that cell–cell adhesion induced by LI-cadherinGPI was homophilic, and that the binding specificity was not affected by the type of membrane attachment. Apparently, the adhesive function of LI-cadherin is independent of any interaction with cytoplasmic components. Thus, it can be concluded that the structure of the extracellular domain alone is capable to support the adhesive properties of LI-cadherin.
What are the physiological implications of a cadherin that mediates cell–cell adhesion without binding to the cytoskeleton? LI-cadherin is specifically expressed in liver and intestine, where it is found exclusively on the lateral surface of polarized cells outside of adherens junctions and desmosomes (Berndorff et al., 1994). In contrast, E-cadherin is found in the same cells preferentially in adherens junctions, but to some extent also on the basolateral surface (Boller et al., 1985). Enterocytes of the intestinal epithelium are derived from highly proliferative stem cells residing in the crypts of Lieberkühn and differentiate as they migrate into the villus region (Gordon, 1989). Interestingly, undifferentiated crypt cells from the adult chicken small intestine contain 15-fold higher levels of tyrosine phosphorylated proteins than do differentiated enterocytes (Burgess et al., 1989). Furthermore, a high level of pp60c-src activity has been observed in dividing intestinal crypt cells, and the activity of this tyrosine kinase decreases during migration of enterocytes to the apex of the villus (Cartwright et al., 1993). Tyrosine phosphorylation of catenins by members of the src-family, which are found enriched in adherens junctions (Tsukita et al., 1991), is correlated with the inhibition of cell–cell adhesion mediated by classical cadherins (Matsuyoshi et al., 1992; Behrens et al., 1993; Hamaguchi et al., 1993), and with disintegration of adherens junctions (Volberg et al., 1992). Consequently, the adhesive function of E-cadherin should be reduced in undifferentiated enterocytes. Since LI-cadherin lacks cytoplasmic tyrosine residues and mediates cell–cell adhesion independent of catenin binding, its adhesive function should neither be affected by cadherin nor by catenin tyrosine phosphorylation. We thus propose a model in which the adhesive function of LI-cadherin is complementary to that of classical cadherins ensuring cell–cell adhesion throughout the entire enterocyte differentiation pathway even under conditions that cause downregulation of classical cadherins. This view is supported by the analysis of transgenic mice, which developed inflammatory bowel disease as a result of intestinal epithelial-specific expression of a mutated N-cadherin lacking the extracellular domain (Hermiston and Gordon, 1995a,b). Despite the complete loss of E-cadherin function, the intestinal epithelium was only partially disrupted, and the enterocytes remained attached at their lateral sides.
Still, it cannot be excluded that LI-cadherin is able to laterally associate with yet unknown proteins. This consideration gains further support by the recent finding that LIcadherin is the rat homologue (Böttinger, A., A. Volz, B. Kreft, C. Fieger, D. Patschan, N. Schnoy, R. Geßner, and R. Tauber, manuscript in preparation) of HPT-1, a protein involved in proton-dependent peptide transport across the intestinal epithelium (Dantzig et al., 1994). Moreover, a second cadherin with homologous structure, Ksp-cadherin, has been reported to be associated with a renal Na+/HCO3– cotransporter (Thomson et al., 1995). This opens the possibility that LI-cadherin, in addition to its adhesive function, might be associated with other transport proteins. Together with its apparent complementary function and its different extracellular structure, this clearly distinguishes LI-cadherin from GPI-anchored chicken T-cadherin (Ranscht and Dours-Zimmermann, 1991), the only other known cadherin that mediates Ca2+-dependent cell– cell adhesion independent of interactions with the cytoskeleton (Vestal and Ranscht, 1992).
In summary, we were able to show that LI-cadherin is neither associated with catenins nor firmly linked to or dependent on an intact actin cytoskeleton. In sharp contrast to classical cadherins, cell–cell adhesion mediated by LIcadherin is independent of any interaction with cytoplasmic components. We postulate that the adhesive function of LI-cadherin is complementary to that of coexpressed classical cadherins, and therefore may be important in the formation and maintenance of epithelial integrity in liver and intestine.
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Acknowledgments |
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This work was supported by a graduate scholarship (NaFöG) to Bertolt Kreft and grants from the Deutsche Forschungsgemeinschaft (SFB 366, Teilprojekt C2) and the Sonnenfeld-Stiftung.
Submitted: 18 June 1996
Revised: 22 November 1996
Please address all correspondence to Dr. R. Geßner, Institut für Klinische Chemie und Biochemie, Virchow-Klinikum der Humboldt-Universität zu Berlin, Augustenberger Platz 1, D-13353 Berlin, Germany. Tel.: 49 30 450 69007. Fax: 49 30 450 69900. E-Mail: gessner{at}ukrv.de
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