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First published online December 5, 2007
doi: 10.1242/10.1242/jcs.03489

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A dileucine motif in its cytoplasmic domain directs β-catenin-uncoupled E-cadherin to the lysosome

Department of Biochemistry and Molecular Biology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan

View larger version (51K): [in this window] [in a new window]   Fig. 1. E-cadherin constructs that do not bind β-catenin accumulate in intracellular compartments in MDCK cells. (A) Schematic representations of HA-tagged wild-type E-cadherin (Ecad) and two Ecad mutants: ESA, full-length E-cadherin in which eight conserved serine residues within the catenin-binding site are substituted with alanine; EC81, E-cadherin in which 70 amino acids of the C-terminal are deleted. (B) Immunofluorescence staining of MDCK cells immunostained for Ecad, ESA and EC81 using anti-HA mAbs. Analysis of two to four other clones expressing each construct gave essentially the same results. (C) Trypsin digestion of cells expressing Ecad, ESA or EC81 with or without free Ca2+. Cells were incubated with 0.01% trypsin for 10 minutes at 37°C in the presence of 2 mM Ca2+ (TC) or 1 mM EGTA (TE) and immunostaining with anti-HA mAb showed that a significant percentage of ESA and EC81 remain inside the cells. Protein bands of high molecular mass (marked by asterisks) correspond to the intracellular, incompletely processed proteins that retain the precursor segment.

View larger version (97K): [in this window] [in a new window]   Fig. 2. Localization of E-cadherin constructs. Immunofluorescence images of stably transfected cells expressing E-cadherin constructs (anti-HA antibody) and endogenous Na+,K+-ATPase. (Top panels) Cross sections of monolayers. (Bottom panels) xz sections of cells. Yellow lines in the top panels indicate the planes from which the xz images were optically reconstructed.

View larger version (57K): [in this window] [in a new window]   Fig. 3. EC81 colocalizes with Golgi and post-Golgi markers. MDCK cells stably expressing EC81 were fixed and incubated with rat monoclonal antibodies against HA, the ER marker GRP78, the Golgi markers GM130 or golgin-97, post-Golgi compartment markers EEA1 or LysoTracker Red DND-99, or β-catenin.

View larger version (46K): [in this window] [in a new window]   Fig. 4. β-catenin-uncoupled E-cadherin is targeted to lysosomes. (A) Intracellular accumulation of EC81 does not depend on clathrin-mediated endocytosis. MDCK cells stably expressing EC81 were transfected with (upper panel) wild-type or (lower panel) dominant-negative dynamin and analyzed for their transient expression. Asterisks indicate cells expressing GFP-tagged dynamin. (B) Newly synthesized Ecad is expressed at the cell surface but EC81GFP is not. MDCK cells stably expressing Ecad or EC81GFP were incubated with 10 µM cycloheximide for 12 hours to deplete Ecad or EC81GFP from the protein synthesis pathway and the cell surface. Cycloheximide was washed out, and cells were returned to normal medium for the indicated times before fixation. Cell-surface localization of Ecad is already detectable after 4 hours, increasing over time. EC81GFP was never detected at the surface. (C) ESA and EC81 decreased upon treatment with cycloheximide. Cells were incubated for 2 hours in the presence or absence of 10 µM cycloheximide (CHX) and then subjected to immunoblot analysis using anti-HA antibody. (D) Effect of lysosome and proteasome inhibitors on ESA and EC81 stability. Cells were incubated for 3 hours with the lysosome inhibitor chloroquine (CQ, 200 µM) or the proteasome inhibitor MG132 (MG, 10 µM). Cell lysates were prepared and analyzed by western blotting using anti-HA antibodies to detect ESA and EC81. (E) Quantitative analysis of C and D, indicating that ESA and EC81 levels are increased in lysosomes in cells treated with chloroquine. (F) Accumulation of EC81 in lysosomes after chloroquine treatment. Cells were incubated for 3 hours in the presence (+CQ) or absence (–CQ) of chloroquine and processed for immunofluorescence staining with anti-HA. Images show a marked accumulation of EC81 after treatment.

View larger version (54K): [in this window] [in a new window]   Fig. 5. The tail-less construct EC0 is detected on the cell surface. (A) Schematic representation of HA-tagged wild-type E-cadherin (Ecad) and two mutant constructs: EC0, lacking the entire cytoplasmic domain; Ex, consisting of the extracellular domain but lacking the cytoplasmic and TM domains. (B) Confocal imaging of basolateral targeting of the tail-less EC0 construct. E-cadherin and endogenous Na+,K+-ATPase were detected by using DECMA-1and anti-Na+,K+-ATPase mAb. (C) Expression and basolateral targeting of EC0. (Upper panel) Immunoblot analysis of total cell lysates using DECMA-1 revealed that five times more EC0 was expressed than endogenous E-cadherin. (Lower panel) Cells expressing EC0 were grown on Transwell filters and their (a) apical or (b) basolateral membranes were biotinylated. After precipitating the biotinylated proteins using immobilized streptavidin, EC0 and endogenous E-cadherin were detected using DECMA-1. (D) No EC0 labeling was detected on the apical membrane. Cells grown on Transwell filters were fixed and incubated with DECMA-1 without permeabilization to detect E-cadherin on the cell surface. Optical data were obtained as described in B. (E) EC0 does not form lateral dimers with endogenous E-cadherin. Cells expressing EC0 were lysed and incubated with either anti-HA or mAbs against the cytoplasmic domain of E-cadherin (C20820) preabsorbed with protein G-Sepharose. Proteins were separated by SDS-PAGE and detected using DECMA-1. (F) Immunofluorescence labeling of MDCK cells expressing EC81GFP with pan-cadherin antibodies. (G) The Ecad mutant construct Ex is released into both the apical and basolateral media. Cells expressing Ex were grown on Transwell filters. Secretion of Ex in the apical and basolateral media was assessed by immunoprecipitation with rabbit anti-E-cadherin antibodies and immunoblotting with DECMA-1. The barrier function of the transfectants was confirmed by the observation that mouse immunoglobulin was detected in the apical medium when added to the apical medium, but not when added to the basolateral medium (not shown). (H) Schematic representation of the full-length IL2 receptor chain construct (IL2R) and its derivatives. IL2RC: a construct lacking the entire cytoplasmic domain; IRCTME: a tail-less construct whose transmembrane domain was replaced with that of E-cadherin; IL2RECT: a construct whose cytoplasmic domain was replaced with that of E-cadherin; IL2RECTLA: a construct whose cytoplasmic domain was replaced with that of E-cadherin with the LA substitution. (I) Detection of IL2RC and IL2RCTME but not IL2RECT and IL2RECTLA on the apical membrane. Optical data were obtained as described in Fig. 5D except that anti-IL2R antibodies were used.

View larger version (41K): [in this window] [in a new window]   Fig. 6. The N-terminal repeats of the E-cadherin extracellular domain are required for the basolateral localization. (A) Schematic representation of EC0 and its derivatives each carrying a deletion of one of the five extracellular domains. (B) Expression of the EC0 derivatives. Constructs were detected using antibodies as indicated. (C) Basolateral localization of the EC0 derivatives. Cells were grown on Transwell filters, and either the (a) apical or (b) basolateral membranes were biotinylated. After the biotinylated proteins were precipitated using immobilized streptavidin, the constructs were detected using DECMA-1 except for EC0EC4, which was detected with ECCD-2. Constructs lacking either EC1, EC2 or EC3 were detected on the apical membrane. (D) Detection of EC1, EC2 and EC3, but not EC4 and EC5 on the apical membrane. Cells grown on Transwell filters were fixed and incubated with DECMA-1 without permeabilization to detect E-cadherin on the cell surface. To detect the EC0 deletion constructs EC0EC4 and EC0EC5, ECCD-2 was used because DECMA-1 cannot recognize these constructs on the cell surface. Constructs EC0EC1, EC0EC2 and EC0EC3 were detected on the apical membrane. Optical data were obtained as described in Fig. 5D. (E) Quantitative analysis of the apical membrane targeting. Relative amounts of the pool of E-cadherin at the apical membrane (not shown) and of its derivatives as described in Fig. 5D, Fig. 6D,G, and Fig. 8D were quantified using NIH Image and expressed as a percentage of the total cell surface pool of the protein. In addition to EC0 derivatives lacking EC1, EC2 or EC3, another EC0 construct containing a W2A substitution (EC0WA) was detected on the apical membrane. (F) Immunoblot detection of EC0WA and EWA. Total cell lysates were analyzed by using DECMA-1. (G). Detection of EC0WA but not EWA on the apical membrane. Optical data were obtained as in Fig. 5D.

View larger version (48K): [in this window] [in a new window]   Fig. 7. The dileucine motif is crucial for the lysosomal targeting of β-catenin-uncoupled E-cadherin constructs. (A) Schematic representations of construct EC81 and its derivatives: EC81MP14 and EC81MP39, EC81 derivatives with additional internal deletions of residues 602-615 or 578-616, respectively; EC20: a construct lacking 131 amino acids at its C-terminal. (B) Immunofluorescence images reveal that that the 20 amino-acid residues within the JM region of the E-cadherin cytoplasmic domain contain the signal for lysosomal targeting of β-catenin-uncoupled E-cadherin constructs. Cells expressing the indicated constructs were labeled with anti-HA mAbs. (C) TC and TE treatment revealed that a significant percentage of EC20 and EC81MP14 remained inside the cells. Cells expressing the indicated constructs were incubated with 0.01% trypsin for 10 minutes at 37°C in the presence of 2 mM Ca2+ (TC) or 1 mM EGTA (TE). Proteins were detected with anti-HA mAbs. Protein bands of high molecular mass (marked by asterisks) correspond to intracellular, incompletely processed proteins that retain the precursor segment. (D) The sequence of the 20 amino-acid residues in the JM region of the E-cadherin cytoplasmic domain and of those substituted in the mutant constructs. (E) Immunofluorescence images showing localization of the constructs. Cells expressing the indicated constructs were labeled with anti-HA mAb. EC81LA was detected at the cell surface, whereas other constructs were detected in the intracellular compartments. (F) Quantification of the intracellular pool of EC81 and its derivatives. Relative amounts of the intracellular pools obtained after TE treatment were quantified using NIH Image and expressed as a percentage of the pool of total protein obtained after TC treatment. Before trypsinization, cells were incubated for 30 minutes at 37°C in medium with or without 0.45 M sucrose (+suc or –suc, respectively). Inhibition of endocytosis by hypertonic medium did not change the intracellular pool of EC81, EC81PA and EC81DA but reduced that of EC81KR, EC81EPA and EC81LA.

View larger version (84K): [in this window] [in a new window]   Fig. 8. The dileucine motif is not required for basolateral targeting. (A) Basolateral targeting of EC81LA in MDCK and DLD1 cells. Cells were grown on Transwell filters and either the (a) apical or (b) basolateral membranes were biotinylated. After precipitation of the biotinylated protein using immobilized streptavidin, EC81LA was detected using anti-HA mAbs. (B) Confocal imaging of basolateral targeting of EC81LA. EC81LA and endogenous Na+,K+-ATPase were detected using anti-HA antibody and antibody against Na+,K+-ATPase, respectively. (C) EC81LA and ELA do not colocalize with the apical membrane marker gp135. EC81LA and ELA were detected using anti-HA antibody, whereas endogenous gp135 was detected using 3F2 mAb. (D) EC81LA, ELA and EWALA are detected at the basolateral membrane but not the apical membrane. Optical data were obtained as described in Fig. 5D.

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