Coupling assembly of the E-cadherin/beta-catenin complex to efficient endoplasmic reticulum exit and basal-lateral membrane targeting of E-cadherin in polarized MDCK cells

Abstract

The E-cadherin/catenin complex regulates Ca++-dependent cell-cell adhesion and is localized to the basal-lateral membrane of polarized epithelial cells. Little is known about mechanisms of complex assembly or intracellular trafficking, or how these processes might ultimately regulate adhesion functions of the complex at the cell surface. The cytoplasmic domain of E-cadherin contains two putative basal-lateral sorting motifs, which are homologous to sorting signals in the low density lipoprotein receptor, but an alanine scan across tyrosine residues in these motifs did not affect the fidelity of newly synthesized E-cadherin delivery to the basal-lateral membrane of MDCK cells. Nevertheless, sorting signals are located in the cytoplasmic domain since a chimeric protein (GP2CAD1), comprising the extracellular domain of GP2 (an apical membrane protein) and the transmembrane and cytoplasmic domains of E-cadherin, was efficiently and specifically delivered to the basal-lateral membrane. Systematic deletion and recombination of specific regions of the cytoplasmic domain of GP2CAD1 resulted in delivery of <10% of these newly synthesized proteins to both apical and basal-lateral membrane domains. Significantly, >90% of each mutant protein was retained in the ER. None of these mutants formed a strong interaction with beta-catenin, which normally occurs shortly after E-cadherin synthesis. In addition, a simple deletion mutation of E-cadherin that lacks beta-catenin binding is also localized intracellularly. Thus, beta-catenin binding to the whole cytoplasmic domain of E-cadherin correlates with efficient and targeted delivery of E-cadherin to the lateral plasma membrane. In this capacity, we suggest that beta-catenin acts as a chauffeur, to facilitate transport of E-cadherin out of the ER and the plasma membrane.

Figure 1

Amino acid sequence comparison of the basal-lateral membrane sorting determinants in human LDL receptor (Matter et al., 1992), and putative basal-lateral sorting determinant(s) in the cytoplasmic domain of canine E-cadherin.

Figure 4

Truncations of the cytoplasmic domain of E-cadherin result in accumulation of mutant proteins in the ER. Diagrams show schematically the structure of GP2/E-cadherin cytoplasmic domain chimeric constructs (GP2CAD1– 10). E-cadherin and GP2CAD1 diagrams are included for comparison. E-cadherin and GP2CAD1 data from Figs. 2 and 3 are included for comparison. Immunofluorescence (IF) revealed plasma membrane (PM) staining for E-cadherin and GP2CAD1 while all other chimeric proteins (GP2CAD2–10) gave an intracellular reticular staining pattern consistent with localization in the endoplasmic reticulum (ER). Examples of staining are given in Fig. 5. 1 h labeling with ³⁵S-Met/Cys and cell surface biotinylation show delivery of newly synthesized chimeric protein to both apical (Ap) and basal-lateral (Bl) membrane domains. β-catenin binding to GP2CAD1 and derived mutant proteins was assessed by steady state ³⁵S-Met/Cys labeling for 24 h and immunoprecipitation using anti-GP2 antibody. Examples of immunoprecipitates of GP2CAD1, 3, 7, 8, and 10 are given in Fig. 9. Catenin binding was not determined (ND) for GP2CAD2, 4, and 6, because these proteins completely lacked the minimal β-catenin binding domain (see text).

Figure 2

Delivery of newly synthesized KT3 tagged E-cadherin mutants to the basal-lateral membrane domain, and secretion of E-cad^sol into both apical and basal-lateral media. The diagram shows schematically the structure of each mutant constructs. The scale shows the number of amino acid residues in the cytoplasmic domain (CYT); the extracellular domain is not drawn to the same scale. To examine plasma membrane delivery, cells expressing E-cadherin (E-cad), or tyrosine to alanine substitution mutants (E-cad BL1, BL2, and BL12) were grown on Transwell™ filters for 7 d, labeled with ³⁵S-Met/Cys for 1 h, cell surface biotinylated on either the apical (Ap) or basal-lateral (Bl) membrane, and then processed for immunoprecipitation using the specified antibodies (mAb KT3 against epitope tag, or 3G8 against the extracellular domain of canine E-cadherin) followed by immobilized avidin. Secretion of E-cad^sol was assessed in confluent cell monolayers grown on Transwell™ filters by sampling the apical and basal-lateral media for ³⁵S-Met/Cys protein.

Figure 3

Dominant basal-lateral sorting activity of E-cadherin is located in the transmembrane/ cytoplasmic domains. (A) GPI modification is the sole determinant for targeting GP2 to the apical membrane. GP2 has been shown previously to be preferentially delivered to the apical domain of MDCK clone II/G cells (Mays et al., 1995). Diagrams show schematically the structure of GP2 lacking the GPI modification signal (GP2ΔGPI). GP2ΔGPI delivery was determined as described for E-cad^sol (see Fig. 1 legend, and Materials and Methods). (B) Newly synthesized GP2CAD1, labeled for 1 h, is sorted to the basal-lateral membrane domain, similar to endogenous E-cadherin. Diagrams show schematically the structure of GP2CAD1 compared with E-cadherin. GP2CAD1 delivery to the cell surface was assessed by ³⁵S-Met/Cys labeling and cell surface biotinylation of the apical (Ap) or the basal-lateral (Bl) membrane domain. Note that the autoradiogram showing the E-cadherin targeting is the same as that shown in Fig. 2, and is included here for comparison.

Figure 5

GP2CAD1 and GP2CAD10 show differences in sub-cellular localization by immunofluorescence staining. Reticular staining of GP2CAD10 (B) is distinct from cell surface staining of GP2CAD1 (A), Golgi staining (C), and lysosomal staining (D). Monoclonal antibody against rat GP2 was used to stain GP2CAD1 (A) and GP2CAD10 (B) expressing MDCK cells; (C) NBD-ceramide staining of paraformaldehyde-fixed MDCK cells; (D) Acridine orange staining of living MDCK cells. Bar, 50 μm.

Figure 9

Catenins bind to GP2CAD1 but not GP2CAD3, GP2CAD7, GP2CAD8, or GP2CAD10. MDCK cells expressing individual constructs were grown on Transwell™ filters for 7 d, labeled with ³⁵S-Met/Cys for 24 h, extracted, and proteins were immunoprecipitated with GP2 antibody. Immunoprecipitates were resolved by 10% SDS-PAGE. Markings to the right of columns for GP2CAD10 and GP2CAD8, and those to the left of columns of GP2CAD3 and GP2CAD7 show the positions of molecular mass markers; 116, 97, and 66 kD.

Figure 12

Intracellular localization and size exclusion column elution profile of E-cad8 are distinctively different from those of full-length E-cadherin. (A) Schematic diagram of E-cad8 compared with full-length E-cadherin. The COOH-terminal 36 aa of E-cadherin are deleted in E-cad8. This deletion is equivalent to the deletion in GP2CAD8 (Fig. 4). The KT3 epitope tag was fused at the COOH terminus of the cytoplasmic domain of E-cad8. EXT, extracellular domain; TM, transmembrane domain; BB, β-catenin binding domain. Note the diagram is not to scale. (B) Immunofluorescence staining of E-cad8 in MDCK cells using monoclonal antibody KT3. Bar, 50 μm. (C) Elution profile of full-length E-cadherin, E-cad8, and β-catenin after Superose 6 size exclusion chromatography. Individual fractions were resolved by SDS-PAGE and proteins were probed with a monoclonal antibody against the extracellular domain of E-cadherin or a monoclonal antibody against β-catenin. The numbers given on top of the gels are the fraction numbers. Note that there are three polypeptide bands in the MDCK E-cad8/E-cadherin Western blot. E-cad8 has an electrophoretic mobility faster than that of full-length E-cadherin, which is consistent with the cytoplasmic domain deletion. Full-length E-cadherin is the middle polypeptide band. A protein band with an electrophoretic mobility slower than that of the full-length E-cadherin probably represents the precursor form of E-cad8. T, total cell lysate.

Figure 6

The majority of GP2CAD10 is sensitive to endoglycosidase H digestion. GP2CAD10 expressing MDCK cells were grown on Transwell™ filters for 7 d, labeled with ³⁵S-Met/Cys for 24 h, extracted and immunoprecipitated with GP2 antibody. Immunoprecipitates were incubated in the presence (+) or absence (−) of endo H, and were subsequently resolved by SDS-PAGE and detected by fluorography. Only a small fraction (∼10%) of GP2CAD10 (marked with asterisk) does not display an increase in electrophoretic mobility upon endo H treatment. Numbers at the left show molecular mass in kD.

Figure 7

Rate of E-cadherin and GP2CAD1 delivery to the cell surface are similar, while GP2CAD10 is delivered to the cell surface at a reduced rate. At steady state, the majority of GP2CAD1 is on plasma membrane, while the majority of GP2CAD10 is intracellular. (A) Arrival of newly synthesized proteins on cell surface monitored by cell surface biotinylation. Cells were labeled with ³⁵S-Met/Cys for 20 min, chased for 40 min, cell surface biotinylated, lysed, and immunoprecipitated with antibodies to either E-cadherin (E-cad) or GP2. Aliquots of cell surface protein (S, biotinylated, open columns) were normalized to the amount of protein in total cell lysate (T, shaded columns). Error bars show standard deviation (n = 3). (B) Steady state distribution of GP2CAD1 and GP2CAD10. Cells were labeled with ³⁵S-Met/Cys for 24 h, surface biotinylated on both apical and basal membranes, extracted and immunoprecipitated; biotinylated proteins were retrieved with immobilized avidin. Results show that 91.3% (±1.3%, n = 2) of GP2CAD1 is on the cell surface, but ∼7.2% (±0.8%, n = 2) of GP2CAD10 is on cell surface.

Figure 7

Rate of E-cadherin and GP2CAD1 delivery to the cell surface are similar, while GP2CAD10 is delivered to the cell surface at a reduced rate. At steady state, the majority of GP2CAD1 is on plasma membrane, while the majority of GP2CAD10 is intracellular. (A) Arrival of newly synthesized proteins on cell surface monitored by cell surface biotinylation. Cells were labeled with ³⁵S-Met/Cys for 20 min, chased for 40 min, cell surface biotinylated, lysed, and immunoprecipitated with antibodies to either E-cadherin (E-cad) or GP2. Aliquots of cell surface protein (S, biotinylated, open columns) were normalized to the amount of protein in total cell lysate (T, shaded columns). Error bars show standard deviation (n = 3). (B) Steady state distribution of GP2CAD1 and GP2CAD10. Cells were labeled with ³⁵S-Met/Cys for 24 h, surface biotinylated on both apical and basal membranes, extracted and immunoprecipitated; biotinylated proteins were retrieved with immobilized avidin. Results show that 91.3% (±1.3%, n = 2) of GP2CAD1 is on the cell surface, but ∼7.2% (±0.8%, n = 2) of GP2CAD10 is on cell surface.

Figure 8

Effects of chloroquine phosphate (top panels) and aLLN (bottom panels) on the degradation of GP2CAD1 (left panels) and GP2CAD10 (right panels). Chloroquine phosphate (CQ, 0.1 mM) treatment of cells started 1 h before pulse labeling with ³⁵S-Met/Cys (15 min), and aLLN (25 μM) treatment started 3 h before pulse labeling with ³⁵S-Met/Cys (15 min). At the end of the pulse labeling (15 min) or chase period (240 min, 15-min pulse labeling, and 225-min chase), cells were extracted with 1× NDET, and protein was immunoprecipitated with an antibody against rat GP2. Data for each column were from two 24-mm Transwell™ filters and the error bars show the range of the data.

Figure 10

Catenins bind to E-cadherin and epitope tagged (Tyr→ Ala) E-cadherin mutants. Parental MDCK cells or cells expressing individual mutant E-cadherins were grown on Transwell™ filters for 7 d, labeled for 24 h with ³⁵S-Met/Cys, extracted, and immunoprecipitated with either mAb 3G8 (against the extracellular domain of canine E-cadherin) or mAb KT3 (against KT3 tag). Note that KT3 antibody immunoprecipitated nonspecifically a protein band with apparent molecular mass of ∼112 kD in untransfected cells (marked with asterisk). In cells expressing KT3 tagged E-cadherin mutants, specific bands corresponding to E-cadherin, α-catenin, and β-catenin were detected.

Figure 11

Catenins bind weakly to CD7BB1. MDCK cells expressing human CD7, or the chimeric protein CD7BB1 were grown on Transwell™ filters for 7 d, labeled for 24 h with ³⁵S-Met/ Cys, extracted with Triton X-100 lysis buffer, and immunoprecipitated with monoclonal antibody T3.3A1 (against the extracellular domain of human CD7). Identical samples were resolved by 7.5% or 10% SDS-PAGE to obtain better separation of CD7/CD7BB1 and catenins, respectively. Numbers at the right mark the positions of molecular mass in kD.