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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.

Chen YT, Stewart DB, Nelson WJ - J. Cell Biol. (1999)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305-5435, USA.

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.

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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).
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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).

Mentions: GP2CAD2, 4, 6, 7, 8, and 10 are simple truncation mutants of GP2CAD1. GP2CAD1 was used as the PCR template. Individual antisense oligonucleotides hybridizing with the 6 aa upstream to the desired truncation, with a stop codon and Not1 site, were used with the oligonucleotide primer GP2-3S (5′-CGC GGG CAA GTC GAC TTC GCA GTA GTG AAC C-3′), which hybridizes to the region of the endogenous Sal1 site (underlined in the sequence) close to the COOH terminus of GP2 coding region, for individual PCR reactions to amplify parts of the cytoplasmic domain of E-cadherin. The resulting PCR products were cloned into GP2CAD1 through Sal1-Not1 sites. The cytoplasmic domain carried by each chimeric protein is shown in the diagram in Fig. 4, and described in detail in the Results section.


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.

Chen YT, Stewart DB, Nelson WJ - J. Cell Biol. (1999)

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).
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Related In: Results  -  Collection

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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).
Mentions: GP2CAD2, 4, 6, 7, 8, and 10 are simple truncation mutants of GP2CAD1. GP2CAD1 was used as the PCR template. Individual antisense oligonucleotides hybridizing with the 6 aa upstream to the desired truncation, with a stop codon and Not1 site, were used with the oligonucleotide primer GP2-3S (5′-CGC GGG CAA GTC GAC TTC GCA GTA GTG AAC C-3′), which hybridizes to the region of the endogenous Sal1 site (underlined in the sequence) close to the COOH terminus of GP2 coding region, for individual PCR reactions to amplify parts of the cytoplasmic domain of E-cadherin. The resulting PCR products were cloned into GP2CAD1 through Sal1-Not1 sites. The cytoplasmic domain carried by each chimeric protein is shown in the diagram in Fig. 4, and described in detail in the Results section.

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305-5435, USA.

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.

Show MeSH
Related in: MedlinePlus