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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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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.
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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.

Mentions: We placed the putative minimal β-catenin binding domain (aa 83–129, BB1) of E-cadherin cytoplasmic domain on the COOH terminus of a unrelated type-I transmembrane protein, human CD7. The resulting chimeric protein (CD7BB1) bound α-catenin and β-catenin, whereas CD7 did not (Fig. 11; note that aliquots of the sample were resolved by either 7.5% or 10% SDS-PAGE in order to obtain better separation of α-catenin/β-catenin and CD7/ CD7BB1, respectively). However, quantification of the amounts of α- and β-catenins bound to CD7BB1 revealed that the stoichiometry of CD7BB1/β-catenin/α-catenin was 5:1:0.8 when normalized to the number of Met and Cys residues in CD7BB1 and catenins; this compared with the ratio of ∼1:1:1 for E-cadherin/β-catenin/α-catenin or GP2CAD1/β-catenin/α-catenin (see above). Most of CD7 and CD7BB1 was localized at the cell surface (Y.-T. Chen, unpublished results); presumably, the cytoplasmic domain of CD7 in the CD7BB1 chimera contains signal(s) for its entry into the secretory pathway regardless of whether β-catenin is bound or not. Trafficking patterns of CD7 and CD7BB1 were similar in polarized MDCK cells. Approximately 60% of the cell surface delivery of both proteins was to the basal-lateral membrane (Y.-T. Chen, unpublished results). We conclude that a domain containing aa 83–129 is a transportable β-catenin binding domain in E-cadherin, but the binding strength of β-catenin/E-cadherin, based on high stringency washes, is much lower than that of β-catenin bound to the complete cytoplasmic domain of E-cadherin. Neither the BB1 domain alone nor β-catenin binding, per se, specifies protein delivery to the basal-lateral membrane.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132940&req=5

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.
Mentions: We placed the putative minimal β-catenin binding domain (aa 83–129, BB1) of E-cadherin cytoplasmic domain on the COOH terminus of a unrelated type-I transmembrane protein, human CD7. The resulting chimeric protein (CD7BB1) bound α-catenin and β-catenin, whereas CD7 did not (Fig. 11; note that aliquots of the sample were resolved by either 7.5% or 10% SDS-PAGE in order to obtain better separation of α-catenin/β-catenin and CD7/ CD7BB1, respectively). However, quantification of the amounts of α- and β-catenins bound to CD7BB1 revealed that the stoichiometry of CD7BB1/β-catenin/α-catenin was 5:1:0.8 when normalized to the number of Met and Cys residues in CD7BB1 and catenins; this compared with the ratio of ∼1:1:1 for E-cadherin/β-catenin/α-catenin or GP2CAD1/β-catenin/α-catenin (see above). Most of CD7 and CD7BB1 was localized at the cell surface (Y.-T. Chen, unpublished results); presumably, the cytoplasmic domain of CD7 in the CD7BB1 chimera contains signal(s) for its entry into the secretory pathway regardless of whether β-catenin is bound or not. Trafficking patterns of CD7 and CD7BB1 were similar in polarized MDCK cells. Approximately 60% of the cell surface delivery of both proteins was to the basal-lateral membrane (Y.-T. Chen, unpublished results). We conclude that a domain containing aa 83–129 is a transportable β-catenin binding domain in E-cadherin, but the binding strength of β-catenin/E-cadherin, based on high stringency washes, is much lower than that of β-catenin bound to the complete cytoplasmic domain of E-cadherin. Neither the BB1 domain alone nor β-catenin binding, per se, specifies protein delivery to the basal-lateral membrane.

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