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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 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.
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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.

Mentions: The GP2CAD1/catenin complex was coimmunoprecipitated with GP2 antibody and subjected to a high stringency wash in buffer containing 0.1% SDS, 1% sodium deoxycholate, and 1 M NaCl (High Stringency Wash Buffer, see Materials and Methods). Note that under this wash condition the stoichiometry of the coimmunoprecipitated complex of GP2CAD1/β-catenin/α-catenin was ∼1: 1:1 (Fig. 9), similar to that of the endogenous E-cadherin/ catenin complex (Ozawa and Kemler, 1992; Hinck et al., 1994a; see also Fig. 10). Surprisingly, we found that neither GP2CAD10 nor GP2CAD7 bound catenins under these conditions (Fig. 9), although both chimeras contained the previously mapped minimal β-catenin binding domain of E-cadherin (see Fig. 4). Similar results were obtained with GP2CAD5 and GP2CAD9 (Y.-T. Chen, unpublished results). Catenins were not coimmunoprecipitated in a complex with either GP2CAD8 (Fig. 9) in which the COOH-terminal half of the β-catenin binding domain had been deleted, or GP2CAD3 (Fig. 9) in which the β-catenin binding domain had been completely deleted. Overall, there is a strong correlation between lack of β-catenin binding to the cytoplasmic domain of E-cadherin, and retention of the majority of each GP2CAD1 mutant in the ER. Note that Fig. 9 also reveals a difference in the apparent molecular mass of GP2CAD1 and all other GP2CAD mutants. It is likely that this is due to differences in complex carbohydrate modifications between the proteins. GP2 is known to undergo extensive N-linked glycosylation (Hoops and Rindler, 1991). We show here that GP2CAD1 is efficiently delivered to cell surface and, thereby, receives extensive carbohydrate modifications during trafficking through the secretory pathway. In contrast, all other GP2CAD mutants are retained in the ER and, therefore, do not undergo complex carbohydrate modifications.


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 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.
© Copyright Policy
Related In: Results  -  Collection

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

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.
Mentions: The GP2CAD1/catenin complex was coimmunoprecipitated with GP2 antibody and subjected to a high stringency wash in buffer containing 0.1% SDS, 1% sodium deoxycholate, and 1 M NaCl (High Stringency Wash Buffer, see Materials and Methods). Note that under this wash condition the stoichiometry of the coimmunoprecipitated complex of GP2CAD1/β-catenin/α-catenin was ∼1: 1:1 (Fig. 9), similar to that of the endogenous E-cadherin/ catenin complex (Ozawa and Kemler, 1992; Hinck et al., 1994a; see also Fig. 10). Surprisingly, we found that neither GP2CAD10 nor GP2CAD7 bound catenins under these conditions (Fig. 9), although both chimeras contained the previously mapped minimal β-catenin binding domain of E-cadherin (see Fig. 4). Similar results were obtained with GP2CAD5 and GP2CAD9 (Y.-T. Chen, unpublished results). Catenins were not coimmunoprecipitated in a complex with either GP2CAD8 (Fig. 9) in which the COOH-terminal half of the β-catenin binding domain had been deleted, or GP2CAD3 (Fig. 9) in which the β-catenin binding domain had been completely deleted. Overall, there is a strong correlation between lack of β-catenin binding to the cytoplasmic domain of E-cadherin, and retention of the majority of each GP2CAD1 mutant in the ER. Note that Fig. 9 also reveals a difference in the apparent molecular mass of GP2CAD1 and all other GP2CAD mutants. It is likely that this is due to differences in complex carbohydrate modifications between the proteins. GP2 is known to undergo extensive N-linked glycosylation (Hoops and Rindler, 1991). We show here that GP2CAD1 is efficiently delivered to cell surface and, thereby, receives extensive carbohydrate modifications during trafficking through the secretory pathway. In contrast, all other GP2CAD mutants are retained in the ER and, therefore, do not undergo complex carbohydrate modifications.

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