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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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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.
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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.

Mentions: To confirm biochemically that GP2CAD1 deletion mutants were retained in the ER, we examined the sensitivity of GP2CAD10 to digestion by endoglycosidase H (endo H). GP2CAD10 MDCK cells were incubated with 35S- Met/Cys for 24 h to label proteins to steady state. Total cell lysates were prepared, and GP2CAD10 was immunoprecipitated with anti-GP2 antibody and then subjected to endo H digestion. As shown in Fig. 6, endo H digestion resulted in a significant increase in electrophoretic mobility of the major GP2CAD10 protein band. Note that the electrophoretic mobility of a minor protein band (Fig. 6, asterisk) was not affected by incubation with endo H. This upper band appears to be specific to the GP2 antibody as a minor protein with a slower electrophoretic mobility was also present in other GP2 chimeric protein immunoprecipitates. It is likely that this protein band represents a small portion (∼10%) of the chimeric protein that exited the ER and had undergone processing in the Golgi to remove the high mannose carbohydrate chain. From the data on the sensitivity of GP2CAD10 to endo H digestion, we conclude that at steady state >85% of GP2CAD10 retained high mannose carbohydrate chains and had not undergone further processing consistent with its location in the ER.


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)

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.
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Related In: Results  -  Collection

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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.
Mentions: To confirm biochemically that GP2CAD1 deletion mutants were retained in the ER, we examined the sensitivity of GP2CAD10 to digestion by endoglycosidase H (endo H). GP2CAD10 MDCK cells were incubated with 35S- Met/Cys for 24 h to label proteins to steady state. Total cell lysates were prepared, and GP2CAD10 was immunoprecipitated with anti-GP2 antibody and then subjected to endo H digestion. As shown in Fig. 6, endo H digestion resulted in a significant increase in electrophoretic mobility of the major GP2CAD10 protein band. Note that the electrophoretic mobility of a minor protein band (Fig. 6, asterisk) was not affected by incubation with endo H. This upper band appears to be specific to the GP2 antibody as a minor protein with a slower electrophoretic mobility was also present in other GP2 chimeric protein immunoprecipitates. It is likely that this protein band represents a small portion (∼10%) of the chimeric protein that exited the ER and had undergone processing in the Golgi to remove the high mannose carbohydrate chain. From the data on the sensitivity of GP2CAD10 to endo H digestion, we conclude that at steady state >85% of GP2CAD10 retained high mannose carbohydrate chains and had not undergone further processing consistent with its location in the ER.

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