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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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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.
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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.

Mentions: The difference in staining patterns of GP2CAD1 and GP2CAD10 (Fig. 5) might reflect differences in the efficiency of protein passage through the secretory pathway. Therefore, we compared the kinetics of cell surface delivery of newly synthesized GP2CAD1, GP2CAD10, and endogenous E-cadherin (Fig. 7). MDCK cells stably expressing GP2CAD1 or GP2CAD10 were grown on Transwell™ filters for 7 d, pulse labeled with 35S-Met/Cys for 20 min, and chased in DMEM containing an excess of nonradioactive Met/Cys for 40 min. At the end of the chase period, either whole cell lysates were collected, or cells were treated with the plasma membrane-impermeant biotinylating agent NHS-SS-biotin before lysis. Cell lysates were immunoprecipitated with antibody specific to rat GP2; biotinylated proteins were further precipitated with immobilized avidin. Identical experiments were performed with parental MDCK cells to examine the kinetics of endogenous E-cadherin delivery to the plasma 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)

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.
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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.
Mentions: The difference in staining patterns of GP2CAD1 and GP2CAD10 (Fig. 5) might reflect differences in the efficiency of protein passage through the secretory pathway. Therefore, we compared the kinetics of cell surface delivery of newly synthesized GP2CAD1, GP2CAD10, and endogenous E-cadherin (Fig. 7). MDCK cells stably expressing GP2CAD1 or GP2CAD10 were grown on Transwell™ filters for 7 d, pulse labeled with 35S-Met/Cys for 20 min, and chased in DMEM containing an excess of nonradioactive Met/Cys for 40 min. At the end of the chase period, either whole cell lysates were collected, or cells were treated with the plasma membrane-impermeant biotinylating agent NHS-SS-biotin before lysis. Cell lysates were immunoprecipitated with antibody specific to rat GP2; biotinylated proteins were further precipitated with immobilized avidin. Identical experiments were performed with parental MDCK cells to examine the kinetics of endogenous E-cadherin delivery to the plasma 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