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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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Effects of chloroquine phosphate (top panels) and  aLLN (bottom panels) on the degradation of GP2CAD1 (left  panels) and GP2CAD10 (right panels). Chloroquine phosphate  (CQ, 0.1 mM) treatment of cells started 1 h before pulse labeling  with 35S-Met/Cys (15 min), and aLLN (25 μM) treatment started  3 h before pulse labeling with 35S-Met/Cys (15 min). At the end of  the pulse labeling (15 min) or chase period (240 min, 15-min  pulse labeling, and 225-min chase), cells were extracted with 1×  NDET, and protein was immunoprecipitated with an antibody  against rat GP2. Data for each column were from two 24-mm  Transwell™ filters and the error bars show the range of the data.
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Figure 8: Effects of chloroquine phosphate (top panels) and aLLN (bottom panels) on the degradation of GP2CAD1 (left panels) and GP2CAD10 (right panels). Chloroquine phosphate (CQ, 0.1 mM) treatment of cells started 1 h before pulse labeling with 35S-Met/Cys (15 min), and aLLN (25 μM) treatment started 3 h before pulse labeling with 35S-Met/Cys (15 min). At the end of the pulse labeling (15 min) or chase period (240 min, 15-min pulse labeling, and 225-min chase), cells were extracted with 1× NDET, and protein was immunoprecipitated with an antibody against rat GP2. Data for each column were from two 24-mm Transwell™ filters and the error bars show the range of the data.

Mentions: The data presented above show that nearly all of GP2CAD10 is retained in the ER, whereas nearly all GP2CAD1 is localized in the plasma membrane. It is generally acknowledged that misfolded secretory proteins retained in the ER are degraded through the proteasome pathway (for a recent review, see Coux et al., 1996). To examine degradative pathways, cells were pulse-labeled with 35S-Met/Cys for 15 min and then chased in an excess of nonradioactive Met/Cys for 225 min in the presence of either chloroquine phosphate (a lysosome inhibitor) or N-acetyl-Leu-Leu-norleucinal (aLLN, a 26S proteasome inhibitor). As shown in Fig. 8 (upper panels), 70% of newly synthesized GP2CAD1 remained at the end of the chase period in the presence of chloroquine, compared with 40% in its absence. By comparison, ∼50% of GP2CAD10 remained in the presence of chloroquine compared with 42% in its absence. In the presence of aLLN (Fig. 8, bottom panels), 82% of GP2CAD1 remained compared with 50% in its absence; 83% of GP2CAD10 remained in the presence of aLLN, compared with 60% in its absence. These results show that chloroquine phosphate suppressed degradation of both GP2CAD1 and GP2CAD10 but the effect on GP2CAD1 was greater. On the other hand, aLLN had a more inhibitory effect on the degradation of GP2CAD10 than did chloroquine, while both aLLN and chloroquine suppressed the degradation of GP2CAD1.


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)

Effects of chloroquine phosphate (top panels) and  aLLN (bottom panels) on the degradation of GP2CAD1 (left  panels) and GP2CAD10 (right panels). Chloroquine phosphate  (CQ, 0.1 mM) treatment of cells started 1 h before pulse labeling  with 35S-Met/Cys (15 min), and aLLN (25 μM) treatment started  3 h before pulse labeling with 35S-Met/Cys (15 min). At the end of  the pulse labeling (15 min) or chase period (240 min, 15-min  pulse labeling, and 225-min chase), cells were extracted with 1×  NDET, and protein was immunoprecipitated with an antibody  against rat GP2. Data for each column were from two 24-mm  Transwell™ filters and the error bars show the range of the data.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Effects of chloroquine phosphate (top panels) and aLLN (bottom panels) on the degradation of GP2CAD1 (left panels) and GP2CAD10 (right panels). Chloroquine phosphate (CQ, 0.1 mM) treatment of cells started 1 h before pulse labeling with 35S-Met/Cys (15 min), and aLLN (25 μM) treatment started 3 h before pulse labeling with 35S-Met/Cys (15 min). At the end of the pulse labeling (15 min) or chase period (240 min, 15-min pulse labeling, and 225-min chase), cells were extracted with 1× NDET, and protein was immunoprecipitated with an antibody against rat GP2. Data for each column were from two 24-mm Transwell™ filters and the error bars show the range of the data.
Mentions: The data presented above show that nearly all of GP2CAD10 is retained in the ER, whereas nearly all GP2CAD1 is localized in the plasma membrane. It is generally acknowledged that misfolded secretory proteins retained in the ER are degraded through the proteasome pathway (for a recent review, see Coux et al., 1996). To examine degradative pathways, cells were pulse-labeled with 35S-Met/Cys for 15 min and then chased in an excess of nonradioactive Met/Cys for 225 min in the presence of either chloroquine phosphate (a lysosome inhibitor) or N-acetyl-Leu-Leu-norleucinal (aLLN, a 26S proteasome inhibitor). As shown in Fig. 8 (upper panels), 70% of newly synthesized GP2CAD1 remained at the end of the chase period in the presence of chloroquine, compared with 40% in its absence. By comparison, ∼50% of GP2CAD10 remained in the presence of chloroquine compared with 42% in its absence. In the presence of aLLN (Fig. 8, bottom panels), 82% of GP2CAD1 remained compared with 50% in its absence; 83% of GP2CAD10 remained in the presence of aLLN, compared with 60% in its absence. These results show that chloroquine phosphate suppressed degradation of both GP2CAD1 and GP2CAD10 but the effect on GP2CAD1 was greater. On the other hand, aLLN had a more inhibitory effect on the degradation of GP2CAD10 than did chloroquine, while both aLLN and chloroquine suppressed the degradation of GP2CAD1.

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