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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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Delivery of newly synthesized KT3  tagged E-cadherin mutants to the basal-lateral  membrane domain, and secretion of E-cadsol into  both apical and basal-lateral media. The diagram  shows schematically the structure of each mutant  constructs. The scale shows the number of amino  acid residues in the cytoplasmic domain (CYT);  the extracellular domain is not drawn to the  same scale. To examine plasma membrane delivery, cells expressing E-cadherin (E-cad), or tyrosine to alanine substitution mutants (E-cad  BL1, BL2, and BL12) were grown on Transwell™  filters for 7 d, labeled with 35S-Met/Cys for 1 h,  cell surface biotinylated on either the apical  (Ap) or basal-lateral (Bl) membrane, and then  processed for immunoprecipitation using the  specified antibodies (mAb KT3 against epitope  tag, or 3G8 against the extracellular domain of  canine E-cadherin) followed by immobilized avidin. Secretion of E-cadsol was assessed in confluent cell monolayers grown on Transwell™ filters  by sampling the apical and basal-lateral media  for 35S-Met/Cys protein.
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Figure 2: Delivery of newly synthesized KT3 tagged E-cadherin mutants to the basal-lateral membrane domain, and secretion of E-cadsol into both apical and basal-lateral media. The diagram shows schematically the structure of each mutant constructs. The scale shows the number of amino acid residues in the cytoplasmic domain (CYT); the extracellular domain is not drawn to the same scale. To examine plasma membrane delivery, cells expressing E-cadherin (E-cad), or tyrosine to alanine substitution mutants (E-cad BL1, BL2, and BL12) were grown on Transwell™ filters for 7 d, labeled with 35S-Met/Cys for 1 h, cell surface biotinylated on either the apical (Ap) or basal-lateral (Bl) membrane, and then processed for immunoprecipitation using the specified antibodies (mAb KT3 against epitope tag, or 3G8 against the extracellular domain of canine E-cadherin) followed by immobilized avidin. Secretion of E-cadsol was assessed in confluent cell monolayers grown on Transwell™ filters by sampling the apical and basal-lateral media for 35S-Met/Cys protein.

Mentions: The primary structure of the cytoplasmic domain of E-cadherin contains two regions with amino acid sequences and spacing that are similar to previously identified basal-lateral sorting signals in the cytoplasmic domain of the LDL-R (Matter et al., 1992; see Fig. 1). We assessed whether these two motifs function as basal-lateral sorting determinants for E-cadherin. All tyrosine residues in each motif of canine E-cadherin were mutated into alanine in region 1 (BL1), or region 2 (BL2), or both (BL12); similar mutations in both motifs of LDL-R resulted in random sorting to the cell surface in MDCK cells (Matter et al., 1992). Each mutant was epitope-tagged with the SV-40 large T antigen epitope (KT3 tag, amino acid sequence KPPTPPPEPET; see MacArthur and Walter, 1984) and stably transfected into MDCK cells (Fig. 2). Confluent monolayers of cells were grown on Transwell™ filters for 7 d, and then labeled with 35S-Met/Cys for 1 h. At the end of the 1 h labeling period, the first wave of newly synthesized E-cadherin that arrived at the apical or basal-lateral membrane (Shore and Nelson, 1991) was detected by cell surface biotinylation. Mutant E-cadherin was distinguished from endogenous E-cadherin with the KT3 monoclonal antibody to the epitope tag.


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)

Delivery of newly synthesized KT3  tagged E-cadherin mutants to the basal-lateral  membrane domain, and secretion of E-cadsol into  both apical and basal-lateral media. The diagram  shows schematically the structure of each mutant  constructs. The scale shows the number of amino  acid residues in the cytoplasmic domain (CYT);  the extracellular domain is not drawn to the  same scale. To examine plasma membrane delivery, cells expressing E-cadherin (E-cad), or tyrosine to alanine substitution mutants (E-cad  BL1, BL2, and BL12) were grown on Transwell™  filters for 7 d, labeled with 35S-Met/Cys for 1 h,  cell surface biotinylated on either the apical  (Ap) or basal-lateral (Bl) membrane, and then  processed for immunoprecipitation using the  specified antibodies (mAb KT3 against epitope  tag, or 3G8 against the extracellular domain of  canine E-cadherin) followed by immobilized avidin. Secretion of E-cadsol was assessed in confluent cell monolayers grown on Transwell™ filters  by sampling the apical and basal-lateral media  for 35S-Met/Cys protein.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132940&req=5

Figure 2: Delivery of newly synthesized KT3 tagged E-cadherin mutants to the basal-lateral membrane domain, and secretion of E-cadsol into both apical and basal-lateral media. The diagram shows schematically the structure of each mutant constructs. The scale shows the number of amino acid residues in the cytoplasmic domain (CYT); the extracellular domain is not drawn to the same scale. To examine plasma membrane delivery, cells expressing E-cadherin (E-cad), or tyrosine to alanine substitution mutants (E-cad BL1, BL2, and BL12) were grown on Transwell™ filters for 7 d, labeled with 35S-Met/Cys for 1 h, cell surface biotinylated on either the apical (Ap) or basal-lateral (Bl) membrane, and then processed for immunoprecipitation using the specified antibodies (mAb KT3 against epitope tag, or 3G8 against the extracellular domain of canine E-cadherin) followed by immobilized avidin. Secretion of E-cadsol was assessed in confluent cell monolayers grown on Transwell™ filters by sampling the apical and basal-lateral media for 35S-Met/Cys protein.
Mentions: The primary structure of the cytoplasmic domain of E-cadherin contains two regions with amino acid sequences and spacing that are similar to previously identified basal-lateral sorting signals in the cytoplasmic domain of the LDL-R (Matter et al., 1992; see Fig. 1). We assessed whether these two motifs function as basal-lateral sorting determinants for E-cadherin. All tyrosine residues in each motif of canine E-cadherin were mutated into alanine in region 1 (BL1), or region 2 (BL2), or both (BL12); similar mutations in both motifs of LDL-R resulted in random sorting to the cell surface in MDCK cells (Matter et al., 1992). Each mutant was epitope-tagged with the SV-40 large T antigen epitope (KT3 tag, amino acid sequence KPPTPPPEPET; see MacArthur and Walter, 1984) and stably transfected into MDCK cells (Fig. 2). Confluent monolayers of cells were grown on Transwell™ filters for 7 d, and then labeled with 35S-Met/Cys for 1 h. At the end of the 1 h labeling period, the first wave of newly synthesized E-cadherin that arrived at the apical or basal-lateral membrane (Shore and Nelson, 1991) was detected by cell surface biotinylation. Mutant E-cadherin was distinguished from endogenous E-cadherin with the KT3 monoclonal antibody to the epitope tag.

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