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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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Amino acid sequence comparison of the basal-lateral  membrane sorting determinants in human LDL receptor (Matter  et al., 1992), and putative basal-lateral sorting determinant(s) in  the cytoplasmic domain of canine E-cadherin.
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Figure 1: Amino acid sequence comparison of the basal-lateral membrane sorting determinants in human LDL receptor (Matter et al., 1992), and putative basal-lateral sorting determinant(s) in the cytoplasmic domain of canine E-cadherin.

Mentions: We are interested in the relationship between intracellular trafficking of membrane proteins and cell–cell adhesion. Previous studies reported that β-catenin binds to the cytoplasmic domain of E-cadherin shortly after E-cadherin synthesis, while α-catenin joins the complex later, probably after E-cadherin has reached the cell surface (Ozawa et al., 1990; Hinck et al., 1994a). Thus, unlike other type-I membrane proteins studied (see above), E-cadherin is sorted and targeted to the basal-lateral membrane in a stoichiometric complex with a cytosolic protein, β-catenin. This prompted us to explore the relationship between E-cadherin sorting to the basal-lateral membrane domain and assembly of the E-cadherin/β-catenin complex. There are two regions of the cytoplasmic domain of E-cadherin which are similar in amino acid sequence to previously identified basal-lateral sorting signals of LDL-R (Matter et al., 1992), i.e., a sequence motif of Y---XXX, where X is an acidic amino acid residue. The first putative sorting signal in E-cadherin is immediately proximal to the transmembrane domain and the other is at the extreme COOH terminus, also similar to the locations of the two sorting signals in LDL-R cytoplasmic domain (see Fig. 1). We show that despite these similarities, mutations of tyrosine to alanine in those two potential basal-lateral sorting signals in E-cadherin do not affect sorting of newly synthesized mutant E-cadherin in MDCK cells. Further dissection of the cytoplasmic domain of E-cadherin using a chimeric protein approach revealed that E-cadherin exit from the ER and efficient sorting to the basal-lateral membrane are coupled to assembly of the E-cadherin/β-catenin complex shortly after E-cadherin synthesis.


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)

Amino acid sequence comparison of the basal-lateral  membrane sorting determinants in human LDL receptor (Matter  et al., 1992), and putative basal-lateral sorting determinant(s) in  the cytoplasmic domain of canine E-cadherin.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Amino acid sequence comparison of the basal-lateral membrane sorting determinants in human LDL receptor (Matter et al., 1992), and putative basal-lateral sorting determinant(s) in the cytoplasmic domain of canine E-cadherin.
Mentions: We are interested in the relationship between intracellular trafficking of membrane proteins and cell–cell adhesion. Previous studies reported that β-catenin binds to the cytoplasmic domain of E-cadherin shortly after E-cadherin synthesis, while α-catenin joins the complex later, probably after E-cadherin has reached the cell surface (Ozawa et al., 1990; Hinck et al., 1994a). Thus, unlike other type-I membrane proteins studied (see above), E-cadherin is sorted and targeted to the basal-lateral membrane in a stoichiometric complex with a cytosolic protein, β-catenin. This prompted us to explore the relationship between E-cadherin sorting to the basal-lateral membrane domain and assembly of the E-cadherin/β-catenin complex. There are two regions of the cytoplasmic domain of E-cadherin which are similar in amino acid sequence to previously identified basal-lateral sorting signals of LDL-R (Matter et al., 1992), i.e., a sequence motif of Y---XXX, where X is an acidic amino acid residue. The first putative sorting signal in E-cadherin is immediately proximal to the transmembrane domain and the other is at the extreme COOH terminus, also similar to the locations of the two sorting signals in LDL-R cytoplasmic domain (see Fig. 1). We show that despite these similarities, mutations of tyrosine to alanine in those two potential basal-lateral sorting signals in E-cadherin do not affect sorting of newly synthesized mutant E-cadherin in MDCK cells. Further dissection of the cytoplasmic domain of E-cadherin using a chimeric protein approach revealed that E-cadherin exit from the ER and efficient sorting to the basal-lateral membrane are coupled to assembly of the E-cadherin/β-catenin complex shortly after E-cadherin synthesis.

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