Limits...
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.

Show MeSH

Related in: MedlinePlus

Dominant basal-lateral sorting activity  of E-cadherin is located in the transmembrane/ cytoplasmic domains. (A) GPI modification is  the sole determinant for targeting GP2 to the  apical membrane. GP2 has been shown previously to be preferentially delivered to the apical  domain of MDCK clone II/G cells (Mays et al.,  1995). Diagrams show schematically the structure of GP2 lacking the GPI modification signal  (GP2ΔGPI). GP2ΔGPI delivery was determined as described for E-cadsol (see Fig. 1 legend, and Materials and Methods). (B) Newly  synthesized GP2CAD1, labeled for 1 h, is sorted  to the basal-lateral membrane domain, similar to  endogenous E-cadherin. Diagrams show schematically the structure of GP2CAD1 compared  with E-cadherin. GP2CAD1 delivery to the cell  surface was assessed by 35S-Met/Cys labeling and cell surface biotinylation of the apical (Ap) or the basal-lateral (Bl) membrane domain. Note that the autoradiogram showing the E-cadherin targeting is the same as that shown in Fig. 2, and is included here for comparison.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132940&req=5

Figure 3: Dominant basal-lateral sorting activity of E-cadherin is located in the transmembrane/ cytoplasmic domains. (A) GPI modification is the sole determinant for targeting GP2 to the apical membrane. GP2 has been shown previously to be preferentially delivered to the apical domain of MDCK clone II/G cells (Mays et al., 1995). Diagrams show schematically the structure of GP2 lacking the GPI modification signal (GP2ΔGPI). GP2ΔGPI delivery was determined as described for E-cadsol (see Fig. 1 legend, and Materials and Methods). (B) Newly synthesized GP2CAD1, labeled for 1 h, is sorted to the basal-lateral membrane domain, similar to endogenous E-cadherin. Diagrams show schematically the structure of GP2CAD1 compared with E-cadherin. GP2CAD1 delivery to the cell surface was assessed by 35S-Met/Cys labeling and cell surface biotinylation of the apical (Ap) or the basal-lateral (Bl) membrane domain. Note that the autoradiogram showing the E-cadherin targeting is the same as that shown in Fig. 2, and is included here for comparison.

Mentions: To locate the basal-lateral sorting signal(s) in E-cadherin, we constructed a chimeric protein comprising the transmembrane and specific regions of the cytoplasmic domain of E-cadherin fused to the extracellular domain of GP2, a highly glycosylated protein of zymogen granules in pancreatic exocrine cells (Hoops and Rindler, 1991). Normally, GP2 is attached to the membrane via a GPI anchor, and is delivered exclusively to the apical domain of MDCK clone II/G cells (Mays et al., 1995). A deletion mutant of GP2 that lacked the GPI modification signal (Fig. 3 A, GP2ΔGPI) was expressed in MDCK cells. As shown in Fig. 3 A, newly synthesized GP2ΔGPI was secreted equally into both the apical and basal-lateral medium. This result confirms that GP2 sorting to the apical membrane requires the GPI-anchor (see Mays et al., 1995), and reveals that the extracellular domain of GP2 does not contain specific sorting signals for the basal-lateral 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)

Dominant basal-lateral sorting activity  of E-cadherin is located in the transmembrane/ cytoplasmic domains. (A) GPI modification is  the sole determinant for targeting GP2 to the  apical membrane. GP2 has been shown previously to be preferentially delivered to the apical  domain of MDCK clone II/G cells (Mays et al.,  1995). Diagrams show schematically the structure of GP2 lacking the GPI modification signal  (GP2ΔGPI). GP2ΔGPI delivery was determined as described for E-cadsol (see Fig. 1 legend, and Materials and Methods). (B) Newly  synthesized GP2CAD1, labeled for 1 h, is sorted  to the basal-lateral membrane domain, similar to  endogenous E-cadherin. Diagrams show schematically the structure of GP2CAD1 compared  with E-cadherin. GP2CAD1 delivery to the cell  surface was assessed by 35S-Met/Cys labeling and cell surface biotinylation of the apical (Ap) or the basal-lateral (Bl) membrane domain. Note that the autoradiogram showing the E-cadherin targeting is the same as that shown in Fig. 2, and is included here for comparison.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Dominant basal-lateral sorting activity of E-cadherin is located in the transmembrane/ cytoplasmic domains. (A) GPI modification is the sole determinant for targeting GP2 to the apical membrane. GP2 has been shown previously to be preferentially delivered to the apical domain of MDCK clone II/G cells (Mays et al., 1995). Diagrams show schematically the structure of GP2 lacking the GPI modification signal (GP2ΔGPI). GP2ΔGPI delivery was determined as described for E-cadsol (see Fig. 1 legend, and Materials and Methods). (B) Newly synthesized GP2CAD1, labeled for 1 h, is sorted to the basal-lateral membrane domain, similar to endogenous E-cadherin. Diagrams show schematically the structure of GP2CAD1 compared with E-cadherin. GP2CAD1 delivery to the cell surface was assessed by 35S-Met/Cys labeling and cell surface biotinylation of the apical (Ap) or the basal-lateral (Bl) membrane domain. Note that the autoradiogram showing the E-cadherin targeting is the same as that shown in Fig. 2, and is included here for comparison.
Mentions: To locate the basal-lateral sorting signal(s) in E-cadherin, we constructed a chimeric protein comprising the transmembrane and specific regions of the cytoplasmic domain of E-cadherin fused to the extracellular domain of GP2, a highly glycosylated protein of zymogen granules in pancreatic exocrine cells (Hoops and Rindler, 1991). Normally, GP2 is attached to the membrane via a GPI anchor, and is delivered exclusively to the apical domain of MDCK clone II/G cells (Mays et al., 1995). A deletion mutant of GP2 that lacked the GPI modification signal (Fig. 3 A, GP2ΔGPI) was expressed in MDCK cells. As shown in Fig. 3 A, newly synthesized GP2ΔGPI was secreted equally into both the apical and basal-lateral medium. This result confirms that GP2 sorting to the apical membrane requires the GPI-anchor (see Mays et al., 1995), and reveals that the extracellular domain of GP2 does not contain specific sorting signals for the basal-lateral 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