E-cadherin is a member of the cadherin family of Ca 2؉ -dependent cell-cell adhesion molecules. E-cadherin associates with -catenin at the membrane-distal region of its cytosolic domain and with p120 at the membrane-proximal region of its cytoplasmic domain. It has been shown that a pool of cell surface E-cadherin is constitutively internalized and recycled back to the surface. Further, p120 knockdown by small interference RNA resulted in dose-dependent elimination of cell surface E-cadherin. Consistent with these observations, we found that selective uncoupling of p120 from E-cadherin by introduction of amino acid substitutions in the p120-binding site increased the level of E-cadherin endocytosis. The increased endocytosis was clathrin-dependent, because it was blocked by expression of a dominant-negative form of dynamin or by hypertonic shock. A dileucine motif in the juxtamembrane cytoplasmic domain is required for E-cadherin endocytosis, because substitution of these residues to alanine resulted in impaired internalization of the protein. The alanine substitutions in the p120-uncoupled construct reduced endocytosis of the protein, indicating that this motif was dominant to p120 binding in the control of E-cadherin endocytosis. Therefore, these results are consistent with the idea that p120 regulates E-cadherin endocytosis by masking the dileucine motif and preventing interactions with adaptor proteins required for internalization.Cadherins are a family of structurally and functionally related molecules that mediate Ca 2ϩ -dependent cell-cell adhesion in a homophilic manner (1, 2). Ca 2ϩ protects the extracellular domains of cadherins from proteolytic degradation and is necessary for their function. Cadherin-based adhesion is a central determinant of cell patterning during development and is necessary for the preservation of established tissue architecture in mature organisms. E-cadherin is a prototypic member of the family and is required for the establishment and maintenance of cell-cell adhesion and cell polarity in epithelia and plays key roles in tissue morphogenesis and tumorigenesis. Genetic studies have shown that E-cadherin is essential for epithelial integrity during development in Xenopus and mice (3-5). At the cellular level, E-cadherin facilitates assembly of specialized intercellular junctions (desmosomes, gap, and tight junctions) necessary to link epithelial cells into functional monolayers (6, 7).The adhesive strength of cell-cell contacts depends on factors such as dimerization and lateral clustering of cadherin molecules (8, 9). Cadherins also form high affinity complexes with catenins and other molecules that participate in the overall function and stability of adherens junctions (10, 11). E-cadherin interacts with -catenin and ␣-catenin, which links the complex to the actin cytoskeleton (11, 12). The Rho family GTPases Rho, Rac, and Cdc42 produce different configurations of actin in cells (13) and have been implicated in remodeling actin for the regulation of E-cadherin-mediated adhesi...
2+-dependent cell-cell adhesion and is localized to the basolateral membrane of polarized epithelial cells. Uncoupling -catenin from E-cadherin by deletion or substitution mutations causes accumulation of these proteins in intracellular compartments, including the trans-Golgi network and early endosomes, and degradation in lysosomes. Expression of a dominant-negative dynamin did not change the pattern of the mutant E-cadherin localization, indicating that the endocytosis of the protein from the cell surface does not contribute significantly to the accumulation of the protein in the intracellular compartments. Alternatively, E-cadherin lacking its entire cytoplasmic domain (tail-less E-cadherin) was detected on the surface of cells and targeted to the basolateral membrane. We found that 20 amino acid residues within the juxtamembrane region contain the signal responsible for intracellular accumulation and the lysosomal targeting of E-cadherin. A dileucine motif within this region seems crucial, because substitution of these residues to alanines resulted in efficient surface expression of the protein. The tail-less E-cadherin construct and the dileucinesubstitution construct were detected on the basolateral membranes. Thus, the dileucine motif of E-cadherin is not required for its basolateral targeting.
SummaryIn fulminant hepatic failure, various toxins causing multi-organ failure increase in plasma. As a novel toxin, levels of ceramide, a well-studied lipid mediator of apoptosis, were determined by LC-MS/MS in the liver and plasma of carbon tetrachloride (CCl 4 )-intoxicated rats. After 6 h of oral administration of CCl 4 (4 mL/kg body weight as a 1 : 1 mixture of CCl 4 and mineral oil) to rats, extensive hepatic failure occurred as evidenced by a severe elevation in plasma AST and ALT. The liver concentration of major ceramide components (C16:0, C24:0, C24:1, C18:0, C22:0, and C24:2 in decreasing order), and the sum of these ceramides increased significantly 2 h after CCl 4 intoxication compared to that in the control group given mineral oil. The total ceramide concentration in the plasma was also increased to 4.1 times that in the control 24 h after administration of CCl 4 . In conclusion, the early increase in liver ceramides may contribute to hepatic cell death and the increase in plasma ceramides during fulminant hepatic failure may cause damage in other organs including the brain and kidney.
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