Abstract
Endothelial cell-selective adhesion molecule (ESAM) is an immunoglobulin-like transmembrane protein associated with endothelial tight junctions (TJ). Based on a yeast two-hybrid screen, we have identified the membrane-associated guanylate kinase protein MAGI-1 as an intracellular binding partner of ESAM. MAGI-1 is a multidomain adaptor protein, which binds to transmembrane, cytoskeletal, and signaling molecules, and has been localized to tight junctions in epithelial cells. MAGI-1 associates with the very C-terminal sequence of ESAM most likely through a PDZ domain-mediated interaction. The direct interaction between ESAM and MAGI-1 was confirmed by pull-down experiments. The two proteins formed stable complexes in transfected Chinese hamster ovary (CHO) cells, which could be immunoisolated. We found MAGI-1 to be associated with cell-cell contacts in human umbilical vein endothelial cells (HUVECs) and in mouse endothelium, where it colocalizes with ESAM. In CHO cells, recruitment of MAGI-1 to cell contacts required the presence of ESAM. Hence, ESAM may be involved in anchoring MAGI-1 at endothelial tight junctions.
MeSH terms
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Adaptor Proteins, Signal Transducing / genetics
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Adaptor Proteins, Signal Transducing / metabolism
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Adaptor Proteins, Vesicular Transport / genetics
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Adaptor Proteins, Vesicular Transport / isolation & purification
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Adaptor Proteins, Vesicular Transport / metabolism*
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Alternative Splicing / genetics
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Animals
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Binding Sites / physiology
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CHO Cells
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Cell Adhesion / physiology
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Cell Adhesion Molecules / genetics
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Cell Adhesion Molecules / metabolism*
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Cell Communication / physiology
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Cell Line, Tumor
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Cricetinae
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Endothelial Cells
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Endothelium, Vascular / metabolism
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Guanylate Kinases
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Humans
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Macromolecular Substances
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Membrane Proteins / genetics
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Membrane Proteins / isolation & purification
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Membrane Proteins / metabolism*
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Mice
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Molecular Sequence Data
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Nucleoside-Phosphate Kinase / genetics
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Nucleoside-Phosphate Kinase / isolation & purification
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Nucleoside-Phosphate Kinase / metabolism*
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Phylogeny
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Protein Binding / physiology
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Protein Structure, Tertiary / physiology
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Sequence Homology, Amino Acid
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Sequence Homology, Nucleic Acid
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Tight Junctions / metabolism*
Substances
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Adaptor Proteins, Signal Transducing
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Adaptor Proteins, Vesicular Transport
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CNKSR3 protein, human
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Cell Adhesion Molecules
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Esam protein, mouse
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Macromolecular Substances
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Membrane Proteins
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Nucleoside-Phosphate Kinase
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Guanylate Kinases
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Magi1 protein, mouse