CEACAM1, a cell-cell adhesion molecule, directly associates with annexin II in a three-dimensional model of mammary morphogenesis.
The epithelial cell adhesion molecule CEACAM1 (carcinoembryonic antigen cell adhesion molecule-1) is down-regulated in colon, prostate, breast, and liver cancer. Here we show that CEACAM1-4S, a splice form with four Ig-like ectodomains and a short cytoplasmic domain (14 amino acids), directly associates with annexin II, a lipid raft-associated molecule, which ... is also down-regulated in many cancers. Annexin II was identified using a glutathione S-transferase pull-down assay in which the cytoplasmic domain of CEACAM-4S was fused to glutathione S-transferase, the fusion protein was incubated with cell lysates, and isolated proteins were sequenced by mass spectrometry. The interaction was confirmed first by reciprocal immunoprecipitations using anti-CEACAM1 and anti-annexin II antibodies and second by confocal laser microscopy showing co-localization of CEACAM1 with annexin II in mammary epithelial cells grown in Matrigel. In addition, CEACAM1 co-localized with p11, a component of the tetrameric AIIt complex at the plasma membrane, and with annexin II in secretory vesicles. Immobilized, oriented peptides from the cytoplasmic domain of CEACAM1-4S were shown to directly associate with bovine AIIt, which is 98% homologous to human AIIt, with average KD values of about 30 nM using surface plasmon resonance, demonstrating direct binding of functionally relevant AIIt to the cytoplasmic domain of CEACAM1-4S.
Mesh Terms:
Amino Acid Sequence, Animals, Annexin A2, Antigens, CD, Antigens, Differentiation, Blotting, Western, Cattle, Cell Adhesion, Cell Adhesion Molecules, Cell Line, Cell Line, Tumor, Collagen, Cytoplasm, Down-Regulation, Drug Combinations, Glutathione Transferase, Hela Cells, Humans, Kinetics, Laminin, Mass Spectrometry, Microscopy, Confocal, Molecular Sequence Data, Peptides, Precipitin Tests, Protein Binding, Protein Structure, Tertiary, Proteoglycans, Recombinant Fusion Proteins, Surface Plasmon Resonance, Time Factors
Amino Acid Sequence, Animals, Annexin A2, Antigens, CD, Antigens, Differentiation, Blotting, Western, Cattle, Cell Adhesion, Cell Adhesion Molecules, Cell Line, Cell Line, Tumor, Collagen, Cytoplasm, Down-Regulation, Drug Combinations, Glutathione Transferase, Hela Cells, Humans, Kinetics, Laminin, Mass Spectrometry, Microscopy, Confocal, Molecular Sequence Data, Peptides, Precipitin Tests, Protein Binding, Protein Structure, Tertiary, Proteoglycans, Recombinant Fusion Proteins, Surface Plasmon Resonance, Time Factors
J. Biol. Chem.
Date: Dec. 12, 2003
PubMed ID: 14522961
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