Claudin-2 Forms Homodimers and Is a Component of a High Molecular Weight Protein Complex

• Published in: The Journal of biological chemistry Vol. 286; no. 5; pp. 3442 - 3450
• Main Authors: Van Itallie, Christina M., Mitic, Laura L., Anderson, James M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 04.02.2011; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Cell Biology; Cell Junctions; Claudin; Claudin-1; Claudin-4; Claudins; Dogs; Electrophoresis, Polyacrylamide Gel - methods; Epithelial Cell; Liver - ultrastructure; Membrane Proteins; Membrane Proteins - analysis; Membrane Proteins - metabolism; Mice; Molecular Weight; Multiprotein Complexes - chemistry; Occludin; Protein Cross-linking; Protein Multimerization; Protein-Protein Interactions; Tight Junction; Tight Junctions - metabolism; Membrane Proteins; Claudin; Epithelial Cell; Cell Junctions; Protein Cross-linking; Protein-Protein Interactions; Tight Junction; Occludin
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M110.195578

Tight junctions are multiprotein complexes that form the fundamental physiologic and anatomic barrier between epithelial and endothelial cells, yet little information is available about their molecular organization. To begin to understand how the transmembrane proteins of the tight junction are organized into multiprotein complexes, we used blue native-PAGE (BN-PAGE) and cross-linking techniques to identify complexes extracted from MDCK II cells and mouse liver. In nonionic detergent extracts from MDCK II cells, the tight junction integral membrane protein claudin-2 was preferentially isolated as a homodimer, whereas claudin-4 was monomeric. Analysis of the interactions between chimeras of claudin-2 and -4 are consistent with the transmembrane domains of claudin-2 being responsible for dimerization, and mutational analysis followed by cross-linking indicated that the second transmembrane domains were arranged in close proximity in homodimers. BN-PAGE of mouse liver membrane identified a relatively discrete high molecular weight complex containing at least claudin-1, claudin-2, and occludin; the difference in the protein complex sizes between cultured cells and tissues may reflect differences in tight junction protein or lipid composition or post-translational modifications. Our results suggest that BN-PAGE may be a useful tool in understanding tight junction structure.