title: Claudin-2 forms homodimers and is a component of a high molecular weight protein complex.
abstract: 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.
authors.affiliation: Department of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599, USA. Christina.VanItallie@nih.gov
authors.forename: Christina M
authors.initials: CM
authors.lastname: Van Itallie
authors.forename: Laura L
authors.initials: LL
authors.lastname: Mitic
authors.forename: James M
authors.initials: JM
authors.lastname: Anderson
mesh_terms: D000818:Animals; D062445:Claudin-1; D062506:Claudin-4; D057167:Claudins; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D008099:Liver / Q000648:ultrastructure; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D046912:Multiprotein Complexes / Q000737:chemistry*; D062793:Occludin; D055503:Protein Multimerization; D019108:Tight Junctions / Q000378:metabolism*
chemical_list: D062445:Claudin-1; D062506:Claudin-4; D057167:Claudins; C568113:Cldn1 protein, mouse; C113371:Cldn2 protein, mouse; C568158:Cldn4 protein, mouse; D008565:Membrane Proteins; D046912:Multiprotein Complexes; D062793:Occludin; C568986:Ocln protein, mouse