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.

Comprehensive Cysteine-scanning Mutagenesis Reveals Claudin-2 Pore-lining Residues with Different Intrapore Locations

“…G49C, L50C, W51C, G60C, and P74C were mostly intracellularly localized. Mutations of the conserved motif Gly 49 -Leu 50 -Trp 51 resulted in claudin-2 mislocalization, which is consistent with previous reports (18,19). The mislocalization of G60C and P74C is described for the first time.…”

Autophagy Enhances Intestinal Epithelial Tight Junction Barrier Function by Targeting Claudin-2 Protein Degradation

“…The pore pathway is largely regulated by the TJ membrane proteins claudin, particularly claudin-2, which is a cation-selective, pore-forming protein (21,22). Therefore, we next studied protein expression of selected claudins (claudin-1, 2, 3, 8, and 13) that are known to regulate the paracellular TJ barrier (23). The protein level of claudin-1, 3, 8, and 13 showed no change during starvation (Fig.…”

Phosphorylation of claudin-2 on serine 208 promotes membrane retention and reduces trafficking to lysosomes

“…7D,E). Fourth, vsv-g tagged claudin-2 containing mutations in the highly conserved GLW in first extracellular domain is neither localised at the tight junction (Van Itallie et al, 2011) nor phosphorylated at S208 (Fig. 7D,E).…”

“…Tet-off MDCK I cells were generously provided by Dr Alan Yu, University of Kansas, Kansas City, KS; MDCK II Tet-off cells, cells expressing wild-type mouse claudin-2, claudin-2 knockdown cells (Van Itallie et al, 2008), claudin-2 containing the GLW to AAA mutation, N-terminal FLAG-tag (Van Itallie et al, 2011) and (-) PDZ binding motif (Van Itallie et al, 2009b) have been previously described. N-terminally tagged GFP-and mApple claudin-2 were cloned in frame into C-series vectors by PCR; the mApple vector was a gift from Dr Clare Waterman, NIH.…”

“…Considerable study has been devoted to the role of specific amino acids in the extracellular domains and how they influence paracellular charge selectivity (Colegio et al, 2002;Hou et al, 2005;Angelow and Yu, 2009) and claudin: claudin interactions (Piontek et al, 2008;Piontek et al, 2011). In addition, the transmembrane helices have been implicated in homo- (Van Itallie et al, 2011) and heterodimerisation (Hou et al, 2009). Most claudins share a highly conserved PDZ-binding motif at the extreme carboxyl terminus which mediates binding to the first PDZ domain of the tight junction scaffolding proteins ZO-1, -2 and -3 (Itoh et al, 1999).…”