The Coiled-coil Domain of Occludin Can Act to Organize Structural and Functional Elements of the Epithelial Tight Junction

Abstract

Occludin is an integral membrane protein that has been suggested to play a role in the organization and dynamic function of the epithelial tight junction (TJ). A number of other proteins have also been described to localize to the TJ. We have used a novel bait peptide method to investigate potential protein-protein interactions of the putative coiled-coil domain of occludin with some of these other TJ proteins. A 27-amino acid peptide of the human occludin sequence was synthesized, biotinylated at the N terminus, and modified to contain a photoactive moiety at either its hydrophobic or hydrophilic surface. These bait peptides were ␣-helical in solution, characteristic of coiled-coil structures. Photoactivation studies in the presence and absence of control peptides were used to assess the potential interactions in polarized sheets of a human intestinal cell line T84. Although a large number of proteins associated with the TJ or that are known to be involved in regulatory events of epithelial cells failed to be specifically labeled, occludin itself, ZO-1, protein kinase C-, c-Yes, the regulatory subunit of phosphatidylinositol 3-kinase, and the gap junction component connexin 26 were specifically labeled. Our data demonstrate the potential of one specific domain of occludin, contained within 27 amino acids, to coordinate the binding of proteins that have been previously suggested to modulate TJ structure and function.Over the last decade a number of proteins have been identified that localize to the tight junction (TJ) 1 structures of epithelial cells. Possible functional interactions between these proteins have been described (reviewed in Ref. 1). Critical extracellular interactions in TJs have been attributed to two transmembrane proteins, claudin(s) and occludin (2). Although it has been suggested that claudins recruit occludin to TJ sites (3), several recent studies have suggested instead that occludin dynamically regulates claudin-based TJ strands. Transfection of occludin mutants lacking either the intracellular (4) or extracellular (5) domains induces disruption of epithelial barrier properties. In addition, TJ barrier function is also influenced by incubation with peptides containing the two extracellular loop amino acid sequences (6 -8). As yet, analogous studies have not been described for claudin(s). Finally, disruption of TJ function mediated by the constitutive activation of Raf-1 is associated with down-regulation of occludin and claudin-1 expression, an effect that can be reversed by the reintroduction of occludin expression that in turn restores claudin-1 protein levels (9).Human occludin is approximately 65 kDa with what appears to be a 65-amino acid cytosolic N terminus, two extracellular loops of 46 and 48 amino acids separated by a 10-amino acid cytosolic loop, and a C-terminal tail of approximately 255 amino acids (10). Both the N-and C-terminal domains have a large number of serine and threonine residues, and the functionally active form of the protein localizing to the TJ appears to be hype...

Inflammatory processes have differential effects on claudins 2, 3 and 4 in colonic epithelial cells

“…90 As we did not see reversal of the IL-13-mediated increase in the passage of FITC-dextran with LY294002, this also suggests separable regulation of the TER and the passage of uncharged molecules, the latter possibly regulated by occludin (see above). As PI3K has been shown to bind to occludin C-terminus 90,91 this result was unexpected but it implies that the two mechanisms are separable or may have different sensitivities to PI3K inhibition in T84 cells.…”

Neutrophil-Epithelial Interactions

“…For example, expression of full-length occludin has been shown to reverse epithelial-to-mesenchymal transition in transformed salivary epithelial cells, an effect that is dependent on the coiled coil extracellular loop. 103 Aberrant claudin, CAR, and JAM-A protein expression has been correlated with cancer progression, altered barrier, and certain inflammatory diseases. 99,104,105 These associations, epithelial functions of junction-associated proteins, have been investigated using forward genetic approaches.…”

Tight junctions: from simple barriers to multifunctional molecular gates

“…Occludin, a close relative of MarvelD3, also interacts with a mitogen activated protein (MAP) kinase pathway, but suppresses dissociation of cell junctions by Raf-1 activated Erk signalling via unknown mechanisms 151 . Occludin interacts with multiple signalling proteins that may play a role and has also been linked to TGFβ-induced junction dissociation (Table 1) 42,152,153 .…”