The coiled-coil domain of occludin can act to organize structural and functional elements of the epithelial tight junction

Occludin is an integral membrane protein that has been suggested to play a role in the organization and dynamic function of the epithelial tight junct ion (TJ). A number of other proteins have also been described to localize t o the TJ, We have used a novel bait peptide method to investigate potential protein-protein interactions of the putative coiled-coil domain of occludi n with some of these other TJ proteins. A 27-amino acid peptide of the huma n occludin sequence was synthesized, biotinylated at the N terminus, and mo dified to contain a photoactive moiety at either its hydrophobic or hydroph ilic surface. These bait peptides were alpha-helical in solution, character istic of coiled-coil structures. Photoactivation studies in the presence an d absence of control peptides were used to assess the potential interaction s 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-zeta c-Yes, the regulatory subuni t of phosphatidylinositol 3-kinase, and the gap junction component connexin 26 were specifically labeled. Our data demonstrate the potential of one sp ecific domain of occludin, contained within 27 amino acids, to coordinate t he binding of proteins that have been previously suggested to modulate TJ s tructure and function.