Identification and characterization of proteins that interact with zonula occludens proteins

Abstract

Tight junctions (TJ) are specialized plasma membrane domains that regulate the paracellular transepithelial permeability and prevent the inter-mixing of apical and basolateral plasma membrane domains. TJs consist of integral membrane proteins that are linked via cytoplasmic peripheral proteins to the actin cytoskeleton. The objective of this study is to identify and characterize proteins that interact with zonula occluden-1 (ZO-1). Novel proteins that interact with ZO-1 were identified using a yeast-two-hybrid screen. Connexin 45 and ARVCF were identified and the functional significances of their interaction were analyzed. In addition, ZO-1 was found to interact with claudin-16, and its role in TJ barrier function was analyzed. Mutations in claudin-16 have been linked to familial hypomagnesaemia with hypercalciuria and nephrocalcinosis (FHHNC), where affected individuals excrete excessive magnesium and calcium in the urine, leading to kidney stones and end-stage renal failure. The molecular mechanisms by which these mutations affect CLDN16 function were determined. Interestingly, mutations associated with FHHNC could affect either the correct intracellular transport of CLDN16, or its function in paracellular ion transport.