Macromolecular Complex of CFTR, NHERF2 and iNOS at the Plasma Membrane Contributes to Diarrhea in IBD

Abstract

Macromolecular complex formation is a common theme in regulation of protein function. The cystic fibrosis transmembrane conductance regulator (CFTR), a cGMP/cAMP‐regulated chloride (Cl−) channel, is a well‐known hub protein involved in multiple protein complexes. In this study, we find that a signaling macromolecular complex of CFTR, Na+/H+ exchanger regulatory factor 2 (NHERF2), and inducible Nitric Oxide Synthase (iNOS) is formed at the plasma membrane in Inflammatory Bowel disease (IBD). We demonstrated the protein interactions using biochemical methods (e.g., protein pairwise binding assay, macromolecular complex assembly assay, and co‐immunoprecipitation) and in vitro methods for studying protein interaction in live cell (e.g., FRET). We conclude based on our in vitro (polarized colonic epithelial monolayers) and in vivo (DSS‐induced colitis in mice) functional studies that the interaction is integrative to CFTR dependent diarrhea in IBD. To summarize our findings: (a) CFTR interacts with iNOS mediated by NHERF2 in a PDZ domain‐dependent manner. (b) Upregulation of iNOS increased CFTR channel function by at least two folds in in vivo and in vitro models of inflammation. (c). Diarrhea was attenuated in Nherf2−/− mice compared to Nherf2+/+mice in DSS‐induced colitis model which simulates IBD. Taken together, our results demonstrate that the macromolecular complex of CFTR, NHERF2 and iNOS play important roles in the pathogenic process of diarrhea in IBD. Our study will not only help understand the molecular mechanisms underlying the pathogenic process of diarrhea in IBD, but help to identify novel therapeutic targets and agents for IBD therapy.