This application for continued support will focus on studying how protein-protein interactions among the cystic fibrosis transmembrane conductance regulator (CFTR), multidrug resistance protein 4 (MRP4) and inducible nitric oxide synthase (iNOS) contribute to the diarrheal symptom observed in Ulcerative Colitis (UC). Our unifying hypothesis is that CFTR, NHERF2, MRP4 and iNOS form a macromolecular complex at or near the plasma membrane in gut epithelial in UC and this macromolecular complex is required for the pathogenic process of diarrhea observed in UC. Specifically, we proposed that iNOS is overexpressed at the plasma membrane of gut epithelia in UC, resulting in the production of sustained high level of nitric oxide (NO) which triggers NO-cGMP pathway and generates compartmentalized cGMP. Since iNOS forms a macromolecular complex with CFTR, NHERF2 and MRP4 at or near the plasma membrane, this NO-dependent compartmentalized cGMP triggers the hyperactivation of CFTR chloride channels and thus causes diarrhea phenotype. Disruption of the macromolecular complex will abolish the functional coupling of iNOS-dependent cGMP generation and CFTR Cl- channel function.
The specific aims of this proposal are:
Specific Aim 1. To test the hypothesis that iNOS is overexpressed in UC-affected human gut epithelia and to test whether it is coupled to CFTR and MRP4 and upregulates CFTR Cl- channel function Specific Aim 2. To test the hypothesis that iNOS, CFTR, and MRP4 form a macromolecular complex at or near the plasma membrane of gut epithelial cells and that this macromolecular complex is required for the pathogenic process of diarrhea observed in UC. The proposed studies will not only help us better understand the mechanisms underlying the diarrhea symptom commonly observed in UC, but probably provide novel targets and methods for therapeutic interventions of the disease. Therefore, the studies proposed in this application will have clinical relevance for many individuals suffering from UC and also for those suffering from certain forms of secretory diarrheas.

Public Health Relevance

In this grant proposal, we will address a novel mechanism of how protein-protein interactions can contribute to the progression of ulcerative colitis (UC)-related diarrhea. We hypothesize that the macromolecular complex of iNOS (the inducible Nitric Oxide Synthase), CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), NHERF2 (Na+/H+ exchanger regulatory Factor 2) and MRP4 (Multiple drug related protein 4) is spatially and temporally coupled to cGMP (cyclic guanosine monophosphate) generation which causes CFTR-dependent chloride secretion and thus diarrhea in UC. By targeting this macromolecular complex, we may find ways to control fluid secretion and thereby control or cure the disease.

National Institute of Health (NIH)
Research Project (R01)
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Clinical, Integrative and Molecular Gastroenterology Study Section (CIMG)
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Grey, Michael J
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Sinha, Chandrima; Arora, Kavisha; Moon, Chang Suk et al. (2014) Förster resonance energy transfer - an approach to visualize the spatiotemporal regulation of macromolecular complex formation and compartmentalized cell signaling. Biochim Biophys Acta 1840:3067-72
Arora, Kavisha; Moon, Changsuk; Zhang, Weiqiang et al. (2014) Stabilizing rescued surface-localized ?f508 CFTR by potentiation of its interaction with Na(+)/H(+) exchanger regulatory factor 1. Biochemistry 53:4169-79
Holcomb, Joshua; Jiang, Yuanyuan; Lu, Guorong et al. (2014) Structural insights into PDZ-mediated interaction of NHERF2 and LPA(2), a cellular event implicated in CFTR channel regulation. Biochem Biophys Res Commun 446:399-403
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Ren, Aixia; Zhang, Weiqiang; Yarlagadda, Sunitha et al. (2013) MAST205 competes with cystic fibrosis transmembrane conductance regulator (CFTR)-associated ligand for binding to CFTR to regulate CFTR-mediated fluid transport. J Biol Chem 288:12325-34
Sinha, Chandrima; Ren, Aixia; Arora, Kavisha et al. (2013) Multi-drug resistance protein 4 (MRP4)-mediated regulation of fibroblast cell migration reflects a dichotomous role of intracellular cyclic nucleotides. J Biol Chem 288:3786-94
Arora, Kavisha; Sinha, Chandrima; Zhang, Weiqiang et al. (2013) Compartmentalization of cyclic nucleotide signaling: a question of when, where, and why? Pflugers Arch 465:1397-407
Zhang, Weiqiang; Fujii, Naoaki; Naren, Anjaparavanda P (2012) Recent advances and new perspectives in targeting CFTR for therapy of cystic fibrosis and enterotoxin-induced secretory diarrheas. Future Med Chem 4:329-45
Ray, Ramesh M; Li, Chunying; Bhattacharya, Sujoy et al. (2012) Spermine, a molecular switch regulating EGFR, integrin ýý3, Src, and FAK scaffolding. Cell Signal 24:931-42
Sellers, Zachary M; Naren, Anjaparavanda P; Xiang, Yang et al. (2012) MRP4 and CFTR in the regulation of cAMP and ?-adrenergic contraction in cardiac myocytes. Eur J Pharmacol 681:80-7

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