title: Distribution dynamics and functional importance of NHERF1 in regulation of Mrp-2 trafficking in hepatocytes.
abstract: Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) is a multifunctional scaffolding protein that interacts with receptors and ion transporters in its PDZ domains and with the ezrin-radixin-moesin (ERM) family of proteins in its COOH terminus. The role of NHERF1 in hepatocyte function remains largely unknown. We examine the distribution and physiological significance of NHERF1 and multidrug resistance-associated protein 2 (Mrp-2) in hepatocytes. A WT radixin binding site mutant (F355R) and NHERF1 PDZ1 and PDZ2 domain adenoviral mutant constructs were tagged with yellow fluorescent protein and expressed in polarized hepatocytes to study localization and function of NHERF1. Cellular distribution of NHERF1 and radixin was visualized by fluorescence microscopy. A 5-chloromethylfluorescein diacetate (CMFDA) assay was used to characterize Mrp-2 function. Similar to Mrp-2, WT NHERF1 and the NHERF1 PDZ2 deletion mutant were localized to the canalicular membrane. In contrast, the radixin binding site mutant (F355R) and the NHERF1 PDZ1 deletion mutant, which interacts poorly with Mrp-2, were rarely associated with the canalicular membrane. Knockdown of NHERF1 led to dramatically impaired CMFDA secretory response. Use of CMFDA showed that the NHERF1 PDZ1 and F355R mutants were devoid of a secretory response, while WT NHERF1-infected cells exhibited increased secretion of glutathione-methylfluorescein. The data indicate that NHERF1 interacts with Mrp-2 via the PDZ1 domain of NHERF1 and, furthermore, that NHERF1 is essential for maintaining the localization and function of Mrp-2.
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Medicine, Medical University of South Carolina, Charleston, South Carolina; karvar@musc.edu.
authors.forename: Serhan
authors.initials: S
authors.lastname: Karvar
authors.affiliation: Division of Gastrointestinal and Liver Diseases, Keck School of Medicine, University of Southern California, Los Angeles, California; and.
authors.forename: Jo
authors.initials: J
authors.lastname: Suda
authors.affiliation: Digestive Diseases and Nutrition Center, University at Buffalo, State University of New York, Buffalo, New York.
authors.forename: Lixin
authors.initials: L
authors.lastname: Zhu
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Medicine, Medical University of South Carolina, Charleston, South Carolina;
authors.forename: Don C
authors.initials: DC
authors.lastname: Rockey
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism; D015870:Gene Expression; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D000089762:Multidrug Resistance-Associated Protein 2; D020125:Mutation, Missense; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D051381:Rats; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*; D000097202:ATP-Binding Cassette Sub-Family B Member 4
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D010750:Phosphoproteins; D017923:Sodium-Hydrogen Exchangers; D000097202:ATP-Binding Cassette Sub-Family B Member 4; C000715087:ABCC2 protein, human; C093376:sodium-hydrogen exchanger regulatory factor; C071776:radixin
keywords: EBP-50; bile canaliculus; bile transporter; cytoskeleton; liver