title: The role of Nedd4-1 WW domains in binding and regulating human organic anion transporter 1.
abstract: Human organic anion transporter 1 (hOAT1), expressed at the basolateral membrane of kidney proximal tubule cells, mediates the active renal secretion of a diverse array of clinically important drugs, including anti-human immunodeficiency virus therapeutics, antitumor drugs, antibiotics, antihypertensives, and anti-inflammatories. We have previously demonstrated that posttranslational modification of hOAT1 by ubiquitination is an important mechanism for the regulation of this transporter. The present study aimed at identifying the ubiquitin ligase for hOAT1 and its mechanism of action. We showed that overexpression of neural precursor cell expressed, developmentally downregulated (Nedd)4-1, an E3 ubiquitin ligase, enhanced hOAT1 ubiquitination, decreased hOAT1 expression at the cell surface, and inhibited hOAT1 transport activity. In contrast, overexpression of the ubiquitin ligase-dead mutant Nedd4-1/C867S was without effects on hOAT1. Furthermore, knockdown of endogenously expressed Nedd4-1 by Nedd4-1-specific small interfering RNA reduced hOAT1 ubiquitination. Immunoprecipitation experiments in cultured cells and rat kidney slices and immunofluorescence experiments in rat kidney slices showed that there was a physical interaction between OAT1 and Nedd4-1. Nedd4-1 contains four protein-protein interacting WW domains. When these WW domains were inactivated by mutating two amino acid residues in each of the four WW domains (Mut-WW1: V210W/H212G, Mut-WW2: V367W/H369G, Mut-WW3: I440W/H442G, and Mut-WW4: I492W/H494G, respectively), only Mut-WW2 and Mut-WW3 significantly lost their ability to bind and to ubiquitinate hOAT1. As a result, Mut-WW2 and Mut-WW3 were unable to suppress hOAT1-mediated transport as effectively as wild-type Nedd4-1. In conclusion, this is the first demonstration that Nedd4-1 regulates hOAT1 ubiquitination, expression, and transport activity through its WW2 and WW3 domains.
authors.affiliation: Department of Pharmaceutics, Rutgers University, Piscataway, New Jersey;
authors.forename: Da
authors.initials: D
authors.lastname: Xu
authors.affiliation: Department of Pharmaceutics, Rutgers University, Piscataway, New Jersey;
authors.forename: Haoxun
authors.initials: H
authors.lastname: Wang
authors.affiliation: Department of Pharmacology and Toxicology, Rutgers University, Piscataway, New Jersey;
authors.forename: Carol
authors.initials: C
authors.lastname: Gardner
authors.affiliation: Thoracic Surgery Division, Department of Surgery, The Ohio State University Wexner Medical Center, Columbus, Ohio; and.
authors.forename: Zui
authors.initials: Z
authors.lastname: Pan
authors.affiliation: Department of Anatomic Pathology, William Beaumont Hospital, Royal Oak, Michigan.
authors.forename: Ping L
authors.initials: PL
authors.lastname: Zhang
authors.affiliation: Department of Pharmaceutics, Rutgers University, Piscataway, New Jersey;
authors.forename: Jinghui
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Pharmaceutics, Rutgers University, Piscataway, New Jersey; gyou@rci.rutgers.edu.
authors.forename: Guofeng
authors.initials: G
authors.lastname: You
mesh_terms: D000818:Animals; D019921:Biotinylation; D019556:COS Cells; D002522:Chlorocebus aethiops; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D066298:In Vitro Techniques; D007668:Kidney / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D000075702:Nedd4 Ubiquitin Protein Ligases; D027362:Organic Anion Transport Protein 1 / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D051381:Rats; D017207:Rats, Sprague-Dawley; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; D008565:Membrane Proteins; D027362:Organic Anion Transport Protein 1; C000618479:NEDD4L protein, rat; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618484:Nedd4 protein, rat; D044767:Ubiquitin-Protein Ligases
keywords: WW domain; drug transporter; membrane transporter; regulation; ubiquitin ligase; ubiquitination