A PDZ-Like Motif in the Biliary Transporter ABCB4 Interacts with the Scaffold Protein EBP50 and Regulates ABCB4 Cell Surface Expression

RESEARCH ARTICLE A PDZ-Like Motif in the Biliary Transporter ABCB4 Interacts with the Scaffold Protein EBP50 and Regulates ABCB4 Cell Surface Expression Quitterie Venot1, Jean-Louis Delaunay1, Laura Fouassier1, Danièle Delautier1, Thomas Falguières1, Chantal Housset1,2, Michèle Maurice1, Tounsia Aït-Slimane1* a11111 1 Sorbonne Universités, UPMC Univ Paris 06, INSERM, UMR_S 938, Centre de Recherche Saint-Antoine, Paris, France, 2 Assistance Publique-Hôpitaux de Paris, Hôpital Saint-Antoine, Centre de Référence Maladies Rares Maladies Inflammatoires des Voies Biliaires & Service d’Hépatologie, Paris, France * OPEN ACCESS Citation: Venot Q, Delaunay J-L, Fouassier L, Delautier D, Falguières T, Housset C, et al. (2016) A PDZ-Like Motif in the Biliary Transporter ABCB4 Interacts with the Scaffold Protein EBP50 and Regulates ABCB4 Cell Surface Expression. PLoS ONE 11(1): e0146962. doi:10.1371/journal. pone.0146962 Editor: Laszlo Buday, Hungarian Academy of Sciences, HUNGARY Received: August 3, 2015 Accepted: December 23, 2015 Published: January 20, 2016 Copyright: © 2016 Venot et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract ABCB4/MDR3, a member of the ABC superfamily, is an ATP-dependent phosphatidylcholine translocator expressed at the canalicular membrane of hepatocytes. Defects in the ABCB4 gene are associated with rare biliary diseases. It is essential to understand the mechanisms of its canalicular membrane expression in particular for the development of new therapies. The stability of several ABC transporters is regulated through their binding to PDZ (PSD95/ DglA/ZO-1) domain-containing proteins. ABCB4 protein ends by the sequence glutamineasparagine-leucine (QNL), which shows some similarity to PDZ-binding motifs. The aim of our study was to assess the potential role of the QNL motif on the surface expression of ABCB4 and to determine if PDZ domain-containing proteins are involved. We found that truncation of the QNL motif decreased the stability of ABCB4 in HepG2-transfected cells. The deleted mutant ABCB4-ΔQNL also displayed accelerated endocytosis. EBP50, a PDZ protein highly expressed in the liver, strongly colocalized and coimmunoprecipitated with ABCB4, and this interaction required the QNL motif. Down-regulation of EBP50 by siRNA or by expression of an EBP50 dominant-negative mutant caused a significant decrease in the level of ABCB4 protein expression, and in the amount of ABCB4 localized at the canalicular membrane. Interaction of ABCB4 with EBP50 through its PDZ-like motif plays a critical role in the regulation of ABCB4 expression and stability at the canalicular plasma membrane. Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by grants from the French association for the study of the liver (AFEF), and the Association Mucoviscidose – ABCF2. QV received a fellowship from the “Ministère de l’Enseignement Supérieur et de la Recherche” and “Association pour la lutte contre les maladies inflammatoires du foie et des voies biliaires (Albi)”. TF was supported by fellowships from INSERM and Fondation pour la Recherche Médicale (FRM). Introduction The superfamily of ABC (ATP-binding cassette) transporters comprises a large number of membrane proteins, which mediate the translocation of a wide variety of molecules across cellular membranes. ABCB4, also called MDR3 (multidrug resistance 3) is a transporter expressed at the canalicular membrane of hepatocytes, where it translocates phosphatidylcholine (PC) PLOS ONE | DOI:10.1371/journal.pone.0146962 January 20, 2016 1 / 17 Regulation of ABCB4 Stability by Its C-Terminal PDZ-Like Motif Competing Interests: The authors have declared that no competing interests exist. into bile [1, 2]. In the aqueous environment of bile, phospholipids form mixed micelles with cholesterol and bile acids, thereby preventing the formation of cholesterol gallstones and the detergent action of free bile acids [3, 4]. Pathogenic mutations in the ABCB4 gene sequence are associated with rare biliary diseases, in particular progressive familial intrahepatic cholestasis type 3 (PFIC3), which develops early in childhood and may be lethal in the absence of liver transplantation [5–7]. Perspectives to treat PFIC3 patients by pharmacological means have been recently opened with the observation that cyclosporin A was able to partially rescue an ABCB4 misfolded mutant retained in the endoplasmic reticulum [8]. However, rescued mutants may remain conformationally unstable after having reached their proper localization [9]. Therefore, therapeutic efforts to correct a folding defect must also aim at strengthening the stability of the mutant protein at the plasma membrane. The mechanisms that control the stability of ABCB4 at the canalicular membrane are poorly known. PDZ (post-synaptic density 95/disks large/zonula occludens-1) domain- containing proteins act as scaffolds by linking transmembrane proteins to the cytoskeleton, and thus regulate their subcellular localization, activity, stability and mobility in the membrane [10, 11]. PDZ protein NHERF-1 (sodium-hydrogen exchanger regulatory factor-1), also known as EBP50 (ezrin-radixin-moesin (ERM)-binding phosphoprotein 50) is highly expressed in the liver, at the apical membrane of biliary epithelial cells, and at the canalicular membrane of hepatocytes [12, 13] and has been shown to control the membrane localization, stability and function of the ABC transporters ABCC7/CFTR and ABCC2/MRP2 [14, 15]. EBP50 is a multifunctional scaffolding protein, with two PDZ-domains at its N-terminus and a C-terminal domain that binds the ERM family of cytoskeletal proteins [16]. PDZ domains comprise 70–90 amino acids that bind preferentially to short sequences at the C-termini of target proteins [17]. They are grouped into three classes based on the target sequence. Class I PDZ domains recognizes the sequence motif -x-[S/T]-x-F, where x represents any residue, and ϕ a hydrophobic residue [18]. Class II recognizes the consensus motif (-x-ϕ-x-ϕ) [19], whereas class III prefers negatively charged amino acid at the -2 position and recognizes the consensus motif -x-[D/E]-x-ϕ [20]. The C-terminal regions of ABCB4 and of the drug transporter ABCB1/MDR1 are conserved, except for the last three amino acids. ABCB4 ends by the sequence glutamineasparagine-leucine (QNL), while the last three amino acids of ABCB1 are lysine-arginineglutamine (KRQ). Although the QNL motif of ABCB4 does not perfectly match any of the three classes of PDZ binding motifs, the presence of a hydrophobic amino acid at the extreme C-terminus suggests properties of a PDZ-binding-like motif. The aim of the work was to study the role of the QNL motif, and its potential binding to the PDZ prote (...truncated)