title: PDZK1 and NHERF1 regulate the function of human organic anion transporting polypeptide 1A2 (OATP1A2) by modulating its subcellular trafficking and stability.
abstract: The human organic anion transporting polypeptide 1A2 (OATP1A2) is an important membrane protein that mediates the cellular influx of various substances including drugs. Previous studies have shown that PDZ-domain containing proteins, especially PDZK1 and NHERF1, regulate the function of related membrane transporters in other mammalian species. This study investigated the role of PDZK1 and NHERF1 in the regulation of OATP1A2 in an in vitro cell model. Transporter function and protein expression were assessed in OATP1A2-transfected HEK-293 cells that co-expressed PDZK1 or NHERF1. Substrate (estrone-3-sulfate) uptake by OATP1A2 was significantly increased to ∼1.6- (PDZK1) and ∼1.8- (NHERF1) fold of control; this was dependent on the putative PDZ-binding domain within the C-terminus of OATP1A2. The functional increase of OATP1A2 following PDZK1 or NHERF1 over-expression was associated with increased transporter expression at the plasma membrane and in the whole cell, and was reflected by an increase in the apparent maximal velocity of estrone-3-sulfate uptake (V(max): 138.9±4.1 (PDZK1) and 181.4±16.7 (NHERF1) versus 55.5±3.2 pmol*(µg*4 min)⁻¹ in control; P<0.01). Co-immunoprecipitation analysis indicated that the regulatory actions of PDZK1 and NHERF1 were mediated by direct interaction with OATP1A2 protein. In further experiments PDZK1 and NHERF1 modulated OATP1A2 expression by decreasing its internalization in a clathrin-dependent (but caveolin-independent) manner. Additionally, PDZK1 and NHERF1 enhanced the stability of OATP1A2 protein in HEK-293 cells. The present findings indicated that PDZK1 and NHERF1 regulate the transport function of OATP1A2 by modulating protein internalization via a clathrin-dependent pathway and by enhancing protein stability.
authors.affiliation: Alkali Soil Natural Environmental Science Center, Northeast Forestry University/Key Laboratory of Saline-alkali Vegetation Ecology Restoration in Oil Field, Ministry of Education, Harbin, China; Faculty of Pharmacy, The University of Sydney, Sydney, New South Wales, Australia.
authors.forename: Jian
authors.initials: J
authors.lastname: Zheng
authors.affiliation: Faculty of Pharmacy, The University of Sydney, Sydney, New South Wales, Australia.
authors.forename: Ting
authors.initials: T
authors.lastname: Chan
authors.affiliation: Faculty of Pharmacy, The University of Sydney, Sydney, New South Wales, Australia.
authors.forename: Florence Shin Gee
authors.initials: FS
authors.lastname: Cheung
authors.affiliation: Retinal Therapeutics Research Group, Save Sight Institute, The University of Sydney, Sydney, New South Wales, Australia.
authors.forename: Ling
authors.initials: L
authors.lastname: Zhu
authors.affiliation: Discipline of Pharmacology, School of Medical Sciences, The University of Sydney, Sydney, New South Wales, Australia.
authors.forename: Michael
authors.initials: M
authors.lastname: Murray
authors.affiliation: Faculty of Pharmacy, The University of Sydney, Sydney, New South Wales, Australia.
authors.forename: Fanfan
authors.initials: F
authors.lastname: Zhou
mesh_terms: D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002966:Clathrin / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D008565:Membrane Proteins; D027361:Organic Anion Transporters / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D055550:Protein Stability; D021381:Protein Transport; D015398:Signal Transduction; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D002966:Clathrin; D008565:Membrane Proteins; D027361:Organic Anion Transporters; C484659:PDZK1 protein, human; D010750:Phosphoproteins; C512988:SLCO1A2 protein, human; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor
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