title: LAPTM4A interacts with hOCT2 and regulates its endocytotic recruitment.
abstract: Human organic cation transporter 2 (hOCT2) is involved in the transport of endogenous and exogenous organic cations mainly in cells of the kidney and the brain. Here, we focus on the regulation of hOCT2 by direct protein-protein interaction. Screening within a mating-based split-ubiquitin-yeast-two-hybrid system (mBSUS) revealed the lysosomal-associated protein transmembrane 4 alpha (LAPTM4A) as a potential interacting protein. Interaction of LAPTM4A and hOCT2 was confirmed by pulldown assays, FRET microscopy analysis and immunofluorescence microscopy. Functionally, overexpression of LAPTM4A significantly decreased ASP(+) uptake in HEK293 cells stably transfected with hOCT2, suggesting a negative regulation of hOCT2-mediated transport. Furthermore, overexpression of LAPTM4A leads to a significantly decreased hOCT2 plasma membrane expression in surface biotinylation experiments. In addition, significant expression of LAPTM4A in human kidney was demonstrated by immunoblotting and immunofluorescence.In this work, LAPTM4A has been identified as interaction partner of hOCT2. LAPTM4A regulates the function of hOCT2 by influencing its trafficking to/from the cell membrane and processing it via the intracellular sorting machinery.
authors.affiliation: Medizinische Klinik und Poliklinik D, Abteilung für Experimentelle Nephrologie, Universitätsklinikum Münster, Domagkstrasse 3A, 48149 Münster, Germany. alexander.grabner@ukmuenster.de
authors.forename: A
authors.initials: A
authors.lastname: Grabner
authors.forename: S
authors.initials: S
authors.lastname: Brast
authors.forename: S
authors.initials: S
authors.lastname: Sucic
authors.forename: S
authors.initials: S
authors.lastname: Bierer
authors.forename: B
authors.initials: B
authors.lastname: Hirsch
authors.forename: H
authors.initials: H
authors.lastname: Pavenstädt
authors.forename: H H
authors.initials: HH
authors.lastname: Sitte
authors.forename: E
authors.initials: E
authors.lastname: Schlatter
authors.forename: G
authors.initials: G
authors.lastname: Ciarimboli
mesh_terms: D001692:Biological Transport; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D027701:Organic Cation Transport Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D000075102:Organic Cation Transporter 2; D025941:Protein Interaction Mapping; D012333:RNA, Messenger / Q000378:metabolism
chemical_list: C569342:LAPTM4A protein, human; D026901:Membrane Transport Proteins; D027701:Organic Cation Transport Proteins; D000075102:Organic Cation Transporter 2; D012333:RNA, Messenger; C491013:SLC22A2 protein, human