title: Regulation of albumin endocytosis by PSD95/Dlg/ZO-1 (PDZ) scaffolds. Interaction of Na+-H+ exchange regulatory factor-2 with ClC-5.
abstract: The constitutive reuptake of albumin from the glomerular filtrate by receptor-mediated endocytosis is a key function of the renal proximal tubules. Both the Cl- channel ClC-5 and the Na+-H+ exchanger isoform 3 are critical components of the macromolecular endocytic complex that is required for albumin uptake, and therefore the cell-surface levels of these proteins may limit albumin endocytosis. This study was undertaken to investigate the potential roles of the epithelial PDZ scaffolds, Na+-H+ exchange regulatory factors, NHERF1 and NHERF2, in albumin uptake by opossum kidney (OK) cells. We found that ClC-5 co-immunoprecipitates with NHERF2 but not NHERF1 from OK cell lysate. Experiments using fusion proteins demonstrated that this was a direct interaction between an internal binding site in the C terminus of ClC-5 and the PDZ2 module of NHERF2. In OK cells, NHERF2 is restricted to the intravillar region while NHERF1 is located in the microvilli. Silencing NHERF2 reduced both cell-surface levels of ClC-5 and albumin uptake. Conversely, silencing NHERF1 increased cell-surface levels of ClC-5 and albumin uptake, presumably by increasing the mobility of NHE3 in the membrane and its availability to the albumin uptake complex. Surface biotinylation experiments revealed that both NHERF1 and NHERF2 were associated with the plasma membrane and that NHERF2 was recruited to the membrane in the presence of albumin. The importance of the interaction between NHERF2 and the cytoskeleton was demonstrated by a significant reduction in albumin uptake in cells overexpressing an ezrin binding-deficient mutant of NHERF2. Thus NHERF1 and NHERF2 differentially regulate albumin uptake by mechanisms that ultimately alter the cell-surface levels of ClC-5.
authors.affiliation: School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Deanne H
authors.initials: DH
authors.lastname: Hryciw
authors.forename: Jenny
authors.initials: J
authors.lastname: Ekberg
authors.forename: Charles
authors.initials: C
authors.lastname: Ferguson
authors.forename: Aven
authors.initials: A
authors.lastname: Lee
authors.forename: Dongsheng
authors.initials: D
authors.lastname: Wang
authors.forename: Robert G
authors.initials: RG
authors.lastname: Parton
authors.forename: Carol A
authors.initials: CA
authors.lastname: Pollock
authors.forename: Chris C
authors.initials: CC
authors.lastname: Yun
authors.forename: Philip
authors.initials: P
authors.lastname: Poronnik
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000418:Albumins / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D018118:Chloride Channels / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D017931:DNA Primers; D000074288:Discs Large Homolog 1 Protein; D000076122:Disks Large Homolog 4 Protein; D004705:Endocytosis; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D009893:Opossums; D010750:Phosphoproteins / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers; D013347:Subcellular Fractions / Q000378:metabolism; D062826:Zonula Occludens-1 Protein
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000418:Albumins; C097503:CLC-5 chloride channel; D018118:Chloride Channels; D003598:Cytoskeletal Proteins; C513314:DLG1 protein, human; C501444:DLG4 protein, human; D017931:DNA Primers; D000074288:Discs Large Homolog 1 Protein; D000076122:Disks Large Homolog 4 Protein; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D017923:Sodium-Hydrogen Exchangers; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; C093376:sodium-hydrogen exchanger regulatory factor