title: Interaction of endogenous nephrin and CD2-associated protein in mouse epithelial M-1 cell line.
abstract: The interpodocyte slit diaphragm is an essential structure for maintaining the functional glomerular filtration barrier. The slit diaphragm is proposed to consist of an interacting meshwork of nephrin molecules. Earlier studies with tagged proteins have suggested that the intracellular part of nephrin interacts with CD2-associated protein (CD2AP). This study was addressed to show by coimmunoprecipitation and pulldown assays an interaction of endogenously expressed nephrin and CD2AP in the kidney-derived mouse epithelial M-1 cell line, to provide evidence of the domain(s) of CD2AP involved in the interaction, and to show the localization of the respective proteins by immunoelectron microscopy in kidney cortex. In addition, the localization of CD2AP, podocin, alpha-actinin 4, and nephrin was studied in human kidney glomeruli and in M-1 cells by immunofluorescence microscopy. The results indicate an endogenous interaction between nephrin and CD2AP in M-1 cells and suggest that this interaction is mediated by the third Src homology 3 (SH3) domain of CD2AP. We also show by immunoelectron microscopy that nephrin and CD2AP are detected at the slit diaphragm area, supporting their interaction in the glomeruli in vivo. In addition, nephrin was found to partially colocalize with CD2AP and podocin in double immunofluorescence microscopy, confirming the close proximity of these proteins and proposing that these proteins may belong to nephrin-associated protein complex in glomeruli. The existence of nephrin, CD2AP, podocin, and alpha-actinin 4 enables further characterization of their relationship in M-1 cells.
authors.affiliation: Department of Bacteriology and Immunology, University Central Hospital, FIN-00014 Helsinki University, PB 63 (Haartmaninkatu 8), Helsinki, Finland.
authors.forename: Tuula
authors.initials: T
authors.lastname: Palmén
authors.forename: Sanna
authors.initials: S
authors.lastname: Lehtonen
authors.forename: Ari
authors.initials: A
authors.lastname: Ora
authors.forename: Dontscho
authors.initials: D
authors.lastname: Kerjaschki
authors.forename: Corinne
authors.initials: C
authors.lastname: Antignac
authors.forename: Eero
authors.initials: E
authors.lastname: Lehtonen
authors.forename: Harry
authors.initials: H
authors.lastname: Holthöfer
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002460:Cell Line; D003598:Cytoskeletal Proteins; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007668:Kidney / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D007672:Kidney Cortex / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D016253:Microscopy, Immunoelectron; D017434:Protein Structure, Tertiary / Q000502:physiology; D011506:Proteins / Q000737:chemistry / Q000502:physiology*; D014018:Tissue Distribution
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C114545:CD2-associated protein; D003598:Cytoskeletal Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C407223:NPHS2 protein; D011506:Proteins; C113267:nephrin