title: Nephrocystin-conserved domains involved in targeting to epithelial cell-cell junctions, interaction with filamins, and establishing cell polarity.
abstract: Nephrocystin is the protein product of the gene mutated in juvenile nephronophthisis, an autosomal recessive cystic kidney disease afflicting children and young adults. Because the normal cellular function of nephrocystin is largely unknown, the molecular defects underlying disease pathogenesis remain obscure. Analysis of nephrocystin amino acid sequences from human and other species revealed three distinct conserved domains including Src homology 3 and coil-coil domains in the N-terminal region, as well as a large highly conserved C-terminal region bearing no obvious homology to other proteins and hence referred to as the "nephrocystin homology domain" (NHD). The objective of this study was to gain insight into nephrocystin function by defining functional properties of the conserved domains. We analyzed a series of nephrocystin deletion mutants expressed in Madin-Darby canine kidney and COS-7 cells. This analysis revealed previously unrecognized functional attributes of the NHD, including abilities to promote both self-association and epithelial cell-cell junctional targeting. We further observed that Madin-Darby canine kidney cell lines stably expressing a nephrocystin mutant with a deletion of the Src homology 3 domain have reduced ability to establish tight junctions as measured by transepithelial electrical resistance. Finally, from a two-hybrid screen and coimmunoprecipitation studies we identified members of the filamin family of actin-binding proteins as having the capacity to interact with the NHD. These findings support a functional role for nephrocystin as a docking protein involved in organizing a protein complex to regulate the actin cytoskeleton at sites of epithelial cell-cell adhesion and further suggest that these properties are important for establishing epithelial cell polarity.
authors: Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
authors: John C
authors: JC
authors: Donaldson
authors: Rebecca S
authors: RS
authors: Dise
authors: Marylyn D
authors: MD
authors: Ritchie
authors: Steven K
authors: SK
authors: Hanks
mesh_terms: D000199:Actins / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D019556:COS Cells; D002448:Cell Adhesion; D016764:Cell Polarity; D003285:Contractile Proteins / Q000737:chemistry*; D003598:Cytoskeletal Proteins; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism*; D000939:Epitopes; D064448:Filamins; D008565:Membrane Proteins; D008840:Microfilament Proteins / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D013997:Time Factors; D020798:Two-Hybrid System Techniques; D018909:src Homology Domains
chemical_list: D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D003285:Contractile Proteins; D003598:Cytoskeletal Proteins; D000939:Epitopes; D064448:Filamins; D008565:Membrane Proteins; D008840:Microfilament Proteins; C108725:NPHP1 protein, human; D011506:Proteins; D016271:Proto-Oncogene Proteins c-myc