Identification and characterization of a novel tight junction-associated family of proteins that interacts with a WW domain of MAGI-1.
The membrane-associated guanylate kinase protein, MAGI-1, has been shown to be a component of epithelial tight junctions in both Madin-Darby canine kidney cells and in intestinal epithelium. Because we have previously observed MAGI-1 expression in glomerular visceral epithelial cells (podocytes) of the kidney, we screened a glomerular cDNA library to identify the potential binding partners of MAGI-1 and isolated a partial cDNA encoding a novel protein. The partial cDNA exhibited a high degree of identity to an uncharacterized human cDNA clone, KIAA0989, which encodes a protein of 780 amino acids and contains a predicted coiled-coil domain in the middle of the protein. In vitro binding assays using the partial cDNA as a GST fusion protein confirm the binding to full-length MAGI-1 expressed in HEK293 cells, as well as endogenous MAGI-1, and also identified the first WW domain of MAGI-1 as the domain responsible for binding to this novel protein. Although a conventional PPxY binding motif for WW domains was not present in the partial cDNA clone, a variant WW binding motif was identified, LPxY, and found to be necessary for interacting with MAGI-1. When expressed in Madin-Darby canine kidney cells, the full-length novel protein was found to colocalize with MAGI-1 at the tight junction of these cells and the coiled-coil domain was found to be necessary for this localization. Because of its interaction with MAGI-1 and its localization to cell-cell junctions, this novel protein has been given the name MAGI-1-associated coiled-coil tight junction protein (MASCOT).
Patrie
Kevin M
KM
Department of Internal Medicine, University of Michigan, 1580 MSRB II, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0676, USA. kpatrie@umich.edu
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004285:Dogs; D015723:Gene Library; D005982:Glutathione Transferase / Q000235:genetics; D051528:Guanylate Kinases; D006801:Humans; D007413:Intestinal Mucosa / Q000201:enzymology; D007668:Kidney; D007678:Kidney Glomerulus / Q000201:enzymology; D051379:Mice; D009703:Nucleoside-Phosphate Kinase / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D019108:Tight Junctions / Q000201:enzymology*; D014158:Transcription, Genetic
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005982:Glutathione Transferase; D009703:Nucleoside-Phosphate Kinase; D051528:Guanylate Kinases