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Albena Kantardzhieva, Ilse Gosens, Svetlana Alexeeva, Ingrid M. Punte, Inge Versteeg, Elmar Krieger, Carla A. Neefjes-Mol, Anneke I. den Hollander, Stef J. F. Letteboer, Jan Klooster, Frans P. M. Cremers, Ronald Roepman, Jan Wijnholds; MPP5 Recruits MPP4 to the CRB1 Complex in Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2005;46(6):2192-2201. doi: 10.1167/iovs.04-1417.
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purpose. Mutations in the human Crumbs homologue 1 (CRB1) gene are a frequent cause of Leber congenital amaurosis (LCA) and various forms of retinitis pigmentosa. CRB1 is thought to organize an intracellular protein scaffold in the retina that is involved in photoreceptor polarity. This study was focused on the identification, subcellular localization, and binding characteristics of a novel member of the protein scaffold connected to CRB1.
methods. To dissect the protein scaffold connected to CRB1, the yeast two-hybrid approach was used to screen for interacting proteins. Glutathione S-transferase (GST) pull-down analysis and immunoprecipitation were used to verify protein–protein interactions. The subcellular localization of the proteins was visualized by immunohistochemistry and confocal microscopy on human retinas and immunoelectron microscopy on mouse retinas.
results. A novel member of the scaffold connected to CRB1, called membrane palmitoylated protein (MPP) subfamily member 4 (MPP4), a membrane-associated guanylate kinase (MAGUK) protein, was identified. MPP4 was found to exist in a complex with CRB1 through direct interaction with the MPP subfamily member MPP5 (PALS1). 3D homology modeling provided evidence for a mechanism that regulates the recruitment of both homo- and heterodimers of MPP4 and -5 proteins to the complex. Localization studies in the retina showed that CRB1, MPP5, and MPP4 colocalize at the outer limiting membrane (OLM).
conclusions. These data imply that MPP4 and -5 have a role in photoreceptor polarity and, by association with CRB1, pinpoint the cognate genes as functional candidate genes for inherited retinopathies.
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