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Heidi Stöhr, Jelena Stojic, Bernhard H. F. Weber; Cellular Localization of the MPP4 Protein in the Mammalian Retina. Invest. Ophthalmol. Vis. Sci. 2003;44(12):5067-5074. doi: 10.1167/iovs.03-0702.
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purpose. Membrane protein, palmitoylated (MPP)-4 is a novel retina-specific member of the p55-like subfamily of membrane-associated guanylate kinases (MAGUKs). MAGUKs are known to act as scaffolding molecules for multiprotein complexes at specialized regions of the plasma membrane. The goal of this study was to characterize the MPP4 protein and to determine its location in the mammalian retina.
methods. RT-PCR and 5′ and 3′ rapid amplification of cDNA ends (RACE) techniques were used to isolate and sequence the full-length bovine MPP4 cDNA. Polyclonal antisera against the bovine MPP4 protein were generated in rabbits immunized with synthetic peptides. Affinity-purified anti-MPP4 antibodies were used to investigate the properties and distribution of MPP4 in retina and transfected 293-Ebna cells by Western blot analysis and immunofluorescence microscopy.
results. The full-length bovine MPP4 cDNA encodes a putative bovine MPP4 protein of 640 amino acids with a predicted molecular mass of 72.9 kDa. Affinity-purified anti-MPP4 antibodies specifically detected the MPP4 protein in extracts from retina and 293-Ebna cells transfected with the MPP4 cDNA. Immunofluorescence analyses revealed that both the bovine and porcine MPP4 proteins are localized in the connecting cilia and synaptic terminals of cone and rod photoreceptors. In addition, postsynaptic structures in the outer plexiform layer and three distinct bands in the inner plexiform layer were MPP4-positive. In porcine but not bovine retina, a subclass of cone bipolar cells were labeled with anti-MPP4.
conclusions. The concurrent presence of MPP4 in connecting cilia and synaptic structures suggests that MPP4 plays a role at membrane–cytoskeleton interfaces in distinct structural and functional compartments of bovine and porcine retinas. This work provides the basis for further investigations into the function of MPP4 as a retinal scaffolding molecule and its possible role in retinal disease.
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