Cellular localization of the MPP4 protein in the mammalian retina.
OBJECTIVE
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.
Stöhr
Heidi
H
Institute of Human Genetics, Biocentre, University of Würzburg, Am Hubland, D-97074 Würzburg, Germany. heidi.stoehr@mail.uni-wuerzburg.de
Stojic
Jelena
J
Weber
Bernhard H F
BH
D000818:Animals; D002417:Cattle; D002460:Cell Line; D018076:DNA, Complementary / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D005822:Genetic Vectors; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009703:Nucleoside-Phosphate Kinase / Q000235:genetics / Q000378:metabolism*; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism; D017729:Presynaptic Terminals / Q000378:metabolism; D011817:Rabbits; D012160:Retina / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D014162:Transfection
D018076:DNA, Complementary; D005136:Eye Proteins; D008565:Membrane Proteins; D009703:Nucleoside-Phosphate Kinase; C470950:MPP4 protein, human