Cellular and Subcellular Localization of the Retina-specific MPP4 Protein

Abstract

Abstract: : Purpose:MPP4 (membrane protein, palmitoylated 4) has recently been identified as a novel retina-specific gene encoding a putative protein 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. In a first step to gain insight into the function of MPP4 in the retinal tissue and its possible role in retinal disease, the cellular and subcellular localization of MPP4 was determined. Methods:Polyclonal antisera were generated in rabbits immunized with two synthetic peptides and murine monoclonal antibodies were raised against a maltose-binding protein (MBP) fusion protein. MPP4 specific antibodies were used in Western blots and immunofluorescence analysis of cryosectioned bovine and murine eyes. Full-length MPP4 was expressed in 293-EBNA cells and subjected to subcellular characterization. Results:Both polyclonal and monoclonal antibodies specifically detected the 70 kDa MPP4 protein in extracts isolated from dissected retinae and 293-EBNA cells transfected with the MPP4 cDNA. Subcellular fractionation and immunofluorescence microscopy demonstrated that MPP4 is associated with the plasma membrane in transfected 293-EBNA cells. Strong MPP4 immunoreactivity was observed in the outer plexiform layer (OPL) of the bovine and murine retina where it colocalized with postsynaptic density-95 (PSD-95). Conclusions:The expression pattern of MPP4 indicates an important role of MPP4 at the plasma membrane of the synaptic terminals of the OPL where it may be involved in junction organization and maintainance.

Keywords: retina • immunohistochemistry • synapse