Purpose: The role of Als2cr4 (amyotrophic lateral sclerosis 2 [juvenile] chromosome region, candidate 4; also known as hypothetical protein FLJ33282) in the mouse retina was determined by characterizing the molecular structure, cellular interacting partners, and potential biochemical functions. Previous in situ hybridization and gene expression profiles show that the mRNAs encoding Als2cr4 are abundant in the eye, hippocampus, cerebellum, and olfactory bulb.
Methods: From predicted antigenic epitopes of Als2cr4, two novel antibodies were developed to examine protein expression and morphologic localization in retinas from light-adapted and dark-adapted mice by immunohistochemistry, immunoblot analysis, and immunoelectron microscopy, and then immunoprecipitation was performed to identify interacting proteins by mass spectroscopy.
Results: Peptide antibodies with Als2cr4 antigenic epitopes from either the amino- or carboxyl terminus were characterized with Als2cr4 recombinant proteins and peptide competition assays. Als2cr4 is a 45-kDa insoluble protein, highly enriched in retina, and localizes to photoreceptor outer segments, ciliary complex, and horizontal cells in the outer plexiform layer. Immunoelectron microscopy for Als2cr4 verified its expression in the discs of photoreceptor outer segments. Immunoprecipitation and mass spectroscopy identified eight potential interacting partners: vimentin, actin, myosin Va, myosin VI, myosin X, myosin XIV, kinesin 1, Als2cr4, and lamin B-1.
Conclusions: Als2cr4 is a novel protein, with a probable tetraspanin-like membrane structure, that is localized in photoreceptors and in the postsynaptic outer plexiform layer and that interacts with cytoskeletal proteins. Als2cr4 may be involved in membrane transport between the photoreceptor inner and outer segments and may be a key component in maintaining the structural integrity of the outer segment.