Characterization of the Cone Photoreceptor– associated Protein, FASH3B

Abstract

Abstract: : Purpose: To characterize a novel, cone photoreceptor–associated protein, FASH3B. Methods: FASH3B was expressed in bacteria and used to make a rabbit polyclonal antibody. Affinity purified FASH3B antibody was characterized and used in Western blot analysis of 1D and 2D gels to determine the localization of FASH3B in whole retina lysates, cultured cell lysates, and subcellular fractions of these lysates. The change in the subcellular localization of FASH3B in response to light was studied using the murine photoreceptor–derived 661W cells. The phosphorylation state of the recombinant protein was studied using kinase and phosphatase reactions followed by Western blot analysis using anti–phosphotyrosine and analysis by MALDI–TOF. Potential binding partners of FASH3B were isolated using Pull–Down assays and FPLC fractionation of lysates, followed by Western blot analysis of the fractions. Results: The FASH3B protein was detected in the retina of human, pig, rat and mouse. A 4mm trephine punch of the human retina from the macula region had higher levels of FASH3B than an equivalent punch from the peripheral retina. Of the cell lines tested (MCF7, COS7, Y79 and 661W) only the 661W cell line had detectable levels of FASH3B. FASH3B was present in the cytoplasm and the nucleus of cells but not in the membrane. There was more FASH3B present in the nucleus of dark–adapted 661W cells than after the cells had been exposed to light. Studies of the recombinant FASH3B showed that the peptide, QWMYK, can be phosphorylated on the tyrosine residue, pY50. Although several potential binding partners were identified these interactions still need to be substantiated. Conclusions: FASH3B is a highly conserved, 12.3kDa protein expressed in cone photoreceptors and cone photoreceptor–derived cell lines. Although its function in the retina remains unclear, its role in signal transduction in the cone photoreceptors is supported by this work demonstrating a light dependent sub–localization in 661W cells.

Keywords: protein purification and characterization • photoreceptors • signal transduction