Nuclear and plasma membrane localization of SH3BP4 in retinal pigment epithelial cells

Abstract

Purpose: The SH3BP4 protein contains domains belonging to the Eps15-Homology (EH) network family of endocytosis proteins and a C-terminal death domain. The purpose of this study was to determine the expression of SH3BP4 in ARPE-19, Y79 and COS-7 cell lines and to determine SH3BP4 subcellular localization within ARPE-19 cells. Methods: A chicken anti-human SH3BP4 antibody was generated that specifically immunostains SH3BP4 fusion proteins and a corresponding endogenous protein band at 120 kDa. Protein expression of SH3BP4 was determined using western analysis of multiple cell lines and dissected retinal tissue. Intracellular localization of both endogenous SH3BP4 and SH3BP4 fusion proteins were determined using subcellular fractionation and microscopy studies using ARPE-19 and COS-7 cells. Results: The retinal pigment epithelial (RPE) cell line ARPE-19 was found to express SH3BP4 protein at more than 7 fold the levels in Y79 retinoblastoma cells and more than 2.5 fold the levels in COS-7 cells. Both the RPE and neural retinal layers of the eye were also found to express the SH3BP4 protein. SH3BP4 endogenous and fusion proteins were found to localize to both membrane and nuclear fractions but not the cytosol in subcellular fractionation experiments. Subsequent microscopy analyses show that SH3BP4 fusion proteins localize to the plasma membrane and the nuclear periphery. Conclusions: These studies show that SH3BP4 is expressed in the RPE and neural retina in vivo, and in ARPE-19, Y79, and COS-7 cell lines. Compared to other EH network and death domain proteins, SH3BP4 fusion proteins have an unusual intracellular localization to the plasma membrane and the nuclear periphery. The present demonstration of the suborganelle localization in conjunction with the unique domain combinations belonging to both endocytosis and cell death pathways suggests that SH3BP4 has physiological significance for RPE cells. ©2004 Molecular Vision.