Cx43 internalization is mediated by the ubiquitin‐binding adaptor protein Eps15 in retinal endothelial cells

Purpose Intercellular communication through gap junctions (GJIC) is important in maintaining the integrity of the blood‐retinal barrier. GJ are multimeric structures composed of proteins called connexins. Modifications on stability or subcellular distribution of connexins have a direct impact on the extent of GJIC. The purpose of this study is to investigate the molecular mechanisms involved in the regulation of connexin 43 (Cx43) stability at plasma membrane by eps15 in retinal endothelial cells. Methods To investigate the interaction of Cx43 with eps15, Cx43 was selectively immunoprecipitated (IP), followed by western blot (WB) and probing with antibodies against eps15. To investigate the role of eps15 in Cx43 stability and subcellular distribution cells were transfected with a dominant negative form of eps15 or siRNA targeted against eps15, cell extracts were WB and probed with antibodies against Cx43. Alternatively, cells were fixed and simultaneously stained with antibodies against Cx43 and eps15, and imaged by confocal microscopy. Results Data obtained in this study indicates that eps15 interacts with Cx43 mediating its internalization from the plasma membrane. When the normal function of eps15 is disrupted by the presence of a dominant negative form of the protein or through silencing of the eps15 gene with siRNA, Cx43 accumulates at the plasma membrane. Conclusion Data suggests a novel protein interaction between Cx43 and eps15 that mediates the plasma membrane stability of Cx43. Disruption of this interaction may impact on GJIC, contributing to endothelial cell dysfunction associated with the breakdown of the blood‐retinal barrier, as observed in diabetic retinopathy. Supported by POCI/SAU‐MMO/57216/2004