Abstract
Purpose: :
Intercellular communication through gap junctions (GJIC) is important in maintaining the integrity of the blood-retinal barrier. GJ are multimeric structures composed of 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 Nedd4 and eps15 in retinal endothelial cells.
Methods: :
To investigate the involvement of Nedd4 and eps15 on Cx43 internalization, cells were transfected with mutant or inactive forms of Cx43, eps15 and Nedd4 and where appropriate proteins were silenced by siRNA. Cell extracts were then selectively immunoprecipitated (IP) against the protein of interest, followed by western blot (WB) and probing with antibodies against Cx43, Nedd4, eps15 or ubiquitin. Alternatively, cells were fixed and simultaneously stained with antibodies against Cx43 and eps15 or Nedd4, and subsequently imaged by confocal microscopy.
Results: :
Data indicates that both Nedd4 and eps15 interact with Cx43. More importantly, interaction between eps15 and Cx43 is required for Cx43 internalization. When cells are transfected with inactive Nedd4 or eps15DUIM, Cx43 accumulates at the plasma membrane. Moreover, disruption of the association between Cx43 and Nedd4, inhibits Cx43 ubiquitination and interaction with Eps15.
Conclusions: :
Data suggests a new molecular mechanism for internalization of Cx43 in retinal endothelial cells. It is suggested that Nedd4 mediated ubiquitination of Cx43 is required for subsequent binding of the adaptor protein eps15 leading to rapid internalization of Cx43. Disruption of the mechanism of Cx43 internalization and degradation is likely to have a direct impact in endothelial dysfunction associated with breakdown of BRB.
Keywords: gap junctions/coupling • proteolysis