Regulation of Gap Junction Internalization and Degradation

Abstract

Gap junctions (GJs) are channels that traverse the plasma membrane of neighboring cells and provide direct intercellular communication (GJIC) in multi-cellular organisms. GJIC is a prerequisite for coordinated development, differentiation and tissue function; disruption of which can cause disease. Post-translational modification is essential for regulation of GJ trafficking, gating, internalization, and degradation. In the dissertation work presented here (1) I show the role of clathrin-mediated endocytosis (CME) on acute Cx43 (a GJ protein) GJ internalization. I treated primary pulmonary artery endothelial cells (pPAECs) with CME inhibitors after vascular endothelial growth factor (VEGF) stimulation and analyzed internalization patterns of GJs. VEGF treatment of endothelial cells leads to rapid internalization of GJs. I found that pharmacological inhibition of CME during VEGF treatment leads to GJs remaining in the plasma membrane, suggesting CME as the acute GJ internalization pathway. (2) I elucidated the ubiquitin-mediated regulatory mechanism of Cx43 GJ internalization and degradation. I utilized mutational analysis to identify two lysine residues that are K63-polyubiquitinated in the Cx43 C-terminal domain that are necessary for GJ internalization. Mutating these residues results in loss of K63-polyubiquitination, accumulation of GJs in the plasma membrane, and longer Cx43 protein half-life. My analysis also revealed a link between phosphorylations known to decrease GJIC (pS368, pS279/pS282, and pS255) and K63-polyubiquitination that regulates GJ internalization. (3) Additionally, I used knockdowns of key autophagy proteins and immuno-colocalization analysis to identify autophagy as the pathway that degrades internalized Cx43 GJs. I found that Beclin-1 and p62/SQSTM1 knockdowns leads to increased AGJs and decreased colocalization of Cx43 AGJs with the autophagic membrane protein, LC3. p62 (a ubiquitin binding protein) colocalization of Cx43 at GJs and AGJs strongly suggests a link between ubiquitination of GJs and autophagic degradation. Additionally, I found that p62 and LC3 robustly colocalize with Cx43 in endogenous Cx43 expressing PAECs. Work during my Ph.D. contributed to four primary research articles (three published, one submitted), four review articles and one book chapter