Doctor of Philosophy
Falk, Matthias M.
Other advisers/committee members
Cassimeris, Lynne; Iovine, M. Kathryn; Auerbach, Wojtek
A gap junction (GJ) is formed when two hemi-channels from two apposed cells dock in the extracellular space. GJs mediate cell-cell communication by allowing direct transfer of molecules (<1.5 kDa) from one cell to the other through these channels. Direct intercellular communication mediated by gap junction channels is a hallmark of normal cell and tissue physiology. In addition, GJs are likely to significantly contribute to physical cell-cell coupling. Clearly, these cellular functions require precise modulation. Defects in GJ intercellular communication lead to serious illnesses. Interestingly, docked GJ channels cannot be separated under physiological conditions. We previously described that cells efficiently internalize their GJs in a clathrin-mediated endocytic process to form double-membrane vesicles termed annular gap junctions (AGJs) or connexosomes (Baker et al., 2008; Gumpert et al., 2008; Piehl et al., 2007). How this complex process is achieved mechanistically in a cell is still unclear. In this dissertation, (1) I elucidate the signals and molecular mechanisms that are involved in GJ internalization. By mutagenesis, I identified two functional tyrosine-based sorting signals (YXX) in the Cx43 (a GJ protein) C-terminal domain that interact with the classical clathrin adaptor protein, adaptor protein complex-2 (AP-2), to recruit clathrin to the plasma membrane (PM), followed by GJ endocytosis. Mutating these two YXX sorting motifs results in inhibition of AP-2 binding, increased accumulation of GJ channels in the PM, impairment of GJ internalization, and increased Cx43 protein half-life. (2) In order to understand the Cx43 protein modifications that initiate GJ endocytosis, I treated mouse embryonic stem cells (mES) with epidermal growth factor (EGF) and analyzed Cx43 phosphorylation. I found that EGF activates MAPK and PKC to phosphorylate Cx43 at Ser262, Ser279/282, and Ser368. These phosphorylation events induce inhibition of GJIC and trigger GJ internalization. Pharmacological inhibition of MAPK counteracts with EGF to maintain GJIC and to inhibit EGF-induced GJ internalization. Work conducted during my PhD contributed to three primary research papers (two published, one submitted) and three review articles.
Fong, John, "Signals and Molecular Mechanisms Involved in Gap Junction Internalization" (2014). Theses and Dissertations. 1485.