Long range intercellular communication throughout the liver is in large part mediated by gap junction channels, through which ions and metabolites pass directly from one cell to the next. The pores of hepatocyte gap junction channels are formed primarily of the gap junction protein connexin32 (Cx32) and Cx26. Recent evidence indicates that other proteins are associated with connexins at gap junctions, and we have termed this macromolecular complex the Nexus, and the new term 'connexosome' has been coined to refer to the internalized gap junction on its way to degradation. We hypothesize that the Nexus components may regulate both the properties of the gap junction channels and also may function in intracellular signal transduction and that Cx32 binding partners in connexosomes dictate their ultimate fate. In order to test this hypothesis, we will determine the identities of the other proteins that bind to Cx32 in liver vesicle fractions and primary cultured hepatocytes and cell lines (interacting with Project 1 to isolate vesicle fractions and Project 3 to study endocytosed connexosomes), measure the strengths of interaction using surface plasmon resonance, determine whether interactions are affected by post-translational connexin modifications, and use in vitro motilitiy assays (with the assistance of Project 1) to reconstruct the mechanisms responsible for vesicular Cx32 trafficking within cells. These studies use a multidisciplinary approach highly dependent on both Cores B and C involving live cell imaging, spectroscopic, biochemical, proteomic and mutational approaches that are directed at exploring a new concept in the field. As such, these studies are expected to lead to novel understanding of roles that gap junctions play in the liver and elsewhere as well as mechanisms of gap junction delivery and retrieval from the cell membrane.
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