The disruption of intercellular coupling through gap junctions has long been associated with cell transformation and that the promotion phase of tumorigenesis. Transient uncoupling also typically accompanies cases where acute activation of cell growth occurs (e.g. responses to growth factors, tissue regeneration, etc.). Despite this strong of correlation between cell coupling and growth suppression, the mechanism by which this is achieved remains obscure. The problem lies in the broad range of signaling activities that can involve intercellular traffic through gap junctions. Thus, our approach has been reductionistic-first define the properties of different connexins, and their mode of regulation by growth promoting factors such as oncogenes. This in turn will provide the tools with which to investigate the specific mitogenic pathways that are influenced by gap junctional coupling in well defined models of tumorigenesis, such as oncogene induced cell transformation. Given that almost all tissues express multiple connexins, Aim #1 will examine interactions between different connexins, both heterotypic (between cells) and heteromeric (within a cell), how they influence one another and the channel properties that result. We also propose a systematic mapping of exposed residues in the extracellular docking domains of connexins to refine our current model of the structural basis of their heterotypic interactions.
Aim #2 seeks to define the gating mechanisms of gap junction channels in response to both voltage and metabolic signals (i.e. phosphorylation). Particular emphasis will be placed on placing the defining mechanism of gap junctional gating by the oncogene v-src. Identification of both the signaling cascades involved, and the specific molecular steps in the gating is proposed. Both of these aims should provide a significantly better understanding of the properties of gap junctions in situ, and should produce tools for Aim #3, in which the molecular mechanisms by which gap junctions suppress cell transformation will be investigated. To provide a define system where the cause of cell transformation is known, and where connexin expression can be controlled for these studies, we have developed an immortalized cell line from Cx43-/- mice. Transformation will be effected by different oncogenes that activate known mitogenic pathways. The efficiency of different connexins in abrogating or preventing this transformation will be assessed, and the specific mitogenic steps that are inhibited by this coupling will be identified. Ultimately, these effects will be correlated with the ability of the different connexins to pass candidate signaling molecules. These strategies are designed to identify both the targets of growth suppressive intercellular signaling, and eventually the signals themselves.
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