The overall objective of this project is to study the involvement of the G- protein Gialpha2, and related G-protein subunits, in cellular differentiation, using the hematopoietic cell lines K562 and U937 as experimental paradigms. Differentiation will be induced in these cells with one or more of the following cell-differentiation-inducing agents, sodium butyrate, retinoic acid and phorbol myristic acetate, so as to achieve different cell phenotypes. The project will accomplish four specific objectives. Firstly, using pertussis toxin and antisense methods to inactive Gialpha2, the hypothesis will be tested that cell differentiation in K562 cells (measured by hemoglobin expression) requires Gialpha2. Secondly, the idea that transcriptional mechanisms underlie the previously reported robust changes in Gialpha2 levels will be verified and confirmed. Thirdly and fourthly, it will be established whether the mechanism of the participation of Gialpha2, and related G-protein subunits, in cell differentiation involves their phosphorylation and/or translocation to the cell nucleus. The G-protein subunits will be detected by immunoblotting with affinity-purified antibodies. Detection of their translocation to the nucleus will also be accomplished by using immunofluorescence and confocal microscopy. Conventional wisdom of how G proteins mediate signal transduction has not traditionally considered their translocation to the cell's nuclear compartment as a potential mode of action. Therefore, this project has the potential to uncover innovative roles of G-protein signaling molecules which do not involve their traditional role of transmembrane effector activation immediately resulting in the formation of second messenger(s).
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