The objective of the proposed research is to determine the extent to which GTP-binding regulatory proteins (G proteins) participate in pathways of signal transduction relevant to normal and aberrant cell proliferation. A primary focus of this research is the extent to which G proteins mediate the mitogenic actions of growth factors: G proteins that participate in pathways of transduction utilized by growth factors will be identified, and the regulation of G proteins serving these and other forms of transduction will be assessed. Studies will extend to malignant transformation, a model of oncogenesis wherein pronounced alterations in second- messenger systems occur. The first goal of the proposed research is to develop monoclonal and peptide-specific antibodies recognizing the 35-kDa form of the beta subunit and the gamma subunit(s) common to G proteins; these will complete the array of immunological tools required for the analysis of G protein regulation. A subsequent goal is the definition of G proteins that exist within cells to be investigated (i.e., mouse 3T3, normal rat kidney, and rat embryo fibroblasts). This will be achieved by employment of immunoadsorption chromatography in conjunction with various indices of primary structure. The third goal is the isolation and definition of G proteins that physically interact with receptors for platelet-derived growth factor, bombesin, insulin, epidermal growth factor, and transforming growth factor- beta. Receptor/G protein complexes pre-existing within membrane will be isolated by affinity chromatography under conditions previously demonstrated to stabilize noncovalent interactions; a G protein(s) sensitive to Islet-activating protein mediates the actions of at least bombesin, and this will be identified by procedures of concomitant (32P)ADP-ribosylation. A fourth goal entails assessment of the extent to which G proteins serve as targets for growth factor-elicited enzymic activities or regulation of transcription. Alterations in the quantities of G proteins and the occurrence of post-translational modifications will be examined by means of immunotransfer blotting and radiolabeling in conjunction with electrophoretic procedures. The final goal of this proposal is the assessment of G protein regulation in relation to malignant transformation effected by Rous and Kirsten sarcoma viruses; the obtained results will be integrated with those previously acquired in this laboratory. The proposed research addresses the extent to which G proteins are either engaged or modified within the context of cell proliferation. Accordingly, this research will provide information relevant to the basic actions of growth factors and the sensitivity of cells to external stimuli during normal and oncogenic growth.
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