The objective of the proposed research is to determine the extent to which modifications of the guanine nucleotide-binding proteins G?s? and G?i? participate in the regulation of adenylate cyclase coincident with malignant transformation. Transformation of various cells by Rous and Kirsten sarcoma viruses will be empoloyed as models for investigation. An initial aim of the proposed research is the analysis of G?s? and G?i? function in relation to transformation. The ability of G?s? and G?i? to mediate activation and inhibition of adenylate cyclase in native plasma membranes and in reconstituted systems will be investigated. A subsequent aim is the development of antibodies specific for subunits of G?s? and G?i?. Antibodies will be elicited by immunization with conjugated synthetic peptides that correspond to hydrophilic regions of these proteins. Serum titer and specificity will be monitored by solid-phase techniques and procedures of immunoprecipitation.
The third aim i s the assessment of regulatory events that may impinge upon G?s? and G?i? in relation to transformation. Events to be investigated include alterations in level of G?s? and G?i?, alterations in relative levels of isozymic forms of these proteins, post-translational modification, and physical interactions with other proteins.
This aim will be achieved by employment of radiolabel and immunological procedures. Alterations in basal activities of adenylate cyclase, and in the responsiveness of this enzyme to extracellular agents, have been suggested as participating in transformation, metastasis, differentiation, and progression of the cell cycle. The ultimate objective of the proposed research is to contribute to the understanding of the relationship between these alterations and the general phenomenon of cell proliferation. (N)
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