The long term goal of this project is to determine the molecular mechanism by which EGF generates intracellular signals. Both angiotensin II and EGF increase the breakdown of phosphatidylinositol 4.5 bisphosphate in the hepatocyte, stimulating formation of inositol triphosphate and mobilization of Ca2+ ion. However, only the effects of EGF are blocked by pretreatment of the cells with pertussis toxin. The possibility exists that the pertussis toxin substrate involved in EGF action may be an uncharacterized G protein having at least some unique properties. The major goal of this proposal is to examine the nature of the pertussis toxin sensitive G proteins in the hepatocyte to determine which of these proteins couples the EGF receptor to phospholipase C. The path to the goal will combine the experimental approaches of protein purification and molecular cloning. The strategy proposed takes advantage of the high degree of protein and nucleic acid sequence homology between the GTP- binding and pertussis toxin ADP-ribosylation sites in the known G proteins. This sequence information will be used to design specific probes to search cDNA libraries for clones corresponding to other putative G proteins. When clones with the appropriate properties are found, the corresponding G proteins will be purified from liver and their physical and biochemical properties studied. The project will begin with molecular cloning because of the high probability of a successful result and because the sequence information obtained from new clones will provide useful knowledge and reagents such as anti-peptide antibodies that will greatly improve the methods used for purification of the protein. To this end, the applicant's laboratory has isolated full length clones for the alpha subunits of Gs and Gi from a rat kidney lambda gt10 library. Using these clones as reagents, the strategy is to prepare probes to screen rat brain and hepatocyte cDNA libraries for two types of clones: (1) those that hybridize with a well conserved region in the carboxyl corresponding to the site of modification by pertussis toxin; and (2) those that hybridize with the highly conserved region corresponding to one of the GTP-binding sites, a general property of the alpha subunits. The promising clones will be characterized and sequenced. If a unique clone is obtained, anti-peptide antisera will be prepared against regions of the molecule predicted to be antigenic and with the aid of these reagents, the new G protein be purified from rat hepatic membranes. CNBr or tryptic fragments of the protein will be sequenced to compare with the cDNA structure. Finally, the physical and biochemical properties of the new G protein will be studied.
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