The overall goal of this project is to understand at a molecular level the interactions of the components of the cell surface signal transducing systems in the liver. The studies are focused on those systems that use heterotrimeric GTP binding proteins (G-proteins) as transducers. In the coming term we plan to analyze the phospholipase C signal transduction pathway of the liver. This pathway is used by a variety of hormones such as epinephrine acting through the alpha1-adrenergic receptor, vasopressin and angiotensin to modulate carbohydrate metabolism in the liver. Our studies will focus on determining the identity of the pertussis toxin insensitive G-protein that functions in the phospholipase C pathway in the liver and its mechanism of action. We will use both biochemical and molecular biologic approaches. We will use the IP3 mediated, Ca2+ dependent Cl- current in Xenopus oocyte as an assay for IP3 for production. We will inject mRNA encoding the alpha1-adrenergic receptor in Xenopus oocytes and then study the ability of various purified G-proteins from liver to couple the expressed alpha1-adrenergic receptor to the Cl- current. The G-protein that is capable of functioning in the phospholipase C pathway will be purified to apparent homogeneity. We will also attempt to isolate the cDNA for the pertussis toxin insensitive G-protein in the phospholipase C pathway. For this purpose we will construct oligonucleotides based on the predicted structures of this G-protein alpha- subunit and use them to amplify a portion of the putative cDNA by polymerase chain reaction from liver cDNA library to isolate the cDNA for the G-protein of interest. By site directed mutagenesis we will engineer a pertussis toxin insensitive G-protein, by conversion of the Cys in the fourth position from the C-terminus to a Ser. This model pertussis toxin insensitive G-protein will also be studied for its function in phosphatidyl inositol pathway. We will map the regions of the G-protein alpha-subunit involved in phospholipase C interactions. For this we will rely on our current finding that Go serves as the signal transducer in the pertussis toxin sensitive pathway. We will construct chimeras of the alphaO and alphaS and study the capabilities of these chimeras to stimulate phospholipase C and adenylyl cyclase. From a series of chimeras we will determine which region of alphaO interacts with phospholipase C. It is hoped that these studies will serve as a solid foundation for understanding G-protein-effector interactions and the regulation of liver metabolism.
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