Elevation of cytosolic Ca2+ by Alpha-1-adrenergic agents and vasopressin in liver, leading to increased glycogenolysis, is associated with receptor-mediated hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate (PIP2) to produce the intracellular messengers, inositol 1,4,5 trisphosphate (IP3) and 1,2-diacylglycerol. IP3 mobilizes Ca2+ from intracellular stores, and diacylglycerol, through a phospholipid-dependent protein kinase (protein kinase C) can effect changes in protein phosphorylation. The overall objective is to assess the significance of protein kinase C in the actions of Ca2+-mobilizing hormones, and potential cross-relationships with the actions of glucagon and insulin. These studies will focus in particular on the mechanism whereby the tumor-promoting phorbol ester (PMA), which can directly activate protein kinase C, inhibits Alpha 1-agonist-induced Ca2+ mobilization, but has little effect on that induced by vasopressin. The main hypothesis to be tested is that phosphorylation by protein kinase C of a particular protein, possibly closely associated with the Alpha-1-receptor, inhibits the signal transduction process. The particular aims addressed are: (1) Delineation of the inhibitory site of action of PMA on Alpha-1-agonist-induced Ca2+ mobilization; (2) Characterization of protein kinase C and its substrate proteins in liver; (3) Identification of the phosphoprotein(s) responsible for Alpha-1-receptor down-regulation; (4) Determination of changes in hepatocyte 32-P-protein labeling by insulin and possible relationships with PMA-induced phosphorylation. The locus of the PMA effect on Alpha-1-events will be addressed by examining effects of PMA wiht or without exogenous protein kinase C on [3H] prazosin binding, and on breakdown of [32P] PIP2 in membranes prepared from hepatocytes prelabeled with 32P. The five proteins thus far identified as protein kinase C substrates (Mr values of 68,000, 52,000, 36,000 and two 16,000 dalton proteins) will be further characterized and the regulatory properties of purified liver protein kinase C will be examined. Insulin-induced changes in 32P hepatocyte protein labeling will be examined by two-dimensional SDS-PAGE. This study should provide new information regarding early events associated with the actions of a tumor-promoting agent, clarify the mechanism of receptor-mediated inositol lipid breakdown and indicate whether the inhibitory effects of insulin and PMA on Alpha-1-adrenergic actions occur through a similar mechanism, involving phosphorylation of a key regulatory protein albeit mediated by different protein kinases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
7R01DK033795-02
Application #
3232198
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1987-04-01
Project End
1989-03-31
Budget Start
1987-04-01
Budget End
1988-03-31
Support Year
2
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of South Carolina at Columbia
Department
Type
Schools of Medicine
DUNS #
111310249
City
Columbia
State
SC
Country
United States
Zip Code
29208
Beeler, J F; Cooper, R H (1995) Regulation of hepatocyte plasma membrane alpha 1-adrenergic receptors by 4 beta-phorbol 12-myristate 13-acetate. Biochem J 305 ( Pt 1):73-9
Beeler, J F; Cooper, R H (1993) Rapid and reversible uncoupling of the hepatic alpha 1-adrenergic receptor and guanine-nucleotide-binding protein by phorbol 12,13-dibutyrate. Biochim Biophys Acta 1176:339-42
Chuang, L F; Cooper, R H; Yau, P et al. (1987) Protein kinase C phosphorylates leukemia RNA polymerase II. Biochem Biophys Res Commun 145:1376-83