title: Increased cyclic adenosine 3',5'-monophosphate inhibits G protein-coupled activation of phospholipase C in rat FRTL-5 thyroid cells.
abstract: Thyroid cell growth and function are regulated by several hormones and growth factors that bind to cell surface receptors coupled via G proteins, Gs and Gq, to stimulation of adenylyl cyclase and phospholipase C (PLC), respectively. We created a permanently transfected FRTL-5 cell line (TG8) in which the thyroglobulin gene promoter directs expression of the cholera toxin (CT) A1 subunit (CTA1). CTA1 catalyzes ADP ribosylation of Gs alpha, which results in persistent activation of Gs alpha. Activated Gs alpha causes constitutive stimulation of adenylyl cyclase and increases levels of intracellular cAMP. Because G protein-coupled signaling pathways exhibit cross-talk, we compared TG8 cells to FRTL-5 cells transfected with the neomycin resistance gene (TG4) to determine whether constitutive stimulation of adenylyl cyclase influences the PLC pathway. PLC activity was assessed by measuring levels of total inositol phosphates (IPs) in TG4 and TG8 cells that had been preincubated with myo-[3H]inositol for 2 days. Baseline values of [3H]IP production were similar for the two cell lines. Incubation of TG4 control cells with 10(-8) M TSH, 300 microM ATP, and 100 microM norepinephrine for 60 min stimulated 2.5-, 8.1-, and 3.4-fold increases, respectively, in [3H]IP production over the control value. By contrast, there was no [3H]IP response to any of these ligands in TG8 cells. TG8 cells exhibit a decrease in [35S]adenosine 5'-(gamma-thio)triphosphate binding to their cell surface compared to TG4 control cells counterparts, but no decrease in [125I]TSH binding. Treatment of TG4 cells with 100 ng/ml CT, 50 microM forskolin, or 1 mM 8-bromo-cAMP for 2 days reproduced the loss of ligand-stimulated [3H]IP synthesis present in TG8 cells. Although levels of immunoreactive Gq alpha and Gq alpha 11 were normal in TG8 cells, sodium fluoride-induced [3H]IP production was also inhibited. Levels of immunoreactive PLC beta 3, the dominant subtype of PLC beta in FRTL-5 cells, were not altered in TG8 cells or by CT treatment of TG4 cells. These data indicate that elevated levels of cAMP can inhibit the activity of G protein-coupled PLC. Further study of this model will elucidate our understanding of the exact mechanism responsible for this interaction.
authors: Section on Cell Regulation, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors: G
authors: G
authors: Laglia
authors: M A
authors: MA
authors: Zeiger
authors: A
authors: A
authors: Leipricht
authors: P
authors: P
authors: Caturegli
authors: M A
authors: MA
authors: Levine
authors: L D
authors: LD
authors: Kohn
authors: M
authors: M
authors: Saji
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002461:Cell Line, Transformed; D002772:Cholera Toxin / Q000235:genetics / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D019204:GTP-Binding Proteins / Q000502:physiology*; D007295:Inositol Phosphates / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D051381:Rats; D013961:Thyroid Gland / Q000166:cytology / Q000378:metabolism*; D013972:Thyrotropin / Q000378:metabolism; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*
chemical_list: D007295:Inositol Phosphates; D007527:Isoenzymes; D000255:Adenosine Triphosphate; D013972:Thyrotropin; D002772:Cholera Toxin; D000242:Cyclic AMP; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins