Laglia G, Zeiger M A, Leipricht A, Caturegli P, Levine M A, Kohn L D, Saji M
Section on Cell Regulation, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Endocrinology. 1996 Aug;137(8):3170-6. doi: 10.1210/endo.137.8.8754735.
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.
甲状腺细胞的生长和功能受多种激素和生长因子调节，这些激素和生长因子与通过G蛋白（Gs和Gq）偶联的细胞表面受体结合，分别刺激腺苷酸环化酶和磷脂酶C（PLC）。我们构建了一个永久转染的FRTL-5细胞系（TG8），其中甲状腺球蛋白基因启动子指导霍乱毒素（CT）A1亚基（CTA1）的表达。CTA1催化Gsα的ADP核糖基化，导致Gsα持续激活。激活的Gsα导致腺苷酸环化酶的组成性刺激并增加细胞内cAMP水平。由于G蛋白偶联信号通路存在相互作用，我们将TG8细胞与转染了新霉素抗性基因的FRTL-5细胞（TG4）进行比较，以确定腺苷酸环化酶的组成性刺激是否影响PLC通路。通过测量预先用肌醇-[3H]肌醇预孵育2天的TG4和TG8细胞中总肌醇磷酸（IPs）的水平来评估PLC活性。两种细胞系的[3H]IP产生的基线值相似。将TG4对照细胞与10^(-8) M促甲状腺激素（TSH）、300 microM三磷酸腺苷（ATP）和100 microM去甲肾上腺素孵育60分钟，[3H]IP产生分别比对照值增加2.5倍、8.1倍和3.4倍。相比之下，TG8细胞对这些配体中的任何一种均无[3H]IP反应。与TG4对照细胞相比，TG8细胞与其细胞表面的[35S]腺苷5'-（γ-硫代）三磷酸结合减少，但[125I]TSH结合无减少。用100 ng/ml CT、50 microM毛喉素或1 mM 8-溴-cAMP处理TG4细胞2天，重现了TG8细胞中存在的配体刺激的[3H]IP合成的丧失。尽管TG8细胞中免疫反应性Gqα和Gqα11的水平正常，但氟化钠诱导的[3H]IP产生也受到抑制。FRTL-5细胞中PLCβ的主要亚型免疫反应性PLCβ3的水平在TG8细胞中或用CT处理TG4细胞后未改变。这些数据表明，cAMP水平升高可抑制G蛋白偶联PLC的活性。对该模型的进一步研究将阐明我们对负责这种相互作用的确切机制的理解。
