ACTIVATION OF CYCLIC-NUCLEOTIDE PHOSPHODIESTERASES IN FRTL-5 THYROID-CELLS EXPRESSING A CONSTITUTIVELY ACTIVE GS-ALPHA
ACTIVATION OF CYCLIC-NUCLEOTIDE PHOSPHODIESTERASES IN FRTL-5 THYROID-CELLS EXPRESSING A CONSTITUTIVELY ACTIVE GS-ALPHA
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DOI：
10.1210/me.9.10.1279
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发表时间：
1995-10-01
影响因子：
--
通讯作者：
CONTI, M
中科院分区：
文献类型：
--
作者：
NEMOZ, G;SETTE, C;CONTI, M
在大鼠甲状腺细胞系FRTL-5中组成型激活的Gs α蛋白的表达引起非TSH依赖性腺苷酸环化酶活性的增加，并促进细胞的非TSH依赖性生长。尽管腺苷酸环化酶的组成性激活，但这些细胞中的基础cAMP水平仅略微增加。为了确定磷酸二酯酶（PDE）在该表型发生中的作用，在表达突变的Gs α（Q227 L）的两个细胞系中测定环核苷酸水解。在这些细胞中，cAMP和cGMP的水解与正常细胞相比显着增加。这种增加是不同形式PDE激活的结果。cGMP水解和Ca++/钙调素刺激PDE活性的分析表明，Ca++/钙调素刺激的PDE的活性在两种细胞系中均增加。此外，两种细胞系中高亲和力、咯利普兰敏感性cAMP-PDE活性的增加与67-68千道尔顿（kDa）蛋白的出现相关，该蛋白与两种抗cAMP-PDE的抗体交叉反应。这种形式具有大鼠PDE 3.2/PDE 4D 2的特性，这是一种在野生型细胞中可由TSH诱导的cAMP-PDE。cAMP特异性、咯利普兰敏感性PDE的增加在由Q227 L Gs α诱导的表型中起作用，这通过促有丝分裂活性的测量得到证实。与咯利普兰孵育，这对野生型细胞没有影响，引起cAMP水平的增加，并进一步刺激TSH非依赖性增殖的两种细胞系携带突变。这些数据表明，FRTL-5中PDE系统的激活至少部分抵消了由Gs α中的激活突变引起的表型。
The expression of a constitutively activated Gs alpha protein in the rat thyroid cell line FRTL-5 causes an increase in the hormone-independent adenylyl cyclase activity and promotes TSH-independent growth of the cells. In spite of the constitutive activation of the adenylyl cyclase, the basal cAMP levels in these cells are only marginally increased. To define the role of phosphodiesterases (PDEs) in the genesis of this phenotype, cyclic nucleotide hydrolysis was determined in two cell lines expressing a mutated Gs alpha (Q227L). In these cells, the hydrolysis of both cAMP and cGMP was markedly increased in comparison with normal cells. This increase is the result of the activation of different forms of PDEs. Analysis of the cGMP hydrolysis and Ca++/calmodulin stimulation of the PDE activity indicated that the activity of a Ca++/calmodulin stimulated PDE is increased in both cell lines. In addition, an increase in high-affinity, rolipram-sensitive cAMP-PDE activity was associated in both cell lines with the appearance of a 67-68 kilodalton (kDa) protein that cross-reacts with two antibodies against cAMP-PDEs. This form had the properties of ratPDE3.2/PDE4D2, a cAMP-PDE that is inducible by TSH in wild type cells. That an increase in cAMP-specific, rolipram-sensitive PDE plays a role in the phenotype induced by Q227L Gs alpha was confirmed by measurements of the mitogenic activity. Incubation with rolipram, which had no effect on wild type cells, caused an increase in cAMP levels and further stimulated TSH-independent proliferation in both cell lines carrying the mutation. These data demonstrate that activation of the PDE system in FRTL-5 counteracts, at least in part, the phenotype caused by an activating mutation in Gs alpha.