Thyroid hormone signaling

最近发现，甲状腺激素对大鼠垂体细胞Kv11.1通道活性的快速影响(Storey Et Al 2006)是由经典的甲状腺激素核受体(Tr)介导的，它通过磷脂酰肌醇3激酶(PI3K)和Rac GTP酶作用于质膜，Rac GTP酶是众所周知的依赖于PIP3的RAC交换因子的效应器。因此，用Wortmannin或显性负性Rac 17N抑制PI3K或显性负性Rac 17N可阻断甲状腺激素对Kv11.1通道的刺激，外源性PI3K作用的直接产物3，4，5-PIP3或Rac 61L可恢复Kv11.1通道的兴奋。更重要的是，通过人Kv11.1通道和人受体的异源表达，CHO细胞中的信号被重组，但不是受体。我们继续研究TR刺激PI3K的机制，以及PI3K信号对甲状腺激素生理效应的影响。我们已经用免疫沉淀法证明，在没有配体的情况下，TR&946；与PI3K的调节P85亚单位结合，但在甲状腺激素存在的情况下，它与PI3K的调节P85亚单位结合。我们还观察到了甲状腺激素依赖的Akt蛋白激酶的快速磷酸化和RAC在质膜上的募集，证实了甲状腺激素刺激PI3K依赖的效应分子。其他蛋白质通过识别磷酸酪氨酸的两个Src同源(SH2)结构域与P85相互作用。TR&946；而不是tr&945；含有与p85高亲和力的SH2结合结构域。在CHO细胞中，将该共有位点的酪氨酸突变为苯丙氨酸可阻止甲状腺激素对Kv11.1调节的重建。

The rapid effects of thyroid hormone on the activity of Kv11.1 channels in rat pituitary cells were recently shown (Storey et al 2006) to be mediated by the classical nuclear receptor for thyroid hormone, TRβ, acting at the plasma membrane through the phosphoinositide 3 kinase (PI3K) and the Rac GTPase, which is a well-known effector of PIP3 dependent Rac exchange factors. Thus, Kv11.1 channel stimulation by thyroid hormone was blocked by inhibiting PI3K with wortmannin or dominant negative Rac 17N and restored by exogenous application of 3,4,5-PIP3, the immediate product of PI3K action, or by constitutively active Rac 61L. More importantly signaling was reconstituted in CHO cells by heterologous expression of human Kv11.1 channels and the human TRβ but not the TRα receptor. We have continued to investigate the mechanism of PI3K stimulation by TRβ and the consequences of PI3K signaling for the physiological effects of thyroid hormone. We have used immunoprecipitation to show that TRβ associates with the regulatory p85 subunit of PI3K in the absence of ligand, but dissociates in the presence of thyroid hormone. We have also observed rapid thyroid hormone-dependent phosphorylation of the Akt protein kinase and recruitment of Rac to the plasma membrane confirming that thyroid hormone stimulates PI3K-dependent effectors. Other proteins interact with p85 through two Src homology (SH2) domains which recognize phosphotyrosine. TRβ but not TRα contains a consensus SH2-binding domain with high affinity for p85. Mutating the tyrosine in that consensus site to phenylalanine prevents reconstitution of Kv11.1 regulation by thyroid hormone in CHO cells.