Ubiquitination and proteasomal degradation of endogenous and exogenous inositol 1,4,5-trisphosphate receptors in alpha T3-1 anterior pituitary cells.
Ubiquitination and proteasomal degradation of endogenous and exogenous inositol 1,4,5-trisphosphate receptors in alpha T3-1 anterior pituitary cells.
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α T3-1 垂体前叶细胞内源性和外源性肌醇 1,4,5-三磷酸受体的泛素化和蛋白酶体降解。
DOI：
10.1074/jbc.m206607200
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Gao,Chen
中科院分区：
文献类型：
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作者：
Wojcikiewicz,RichardJH;Xu,Qun;Webster,JackM;Alzayady,Kamil;Gao,Chen
在αT3-1小鼠垂体前叶促性腺激素中，促性腺激素释放激素（GnRH）受体的慢性激活导致1，4，5-三磷酸肌醇（InsP 3）受体下调（Willars，G. B.，罗亚尔，J.E.，Nahorski，S. R.，El-Gehani，F.，珠峰、H.和McArdle，C. A.（2001）J.Biol.Chem.276，3123-3129）。在当前的研究中，我们试图定义这种适应性反应背后的机制。我们发现，GnRH诱导InsP 3受体多聚泛素化的快速和显着增加，蛋白酶体抑制剂阻断InsP 3受体下调，并导致多聚泛素化受体的积累。因此，泛素/蛋白酶体途径在αT3-1细胞中是活跃的，GnRH通过该机制调节InsP 3受体的水平。鉴于这些发现和对该系统的进一步表征，我们还研究了αT3-1细胞可用于检测通过cDNA转染引入的外源InsP 3受体的泛素化的可能性。这是因为外源性野生型InsP 3受体，而不是结合缺陷的突变体受体，以GnRH依赖的方式被多聚泛素化，抑制内源性受体的多聚泛素化的药物也抑制外源性受体的多聚泛素化。此外，我们使用该系统来确定是否磷酸化参与触发InsP 3受体聚泛素化。但事实并非如此，因为丝氨酸残基1588和1755（I型受体中主要的磷酸化位点）的突变并不抑制聚泛素化。总之，这些数据表明，泛素/蛋白酶体途径在垂体前叶细胞中是活跃的，该途径靶向GnRH刺激的αT3-1细胞中的内源性和外源性InsP 3受体，并且与许多其他底物的情况相反，磷酸化不会触发InsP 3受体聚泛素化。
In αT3-1 mouse anterior pituitary gonadotropes, chronic activation of gonadotropin-releasing hormone (GnRH) receptors causes inositol 1,4,5-trisphosphate (InsP3) receptor down-regulation (Willars, G. B., Royall, J. E., Nahorski, S. R., El-Gehani, F., Everest, H. and McArdle, C. A. (2001)J. Biol. Chem. 276, 3123–3129). In the current study, we sought to define the mechanism behind this adaptive response. We show that GnRH induces a rapid and dramatic increase in InsP3receptor polyubiquitination and that proteasome inhibitors block InsP3receptor down-regulation and cause the accumulation of polyubiquitinated receptors. Thus, the ubiquitin/proteasome pathway is active in αT3-1 cells, and GnRH regulates the levels of InsP3receptors via this mechanism. Given these findings and further characterization of this system, we also examined the possibility that αT3-1 cells could be used to examine the ubiquitination of exogenous InsP3receptors introduced by cDNA transfection. This was found to be the case, since exogenous wild-type InsP3receptors, but not binding-defective mutant receptors, were polyubiquitinated in a GnRH-dependent manner, and agents that inhibited the polyubiquitination of endogenous receptors also inhibited the polyubiquitination of exogenous receptors. Further, we used this system to determine whether phosphorylation was involved in triggering InsP3receptor polyubiquitination. This was not the case, since mutation of serine residues 1588 and 1755 (the predominant phosphorylation sites in the type I receptor) did not inhibit polyubiquitination. In total, these data show that the ubiquitin/proteasome pathway is active in anterior pituitary cells, that this pathway targets both endogenous and exogenous InsP3receptors in GnRH-stimulated αT3-1 cells, and that, in contrast to the situation for many other substrates, phosphorylation does not trigger InsP3receptor polyubiquitination.