Knock-down of RGS4 and beta tubulin in CHO cells expressing the human MT1 melatonin receptor prevents melatonin-induced receptor desensitization.
Knock-down of RGS4 and beta tubulin in CHO cells expressing the human MT1 melatonin receptor prevents melatonin-induced receptor desensitization.
复制标题
在表达人 MT1 褪黑激素受体的 CHO 细胞中敲低 RGS4 和 β 微管蛋白可防止褪黑激素诱导的受体脱敏。
DOI：
10.1016/j.lfs.2004.08.002
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Melan,MA
中科院分区：
文献类型：
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作者：
Witt-Enderby,PA;Jarzynka,MJ;Krawitt,BJ;Melan,MA
以前，它已被证明，慢性褪黑激素暴露在MT 1-CHO细胞的结果在受体脱敏，而在同一时间产生剧烈的形态变化。在褪黑激素预处理期间加入解聚剂可防止MT 1受体脱敏和细胞形态学变化。在解聚剂存在下缺乏形态学变化很容易被解释为微管不能活化，然而，受体脱敏的预防稍微复杂一点，可能涉及G蛋白活化。本研究的目的是确定褪黑激素诱导的MT 1受体脱敏是否受到已知调节G蛋白激活状态的蛋白质β-微管蛋白和RGS 4的调节，使用反义敲除方法。使用RT PCR证实RGS 4 mRNA在CHO细胞中的表达，并且通过蛋白质印迹分析或定量PCR证实每种的成功敲低。MT 1-CHO细胞的预处理，转染无义探针和暴露于褪黑激素，导致受体的脱敏，增加毛喉素诱导的cAMP积累，增加2-[125 I]-碘褪黑激素结合和褪黑激素对MT 1受体的亲和力没有变化。然而，在MT 1-CHO细胞中敲低β−微管蛋白或RGS 4，然后用褪黑激素预处理，减弱了褪黑激素受体的脱敏作用，降低了总的2-[125 I]-碘褪黑激素结合，并且既不影响毛喉素反应，也不影响褪黑激素对MT 1受体的亲和力。RGS 4和β-tubulin可能通过调节Gα-GDP和Gα-GTP状态，从而调节MT 1褪黑素受体的功能。
Previously, it has been shown that chronic melatonin exposure in MT1-CHO cells results in receptor desensitization while at the same time producing drastic morphological changes. The addition of a depolymerizing agent during the melatonin pretreatment period prevents MT1receptor desensitization and the changes in cellular morphology. The lack of morphological change in the presence of a depolymerizing agent is easily explained by the inability of the microtubules to polymerize, however, the prevention of receptor desensitization is a little more complex and may involve G-protein activation. The goal of this study was to determine whether melatonin-induced MT1receptor desensitization is regulated by proteins known to regulate G-protein activation states, β-tubulin and RGS4,using anti sense knockdown approaches. The expression of RGS4mRNA in CHO cells was confirmed using RT PCR and successful knockdown of each was confirmed by western blot analysis or quantitative PCR. Pretreatment of MT1-CHO cells, transfected with the nonsense probes and exposed to melatonin, resulted in a desensitization of the receptor, an increase in forskolin-induced cAMP accumulation, an increase in 2-[125I]-iodomelatonin binding and no change in the affinity of melatonin for the MT1receptor. However, knockdown of either β−tubulin or RGS4in MT1-CHO cells followed by pretreatment with melatonin attenuated the desensitization of melatonin receptors, decreased total 2-[125I]-iodomelatonin binding, and did not affect neither the forskolin response nor the affinity of melatonin for the MT1receptor. Perhaps RGS4and β-tubulin modulate Gα-GDP and Gα-GTP states thus modulating MT1melatonin receptor function.