Jarzynka Michael J, Passey Deepshikha K, Ignatius Paul F, Melan Melissa A, Radio Nicholas M, Jockers Ralf, Rasenick Mark M, Brydon Lena, Witt-Enderby Paula A
Hillman Cancer Center, Research Pavilion, University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA.
J Pineal Res. 2006 Nov;41(4):324-36. doi: 10.1111/j.1600-079X.2006.00371.x.
Chronic melatonin exposure produces microtubule rearrangements in Chinese hamster ovary (CHO) cells expressing the human MT1 melatonin receptor while at the same time desensitizing MT1 receptors. Because microtubule rearrangements parallel MT1 receptor desensitization, we tested whether microtubules modulate receptor responsiveness. We determined whether depolymerization of microtubules by Colcemid, which prevents melatonin-induced outgrowths in MT1-expressing CHO cells, also prevents MT1 receptor desensitization by affecting G(alpha)-GTP exchange on G-proteins. In this study, we found that depolymerization of microtubules in MT1 receptor expressing cells, prevented melatonin-induced receptor desensitization reflected by an increase in the number of high potency sites when compared with melatonin-treated cells. Further examination of the mechanism(s) underlying this desensitization suggested that these effects occurred at the level of G-proteins. Depolymerization of microtubules during melatonin-induced desensitization, attenuated forskolin-induced cAMP accumulation, the opposite of which usually occurs following melatonin exposure alone. Concomitant to this attenuation in the forskolin response was a reduction in the amount of G(i alpha) protein coupled to MT1 receptors and an increase in [32P] azidoanilido GTP incorporation into G(i) proteins. These data are consistent with the findings that microtubule depolymerization did not affect MT1/G(q) coupling nor did it affect melatonin-induced phosphoinositide hydrolysis following melatonin exposure. However, interestingly, microtubule depolymerization enhanced melatonin-induced protein kinase C activation that was blocked in the presence of pertussis toxin. These data demonstrate that microtubule dynamics can modulate melatonin receptor function through their actions on G(i) proteins and impact on downstream signaling cascades.
长期暴露于褪黑素会使表达人MT1褪黑素受体的中国仓鼠卵巢（CHO）细胞中的微管发生重排，同时使MT1受体脱敏。由于微管重排与MT1受体脱敏平行，我们测试了微管是否调节受体反应性。我们确定秋水仙酰胺使微管解聚（这可阻止褪黑素诱导表达MT1的CHO细胞生长）是否也通过影响G蛋白上的G（α）-GTP交换来阻止MT1受体脱敏。在本研究中，我们发现，与褪黑素处理的细胞相比，表达MT1受体的细胞中微管解聚可阻止褪黑素诱导的受体脱敏，这表现为高效能位点数量增加。对这种脱敏潜在机制的进一步研究表明，这些效应发生在G蛋白水平。褪黑素诱导脱敏过程中微管解聚减弱了福斯高林诱导的cAMP积累，而单独暴露于褪黑素后通常会出现相反的情况。与福斯高林反应减弱同时出现的是，与MT1受体偶联的G（iα）蛋白量减少，以及[32P]叠氮苯胺基GTP掺入G（i）蛋白增加。这些数据与以下发现一致：微管解聚不影响MT1/G（q）偶联，也不影响褪黑素暴露后褪黑素诱导的磷酸肌醇水解。然而，有趣的是，微管解聚增强了褪黑素诱导的蛋白激酶C激活，而百日咳毒素存在时这种激活被阻断。这些数据表明，微管动力学可通过其对G（i）蛋白的作用来调节褪黑素受体功能，并影响下游信号级联反应。
