Gbahou Florence, Cecon Erika, Viault Guillaume, Gerbier Romain, Jean-Alphonse Frederic, Karamitri Angeliki, Guillaumet Gérald, Delagrange Philippe, Friedlander Robert M, Vilardaga Jean-Pierre, Suzenet Franck, Jockers Ralf
Inserm, U1016, Institut Cochin, Paris, France.
CNRS UMR 8104, Paris, France.
Br J Pharmacol. 2017 Jul;174(14):2409-2421. doi: 10.1111/bph.13856. Epub 2017 Jun 11.
The paradigm that GPCRs are able to prolong or initiate cellular signalling through intracellular receptors recently emerged. Melatonin binds to G protein-coupled MT and MT receptors. In contrast to most other hormones targeting GPCRs, melatonin and its synthetic analogues are amphiphilic molecules easily penetrating into cells, but the existence of intracellular receptors is still unclear mainly due to a lack of appropriate tools.
We therefore designed and synthesized a series of hydrophilic melatonin receptor ligands coupled to the Cy3 cyanin fluorophore to reliably monitor its inability to penetrate cells. Two compounds, one lipophilic and one hydrophilic, were then functionally characterized in terms of their affinity for human and murine melatonin receptors expressed in HEK293 cells and their signalling efficacy.
Among the different ligands, ICOA-13 showed the desired properties as it was cell-impermeant and bound to human and mouse MT and MT receptors. ICOA-13 showed differential activities on melatonin receptors ranging from partial to full agonistic properties for the G /cAMP and ERK pathway and β-arrestin 2 recruitment. Notably, ICOA-13 enabled us to discriminate between G /cAMP signalling of the MT receptor initiated at the cell surface and neuronal mitochondria.
We report here the first cell-impermeant melatonin receptor agonist, ICOA-13, which allows us to discriminate between signalling events initiated at the cell surface and intracellular compartments. Detection of mitochondrial MT receptors may have an important impact on the development of novel melatonin receptor ligands relevant for neurodegenerative diseases, such as Huntington disease.
G蛋白偶联受体（GPCRs）能够通过细胞内受体延长或启动细胞信号传导的模式最近才出现。褪黑素与G蛋白偶联的MT1和MT2受体结合。与大多数靶向GPCRs的其他激素不同，褪黑素及其合成类似物是两亲性分子，易于穿透细胞，但由于缺乏合适的工具，细胞内受体的存在仍不明确。
因此，我们设计并合成了一系列与Cy3花青素荧光团偶联的亲水性褪黑素受体配体，以可靠地监测其无法穿透细胞的情况。然后，对两种化合物（一种亲脂性和一种亲水性）在对HEK293细胞中表达的人和小鼠褪黑素受体的亲和力及其信号传导效能方面进行了功能表征。
在不同的配体中，ICOA-13表现出所需的特性，因为它不能穿透细胞，并与人及小鼠的MT1和MT2受体结合。ICOA-13对褪黑素受体表现出不同的活性，对Gαi/cAMP和ERK途径以及β-抑制蛋白2募集具有从部分激动到完全激动的特性。值得注意的是，ICOA-13使我们能够区分在细胞表面和神经元线粒体启动的MT2受体的Gαi/cAMP信号传导。
我们在此报告了第一种不能穿透细胞的褪黑素受体激动剂ICOA-13，它使我们能够区分在细胞表面和细胞内区室启动的信号事件。线粒体MT2受体的检测可能对与神经退行性疾病（如亨廷顿病）相关的新型褪黑素受体配体的开发产生重要影响。
