Fears Letimicia S, Curtis Mary E, Johnson Terrance L, Fentress Hugh M
Department of Biological Sciences, Tennessee State University, United States of America.
EC Pharmacol Toxicol. 2019 Jan 29;30(Suppl 1):103-111. Epub 2016 Apr 1.
The monoamine neurotransmitter serotonin (5-HT) plays a role in many physiological responses by interacting with various receptor subtypes. The 5-HT receptor subtype is a 7-transmembrane, G protein-coupled receptor (GPCR) that is involved in neuronal excitability, spatial learning, mood, and appetite. The microorganism produces a purple pigment, violacein, which can be extracted and purified. Violacein has antibiotic, antileishmanial, antifungal and antitumoral properties in various cancer cell lines. Violacein is derived from the amino acid tryptophan as is 5-HT and therefore, the two have similar chemical structures. However, no one has reported the activity of violacein at 5-HT receptors. Therefore the Fentress lab decided to investigate whether or not violacein had an effect on 5-HT receptor trafficking. Human Embryonic Kidney (HEK) 293 cells expressing fluorescently-tagged 5-HT receptor were treated with 5-HT, violacein, water or vehicle and then cells were fixed and visualized with fluorescent microscopy. Violacein treatment did not cause receptor internalization. Recent studies suggest that the 5-HT receptor can activate the JAK/STAT pathway. To see if violacein can modulate this pathway, HEK 293 cells expressing 5-HT receptor were treated with either 5-HT, violacein, or pretreated with violacein followed by incubation with 5-HT. Phosphorylation states of JAK2 and STAT3 were examined by immunoblotting. Results determined that 5-HT receptor activation had no effect on JAK2 phosphorylation and that violacein blocked STAT3 phosphorylation. Primary radioligand binding determined that violacein has a low affinity for 5-HT receptor but has a higher affinity for adrenergic receptors. Future studies will examine G protein-coupling by measuring phosphoinositide hydrolysis and cAMP assay to investigate adrenergic pathways.
单胺神经递质5-羟色胺（5-HT）通过与各种受体亚型相互作用，在许多生理反应中发挥作用。5-HT受体亚型是一种7跨膜G蛋白偶联受体（GPCR），参与神经元兴奋性、空间学习、情绪和食欲调节。该微生物产生一种紫色色素——紫菌素，可进行提取和纯化。紫菌素在各种癌细胞系中具有抗生素、抗利什曼原虫、抗真菌和抗肿瘤特性。紫菌素与5-HT一样，都来源于氨基酸色氨酸，因此二者具有相似的化学结构。然而，尚无关于紫菌素对5-HT受体活性的报道。因此，芬特雷斯实验室决定研究紫菌素是否对5-HT受体转运有影响。用5-HT、紫菌素、水或赋形剂处理表达荧光标记5-HT受体的人胚肾（HEK）293细胞，然后固定细胞并用荧光显微镜观察。紫菌素处理未导致受体内化。最近的研究表明，5-HT受体可激活JAK/STAT信号通路。为了观察紫菌素是否能调节该信号通路，用5-HT、紫菌素处理表达5-HT受体的HEK 293细胞，或先用紫菌素预处理，然后与5-HT孵育。通过免疫印迹检测JAK2和STAT3的磷酸化状态。结果表明，5-HT受体激活对JAK2磷酸化无影响，而紫菌素可阻断STAT3磷酸化。初级放射性配体结合实验表明，紫菌素对5-HT受体的亲和力较低，但对肾上腺素能受体的亲和力较高。未来的研究将通过测量磷酸肌醇水解和cAMP检测来研究肾上腺素能信号通路，以检测G蛋白偶联情况。
