Division of Life Science and the Biotechnology Research Institute, Hong Kong University of Science and Technology, Hong Kong, China.
State Key Laboratory of Molecular Neuroscience, Molecular Neuroscience Center, Hong Kong University of Science and Technology, Hong Kong, China.
Int J Mol Sci. 2024 Mar 2;25(5):2919. doi: 10.3390/ijms25052919.
Melatonin is a neuroendocrine hormone that regulates the circadian rhythm and many other physiological processes. Its functions are primarily exerted through two subtypes of human melatonin receptors, termed melatonin type-1 (MT) and type-2 (MT) receptors. Both MT and MT receptors are generally classified as G-coupled receptors owing to their well-recognized ability to inhibit cAMP accumulation in cells. However, it remains an enigma as to why melatonin stimulates cAMP production in a number of cell types that express the MT receptor. To address if MT can dually couple to G and G proteins, we employed a highly sensitive luminescent biosensor (GloSensor) to monitor the real-time changes in the intracellular cAMP level in intact live HEK293 cells that express MT and/or MT. Our results demonstrate that the activation of MT, but not MT, leads to a robust enhancement on the forskolin-stimulated cAMP formation. In contrast, the activation of either MT or MT inhibited cAMP synthesis driven by the activation of the G-coupled β-adrenergic receptor, which is consistent with a typical G-mediated response. The co-expression of MT with G enabled melatonin itself to stimulate cAMP production, indicating a productive coupling between MT and G. The possible existence of a MT-G complex was supported through molecular modeling as the predicted complex exhibited structural and thermodynamic characteristics that are comparable to that of MT-G. Taken together, our data reveal that MT, but not MT, can dually couple to G and G proteins, thereby enabling the bi-directional regulation of adenylyl cyclase to differentially modulate cAMP levels in cells that express different complements of MT, MT, and G proteins.
褪黑素是一种神经内分泌激素，调节昼夜节律和许多其他生理过程。其功能主要通过两种人类褪黑素受体亚型发挥，称为褪黑素 1 型 (MT) 和 2 型 (MT) 受体。由于其公认的抑制细胞内 cAMP 积累的能力，MT 和 MT 受体通常被归类为 G 偶联受体。然而，褪黑素为何在表达 MT 受体的许多细胞类型中刺激 cAMP 产生仍然是一个谜。为了解决 MT 是否可以双重偶联到 G 和 G 蛋白的问题，我们使用了一种高度敏感的发光生物传感器 (GloSensor) 来监测表达 MT 和/或 MT 的完整活 HEK293 细胞中细胞内 cAMP 水平的实时变化。我们的结果表明，MT 的激活而不是 MT 的激活导致福司可林刺激的 cAMP 形成显著增强。相比之下，MT 或 MT 的激活抑制了由 G 偶联的β肾上腺素能受体激活驱动的 cAMP 合成，这与典型的 G 介导的反应一致。MT 与 G 的共表达使褪黑素本身能够刺激 cAMP 的产生，表明 MT 和 G 之间存在有效偶联。通过分子建模支持 MT-G 复合物的可能存在，预测的复合物表现出与 MT-G 相当的结构和热力学特征。总之，我们的数据表明 MT 而不是 MT 可以双重偶联到 G 和 G 蛋白，从而能够双向调节腺苷酸环化酶，以不同方式调节表达不同 MT、MT 和 G 蛋白的细胞中的 cAMP 水平。
