Real-Time Determination of Intracellular cAMP Reveals Functional Coupling of G s Protein to the Melatonin MT 1 Receptor

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.