Real-Time Determination of Intracellular cAMP Reveals Functional Coupling of G_s Protein to the Melatonin MT₁ 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 (MT1) and type-2 (MT2) receptors. Both MT1 and MT2 receptors are generally classified as G(i)-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 MT1 receptor. To address if MT1 can dually couple to G(s) and G(i) proteins, we employed a highly sensitive luminescent biosensor (GloSensor (TM)) to monitor the real-time changes in the intracellular cAMP level in intact live HEK293 cells that express MT1 and/or MT2. Our results demonstrate that the activation of MT1, but not MT2, leads to a robust enhancement on the forskolin-stimulated cAMP formation. In contrast, the activation of either MT1 or MT2 inhibited cAMP synthesis driven by the activation of the G(s)-coupled beta(2)-adrenergic receptor, which is consistent with a typical G(i)-mediated response. The co-expression of MT1 with G(s) enabled melatonin itself to stimulate cAMP production, indicating a productive coupling between MT1 and G(s). The possible existence of a MT1-G(s) complex was supported through molecular modeling as the predicted complex exhibited structural and thermodynamic characteristics that are comparable to that of MT1-G(i). Taken together, our data reveal that MT1, but not MT2, can dually couple to G(s) and G(i) proteins, thereby enabling the bi-directional regulation of adenylyl cyclase to differentially modulate cAMP levels in cells that express different complements of MT1, MT2, and G proteins.