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 β₂-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.