Real-Time Determination of Intracellular cAMP Reveals Functional Coupling of G<sub>s</sub> Protein to the Melatonin MT<sub>1</sub> 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<sub>1</sub>) and type-2 (MT<sub>2</sub>) recept...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2024-03-01
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| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/25/5/2919 |
| Summary: | 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<sub>1</sub>) and type-2 (MT<sub>2</sub>) receptors. Both MT<sub>1</sub> and MT<sub>2</sub> receptors are generally classified as G<sub>i</sub>-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<sub>1</sub> receptor. To address if MT<sub>1</sub> can dually couple to G<sub>s</sub> and G<sub>i</sub> proteins, we employed a highly sensitive luminescent biosensor (GloSensor<sup>TM</sup>) to monitor the real-time changes in the intracellular cAMP level in intact live HEK293 cells that express MT<sub>1</sub> and/or MT<sub>2</sub>. Our results demonstrate that the activation of MT<sub>1</sub>, but not MT<sub>2</sub>, leads to a robust enhancement on the forskolin-stimulated cAMP formation. In contrast, the activation of either MT<sub>1</sub> or MT<sub>2</sub> inhibited cAMP synthesis driven by the activation of the G<sub>s</sub>-coupled β<sub>2</sub>-adrenergic receptor, which is consistent with a typical G<sub>i</sub>-mediated response. The co-expression of MT<sub>1</sub> with G<sub>s</sub> enabled melatonin itself to stimulate cAMP production, indicating a productive coupling between MT<sub>1</sub> and G<sub>s</sub>. The possible existence of a MT<sub>1</sub>-G<sub>s</sub> complex was supported through molecular modeling as the predicted complex exhibited structural and thermodynamic characteristics that are comparable to that of MT<sub>1</sub>-G<sub>i</sub>. Taken together, our data reveal that MT<sub>1</sub>, but not MT<sub>2</sub>, can dually couple to G<sub>s</sub> and G<sub>i</sub> proteins, thereby enabling the bi-directional regulation of adenylyl cyclase to differentially modulate cAMP levels in cells that express different complements of MT<sub>1</sub>, MT<sub>2</sub>, and G proteins. |
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| ISSN: | 1661-6596 1422-0067 |