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: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2024-03-01
|
| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/25/5/2919 |
| _version_ | 1797264348903636992 |
|---|---|
| author | Lap Hang Tse Suet Ting Cheung Seayoung Lee Yung Hou Wong |
| author_facet | Lap Hang Tse Suet Ting Cheung Seayoung Lee Yung Hou Wong |
| author_sort | Lap Hang Tse |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-25T00:27:29Z |
| format | Article |
| id | doaj.art-2c5574f58e124f088d0d5e93f99ca568 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-04-25T00:27:29Z |
| publishDate | 2024-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-2c5574f58e124f088d0d5e93f99ca5682024-03-12T16:46:51ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672024-03-01255291910.3390/ijms25052919Real-Time Determination of Intracellular cAMP Reveals Functional Coupling of G<sub>s</sub> Protein to the Melatonin MT<sub>1</sub> ReceptorLap Hang Tse0Suet Ting Cheung1Seayoung Lee2Yung Hou Wong3Division of Life Science and the Biotechnology Research Institute, Hong Kong University of Science and Technology, Hong Kong, ChinaDivision of Life Science and the Biotechnology Research Institute, Hong Kong University of Science and Technology, Hong Kong, ChinaDivision of Life Science and the Biotechnology Research Institute, Hong Kong University of Science and Technology, Hong Kong, ChinaDivision of Life Science and the Biotechnology Research Institute, Hong Kong University of Science and Technology, Hong Kong, ChinaMelatonin 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.https://www.mdpi.com/1422-0067/25/5/2919adenylyl cyclaseG proteinmelatonin receptormodeling |
| spellingShingle | Lap Hang Tse Suet Ting Cheung Seayoung Lee Yung Hou Wong Real-Time Determination of Intracellular cAMP Reveals Functional Coupling of G<sub>s</sub> Protein to the Melatonin MT<sub>1</sub> Receptor International Journal of Molecular Sciences adenylyl cyclase G protein melatonin receptor modeling |
| title | Real-Time Determination of Intracellular cAMP Reveals Functional Coupling of G<sub>s</sub> Protein to the Melatonin MT<sub>1</sub> Receptor |
| title_full | Real-Time Determination of Intracellular cAMP Reveals Functional Coupling of G<sub>s</sub> Protein to the Melatonin MT<sub>1</sub> Receptor |
| title_fullStr | Real-Time Determination of Intracellular cAMP Reveals Functional Coupling of G<sub>s</sub> Protein to the Melatonin MT<sub>1</sub> Receptor |
| title_full_unstemmed | Real-Time Determination of Intracellular cAMP Reveals Functional Coupling of G<sub>s</sub> Protein to the Melatonin MT<sub>1</sub> Receptor |
| title_short | Real-Time Determination of Intracellular cAMP Reveals Functional Coupling of G<sub>s</sub> Protein to the Melatonin MT<sub>1</sub> Receptor |
| title_sort | real time determination of intracellular camp reveals functional coupling of g sub s sub protein to the melatonin mt sub 1 sub receptor |
| topic | adenylyl cyclase G protein melatonin receptor modeling |
| url | https://www.mdpi.com/1422-0067/25/5/2919 |
| work_keys_str_mv | AT laphangtse realtimedeterminationofintracellularcamprevealsfunctionalcouplingofgsubssubproteintothemelatoninmtsub1subreceptor AT suettingcheung realtimedeterminationofintracellularcamprevealsfunctionalcouplingofgsubssubproteintothemelatoninmtsub1subreceptor AT seayounglee realtimedeterminationofintracellularcamprevealsfunctionalcouplingofgsubssubproteintothemelatoninmtsub1subreceptor AT yunghouwong realtimedeterminationofintracellularcamprevealsfunctionalcouplingofgsubssubproteintothemelatoninmtsub1subreceptor |