Part:BBa_K5267001
Mammalian MT1 melatonin receptor, Gi-coupled GPCR.
The mammalian MT1 melatonin receptor is a G protein-coupled receptor (GPCR). MT1 plays a crucial role in regulating circadian rhythms and sleep-wake cycles by responding to melatonin, a hormone produced by the pineal gland. MT1 has a seven-transmembrane domain structure characteristic of GPCRs. Structural studies of MT1 reveal unique features, such as a "lid-like" structure in the extracellular loop 2 (ECL2) that influences ligand binding and selectivity. Understanding the MT1 receptor is essential for projects involving the study of circadian biology, sleep regulation, and the pharmacological targeting of melatonin receptors.Upon melatonin-mediated activation of the melatonin receptor MT1, several pathways, such as the cAMP-PKA pathways and Ca2+ signaling pathways, are activated, thereby influencing gene expression related to cellular processes such as metabolism, growth, and apoptosis.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 629
Illegal BamHI site found at 809 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Profile
Name: MTNR1a
Base Pairs: 1050bp
Origin: Homo sapiens
Properties: A GPCR that responds to melatonin
Short description: MTNR1a
Full description: The part encodes a 7-transmembrane receptor, which responses to melatonin.
Usage and Biology
This part encodes MT1, one of the two high-affinity forms of the melatonin receptor, the primary hormone secreted by the pineal gland. This receptor is a G-protein coupled, 7-transmembrane receptor, a rhodopsin-like class A receptor responsible for melatonin's effects on mammalian circadian rhythm and reproductive alterations influenced by day length. The receptor is an integral membrane protein that is readily detectable and localized to two specific regions of the brain: the hypothalamic suprachiasmatic nucleus, which is involved in circadian rhythm regulation, and the hypophysial pars tuberalis, which may be responsible for the reproductive effects of melatonin.[1] Introducing this part into mammalian cells enables the cells to express MT1, allowing them to sense the stimulation of melatonin or melatonin receptor-related signals.
In the human body, melatonin (N-acetyl-5-methoxytryptamine) is a widespread neurohormone with roles in circadian rhythm regulation, antioxidative protection, and several other functions. It binds to the ligand-binding pocket of the melatonin receptor with high affinity.[2]The figure from Okamoto, H. H., Cecon, E., Nureki, O., Rivara, S., & Jockers, R. (2024) shows the overall structure of MT1 in both its activated and inactivated forms, and the position of the ligand-binding pocket of MT1, where melatonin binds to activate downstream gene pathways.[2]
In our project, this part enables mammalian cells to respond to drugs that can activate the melatonin receptor, laying the foundational basis for a cell-based screening platform and subsequent experiments for screening melatonin receptor agonists.
Figure 1 Overall structures of MT1 (A: inactive state [PDB ID: 6ME2], E: active state [PDB ID: 7DB6]). (B) Top view (left) and side view (right) of MT1 in an inactive state [PDB ID: 6ME2]. (C) Overall TM6 movement during receptor activation of MT1 (inactive state: [PDB ID: 6ME2] and active state: [PDB ID: 7DB6]). (D) Ligand binding site of crystal structures of MT1 (left top: [PDB ID: 6ME2], left bottom: [PDB ID: 6ME3], right top: [PDB ID: 6ME4], right bottom: [PDB ID: 6ME5]). (F) Ligand binding site of cryo‐EM structures of MT1 (left: [PDB ID: 7DB6], middle: [PDB ID: 7VGY], right: [PDB ID: 7VGZ]).[2]
[2]
Figure 2 (A) Overview of structural changes during activation of MT1 (inactive state: [PDB ID: 6ME2], active state: [PDB ID: 7DB6]). (B) Conformational changes from the ligand binding site to the PIF motif. (C) Conformational changes from the Na+ binding site to the DRY (NRY in MTRs) motif.[2]
The MTNR1a part is cloned after the CMV promotor and before the bGH polyA sequence in the frame of Sleeping Beauty transposable element. After transfection into HEK293 cells, the PCMV->MTNR1a->BGH polyA device could stably exist in the genome of HEK293 cells.
Functional Validation
Method 1
In order to validate the basic part MTNR1a, which can express MTNR1a receptor in mammalian cells, we designed a controlled cell experiment, according to the natural gene pathway in human SCN cells that the activated MTNR1a receptors could activate several gene pathways which all lead to the activation of CREB (cAMP response element binding protein)[2], activating the expression of gene with CRE (cAMP response element) promotors.
We prepared two dishes of HEK-293T cell lines, each with the same passage number, identical viability, and a cell count of 500,000. One dish served as the experimental group, and the other as the control group. The experimental group was co-transfected with pLeo694 plasmids, one carrying the construct PCMV -> MTNR1a -> bGH polyA and the other carrying the construct P4xCRE -> P _ min -> lgK -> Nluc -> bGH polyA, in a 100 ng to 50 ng ratio (the optimal transfection ratio validated by experiments). The control group was transfected only with 50 ng of the pLeo694 plasmid carrying the PCMV -> MTNR1a -> bGH_polyA construct. After transfection, both groups of cells were stimulated with 1 nM melatonin, and samples were collected at 24 and 48 hours to detect Nluc expression.
Result 1
The experimental results are as follows:
Figure 3 The HEK293 stable cell line co-transfected by plasmid A and B, or plasmid A and C at the transfection ratio of 50ng : 100ng, stimulated by 1nM melatonin, and Nluc expression is tested 48 hours later. The results are shown above.
plasmid A: plasmid carrying PCRE4->IgK->Nluc->bGH_polyA in the frame of Sleeping Beauty transposable element
plasmid B: plasmid carrying PCMV->MTNR1a->bGH_polyA in the frame of Sleeping Beauty transposable element
plasmid C: pcDNA3.1(+)
Method 2
We have also validated the part by detecting the change of Ca2+ concentration, for it has also been reported that the Ca2+ is up-regulated after the activation of MT1.
We used GCaMP, an ultra-sensitive fluorescent protein for imaging Ca2+[4], to detect the change of intracellular Ca2+ concentration. And used Thapsigargin to stimulate the cell as a positive control, a drug that is reported to cause endoplasmic reticulum stress, which will lead to the up-regulation of Ca2+ in the cytoplasm[5].
Result 2
The results are as follows:
Figure 4 Real-time Ca2+ fluorescence intensity of different groups of cell after the stimulation of melatonin or thapsigargin.
The yellow group represents the cell only transfected by plasmid carrying PCMV->GCaMP ( BBa_K3755007 ) and stimulated by 100 μM Melatonin, as the blank control.
The blue group represents the cell co-transfected by plasmid carrying PCMV->GCaMP ( BBa_K3755007 ) and plasmid carrying PCMV->MTNR1a, and stimulated by Melatonin, as the experimental group.
The red group represents the cell only transfected by plasmid carrying PCMV->GCaMP ( BBa_K3755007 ) and stimulated by thapsigargin, as the positive control.
The result indicates that the up-regulation of intracellular Ca2+ concentration downstream the MTNR1a activation is strong but temporary, which may explain the reason why MTNR1a - Ca2+ pathway did not work.
The results indicate that the part works well.
References
[1] N. database, "Gene [Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004 – [cited 2024 Sep 01]. Available from: https://www.ncbi.nlm.nih.gov/gene/," 2004.
[2] H. H. Okamoto, E. Cecon, O. Nureki, S. Rivara, and R. Jockers, “Melatonin receptor structure and signaling,” Journal of Pineal Research, vol. 76, no. 3, 2024.
[3] M. A. Ayoub, A. Levoye, P. Delagrange, and R. Jockers, “Preferential Formation of MT1/MT2 Melatonin Receptor Heterodimers with Distinct Ligand Interaction Properties Compared with MT2 Homodimers,” Molecular Pharmacology, vol. 66, no. 2, pp. 312-321, 2004.
[4] T.-W. Chen et al., “Ultrasensitive fluorescent proteins for imaging neuronal activity,” Nature, vol. 499, no. 7458, pp. 295–300, Jul. 2013, doi: 10.1038/nature12354.
[5] A. Abdullahi, M. Stanojcic, A. Parousis, D. Patsouris, and M. G. Jeschke, “Modeling Acute ER Stress in Vivo and in Vitro,” Shock, vol. 47, no. 4, pp. 506–513, Apr. 2017, doi: 10.1097/SHK.0000000000000759.
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