Difference between revisions of "Part:BBa K5267001"
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Figure: Overall structures of MT1 (A: inactive state [PDB ID: 6ME2], H: active state [PDB ID: 7DB6]), J: active state [PDB ID: 7VH0]). (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]). (E) 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]). (I) Ligand binding site of cryo‐EM structures of MT1 (left: [PDB ID: 7DB6], middle: [PDB ID: 7VGY], right: [PDB ID: 7VGZ]).[2] | Figure: Overall structures of MT1 (A: inactive state [PDB ID: 6ME2], H: active state [PDB ID: 7DB6]), J: active state [PDB ID: 7VH0]). (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]). (E) 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]). (I) Ligand binding site of cryo‐EM structures of MT1 (left: [PDB ID: 7DB6], middle: [PDB ID: 7VGY], right: [PDB ID: 7VGZ]).[2] | ||
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Figure: (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] | Figure: (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 PGK promotor and before the bGH polyA sequence in the frame of Sleeping Beauty transposable element. After transfection into HEK293 cells, the P_PGK->MTNR1a->BGH polyA device could stably exist in the genome of HEK293 cells. | The MTNR1a part is cloned after the PGK promotor and before the bGH polyA sequence in the frame of Sleeping Beauty transposable element. After transfection into HEK293 cells, the P_PGK->MTNR1a->BGH polyA device could stably exist in the genome of HEK293 cells. | ||
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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. | 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 P _ PGK -> MTNR1a -> bGH_polyA and the other carrying the construct P _4xCRE -> 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 P _ PGK -> 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. The experimental results are as follows: | 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 P _ PGK -> MTNR1a -> bGH_polyA and the other carrying the construct P _4xCRE -> 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 P _ PGK -> 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. The experimental results are as follows: | ||
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Figure: 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. | Figure: 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. | ||
Revision as of 16:50, 23 September 2024
Mammalian MT1 melatonin receptor, Gi-coupled GPCR.
The mammalian MT1 melatonin receptor is a G protein-coupled receptor (GPCR) primarily coupled to the Gi/o protein family. This receptor plays a crucial role in regulating circadian rhythms and sleep-wake cycles by responding to melatonin, a hormone produced by the pineal gland. The MT1 receptor has a seven-transmembrane domain structure characteristic of GPCRs and is involved in inhibiting adenylate cyclase activity, leading to decreased levels of cAMP. It also participates in other signaling pathways, including the activation of phospholipase C and the regulation of intracellular calcium levels. 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. This part is essential for projects involving the study of circadian biology, sleep regulation, and the pharmacological targeting of melatonin receptors.
Usage and Biology
This part of gene encodes one of two high affinity forms of a receptor for melatonin, the primary hormone secreted by the pineal gland. This receptor is a G-protein coupled, 7-transmembrane receptor, a rhodopsin-like class A receptor that is responsible for melatonin effects on mammalian circadian rhythm and reproductive alterations affected by day length. The receptor is an integral membrane protein that is resadily detectable and localized to two specific regions of the brain. The hypothalamic suprachiasmatic nucleus appears to be involved in circadian rhythm while the hypophysial pars tuberalis may be responsible for the reproductive effects of melatonin.[1] 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 melatonin receptor with high affinity in the human body.[2] The figure from Okamoto, H. H., Cecon, E., Nureki, O., Rivara, S., & Jockers, R. (2024) shows the overall structure of MT1, both activated structure and inactivated structure, and the position of the ligand binding pocket of MT1, where allows melatonin binds to it and activate of downstream gene pathways.[2]
[2] Figure: (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 PGK promotor and before the bGH polyA sequence in the frame of Sleeping Beauty transposable element. After transfection into HEK293 cells, the P_PGK->MTNR1a->BGH polyA device could stably exist in the genome of HEK293 cells.
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 P _ PGK -> MTNR1a -> bGH_polyA and the other carrying the construct P _4xCRE -> 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 P _ PGK -> 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. The experimental results are as follows:
Figure: 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 P_PGK->MTNR1a->bGH_polyA in the frame of Sleeping Beauty transposable element
plasmid C: pcDNA3.1(+)
The results indicate that the part works well.
[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.
Figure: Overall structures of MT1 (A: inactive state [PDB ID: 6ME2], H: active state [PDB ID: 7DB6]), J: active state [PDB ID: 7VH0]). (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]). (E) 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]). (I) Ligand binding site of cryo‐EM structures of MT1 (left: [PDB ID: 7DB6], middle: [PDB ID: 7VGY], right: [PDB ID: 7VGZ]).[2]
Functional Validation
References
Sequence and Features
Illegal BamHI site found at 809