Difference between revisions of "Part:BBa K5267001"
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===Functional Validation=== | ===Functional Validation=== | ||
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. | ||
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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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Revision as of 14:42, 27 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.
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 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]