Difference between revisions of "Part:BBa K4414000"

 
 
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<partinfo>BBa_K4414000 short</partinfo>
 
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As an intelligent cell that senses stress, the most critical step is to detect and respond to changes in stress hormones. We choose cortisol as the detection object.NR3C1 is a cortisol receptor gene, and we only truncated its LBD in response to changes in cortisol concentration.
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This part is the ligand-binding domain (LBD) of the human glucocorticoid receptor (GR).
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==Usage and Biology==
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The human glucocorticoid receptor is encoded by NR3C1 (Nuclear Receptor Subfamily 3, Group C, Member 1) gene. GR functions as a transcription factor that binds to glucocorticoid response elements in the promoters of glucocorticoid responsive genes to activate their transcription (Lu & Cidlowski, 2005). In the absence of glucocorticoids, GR binds to small molecular chaperone proteins in the cytoplasm and stays in the cytoplasm. Following glucocorticoid stimulation, it changes its conformation and dissociates from the chaperone protein, thereby exposing the nuclear localization sequence on the ligand-binding domain, which facilitates the entry of the GR into the nucleus. GR consists of an N-terminus binding domain (NTD), a DNA binding domain (DBD), and a ligand binding domain (LBD). As the major functional domain, the LBD domain is responsible for sensing glucocorticoid signals and translocating the protein into the nucleus (Monsalve et al., 2019). It also has a transactivating domain 2 (τ2) and an activation function domain 2 (AF2) which activates downstream gene expression (Weikum et al., 2017). We obtained this DNA by PCR amplification from human cDNA.This part can be used in level 1 biological laboratory.
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Figure1： Linear domain structure of glucocorticoid receptor (GR) (Weikum et al., 2017)
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===Usage and Biology===
 
  
 
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<span class='h3bb'>Sequence and Features</span>
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===Sequence and Features===
 
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===Functional characterization and result===
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We constructed a plasmid that linked LBD to the fluorescent protein EGFP to verify the function of LBD. Cells are treated with 500 and 1000 nM glucocorticoids 6 h after transfection of plasmids into HEK-293T cells. Cells without glucocorticoid treatment were used as control. The results showed that glucocorticoid-treated cells fluorescence was mainly present in the nucleus compared to non-treated control, suggesting that glucocorticoids can bind to LBD and enter the nucleus.
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Figure2: EGFP-labeled GR<sub>LBD</sub> localization in HEK-293T cells w/wo glucocorticoid stimulation. For wide-field microscopy, cells were transfected with pNC051 (P<sub>CMV</sub>-GR<sub>LBD</sub>-EGFP). For confocal images, cells were co-transfected with F-actin labeling pXQ100 (P<sub>CMV</sub>-lifeact-mcherry) and GR<sub>LBD</sub> labeling pNC051 (P<sub>CMV</sub>-GR<sub>LBD</sub>-EGFP). Photos were taken 48 h post-DMEM or glucocorticoid administration, scale bar: 100 µm for wide-field microscopy and 10 µm for confocal microscopy. Data are representative images of 3 independent experiments.
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===Reference===
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1.Lu, N. Z., & Cidlowski, J. A. (2005). Translational regulatory mechanisms generate N-terminal glucocorticoid receptor isoforms with unique transcriptional target genes. Molecular Cell, 18(3), 331–342. https://doi.org/10.1016/j.molcel.2005.03.025
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2.Monsalve, G. C., Yamamoto, K. R., & Ward, J. D. (2019). A New Tool for Inducible Gene Expression in Caenorhabditis elegans. Genetics, 211(2), 419–430. https://doi.org/10.1534/genetics.118.301705
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3.Weikum, E. R., Knuesel, M. T., Ortlund, E. A., & Yamamoto, K. R. (2017). Glucocorticoid receptor control of transcription: Precision and plasticity via allostery. Nature Reviews. Molecular Cell Biology, 18(3), 159–174. https://doi.org/10.1038/nrm.2016.152

Latest revision as of 03:44, 14 October 2022


Ligand-binding domain (LBD) of NR3C1

This part is the ligand-binding domain (LBD) of the human glucocorticoid receptor (GR).

Usage and Biology

The human glucocorticoid receptor is encoded by NR3C1 (Nuclear Receptor Subfamily 3, Group C, Member 1) gene. GR functions as a transcription factor that binds to glucocorticoid response elements in the promoters of glucocorticoid responsive genes to activate their transcription (Lu & Cidlowski, 2005). In the absence of glucocorticoids, GR binds to small molecular chaperone proteins in the cytoplasm and stays in the cytoplasm. Following glucocorticoid stimulation, it changes its conformation and dissociates from the chaperone protein, thereby exposing the nuclear localization sequence on the ligand-binding domain, which facilitates the entry of the GR into the nucleus. GR consists of an N-terminus binding domain (NTD), a DNA binding domain (DBD), and a ligand binding domain (LBD). As the major functional domain, the LBD domain is responsible for sensing glucocorticoid signals and translocating the protein into the nucleus (Monsalve et al., 2019). It also has a transactivating domain 2 (τ2) and an activation function domain 2 (AF2) which activates downstream gene expression (Weikum et al., 2017). We obtained this DNA by PCR amplification from human cDNA.This part can be used in level 1 biological laboratory.

Figure1： Linear domain structure of glucocorticoid receptor (GR) (Weikum et al., 2017)


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



Functional characterization and result

We constructed a plasmid that linked LBD to the fluorescent protein EGFP to verify the function of LBD. Cells are treated with 500 and 1000 nM glucocorticoids 6 h after transfection of plasmids into HEK-293T cells. Cells without glucocorticoid treatment were used as control. The results showed that glucocorticoid-treated cells fluorescence was mainly present in the nucleus compared to non-treated control, suggesting that glucocorticoids can bind to LBD and enter the nucleus.

Figure2: EGFP-labeled GRLBD localization in HEK-293T cells w/wo glucocorticoid stimulation. For wide-field microscopy, cells were transfected with pNC051 (PCMV-GRLBD-EGFP). For confocal images, cells were co-transfected with F-actin labeling pXQ100 (PCMV-lifeact-mcherry) and GRLBD labeling pNC051 (PCMV-GRLBD-EGFP). Photos were taken 48 h post-DMEM or glucocorticoid administration, scale bar: 100 µm for wide-field microscopy and 10 µm for confocal microscopy. Data are representative images of 3 independent experiments.


Reference

1.Lu, N. Z., & Cidlowski, J. A. (2005). Translational regulatory mechanisms generate N-terminal glucocorticoid receptor isoforms with unique transcriptional target genes. Molecular Cell, 18(3), 331–342. https://doi.org/10.1016/j.molcel.2005.03.025

2.Monsalve, G. C., Yamamoto, K. R., & Ward, J. D. (2019). A New Tool for Inducible Gene Expression in Caenorhabditis elegans. Genetics, 211(2), 419–430. https://doi.org/10.1534/genetics.118.301705

3.Weikum, E. R., Knuesel, M. T., Ortlund, E. A., & Yamamoto, K. R. (2017). Glucocorticoid receptor control of transcription: Precision and plasticity via allostery. Nature Reviews. Molecular Cell Biology, 18(3), 159–174. https://doi.org/10.1038/nrm.2016.152