Difference between revisions of "Part:BBa K4414000"
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Figure2: LBD can enter the nucleus in response to the stimulation of glucocorticoid
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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). Photos were taken 48 h post-DMEM or glucocorticoid administration, scale bar: 100 µm for wide-field microscopy.
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For validation of LBD transcriptional activation capabilities, please refer to our registered composite element ([[Part:BBa_K4414024]]), which we used tetR to fill the DBD section and found that LBD can activate the expression of downstream genes.
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Revision as of 18:35, 11 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).

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). Photos were taken 48 h post-DMEM or glucocorticoid administration, scale bar: 100 µm for wide-field microscopy.


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