What is the genomic and functional structure of the glucocorticoid receptor?
- N-terminal - Exon 2
- DNA binding domain
- Ligand binding domain
What's unique about glucocorticoid receptors compared to other steroid receptors?
- Different areas indicate different functions
- HSP90 binding
- Activation function
Describe glucocorticoid cellular signaling.
- Ligand (cortisol) normally sitting in the cytoplasm
- Comes in plasma
- Need to have a carrier protein b/c hydrophobic
- Crosses membrane b/c looks like a steroid
- Binds to GC receptor
- Changes conformation of protein
- Displaces chaperone protein HSP90
- Dimerization and Nuclear localization signal (NLS) now exposed
- Induces dimerization of GR's
- Dimer works as transcription factor
- Binds to DNA
What is the NLS?
- Nuclear localization signal
- Short sequence within protein, like a tag
- Sends protein to appropriate location in cell
- Sends through nuclear pore, into the nucleus
Why are the GR's not already in the nucleus?
- NLS signal is actually obscured/protected/covered/not seen regularly
- Hidden from view by chaperone protein HSP90
What are the two ways to locate proteins?
- Stain it with antibody
- Tag it with fluorescence to see in real time
What is this image showing us? (GR nuclear translocation)
- How much of the nucleus gets filled with GR's
- 2 minutes, filling
- 10 minutes, pretty concentrated in nucleus
- Lowers after that
Why does the GR nuclear localization lower after about 10 minutes?
- Diffuses out again
- Temporary response to stress
- Ligand interaction with GR is on and off
If a protein is changing, why is it pretty long lasting?
Transcription and translation
What is the downstream effects of GR activation?
- DNA binds to specific areas of protein in the promotor
- DNA -> RNAs -> Membrane Proteins/channels/pumps etc.
What is a promotor?
- Regulatory element that sits near the start of protein
- Inert, needs to be activated
What are the 2 ways to study gene expression?
- By analyzing expression of single genes
- By analyzing the whole transcriptome
What are examples of analyzing expression of single genes?
- In situ hybridization
What are examples of analyzing the whole transcriptome?
What is a transcriptome?
The set of all RNA molecules, produced in one or a population of cells
What does transcriptome analysis reveal?
Widespread, selective and cell-specific gene regulation by GR
What kind of transcription factor is GR?
What are the two ways in which GR genomic effects are mediated?
- GR can interact with cis-DNA elements
- GR can affect transcription via interactions with other transcription factors
What are cis-DNA elements?
Elements that are next to each other on the DNA
What are examples of cis-DNA elements?
- Glucocorticoid Response Elements
- GRE and nGRES
What does the "n" stand for in nGRE?
What is the Hormone Response Element?
- Short sequence of DNA within the promotor of a gene
- Sequence is most commonly a pair of inverted repeats, separated by three nucleotides
- Location where receptor binds as a dimer
What does the Hormone Response Element do?
Able to bind a specific hormone receptor complex and regulate transcription
How is the HRE generally written?
- where n represents any nucleotide.
What are some examples of other transcription factors that GR can interact with?
What are the 2 ways GR-induced gene expression is activated?
- Basal transcription machinery
- Via co-activators
What are the different ways GR-induced gene expression is repressed?
- Displacement of activating transcription factors
- Competitive interactions with activating TF's
- Inhibitory interactions with activating TF's
What are some examples of repressing gene expression by displacement of activating TF's?
What does the genetic organization of GR predict?
Alternative splicing sites
What is alternative splicing?
- One gene - many proteins
- Different parts of the DNA are spliced and turned into mRNA in different combinations
- Make different proteins
How much of the human genome may undergo alternative splicing?
Up to 75%
What is asthma?
A disease in which inflammation of the airways causes airflow into and out of the lungs to be restricted
What happens when an asthma attack occurs?
- Mucus production is increased
- Muscles of the bronchial tree become tight
- Lining of the air passage swells
- Reduces airflow and produces the characteristic wheezing sound
How is chronic severe asthma (and allergy) treated?
- Synthetic steroid prednisone
- Suppresses inflammation
What happens to asthma patients after chronic prolonged treatment with prednisone?
- Become steroid-resistant
- Do not respond to the prednisone treatment anymore
What is the common hypothesis and treatment for asthma patients becoming steroid resistant?
- Hypothesis: Adaptation
- Treatment: Increase the steroid dose
What really happens to asthma patients who become steroid-resistant after prolonged treatment with prednisone?
- mRNA from the lung epithelium of those patients have revealed high levels of GR-beta isoform
- GR-beta dimerizes with GR-alpha but doesn't bind to GREs and can't modulate transcription
- It was discovered that GR-beta antagonizes and competes with GR-alpha, hence blocking the GC effects on the target cells
What was the real conclusion and treatment for asthma patients who have become steroid-resistant?
- Conclusion: Those patients have a switch in alternative splicing in their lungs, and therefore become resistant to GC treatment
- Treatment: Stop steroids!