IB 139 Lec 6 Adrenaline/Sympathetic response

  1. What is the genomic and functional structure of the glucocorticoid receptor?
    • N-terminal - Exon 2
    • DNA binding domain
    • Hinge
    • Ligand binding domain
    • C-terminal
  2. What's unique about glucocorticoid receptors compared to other steroid receptors?
    • Different areas indicate different functions
    • HSP90 binding
    • Activation function
    • Dimerization
  3. 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
  4. 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
  5. 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
  6. What are the two ways to locate proteins?
    • Stain it with antibody
    • Tag it with fluorescence to see in real time
  7. 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
  8. 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
  9. If a protein is changing, why is it pretty long lasting?
    Transcription and translation
  10. 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.
  11. What is a promotor?
    • Regulatory element that sits near the start of protein
    • Inert, needs to be activated
  12. What are the 2 ways to study gene expression?
    • By analyzing expression of single genes
    • By analyzing the whole transcriptome
  13. What are examples of analyzing expression of single genes?
    • RT-PCR
    • In situ hybridization
  14. What are examples of analyzing the whole transcriptome?
    • Microarrays
    • RNAseq
  15. What is a transcriptome?
    The set of all RNA molecules, produced in one or a population of cells
  16. What does transcriptome analysis reveal?
    Widespread, selective and cell-specific gene regulation by GR
  17. What kind of transcription factor is GR?
    Zinc factor
  18. 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
  19. What are cis-DNA elements?
    Elements that are next to each other on the DNA
  20. What are examples of cis-DNA elements?
    • Glucocorticoid Response Elements
    • GRE and nGRES
  21. What does the "n" stand for in nGRE?
  22. 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
  23. What does the Hormone Response Element do?
    Able to bind a specific hormone receptor complex and regulate transcription
  24. How is the HRE generally written?
    • 5'AGAACAnnnTGTTCT3'
    • 3'TCTTGTnnnACAAGA5'
    • where n represents any nucleotide.
  25. What are some examples of other transcription factors that GR can interact with?
    • AP-1
    • NF-kb
    • Smad
    • STAT
  26. What are the 2 ways GR-induced gene expression is activated?
    • Basal transcription machinery
    • Via co-activators
  27. 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
  28. What are some examples of repressing gene expression by displacement of activating TF's?
    • Osteocalcin
    • ROMC
    • CRH
    • 5HT-1A
  29. What does the genetic organization of GR predict?
    Alternative splicing sites
  30. 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
  31. How much of the human genome may undergo alternative splicing?
    Up to 75%
  32. What is asthma?
    A disease in which inflammation of the airways causes airflow into and out of the lungs to be restricted
  33. 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
  34. How is chronic severe asthma (and allergy) treated?
    • Synthetic steroid prednisone
    • Suppresses inflammation
  35. What happens to asthma patients after chronic prolonged treatment with prednisone?
    • Become steroid-resistant
    • Do not respond to the prednisone treatment anymore
  36. What is the common hypothesis and treatment for asthma patients becoming steroid resistant?
    • Hypothesis: Adaptation
    • Treatment: Increase the steroid dose
  37. 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
  38. 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!
Card Set
IB 139 Lec 6 Adrenaline/Sympathetic response
IB 139 Lec 6 Adrenaline/Sympathetic response