L07 – Mechanism of Action of Steroid Hormones Flashcards
How do steroid hormones mediate diverse effects on cell differentiation and body physiology?
Enters cytosolic/nuclear receptor –> conformational change –> turn on transcription in DNA
One example of cytosolic receptor and one example of nuclear receptor?
Cytosolic: Glucocorticoid
Nuclear: Estrogen
How do glucocorticoid activate transcription?
1) Cortisol bind Glucocorticoid receptor, kick out HSP (Heat-shock protein)
2) Conformational change of monomer to dimer
3) NLS (Nuclear Localization Signal) translocates dimer through nuclear pore from cytoplasm to nucleus
4) Bind to hormone response-element, TAD recruit co-activator/co-repressor (interacts with basal transcription complex at TATA box in promoter)
Describe the 3 domains of a hormone receptor (from N-terminal to C-terminal)
Transactivation Domain (TAD): recruit different proteins (e.g. co-activator/co-repressor)
DNA-binding domain (DBD): contain 2 C4 Zinc-finger motif (contains Nucleotide Recognition Signal) –> bind HRE
Ligand-binding domain (LBD): bind Hormone (active) or HSP (inactive)
What does “puffing” in polytene chromosome indicate?
Uncoiling of chromosomes
In an electron micrograph, what can you see during “puffing” of a chromosome?
B: Bands (85% DNA) - decondense then recondense in precise temporal sequence (5 puffs over 22 hours), contain newly synthesized RNA (labelled by 3H-uridine)
I: Interbands (15% DNA) - less condensed
What is the steroid hormone used to activate polytene chromosome “puffing”?
Ecdysone
What does the Ashburner hypothesis indicate?
Explains primary and delayed secondary “puffing” response
Primary: steroid hormone and receptor complex forms early puffs –> primary response protein
Delayed secondary: A primary response protein shuts off primary response, turns on secondary-response genes forming protein
What does 3H labelled hormone show when it binds to a steroid receptor?
3H labelled Steroid hormones shows movement from cytoplasm to nucleus
Outline the different stages of the discovery of steroid hormone receptor.
1950: Clever and Ashburner - ecdysone “puffing” experiment
- -> shows Ecdysone coordinates specific programs of gene expression
1970: Radiolabelled ligands to detect steroid hormone receptors and their association with high-affinity binding sites in chromatin
- -> Labels seen to move from cytoplasm to nucleus suggesting effect on gene expression
What are the different outcomes at different temperatures when 3H labelled dexamethasone binds to LYSATE containing glucocorticoid receptor?
4C:
- No binding to chromatin on filter
- Low mobility in ion-exchange column
37C:
- Binding to chromatin on filter
- Increased mobility in column
Shows steroid hormone receptors undergo transformation/conformational change upon hormone binding
What was the first gene identified as being glucocorticoid-responsive?
Viral gene (e.g. MMTV)
Where does glucocorticoid receptor bind in nucleus?
In the promoter region/HRE of responsive genes
What does a footprint analysis show in the promotor region of DNA?
Presence of Glucocorticoid receptor binds to 4 regions, thus 4 regions are missed out by cut/digestion by DNAse I –> shows up as footprints
How does deletion analysis of the promotor region of DNA show?
Shows binding region of Glucocorticoid receptor
How can a gene be made responsive to a steroid hormone?
HREs can be placed in front of other genes to make them hormone-responsive
How can steroid hormone receptors bind as dimers?
Due to their dyad (rotational) symmetry
Which of the following has a different Hormone Response Element (HRE) to the rest:
a) Glucocorticoid receptor (GR)
b) Aldosterone receptor (MR)
c) Androgen receptor (AR)
d) Progesterone receptor (PR)
e) Estrogen receptor (ER)
e) Estrogen receptor
What does Zinc bind to in a “Zinc finger domain” in Estrogen Receptors?
Binds to 4 cysteine molecules
How is Glucocorticoid receptor (GR) cDNA isolated by expression cloning?
Use antibodies raised against specific GR epitopes in PURIFIED GR (94kD) –> screen expression cDNA library for colony formed by GR-expressing bacteria –>
blot GR onto nitrocellulose filter
How are glucocorticoid receptor purified?
Use radiolabelled binding analogues –> achieve homogeneity with SDS-polyacrylamide gel electrophoresis
Where is NLS (Nuclear Localization Signal) located in a steroid hormone receptor?
Hinge region
How are “genomic” and “non-genomic” estrogen pathways different?
“Non-genomic” only requires ligand-binding domain of receptor and extranuclear localization
Doesn’t go inside nucleus
How do sex steroids/Bisphosphonates prevent apoptosis of osteoblasts?
Binds ER/AR:
1) PI3K/Akt pathway –> phosphorylate BAD protein or activate BCL-2
2) (Bisphosphonates bind Connexin 43) –> Src –> ERK –> Elk-1/CREB –> Transcription
3) Src/ ERK pathway –> BCL-2 (anti-apoptotic)
How does PTH prevent apoptosis of osteoblasts?
Binds PTHr, cAMP/PKA activation:
1) CREB –> Transcription
2) phosphorylate BAD protein
