L07 – Mechanism of Action of Steroid Hormones

Question 1

How do steroid hormones mediate diverse effects on cell differentiation and body physiology?

Answer 1

Enters cytosolic/nuclear receptor –> conformational change –> turn on transcription in DNA

Question 2

One example of cytosolic receptor and one example of nuclear receptor?

Answer 2

Cytosolic: Glucocorticoid
Nuclear: Estrogen

Question 3

How do glucocorticoid activate transcription?

Answer 3

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)

Question 4

Describe the 3 domains of a hormone receptor (from N-terminal to C-terminal)

Answer 4

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)

Question 5

What does “puffing” in polytene chromosome indicate?

Answer 5

Uncoiling of chromosomes

Question 6

In an electron micrograph, what can you see during “puffing” of a chromosome?

Answer 6

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

Question 7

What is the steroid hormone used to activate polytene chromosome “puffing”?

Answer 7

Ecdysone

Question 8

What does the Ashburner hypothesis indicate?

Answer 8

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

Question 9

What does 3H labelled hormone show when it binds to a steroid receptor?

Answer 9

3H labelled Steroid hormones shows movement from cytoplasm to nucleus

Question 10

Outline the different stages of the discovery of steroid hormone receptor.

Answer 10

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

Question 11

What are the different outcomes at different temperatures when 3H labelled dexamethasone binds to LYSATE containing glucocorticoid receptor?

Answer 11

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

Question 12

What was the first gene identified as being glucocorticoid-responsive?

Answer 12

Viral gene (e.g. MMTV)

Question 13

Where does glucocorticoid receptor bind in nucleus?

Answer 13

In the promoter region/HRE of responsive genes

Question 14

What does a footprint analysis show in the promotor region of DNA?

Answer 14

Presence of Glucocorticoid receptor binds to 4 regions, thus 4 regions are missed out by cut/digestion by DNAse I –> shows up as footprints

Question 15

How does deletion analysis of the promotor region of DNA show?

Answer 15

Shows binding region of Glucocorticoid receptor

Question 16

How can a gene be made responsive to a steroid hormone?

Answer 16

HREs can be placed in front of other genes to make them hormone-responsive

Question 17

How can steroid hormone receptors bind as dimers?

Answer 17

Due to their dyad (rotational) symmetry

Question 18

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)

Answer 18

e) Estrogen receptor

Question 19

What does Zinc bind to in a “Zinc finger domain” in Estrogen Receptors?

Answer 19

Binds to 4 cysteine molecules

Question 20

How is Glucocorticoid receptor (GR) cDNA isolated by expression cloning?

Answer 20

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

Question 21

How are glucocorticoid receptor purified?

Answer 21

Use radiolabelled binding analogues –> achieve homogeneity with SDS-polyacrylamide gel electrophoresis

Question 22

Where is NLS (Nuclear Localization Signal) located in a steroid hormone receptor?

Answer 22

Hinge region

Question 23

How are “genomic” and “non-genomic” estrogen pathways different?

Answer 23

“Non-genomic” only requires ligand-binding domain of receptor and extranuclear localization

Doesn’t go inside nucleus

Question 24

How do sex steroids/Bisphosphonates prevent apoptosis of osteoblasts?

Answer 24

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)

Question 25

How does PTH prevent apoptosis of osteoblasts?

Answer 25

Binds PTHr, cAMP/PKA activation:

1) CREB –> Transcription
2) phosphorylate BAD protein