Nuclear (Hormone) Receptors

Question 1

Why is PPAR-alpha targeted therapeutically?

Answer 1

It is activated by drugs to treat human disorders of lipid metabolism

Question 2

How can FAS-dependent activation of PPAR-alpha be used to find a ligand for PPAR-alpha?

Answer 2

The ligand probably produced by FAS. Binding of the ligand increased by increased FAS activity. Ligand displaced by PPAR-alpha agonist. To purify: mouse injected with AdFLAG-virus, liver harvested, hypotonic lysis and extract nuclear fraction, incubate with alpha-FLAG Ab, elute with 3 x FLAG peptide, mass spec

Question 3

How do you find an endogenous ligand for a NR?

Answer 3

Exploit fatty acid synthase (FAS)-dependent activation of PPAR-alpha

Question 4

What are nuclear receptors?

Answer 4

Ligand-modulated transcription factors that bind directly to DNA

Question 5

Give 4 components of a transcriptional regulatory complex

Answer 5

1. transcription factors
2. chromatin remodelling factors
3. co-activators and co-repressors
4. basal transcription machinery

Question 6

How do chromatin remodelling factors generally work?

Answer 6

Generally through PTMs of histones/ remodeling (bending) DNA

Question 7

Nuclear receptors bind to specific sequences in DNA. What are these called?

Answer 7

Nuclear receptor response elements

Question 8

The activity of gene regulatory proteins is modulated in several ways in eukaryotic cells. How are nuclear receptors primarily modulated?

Answer 8

1. Ligand binding

2. Regulated nuclear entry

Question 9

What are histones?

Answer 9

Small basic DNA packaging proteins associated with DNA

Question 10

How does acetylation of lysine residues in histone tails affect transcription?

Answer 10

Increases transcription of that gene

Question 11

There are approximately 40 NRs in total. Give a few examples of NRs that fall into:

1) Class I
2) Class II
3) Class III

Answer 11

1) Thyroid hormone receptors, Retinoic acid receptors, PPARs, Vitamin D receptors
2) RXR (retinoid X receptor)
3) Glucocorticoid receptor, Androgen receptor, Progesterone receptor, Estrogen receptor

Question 12

What type of receptors are NRs?

Answer 12

Intracellular receptors for HYDROPHOBIC signal molecules (e.g. steroid/ thyroid hormones and retinoic acid receptor).
The receptor-ligand complex acts as a TF in the nucleus

Question 13

What sort of ligands do NRs bind?

Answer 13

Hydrophobic

Question 14

How does the Class II NR, Retinoid X Receptor (RXR) generally function?

Answer 14

As a heterodimer with other NRs, e.g. Class I NRs (TR, RAR, PPAR, VDR)

Question 15

What are the 5 functional domains within a NR? (from N-terminal to C-terminal)

Answer 15

1. AF-1 (ligand-INDEPENDENT transactivation domain)
2. DBD (recognises specific DNA sequences)
3. variable linker region
4. LBD
5. AF-2 (ligand-DEPENDENT transcriptional activation domain in C-terminal portion of LBD)

Question 16

What is the function of the AF-1 domain in NRs?

Answer 16

It is a ligand-INDEPENDENT transactivation domain

Question 17

What is the function of the AF-2 domain at the C-terminal end of NRs?

Answer 17

It is a ligand-DEPENDENT transcriptional activation domain

Question 18

What are the three key domains in nuclear receptor co-activators such as CBPs?

Answer 18

1. RID (NR-interacting domain): LxxLL
2. KIX (CREB-binding domain)
3. HAT

Question 19

What are the three key domains of a nuclear receptor co-repressor such as NCoR or SMRT?

Answer 19

1. RD1 (recruits HDACs indirectly via mSin3)
2. RD2/3 (directly recruits HDACs)
3. RID (receptor-interacting domain: LxxI/H IxxxI/L)

Question 20

Which domain is found in both co-activators such as CBP/p300 and co-repressors such as NCoR and SMRT?

Answer 20

RID - the NR interacting domain

Question 21

Which co-repressor interacts with retinoic and thyroid hormone receptors?

Answer 21

SMRT

Question 22

What type of molecule are endogenous nuclear receptor ligands?

Answer 22

Small lipophilic molecules

Question 23

How do Class III NRs (eg glucocorticoid receptor, androgen receptor, progesterone receptor, estrogen receptor) work?

Answer 23

1) Steroid hormone permeates plasma membrane and binds receptor (which is bound to HSP complex)
2) Nuclear receptor undergoes LIGAND-DEPENDENT RELEASE from cytoplasmic sequesteration
3) Liganded receptor DIMERISES & TRANSLOCATES to the nucleus
4) Receptor binds to DNA and recruits co-activators to enhance transcription of specific target genes

Question 24

How do Class I NRs (e.g. thyroid receptor, retinoic acid receptor, PPAR and vitamin D receptor) work?

Answer 24

1. Receptor dimer (typically heterodimer with RXR) is stably bound to DNA in an inactive state (often bound to corepressors)
2. Lipophilic ligand permeates the plasma membrane and nucleus OR ligand precursor is converted intracellularly
3. Ligand binds to receptor, allowing recruitment of co-activators and transcription of specific target genes

Question 25

What is the likely signalling mechanism for ‘orphan’ nuclear receptors?

Answer 25

1. Receptor monomer is stably bound to DNA in an inactive state.
2. Ligand permeates the plasma membrane and nucleus OR other PTM of receptor results in its activation
3. Receptor recruits co-activators and transcription of specific target genes

Question 26

Name 2 diseases that are treated by targeting the Retinoid X Receptor

Answer 26

1. Dermatoses

2. Cancer (as chemotherapy)

Question 27

Name two PPAR agonists that are used as pharmacotherapeutics. What diseases do each of them treat?

Answer 27

1) Fibrates - PPAR-alpha agonists - treat Dyslipidemias

2) Thizolidenediones - PPAR-gamma agonists - treat type II diabetes

Question 28

Which NR do thiazolidenediones (TZDs) target? How are they used to treat Type II diabetes?

Answer 28

PPAR-gamma (agonists). They restore insulin sensitivity

Question 29

Which NRs do Fibrates target? How do they treat dyslipidemias?

Answer 29

PPAR-alpha (agonists). They lower TGCs and VLDL and increase HDL

Question 30

How can the sex hormone receptors AR and ER be targeted? What is this used for?

Answer 30

By estradiol (ER) and testosterone (AR) for hormone replacement and contraception

Question 31

How can NR activity be measured?

Answer 31

1) quantifying changes in the levels of expression of target genes
2) utilising promoter-reporter assays

Question 32

Give an example of a promoter-reporter assay that can be used to measure nuclear receptor activity

Answer 32

Luciferase: light signal = luciferase expression = promoter activity = nuclear receptor binding