L12 Nuclear Receptors

Question 1

Nuclear receptors location and types

Answer 1

Nuclear receptors are receptors located inside the cell. These receptors are found either in the cytoplasm (Type I) or the nucleus (Type II) of a cell.

Question 2

Main effect of stimulation of a nuclear receptor

Answer 2

Altered gene transcription

Question 3

Nuclear Receptor subfamiles

Answer 3

7 subfamiles
48 NRs

Question 4

Nuclear receptor binds to ligands (in or out of cell) and interact with what via recognition of response elements

Answer 4

Intracellularly
DNA

Question 5

2 xenobiotic receptors

Answer 5

• pregnane X receptor (PXR)
• constitutive androstane receptor (CAR)

Question 6

What do xenobiotics to the CYP expression

Answer 6

increase expression

Question 7

Orphan Receptors.

Answer 7

When some NRs do not have a known ligand at this point in time.

Question 8

RXR meaning (nuclear receptors)

Answer 8

Retinoid X Receptor

Question 9

Type 2: RXR heterodimer
Ligand, dimerisation,location and response element

Answer 9

L: fatty acids, retinoic acids, cholesterol
D: heterodimer(RXR + Class II NR).
Lo: Nucleus (main) but also cytoplasm.
RE: Direct repeats

Question 9

Type 1: Steroid hormone NR
Ligand, dimerisation,location and response element

Answer 9

L: Glucocorticoids and estrogen.
D: homodimer.
Lo: cytoplasm (main) but also nucleus.
RE: Inverted repeats

Question 10

RE for inverted repeats how many base pairs

Answer 10

one RE half-site spacer (bp=3 is common)

Question 11

RE for direct repeats how many base pairs

Answer 11

one RE half-site spacer (bp=0-5)

Question 12

Class I in the absence of ligands are located primarily

Answer 12

Cytoplasm

Question 13

Structure of a nuclear receptor - 4 domains

Answer 13

N-terminal (A/B)
DNA-Binding Domain (C)
Hinge Region (D)
Ligand-Binding Domain (E/F)

Question 14

N-terminal domain (A/B)

Answer 14

• least conserved - varies in length and amino acid sequence
• activation function 1 (AF1)
• binds co-regulators in a ligand-independent way to modify binding or regulatory capacity of receptor

Question 15

DBD (DNA binding domain, C)

Answer 15

• highly conserved - responsible for DNA recognition and binding
• two zinc fingers formed by cysteine-rich loops
  1. first zinc finger - recognise specific hormone response elements (HREs)
  2. second zinc finger - role in receptor dimerisation

Question 16

Hinge region (D)

Answer 16

• links the DBD and the LBD (ligand binding domain); highly flexible
• may contain a nuclear localisation signal (NLS) - role in receptor nuclear translocation
• NLS can overlap with the DNA binding domain
  [NLS will guide NR to translocate from the cytoplasm to the nucleus via the binding of a ligand. ]

Question 17

Why fluorescence of proteins

Answer 17

To monitor movement of receptor of interest

Question 18

LBD (ligand binding domain, E/F)

Answer 18

• fairly conserved
• a hydrophobic pocket formed by 12 α-helices
  o helix 12: important role in co-activator/co-repressor switching
• role in receptor dimerisation
• activation function 2 (AF2): binds co-regulators in a ligand-
  dependent manner
• post-translational modifications

Question 19

How an antagonist and agonist inhibits and activates AF2

Answer 19

Antag will bind to the LBD to sterically inhibit binding of AF2 and so there is no attachment of co-activator proteins.

Agonist bind to LBD to allow AF@ to bind which allows binding of co-activators which lets gene transcription proceed.

Question 20

(III) dimeric orphan NR (L,D,Lo,RE)

Answer 20

L: unknown
D: homo or hetero
Lo: nucleus
RE: direct repeat

Question 21

(IV) monomeric orphan NR (L,D,Lo,RE)

Answer 21

L: unknown
D: monomer
Lo: nucleus
RE: binds to one extended RE
half-site

Question 22

The HPA axis

Answer 22

• release of glucocorticoids (GCs; cortisol in human)
• negative feedback mechanisms

Question 23

Glucocorticoids (class 1)

Answer 23

• maintain homeostasis and respond to stress
• regulate metabolic and immune responses
• anti-inflammatory and immunosuppressive
• synthetic GCs to treat inflammatory and autoimmune diseases

Question 24

Glucocorticoids can treat inflammation and immune disease bc of

Answer 24

its ability to * maintain homeostasis and respond to stress * regulate metabolic and immune responses

Question 25

Glucocorticoid receptor (encoded by the NR3C1 gene)

Answer 25

* alternative splicing of exon 9 → GR(a) and GR(ß) * GR(y) contains an arginine insertion in the DBD * other splice variants

Question 26

NLS1 and NLS2 (Hinge region)

Answer 26

* NLS1 - overlaps with the end of DBD and the hinge region * NLS2 - overlaps with the LBD

Question 27

GR-mediated signaling + hsp90

Answer 27

* unliganded monomeric GR resides in the cytoplasm and forms a complex with HSP90 (heat shock protein 90) and other chaperone proteins * ligand binding→ GR dissociates from the complex→ expose nuclear localisation signals * GR is translocated into the nucleus

Question 28

increase or drecrease 1.transactivation: gene transcription 2.transrepression: gene transcription

Answer 28

1.↑ 2.↓

Question 29

GR-mediated regulation of gene transcription

Answer 29

GR dimer will interact with its own response element to regulate gene transcription. GR form homodimer will interact with own to **increase expression** of target gene and react with negative RE like a monomer to **inhibit** the transcription of target gene

Question 30

Direct (direct binding of GR to glucocorticoid response element [GRE])

Answer 30

* GR dimer - binds to GRE (GGAACAnnnTGTTCT) - ↑ gene transcription * bind to negative GRE (IR nGRE, CTCC(n)0-2GGAGA) - ↓ gene transcription

Question 31

activator protein 1 (AP1) and nuclear factor kB (NF-kB)

Answer 31

* transcription factors - ↑ gene transcription in response to stimuli, e.g., cytokines, growth factors * pro-inflammatory - important role in inflammatory diseases

Question 32

interaction between GR and AP-1/NF-kB → transrepression

Answer 32

* tethering - GR binds directly to DNA-bound AP-1 or NF-kB (TSF) * composite - GRE-bound GR interacts with neighbouring DNA-bound AP-1 or NF-kB

Question 33

Non-permissive RXR heterodimer

Answer 33

* e.g., thyroid hormone receptor or vitamin D receptor * heterodimer is activated by ligands of the partner NR while RXR is silenced *activated by RXR ligand attached to the partner RN not the RXR*

Question 34

Permissive RXR heterodimer

Answer 34

* e.g., peroxisome proliferator- activated receptor * heterodimer is activated by ligands of either NR, and synergistic effects when both NRs are activated *activated by retinoic acid or partner's ligand meaning that ligand can bind to either the partner or the RXR and will activate NR*

Question 35

PPAR(y) y=gamma

Answer 35

white and brown adipose tissue - adipogenesis, lipid metabolism and insulin sensitivity

Question 36

What type of NRs: Androgen, Oestrogen, glucocorticoid, progesterone

Answer 36

Type 1 Cyto, homo, translocate to nucleus, recruit co-activators, TSF and other proteins

Question 37

What type of NRs: ReinoidX, retinoic acid, thyroid hormone, peroxisome proliferator, pregnaneX

Answer 37

Type 2 (usually lipids) Nuclear, Hetero with RXR, complexed with co-reperssors that are displaces for co-activators

Question 38

PPAR(y) agonists (e.g., thiazolidinediones) are used clinically to treat **what**

Answer 38

type 2 diabetes

Question 39

PPAR(y) and insulin sensitisation

Answer 39

* regulate adipocyte differentiation and mature adipocyte function * lipid metabolism * glucose homeostasis o Glut4 ↑ o PI3K ↑ o CAP ↑ o IRS-1/IRS2 ↑

Question 40

4 Post-translational modifications of NRs

Answer 40

acetylation (A) phosphorylation (P) - change transcriptional activity SUMOylation (S; small ubiquitin related modifiers) ubiquitination (U) - facilitate degradation

Question 41

PXR and CAR as xenobiotic sensors

Answer 41

nuclear receptors and form RXR heterodimers * PXR - pregnane X receptor * CAR - constitutive androstane receptor ↑ CYP enzymes / Phase 2 enzymes / drug transporters

Question 42

phenobarbital-mediated CYP induction

Answer 42

* EGFR-mediated signaling phosphorylates CAR to inhibit its transcriptional activity * binding of phenobarbital to EGFR attenuates the signaling * CAR is monomerised and dephosphorylated * CAR undergoes nuclear localisation and forms a CAR/RXR heterodimer - ↑ target gene transcription