Mod 1 NR

Question 1

What are nuclear hormone receptors?

Answer 1

• Large family of TFs
• Many are ligand/hormone activated
• Diverse physiological roles

Question 2

What are the physiological roles of NHR?

Answer 2

• Development/cellular regulation
• Reproduction
• Metabolism
• Inflammation
• Cancer

Question 3

Endocrine receptors

Answer 3

High affinity, hormonal lipids

Estrogen, Androgens, Thyroid Hormone, Vitamin D, Retinoids

Question 4

Adopted receptors

Answer 4

When an orphan receptor has found a ligand Low affinity, dietary lipids.

Requires a higher concentration of ligands to be activated

Fibrates (lipid lowering) - PPARa

Thiazolidinediones (insulin sensitizers) - PPARy

Question 5

Orphan receptors

Answer 5

Unknown ligand, similar to other receptors but has no endogenous ligand

Nurr1, Nurr77, NOR1 etc

Question 6

General transcription factors

Answer 6

Bind to DNA and allows recruitment of other transcriptional machinery - allows gene expression

Question 7

NR structure

Answer 7

Modular, has domains:

N-terminal regulatory domain: • contains activation function (AF-1, action independent of presence of ligand) – weak transcription, synergises with AF-2 in E-domain > increased up regulation of gene expression • A/B domain is highly variable in sequence between various NR – can be due to alternative splicing or use of alternative promoters

DNA-binding domain (DBD) • Highly conserved • two zinc fingers that bind to specific seq of DNA (HRE – hormone response elements, e.g. nurRE, NBRE)

Hinge region • Flexible, connects DBD with LBD • Influences intracellular trafficking and subcellular distribution

Ligand binding domain (LBD) • Moderately conserved (seq), highly conserved (struc) • Alpha helical sandwich fold, 3 anti-parallel alpha helices (sandwich filling) flanked by 2 alpha helices on side, 3 on the other (bread). • Ligand binding cavity = interior, below “filling” • Contributes to dimerization interface • Binds coactivator and corepressor proteins • Contains AF-2 – strictly ligand dependent, conserved among members of NR superfamily

C-terminal domain • High variable in seq

Question 8

What determines the functional specificity?

Answer 8

Nuclear localisation – “place of work”

How and when NRs get into the nucleus DNA binding o Target sequence specificity o Mode of binding

LBD

Determines ligand specificity o Protein interactions o Different ligands confer variable functionality

N-terminal domain Co-regulator recruitment o Different co-regulators = different transcriptional profile

Cellular context

Question 9

Heat shock protein chaperones

Answer 9

Group of proteins induced by heat shock, involved in folding and unfolding of other proteins, establishes proper protein conformation and prevents unwanted protein aggregation.

Stabilises and aids transport

Release of HSP by ligand is need to expose the nuclear localisation sequence - enables transport

Question 10

Zinc finger DNA binding domain

Answer 10

D box - forms DNA-induced DBD dimerisation interface (2nd zinc finger)

P box - specificity (establishes the selective base contacts in the major groove, Helix 1)

Recognition box - binds to RE *response element

T and A boxes - monomeric DNA binding

Question 11

Mode of binding of NR to RE

Answer 11

Monomer: RE, bind on their own (Nurr1, Nur77, NOR1)

Homodimer: Palindromic-RE (Steroid receptors)

Heterodimer: Direct repeats-RE (RXR, PPARs)

Heterodimer: Palindromic-RE (Nurr1, Nurr77, COUP-TF)

Heterodimer: Inverted palindromes-RE (VDR - vit d)

Question 12

DR 1-to-5 rule

Answer 12

Different NRs favour different spacer sizes

The length of spacer region determines the specificity of hormonal responses

• PPARy/RXR = DR1
• VDR = DR3 (vitamin d)
• TR = DR4 (thyroid hormone)
• RAR = DR2 & DR5 (retinoic acid)
• TR2 = DR1 to DR5

Question 13

Mouse trap model

Answer 13

AF-2 becomes transcriptional compentent. When a ligand binds, H10-H11 remain the same but H12 swings in and unleaches Ohm loop, which flips over underneath the H6 and carries the N-terminal part of H3. In this position, H12 seals as a “lid” on the ligand-binding cavity which further stabilises ligand binding by contributing additional ligand-protein interactions.

Transconformation of H12 and bending of H3 creates surfaces on apo- and holo-LBD.

Some antagonists force H12 into a 3rd postion, impairing co-activator binding.

Question 14

Basic model of NR ligand-dependent activation

Answer 14

Interaction with a CoR represses target gene activation, conformational change within LBD due to hormone presence boots off CoR, allowing rc to interact with CoA

Question 15

Receptor function

Answer 15

Overall size and shape of ligand binding pockets

Steroid - high affinity to a small no. of ligands, smaler volumes in bidning pockets, extensive polar side chains that can H-bond

Adopted orphans - diverse ligands, larger-volume pockets

Question 16

Chromatin remodelling complexes

Answer 16

Exposes DNA/NRs to target sites via alteration of Histone-DNA contacts

Can’t trigger transcription - lacks a ligand (represses txn via co-repressor complexes)

Post-translational histone modification: destabilises chromatin (acetylation)

Histone acetyl transferase (HAT) = acetylated chromatin, open, co-activators

Histone deacetylase (HDAC) = removes acetyl groups, closes chromatin, co-repressors

Question 17

Chromatin tails

Answer 17

Deacetylated = promotes very strong interaction between protein (positive charge) and DNA (negative charge). Transcription is repressed due to CoRs.

LIgand interaction with NR brings in CoAs. CoAs have HAT and this relieves neg-pos charge - neutralises it - allows DNA to wind away and promote transcription as is easily accessible. CoRs are also booted off.

Question 18

Co-activators

Answer 18

Very large proteins

Only LXLL helical motifs are responsible for NR interaction

X = any a.a., L = lucines

Interacts with H3, H4 region that sits below H12

Contain transactivation domains that recruit transcriptional components to drive trxn response

Either contain HAT or recruit

Interact with co-integrator proteins i.e. CBP/p300 - has HAT, NB for NR trxn. Also binds directly to NRs

Question 19

Mutation of L residues - CoA

Answer 19

Impairs interaction, can be completely gone or greatly reduced

Question 20

Co-repressors

Answer 20

Also v. large, interact via a very small region

LXX(I/H)IXXX(I/L) helical motif

Interact with NRs in absence of ligand/presence of antagonists

Motifs have to lock in w/ H3/4 region, requires H12 to be out to expose this

CoR proteins: NCoR and SMRT - recruits HDAC directly and indirectly to complex

Question 21

CoR vs. CoA

Answer 21

When AF2 domain (H12) is impinging - sitting in active conformation, it is in the way of CoR

Dimer: RXR and THR: NCoR can use two of the interaction domains to interact w/ dimer - which is why there are several interaction domains

When ligand changes position at H12, LXXLL motif of CoA (SRC1) can interact with LBD

Question 22

Specific ____ Receptor Modulators

Answer 22

Estrogen/Androgen - breast/prostate CA

Can’t completely block out functions of ER and AR

Drugs that partially block

Question 23

Tamoxifen

Answer 23

First SERM identified

Antagonist for ER in breast (estrogen drives tumour growth in BRCA - gets shut down)

Agonist in bone

Risks: THromboembolism, increased uterine CA risk

Question 24

Raloxifen

Answer 24

Best SERM

krisAntagonist for bone

Decreases UCA and BRCA

Question 25

Tamoxifen in tissues

Answer 25

Different tissues have different signalling pathways

Tamoxifen: Upregulate Gene A/B/C in bone, downreg. them in breast

Different partial modulator: Cruit CoA, express A/B, no C

Phosphorylated CoA: express A/B, AND D. Altered functional output, stability, localisation etc.

Question 26

HER2

Answer 26

Test for positive in BRCA

Breast tumour amplifies expression, cells = hyper-responsive to EGF (epidermal growth factor)

Not mutation - hyper-responsive

Drugs (antibodies) target receptor and its activation

Question 27

HER2 and tamoxifen

Answer 27

May implicate Tamoxifen pathway

HER2 signals to SRC3 (steroid receptor CoA-3) and tamoxifen recruits this CoA instead. Has agonist effect in breast tissue instead of antagonist.