Mod 1 NR Flashcards
Nuclear hormone receptors
What are nuclear hormone receptors?
- Large family of TFs
- Many are ligand/hormone activated
- Diverse physiological roles
What are the physiological roles of NHR?
- Development/cellular regulation
- Reproduction
- Metabolism
- Inflammation
- Cancer
Endocrine receptors
High affinity, hormonal lipids
Estrogen, Androgens, Thyroid Hormone, Vitamin D, Retinoids
Adopted receptors
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
Orphan receptors
Unknown ligand, similar to other receptors but has no endogenous ligand
Nurr1, Nurr77, NOR1 etc
General transcription factors
Bind to DNA and allows recruitment of other transcriptional machinery - allows gene expression
NR structure
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
What determines the functional specificity?
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
Heat shock protein chaperones
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
Zinc finger DNA binding domain
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
Mode of binding of NR to RE
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)
DR 1-to-5 rule
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
Mouse trap model
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.
Basic model of NR ligand-dependent activation
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
Receptor function
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
Chromatin remodelling complexes
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
Chromatin tails
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.
Co-activators
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
Mutation of L residues - CoA
Impairs interaction, can be completely gone or greatly reduced
Co-repressors
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
CoR vs. CoA
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
Specific ____ Receptor Modulators
Estrogen/Androgen - breast/prostate CA
Can’t completely block out functions of ER and AR
Drugs that partially block
Tamoxifen
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
Raloxifen
Best SERM
krisAntagonist for bone
Decreases UCA and BRCA
Tamoxifen in tissues
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.
HER2
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
HER2 and tamoxifen
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.
