Nuclear Receptors PDF

PHAR2210 Foundations of Pharmacology Lecture 12 Nuclear receptors Dr Ricky Chen Chapter 3, Rang & Dale's Pharmacology (Tenth Edition, 2023)  Learning outcomes After completing this lecture, you should be able to describe four classes of nuclear receptors in ter...

PHAR2210 Foundations of Pharmacology Lecture 12 Nuclear receptors Dr Ricky Chen Chapter 3, Rang & Dale's Pharmacology (Tenth Edition, 2023)  Learning outcomes After completing this lecture, you should be able to describe four classes of nuclear receptors in terms of ligand, dimerisation, receptor location, and response element recognition describe the structure, characteristics, and functions of each domain of a nuclear receptor and the use of fluorescent proteins as reporters of receptor localisation describe the structure of the glucocorticoid receptor (GR), GR-mediated signaling, and GR- mediated regulation of gene transcription describe the structure, dimerisation, and actions of the peroxisome proliferator-activated receptor (PPAR) and the clinical use of PPARg agonists list four types of post translational modification of GRa and PPARg describe the molecular mechanism of phenobarbital-mediated CYP induction and recognise PXR and CAR as xenobiotic sensors Core concepts of pharmacology pharmacodynamics pharmacokinetics Drug-target Drug target Steady-state Drug absorption Drug bioavailability interaction concentration Mechanism of Structure-activity Drug distribution Volume of distribution drug action relationship Zero- and first- order kinetics Drug metabolism Drug clearance Affinity Drug selectivity Drug elimination Drug elimination half-life Potency Efficacy DRUG Dose/concentration- Drug tolerance response relationship Therapeutic index Adverse drug reaction Individual variation Adapted from Guilding et al. (2023) Defining and Drug interaction in drug response unpacking the core concepts of pharmacology: A global initiative. British Journal of Pharmacology, 1-18. Patient outcomes https://doi.org/10.1111/bph.16222  Discoveries in nuclear receptors classification of nuclear receptors structure of a nuclear receptor response elements retinoid X receptor (RXR) and RXR heterodimers xenobiotic receptors cofactors Evans and Mangelsdorf (2014)  Nuclear receptors ligand-activated transcription factors seven subfamilies - 48 nuclear receptors (NRs) bind a ligand intracellularly and interact with DNA directly via the recognition of response elements xenobiotic receptors pregnane X receptor (PXR) constitutive androstane receptor (CAR) xenobiotics → CYP expression ↑ Imai et al. (2013)  Classification of nuclear receptors (I) steroid hormone NR (II) RXR heterodimer e.g., glucocorticoid, e.g., fatty acids, retinoic ligand estrogen acids, cholesterol receptor heterodimer homodimer dimerisation (RXR + Class II NR) receptor cytoplasm (common) or nucleus (common) or location nucleus cytoplasm response inverted repeat (IR) direct repeat (DR) element (RE) inverted repeat AGGTCA TGACCT direct repeat Weikum et al. (2018) AGGTCA TGACCT AGGTCA nnn TGACCT AGGTCA n AGGTCA one RE half-site spacer (bp=3 is common) one RE half-site spacer (bp=0-5)  Classification of nuclear receptors (III) dimeric orphan NR (IV) monomeric orphan NR unknown; unknown; ligand receptor displays receptor displays constitutive activity constitutive activity receptor homo- or hetero-dimer monomer dimerisation receptor nucleus nucleus location response binds to one extended RE direct repeat element (RE) half-site inverted repeat AGGTCA TGACCT direct repeat Weikum et al. (2018) AGGTCA TGACCT AGGTCA nnn TGACCT AGGTCA n AGGTCA one RE half-site spacer (bp=3 is common) one RE half-site spacer (bp=0-5)  Structure of a nuclear receptor N-terminal domain (A/B) least conserved - varies in length and amino acid sequence activation function 1 (AF1) o binds co-regulators in a ligand-independent manner post-translational modifications Imai et al. (2013) glucocorticoid receptor (Oakley and Cidlowski, 2013)  Structure of a nuclear receptor DBD (DNA binding domain, C) highly conserved - responsible for DNA recognition and binding two zinc fingers formed by cysteine-rich loops o first zinc finger - recognise specific hormone response elements (HREs) o second zinc finger - role in receptor dimerisation Nicolaides et al. (2010)  Structure of a nuclear receptor hinge region (D) links the DBD and the LBD (ligand binding domain); highly flexible may contain a nuclear localisation signal (NLS) - role in receptor nuclear translocation o NLS can overlap with the DNA binding domain 5α-dihydrotestosterone stimulation induced time-dependent nuclear translocation of GFP-AR (androgen receptor) expressed in three different cell lines (A: PC3, B: HeLa, C: COS1) (Tyagi et al., 2000)  Structure of a nuclear receptor LBD (ligand binding domain, E/F) 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 LBD-antagonist-co-repressor complex (top left) LBD-agonist-co-activator complex (bottom right) ** note the positioning of helix 12 Schoch et al. (2010)  The HPA axis, glucocorticoids, and receptors The HPA axis release of glucocorticoids (GCs; cortisol in human) negative feedback mechanisms Glucocorticoids maintain homeostasis and respond to stress regulate metabolic and immune responses anti-inflammatory and immunosuppressive o synthetic GCs to treat inflammatory and autoimmune diseases Glucocorticoid receptor (encoded by the NR3C1 gene) alternative splicing of exon 9 → GRa and GRb GRg contains an arginine insertion in the DBD other splice variants CRH, corticotropin-releasing hormone; ACTH, adrenocorticotropic hormone [image from Oakley and Cidlowski (2013)]  The glucocorticoid receptor N-terminal domain (A/B) - multiple post translational modification sites DNA binding domain (C) hinge region (D) o NLS1 - overlaps with the end of DBD and the hinge region o NLS2 - overlaps with the LBD ligand binding domain (E/F) (Oakley and Cidlowski, 2013)  GR-mediated signaling 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 non-genomic kinases transactivation ↑ gene transcription genomic transrepression ↓ gene transcription Oakley and Cidlowski (2013)  GR-mediated regulation of gene transcription Oakley and Cidlowski (2013) Direct (direct binding of GR to glucocorticoid response element [GRE]) GR dimer - binds to GRE (GGAACAnnnTGTTCT) - ↑ gene transcription bind to negative GRE (IR nGRE, CTCC(n)0-2GGAGA) - ↓ gene transcription  GR-mediated regulation of gene transcription activator protein 1 (AP1) and nuclear factor B (NF-B) transcription factors - ↑ gene transcription in response to stimuli, e.g., cytokines, growth factors pro-inflammatory - important role in inflammatory diseases interaction between GR and AP-1/NF-B → transrepression tethering - GR binds directly to DNA-bound AP-1 or NF-B composite - GRE-bound GR interacts with neighbouring DNA-bound AP-1 or NF-B AP-1 (dimer of c-Fox and c-Jun); nuclear factor B (p50 and p65 subunits) Oakley and Cidlowski (2013)  Class II NR - RXR heterodimers Non-permissive RXR heterodimer co-repressor e.g., thyroid hormone receptor or NR RXR vitamin D receptor heterodimer is activated by RE gene co-repressor co-activator ligands of the partner NR L L while RXR is silenced RE gene RE gene Permissive RXR heterodimer e.g., peroxisome proliferator- co-activator L activated receptor heterodimer is activated by co-repressor RE gene co-activator ligands of either NR, and L L NR RXR co-activator synergistic effects when both RE gene L NRs are activated RE gene RE gene  Peroxisome proliferator-activated receptor PPARa predominantly in the liver, heart and brown adipose tissue - fatty acid oxidation PPARb/d ubiquitously expressed - fatty acid oxidation PPARg white and brown adipose tissue - adipogenesis, lipid metabolism and insulin sensitivity Poulsen et al. (2012) PPARg and insulin sensitisation adipocyte differentiation and mature adipocyte function lipid metabolism glucose homeostasis o Glut4 ↑ o PI3K ↑ o CAP ↑ o IRS-1/IRS2 ↑ PPARg agonists (e.g., thiazolidinediones) are used clinically to treat type 2 diabetes Ahmadian et al. (2013)  Post-translational modifications of NRs acetylation (A) phosphorylation (P) - change transcriptional activity SUMOylation (S; small ubiquitin-related modifiers) ubiquitination (U) - facilitate degradation Oakley and Cidlowski (2013) Ahmadian et al. (2013)  PXR and CAR as xenobiotic sensors nuclear receptors and form RXR heterodimers PXR - pregnane X receptor CAR - constitutive androstane receptor ↑ CYP enzymes / Phase 2 enzymes / drug transporters Color Atlas of Pharmacology (2005) phenobarbital-mediated CYP induction 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 Negishi et al. (2020)