nuclear receptor 2024-2.pdf

Lecture 5:-Nuclear receptors Super-family of nuclear receptors 2E Table I. Nuclear Receptor Superfamily Family 0B 1A 1B 1C 1D 1F 1H 1I 2A 2B 2C 2E 2F Common name Dosage-sensitive sex reversal-adrenal hypoplasia congenital critical region on the X chromosome, Gene 1 Short heterodimeric partner Thyroi...

Lecture 5:-Nuclear receptors Super-family of nuclear receptors 2E Table I. Nuclear Receptor Superfamily Family 0B 1A 1B 1C 1D 1F 1H 1I 2A 2B 2C 2E 2F Common name Dosage-sensitive sex reversal-adrenal hypoplasia congenital critical region on the X chromosome, Gene 1 Short heterodimeric partner Thyroid hormone receptor-α Thyroid hormone receptor-β Retinoic acid receptor-α Retinoic acid receptor-β Retinoic acid receptor-γ Peroxisome proliferator-activated receptor-α Peroxisome proliferator-activated receptor-β Peroxisome proliferator-activated receptor-γ Reverse-Erb-α Reverse-Erb-β Retinoic acid-related orphan-α Retinoic acid-related orphan-β Retinoic acid-related orphan-γ Farnesoid X receptor Farnesoid X receptor-β Liver X receptor-α Liver X receptor-β Vitamin D receptor Pregnane X receptor Constitutive androstane receptor Hepatocyte nuclear Factor-4-α Hepatocyte nuclear Factor-4-γ Retinoid X receptor-α Retinoid X receptor-β Retinoid X receptor-γ Testicular Receptor 2 Testicular Receptor 4 Tailless homolog orphan receptor Photoreceptor-cell-specific nuclear receptor Chicken ovalbumin upstream Abbreviation Gene name DAX1 NR0B1 Orphan SHP TRα TRβ RARα RARβ RARγ PPARα PPARβ PPARγ REV-ERBα REV-ERBβ RORα RORβ RORγ FXRα FXRβ LXRα LXRβ VDR PXR NR0B2 THRA THRB RARA RARB RARG PPARA PPARD PPARG NR1D1 NR1D2 RORA RORB RORC NR1H4 NR1H5P NR1H3 NR1H2 VDR NR1I2 NR1I3 HNF4A HNF4G RXRA RXRB RXRG NR2C1 NR2C2 NR2E1 NR2E3 NR2F1 Orphan Thyroid hormones Thyroid hormones Retinoic acids Retinoic acids Retinoic acids Fatty acids Fatty acids Fatty acids Heme Heme Sterols Sterols Sterols Bile Acids Orphan Oxysterols Oxysterols 1α,25-dihydroxyvitamin D3 Endobiotics and xenobiotics Xenobiotics Fatty acids Fatty acids 9-Cis retinoic acid 9-Cis retinoic acid 9-Cis retinoic acid Orphan Orphan Orphan Orphan Orphan HNF4α HNF4γ RXRα RXRβ RXRγ TR2 TR4 TLX PNR COUP-TFα Ligand 2F 3A 3B 3C 4A 5A 6A Testicular Receptor 4 Tailless homolog orphan receptor Photoreceptor-cell-specific nuclear receptor Chicken ovalbumin upstream promoter-transcription factor α Chicken ovalbumin upstream promoter-transcription factor β Chicken ovalbumin upstream promoter-transcription factor γ Estrogen receptor-α Estrogen receptor-β Estrogen-related receptor-α Estrogen-related receptor-β Estrogen-related receptor-γ Androgen receptor Glucocorticoid receptor Mineralocorticoid receptor Progesterone receptor Nerve growth Factor 1B Nurr-related Factor 1 Neuron-derived orphan Receptor 1 Steroidogenic Factor 1 Liver receptor Homolog-1 Germ cell nuclear factor TR4 TLX PNR COUP-TFα NR2C2 NR2E1 NR2E3 NR2F1 Orphan Orphan Orphan Orphan COUP-TFβ NR2F2 Orphan COUP-TFγ NR2F6 Orphan ERα ERβ ERRα ERRβ ERRγ AR GR MR ESR1 ESR2 ESRRA ESRRB ESRRG AR NR3C1 NR3C2 PR NGF1-B NURR1 NOR-1 SF-1 LRH-1 GCNF PGR NR4A1 NR4A2 NR4A3 NR5A1 NR5A2 NR6A1 Estrogens Estrogens Orphan Orphan Orphan Androgens Glucocorticoids Mineralocorticoids and glucocorticoids Progesterone Orphan Unsaturated fatty acids Orphan Phospholipids Phospholipids Orphan Modular Structure of Nuclear Receptors Like most TFs, nuclear receptors have a modular structure. They all contain highly conserved DNA-binding domains, consisting of C4 zinc finger motifs, located near the middle of the polypeptide chain. The second subdomain helix makes non-specific contacts with the DNA backbone. The peptide loop in DNA binding Dimer formation region has the least sequenc between nuclear receptors. Li Contributes to DNA binding specificity Cysteines Bind to zinc NR DNA binding domains. (A) Cartoon representation of NR DBDs indicating important motifs. This Figure domain2.contains subdomains, each one zinc finger. TheDBDs first subdomain residues motif NR DNAtwo binding domains. (A) containing Cartoon representation of NR indicating important interact with the DNA grooveone to zinc makefinger. base- The specific interactionsresidues on genomic response subdomains, eachmajor containing first subdomain interact with the DNA majo elements. The second subdomain participates in DBD dimerization and makes non- specific contacts specific interactions on genomic response elements. The second subdomain participates in DBD dime with the DNA backbone. specific contacts with the DNA backbone. Some NRs, like LRH-1 and GCNF, also contain C-terminal e base-specific contacts with the minor that groove. (B)base-specific Cartoon representation of folded GR DBD highlightin NR contain C-terminal extensions (CTEs) make contacts with the minor groove. (PDB: 3FYL). Zinc atoms are represented as spheres.. : Steroid hormone receptors Characteristic features of steroid receptors – binds palindromic HSE (spacer 3 nt) 2x AGAACA - GR, MR, PR, AR 2x AGGTCA – ER – binds as homodimers—Inverted repeats ER binding ERE In addition to Subdomain 1, the CTEs also contribute to specificity of binding of different Steroid hormone NRs SD 1 interacts with specific base sequences. SD2 interaction Non-specifically with phosphate backbone of DNA to increase stability LIGANDS: Steroid hormones share a common basic cholesterol structure. They all pass through the cell membrane to bind their cognate receptors in the cytoplasm. Homodimeric-NRs-Steroid Receptors Receptors is located the cytoplasm and held in an inactive state by heat shock proteins (HSP). The receptor changes shape after binding hormone, sheds the HSP and migrates into nucleus. The receptor dimerizes, binds DNA as a homodimer and then bind to coactivator proteins Induced nuclear transport Nuclear translocation of GR – Time-dependent nuclear translocation of GFP-GR in COS-1 cells in the presence of dexamethasone (DEX). Heterodimeric- RXR NRs are always located in the nucleus. Under basal state, they are bound to DNA and are not active because they are linked to a corepressor that inhibits transcription. Binding of ligand results in release of corepressor and binding of coactivator which activates transcription These models suggested that LBDs have several conformations: Not bound to Ligand, bound to coactivator and bound to corepressor How do Nuclear receptors activate Transcription or Repress transcription CHROMATIN: Higher Order DNA Compaction Within the Nucleus Histone Core: 2 copies each of H2A, H2B, H3, H4 (all basic proteinsrich in Arg, Lys Dec. 2001 residues) Histone Acetylation/Deacetylation Histone N terminal “tails “have positive amino acids e.g. Lysine that can interact with negative charge on phosphate backbone of DNA and make nucleosome bind tightly Dec. 2001 Acetylation of lysines “neutralizes” positive charge and this decrease binding of nucleosomes to DNA Multiple steps for activation CBP Nuclear receptor Binding of Coactivator increases transcription by recruiting Histone acetylase enzymes Binding of Corepressors decreases transcription by recruiting Histone deacetylase enzymes Modular Structure of Nuclear Receptors The ligand-binding domain is located in the C-terminal sequence region. This region serves as a hormonedependent activation domain in receptors, and in other receptors, as a repression domain in the absence of ligand Figure 1. Modular domain structure of NRs. (A) Basic modular domain stru contains the Activation Function 1 (AF-1) surface, a zinc finger DBD, a flex interacts with co-regulator proteins through the Activation Function 2 (AF-2 The size of the ligand binding pocket varies among the different receptors, being for instance very large in PPARg, which allows binding of very differently sized ligands (273). Several differences are evident when comparing unliganded and ligand-bound receptors. The liganded structures are more compact than the unliganded noted that these motifs represent consensus idealized sequences and that naturally occurring HREs can show significant variation from the consensus. Although some monomeric receptors can bind to a single hexameric motif, most receptors bind as homo- or heterodimers to HREs composed typically of two core hexameric motifs. Hormone binding domain contains 12 alpha helices No ligand Plus ligand Dynamic stabilization model H12 is not in one fixed position, but rather is dynamic and dependent on ligand. FIG. 4. Schematic drawing of the nuclear receptor ligand-binding domain (LBD). On the left, the LBD from the crystal structure of the unliganded RXRa is shown. On the right, the ligand-bound LBD of the RARg is shown. Cylinders represent a-helices that are numbered from 1 to 12. Note the different position of the COOH-terminal helix 12 that contains the core AF-2 domain in both situations. [From Wurtz et al. (294), reprinted by permission from Nature, Macmillan Magazines Ltd.] Can bind Coactivators or corepressors These models suggested that LBDs have several conformations: Not bound to Ligand, bound to coactivator and bound to corepressor These models suggested that LBDs have several conformations: Not bound to Ligand, bound to coactivator and bound to corepressor Receptor bias Ligands bias System bias assay reflects the selectivity of TR:GRIP1 interactions 1 (amino observed in vivo. three NR To measure the affinities of the interactions of NID, econdary Coactivator SRC-1 binds receptor and Histone acetylase CBP NID2−, and NID3− with the TRb LBD and to test for ced these This activates transcription Charged dipole due to negative and positive residues flanking hydrophobic motif assay reflects the selectivity of TR:GRIP1 interactions 1 (amino observed in vivo. three NR To measure the affinities of the interactions of NID, econdary Coactivator SRC-1 binds receptor and Histone acetylase CBP NID2−, and NID3− with the TRb LBD and to test for ced these This activates transcription clear receptors have been solved. These studies have demonstrated that overall structures of the different receptors are rather similar, suggesting a canonical structure for the different members of the nuclear receptor superfamily (for a review see Ref. 178). Figure 4 shows a schematic representation of the crystal structure of a receptor LBD. The LBDs are formed by 12 conserved a-helical regions numbered from H1 to H12. A conserved b-turn is situated between H5 and H6. However, PPARg is unique in its overall structure and contains an extra helix designed H29, and the VDR contains a poorly structured insertion between helices H1 and H3 for which no functional role has been defined (221). The LBDs are folded into a three-layered, antiparallel helical sandwich. A central core layer of three helices is packed between two additional layers to create a cavity, the ligand-binding pocket, which accommodates the ligand. This domain is mainly hydrophobic and is buried within the bottom half of the LBD. Contacts with the ligand can be extensive and include different structural elements through the LBD. The size of the ligand binding pocket varies among the different receptors, being for instance very large in PPARg, which allows binding of very differently sized ligands (273). Several differences are evident when comparing unliganded and ligand-bound receptors. The liganded structures are more compact than the unliganded tors undergo a cle B. Hormone Res Nuclear recep to specific DNA se mone response el located in regulato 59-flanking region HREs are found re some cases they a kilobases upstream The analysis of a l well as synthetic H constitutes the co motifs have been preferentially reco whereas AGG/TTC remaining recepto noted that these sequences and tha significant variatio monomeric recept tif, most receptor HREs composed t tor LB RX LB a-h dif tha tio mi Downloaded from journals.physiology.org/journal/physrev at SAINT LOUIS UNIVERSITY (165 Charged dipole due to negative and positive residues flanking hydrophobic motif Some ligands cause conformation of changes in LBD that favor binding of coactivators Coactivator proteins such as SRC-1 interact with NRs via an alpha-helix containing a short LXXLL motif (L- leucine, X- any amino acid). This motif interacts with the NR AF2 domain surface. The coactivators leucine residues lie within the hydrophobic groove of the AF- 2 surface and the ends of the helical peptide are held in place by a charge clamp formed by a lysine on the NR’s H3 and a glutamic acid on H12 Coactivator proteins such as SRC-1 interact with NRs via an alphahelix containing a short LXXLL motif (L- leucine, X- any amino acid). This motif interacts with the NR AF-2 surface. The co-activators’s leucine residues lie within the hydrophobic groove of the AF- 2 surface and the ends of the helical peptide are generally held in place by a charge clamp formed by a lysine on the NR’s H3 and a glutamic acid on H12 In the absence of ligand some NRs (RXRheterodimers ) favor conformation that promote co-repressor binding. Ligand (agonist) binding causes shift in H12 that promotes coactivator binding. Corepressor binds RXR heterodimers and binds histone Deacetylase-this represses transcription Co-repressors such as NCoR contain the conserved (L/I)XX(I/V)I or LXXX(I/L)XXX(I/L) motif (referred to as CoRNR box) (L- leucine, Iisoleucine, X- any amino acid). These extended motifs interact at the same hydrophobic AF2 surface with H12 in different position Heterodimeric- RXR NRs are always located in the nucleus. Under basal state, they are bound to DNA and are not active because they are linked to a corepressor that represses transcription. Binding of ligand results in release of corepressor and binding of coactivator which activates transcription Ligand-activation - a switch from an active repressor to a full activator Coactivator-link: LxxLL a short helix (green) on SRC Corepressor-link: longer helix (red) on NcoR CBP/p300 are Histone acetylases- acetylate histones The size of the ligand binding pocket varies among the different receptors, being for instance very large in PPARg, which allows binding of very differently sized ligands (273). Several differences are evident when comparing unliganded and ligand-bound receptors. The liganded structures are more compact than the unliganded noted that these motifs represent consensus idealized sequences and that naturally occurring HREs can show significant variation from the consensus. Although some monomeric receptors can bind to a single hexameric motif, most receptors bind as homo- or heterodimers to HREs composed typically of two core hexameric motifs. Hormone binding domain contains 12 alpha helices No ligand Plus ligand H12 can adopt many conformation That can bind corepressor or coactivator FIG. 4. Schematic drawing of the nuclear receptor ligand-binding domain (LBD). On the left, the LBD from the crystal structure of the unliganded RXRa is shown. On the right, the ligand-bound LBD of the RARg is shown. Cylinders represent a-helices that are numbered from 1 to 12. Note the different position of the COOH-terminal helix 12 that contains the core AF-2 domain in both situations. [From Wurtz et al. (294), reprinted by permission from Nature, Macmillan Magazines Ltd.] Receptor bias: Idea that some Steroid receptor LBD (ER) when bound by ligand (Estrogen) greatly favor binding SRC-1 but can also bind some N-Cor R1 System bias: Binding of N-CorR1 can be increased by increasing ratio of N-Cor R1 to SRC-1 Ligand bias: Replacing ligand(Estrogen) with Selective Estrogen receptor modulator (SERM) gives LBD a shape that can either bind SRC-1 or N-CorR1 with equal affinity. Cancers arising from tissues are often "hormone sensitive" and dependent on hormone to continue to grow. Breast Cancer cells Estrogen binds ER LBD and activates coactivators to promote growth Tamoxifen (a SERM) binds ER LDB and can bind corepressors or coactivators. Which binds is determined by how much coactivators or corepressors are present in cell Selective estrogen receptor modulators-SERMs ER ER Inactive Estrogen Conformation of ligand binding domain favor interaction with COACTIVATORS TAMOXIFEN Partial Systems Bias Signaling ? Breast Tumor Cells Active Tumor Growth Maintains Bone density Conformation of ligand binding domain is such it interact with COACTIVATORS or COREPRESSORS ? Bone Selective estrogen receptor modulators-SERMs ER ER Inactive Estrogen Conformation of ligand binding domain favor interaction with COACTIVATORS TAMOXIFEN Partial More co-repressor expressed Inhibition of transcription and cell death Breast Tumor Cells Active Tumor growth Conformation of ligand binding domain is such it interact with COACTIVATORS or COREPRESSORS ? Bone Selective estrogen receptor modulators-SERMs ER ER Inactive Estrogen Conformation of ligand binding domain favor interaction with COACTIVATORS TAMOXIFEN Partial More co-repressor expressed Inhibition of transcription and cell death Breast Tumor Cells Active Tumor growth Conformation of ligand binding domain is such it interact with COACTIVATORS or COREPRESSORS More coactivators expressed Increased of transcription bone density is maintained Bone Multiple modes of transcription activation by estrogen Genomic Signaling and estrogen receptors Non-Genomic Non-Genomic signaling via GPR30 Estrogen independent signaling EGF activation of ERFR results in activation Map kinase pathway that activates ERK. ERK phosphorylates serine in AF1 Domain of ER This results in interaction of AF1 domain with Coactivators which cause Transcription and growth of breast cancer cells