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Nuclear Receptors

Nuclear Receptors. Molecular Mechanisms of Signal Transduction, MPHY 616 Bob Koos Spring, 2005. Nuclear Receptor Ligands : <1000 d, lipophilic*.

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Nuclear Receptors

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  1. Nuclear Receptors Molecular Mechanisms of Signal Transduction, MPHY 616 Bob Koos Spring, 2005

  2. Nuclear Receptor Ligands: <1000 d, lipophilic* * There is some recent evidence that thyroid hormones, and perhaps glucocorticoids, may bind to and be internalized via plasma membrane transport proteins.

  3. Nuclear Receptor Ligands and Their “Receptors” Unknown ligands: “orphan receptors” The cDNAs for all the nuclear receptors were cloned in the mid-1980’s.

  4. Chromosome puffs in insects induced by the steroid hormone ecdysone [3H]uridine labeling shows puffs are sites of active RNA synthesis

  5. The sequential increase in RNA, protein, and DNA after a single administration of estradiol to immature or castrated rats

  6. Walter P, Green S, Greene G, Krust A, Bornert JM, Jeltsch JM, Staub A, Jensen E, Scrace G, Waterfield M, et al. 1985 Cloning of the human estrogen receptor cDNA. Proc Natl Acad Sci U S A 82(23):7889-93.

  7. Classic Model of Nuclear Receptor Action

  8. Nuclear Receptor Domains 12 alpha helices (and dimerization)

  9. Nuclear Receptor - Molecular Chaperone Complexes ~9S, non-DNA binding 4S, DNA-Binding Figure 1. Assembly and Disassembly of Intracellular Hormone Receptors A stepwise process is described in which the hormone receptor is assembled into the aporeceptor state by sequential interactions with different molecular chaperones (modified from Nollen and Morimoto, 2002). Upon ligand binding, the hormone receptor binds to the response element. The association of the DNA bound form of the hormone receptor with p23 and Hsp90 leads to the disassembly of the hormone receptor complex and release from DNA. Abbreviations: AD, activation domain; DNA, DNA binding domain; HBD, hormone binding domain. Morimoto RI 2002 Cell 110:281-4

  10. Nuclear Receptor Domains 12 alpha helices (ligand independent) (and dimerization)

  11. Structural Basis of Nuclear Receptor Ligand Binding C-terminal tail

  12. Phosphorylation of Nuclear Receptors

  13. Growth factor modulation of estrogen receptor activity Regulation of steroid receptor transcriptional complexes by MAPK activation. Growth factors such as EGF and IGF-1 stimulate the Ras-MAPK cascade via activation of their respective tyrosine kinase receptors. Activated MAPK may then phosphorylate nuclear targets including ER and AIB1 to modulate gene transcription in response to growth factor and/or steroid stimulation (Font de Mora J, Brown M 2000 AIB1 is a conduit for kinase-mediated growth factor signaling to the estrogen receptor. Mol Cell Biol 20(14):5041-7).

  14. DNA-Binding Domain Zilliacus et al., 1995

  15. ER DNA-Binding Domain (DBD) P Box P Box

  16. Structure of the Nuclear Receptor DBD P box residues make base-specific contacts with DNA. D box residues involved in dimerization and half site spacing.

  17. Palindromic HRE Direct Repeat HRE

  18. GR DBD bound to GRE Zn Zn Zn Zn

  19. The NR Hormone Response Elelment Six base pairs form the core recognition motif (in most cases). Two General classes: AGAACA: glucocorticoid, mineralocorticoid, progesterone, and androgen receptors AGGTCA or AGTTCA: estrogen, thyroid hormone, retinoic acid, and vitamin D receptors Core recognition motifs in natural genes usually exhibit some variation from these idealized sequences

  20. Half site: AAAGGTCA or AGGTCA (ERE half site) (ERs, PRs?) Palindrome (inverted repeat) (AGAACAnnnTGTTCT) or (AGGTCAnnnTGACCT) often with RXR Direct repeat (AGGTCAnxAGGTCA) Palindrome (AGAACATGTTCT) Inverted palindrome (ACAAGAnxTCTTGT)

  21. Classic Model of Nuclear Receptor Action

  22. Halachmi S, Marden E, Martin G, MacKay H, Abbondanza C, Brown M 1994 Estrogen receptor-associated proteins: possible mediators of hormone-induced transcription. Science 264(5164):1455-8. Association is ligand-dependent • Methods • 35S-labeled MCF-7 cells • Cell extracts incubated with • GST-HBD protein affinity • reagent + or – estradiol • Specifically bound proteins • eluted and electrophoresed

  23. THOMAS E. SPENCER, GUIDO JENSTER … & BERT W. O'MALLEY 1997 Steroid receptor coactivator-1 is a histone acetyltransferase.Nature 389:194 – 198 Steroid receptors and coactivator proteins are thought to stimulate gene expression by facilitating the assembly of basal transcription factors into a stable preinitiation complex1. What is not clear, however, is how these transcription factors gain access to transcriptionally repressed chromatin to modulate the transactivation of specific gene networks in vivo. The available evidence indicates that acetylation of chromatin in vivo is coupled to transcription and that specific histone acetyltransferases (HATs)target histones bound to DNA and overcome the inhibitory effect of chromatin on gene expression2-4. The steroid-receptor coactivator SRC-1 is a coactivator for many members of the steroid-hormone receptor superfamily of ligand-inducible transcription factors5. Here we show that SRC-1 possesses intrinsic histone acetyltransferase activity and that it also interacts with another HAT, p300/CBP-associated factor (PCAF). The HAT activity of SRC-1 maps to its carboxy-terminal region and is primarily specific for histones H3 and H4. Acetylation by SRC-1 and PCAF of histones bound at specific promoters may result from ligand binding to steroid receptors and could be a mechanism by which the activation functions of steroid receptors and associated coactivators enhance formation of a stable preinitiation complex, thereby increasing transcription of specific genes from transcriptionally repressed chromatin templates.

  24. Nuclear Receptor Coactivators and Corepressors (ATP-dependent DNA unwinding) 4. Histone methyl transferases 5. Ubiquitin ligases (E6-AP)

  25. Klinge C 2000 Estrogen receptor interaction with co-activators and co-repressors. Steroids 65:227-251

  26. Font de Mora J, Brown M 2000 AIB1 is a conduit for kinase-mediated growth factor signaling to the estrogen receptor. Mol Cell Biol 20(14):5041-7.

  27. Reversible Modifications of Proteins to Regulate Their Activity HAT Chromosomal DNA is tightly wrapped around nucleosomal units composed of core histone proteins. Acetylation of lysine tails on histones “opens up” this chromatin structure.

  28. Corepressors: Bound to NRs in the absence of ligand or the presence of NR antagonists Nagy L, Kao HY, Chakravarti D, Lin RJ, Hassig CA, Ayer DE, Schreiber SL, Evans RM 1997 Nuclear receptor repression mediated by a complex containing SMRT, mSin3A, and histone deacetylase. Cell. 1997 89:373-80

  29. Structural Basis of Nuclear Receptor Corepressor Recrutiment

  30. Structural Basis of Nuclear Receptor Ligand Binding and Coregulator Recrutiment

  31. McKenna NJ, O'Malley BW. Minireview: nuclear receptor coactivators--an update. Endocrinology. 2002 Jul;143(7):2461-5. Review.

  32. Role of Proteosomal Degradation in Nuclear Receptor Action Nawaz Z, O'Malley BW 2004 Urban renewal in the nucleus: is protein turnover by proteasomes absolutely required for nuclear receptor-regulated transcription? Mol Endocrinol 18:493-9.

  33. ER Induction of Transcription Via Binding to Other Transcription Factors Webb et al. 1999 Mol Endo 13: 1672 Saville B, Wormke M, Wang F, Nguyen T, Enmark E, Kuiper G, Gustafsson JA, Safe S 2000 Ligand-, cell-, and estrogen receptor subtype (alpha/beta)-dependent activation at GC-rich (Sp1) promoter elements. J Biol Chem 275(8):5379-87

  34. Inhibitory NRs Hall JM, McDonnell DP.The estrogen receptor beta-isoform (ERbeta) of the human estrogen receptor modulates ERalpha transcriptional activity and is a key regulator of the cellular response to estrogens and antiestrogens. Endocrinology. 1999 Dec;140(12):5566-78.

  35. Nature Reviews Drug Discovery3; 27-41 (2004); doi:10.1038/nrd1283STEROID-HORMONE RAPID ACTIONS, MEMBRANE RECEPTORS AND A CONFORMATIONAL ENSEMBLE MODEL Pathways for generating biological responses by steroid hormones.   In the genomic pathway (left-hand side), occupancy of the nuclear receptor by the cognate steroid hormone leads to an up- or downregulation of genes subject to hormone-receptor regulation. In the rapid-response pathway (right-hand side), occupancy of a putative membrane receptor (which in some instances might be a membrane-associated nuclear receptor) by the steroid hormone can lead to the initiation of rapid responses that are coupled through appropriate second-messenger systems, either directly to the generation of the end biological response(s) or indirectly through modulation of genomic responses; the examples included in the figure are not intended to be exhaustive. The figure also indicates the possibility of conformationally flexible agonist analogues of the parent steroid hormone binding to the nuclear or proposed membrane receptor(s). Further, it is known that conformationally restricted analogues can occupy the membrane receptor and initiate only rapid responses. These possibilities are further discussed in the text, Fig. 3 and Fig. 4 (examples of conformationally restricted analogues) and Fig. 6. cAMP, cyclic AMP; MAP, mitogen-activated protein; mRNA, messenger RNA; PKC, protein kinase C.

  36. Chambliss KL, Shaul PW 2002 Estrogen modulation of endothelial nitric oxide synthase. Endocr Rev 23(5):665-86.

  37. EndogenousER Colocalizes with Caveolin-1 in the Plasma Membrane of Endothelial Cells IHC Western

  38. Filardo EJ 2002 Epidermal growth factor receptor (EGFR) transactivation by estrogen via the G-protein-coupled receptor, GPR30: a novel signaling pathway with potential significance for breast cancer. J Steroid Biochem Mol Biol 80(2):231-8.

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