nuclear receptors

Preface In 1975 Bert O’Malley and Joel Hardman edited five volumes in the Methods in Enzymology series on the topic of hor mone action. Included in this now classic collection was a volume entitled ‘‘Steroid Hormones,’’ which contained close to fifty papers describing the methods of the day used to study nuclear hormone receptors. Assays for steroid hormone binding, receptor purification, biological response systems, and steroid metabolism constituted the arsenal then available to delve into the myriad functions of nuclear receptors. These methods served the receptor community admirably and their application led to the next revolution in the field, which occurred in the mid-1980s when the laboratories of Pierre Chambon, Ronald Evans and Keith Yamamoto reported the first isolation of cDNAs encoding the mammalian estrogen and glucocorticoid receptors. These remarkable achievements ushered in the current era of nuclear receptor research, and with it came the development of new methods to study receptor structure and function. This volume represents a compilation of these newer methods. They span the gamut of current receptor research , from chemical methods for ligand purification, synthesis and quantitation, to the characterization of mice that lack one or more receptor encoding genes. How to utilize receptor- relevant information in the proliferating genome sequ ences of higher eukaryotic organisms is detailed together with molecular methods to identify networks of receptor-responsive target genes. Newer versions of biochemical methods for receptor purification and assay are found, as are those that report in exquisite detail how receptor interacting proteins such as chaperones and coregulators are obtained. The study of receptor biology is not confined to a single organism, and to this end several articles address the use of species as disparate as flies and fish to gain insight into receptor function. The present group of authors also represents a Who’s Who of the new generation of nuclear receptor researchers and each of the represented laboratories has contributed significantly to our current knowledge base. Although none of the old lions from the earlier Methods in Enzymology volume on receptors were able to contribute to the present collection, a substantial debt is owed them for laying the foundations on which all of us stand. Tomes of this nature are not assembled in a vacuum, and we offer heartfelt thanks to the many authors for their hard work and bonhomie; our xiii editors Shirley Light and Noelle Gracy at Academ ic Press, who, under duress from Keith Yamamoto, John Abelson, and Mel Simon, conceived this volume; and our secretaries, Lidia Galvan and Betsy Layton, who are standing in line for canonization. DAVID W. RUSSELL DAVID J. MANGELSDORF xiv PREFACE Table of Contents CONTRIBUTORS TO VOLUME 364 ix P REFACE xiii V OLUMES IN SERIES xv Section I. Analysis of Nuclear Receptor Ligands 1. Identification of Novel Nuclear Hormone Receptor Ligands by Activity-Guided Purification F ELIX GRU ¨ N AND BRUCE BLUMBERG 3 2. Quantitation of Receptor Ligands by Mass Spectrometry E RIK G. LUND AND ULF DICZFALUSY 24 3. Site-Specific Fluorescent Labeling of Estrogen Receptors and Structure–Activity Relationships of Ligands in Terms of Receptor Dimer Stability A NOBEL TAMRAZI AND JOHN A. KATZENELLENBOGEN 37 4. Cell-Free Ligand Binding Assays for Nuclear Receptors S TACEY A. JONES, D EREK J. PARKS, AND STEVEN A. KLIEWER 53 5. Design and Synthesis of Receptor Ligands H IKARI A. I. YOSHIHARA, NGOC-HA NGUYEN, AND THOMAS S. SCANLAN 71 Section II. Structure/Function Analysis of Nuclear Receptors 6. Bioinformatics of Nuclear Receptors M ARC ROBINSON-RECHAVI AND VINCENT LAUDET 95 7. Application of Random Peptide Phage Display to the Study of Nuclear Hormone Receptors C HING-YI CHANG, JOHN D. NORRIS, M ICHELLE JANSEN, H UEY-JING HUANG, AND DONALD P. MCDONNELL 118 8. Methods for Detecting Domain Interactions in Nuclear Receptors E LIZABETH M. WILSON, BIN HE, AND ELIZABETH LANGLEY 142 9. The Orphan Receptor SHP and the Three-Hybrid Interference Assay Y OON-KWANG LEE AND DAVID D. MOORE 152 v 10. Regulation of Glucocorticoid Receptor Ligand- Binding Activity by the hsp90/hsp70-based Chaperone Machinery KIMON C. KANELAKIS AND WILLIAM B. PRATT 159 11. Analysis of Receptor Phosphorylation B RIAN G. ROWAN, R AMESH NARAYANAN, AND NANCY L. WEIGEL 173 Section III. Analysis of Nuclear Receptor Cofactors and Chromatin Remodeling 12. Acetylation and Methylation in Nuclear Receptor Gene Activation W EI XU,HELEN CHO, AND RONALD M. EVANS 205 13. Steroid Receptor Coactivator Peptidomimetics T IMOTHY R. GEISTLINGER AND R. KIPLIN GUY 223 14. Biochemical Isolation and Analysis of a Nuclear Receptor Corepressor Complex M ATTHEW G. GUENTHER AND MITCHELL A. LAZAR 246 15. Isolation and Functional Characterization of the TRAP/Mediator Complex S OHAIL MALIK AND ROBERT G. ROEDER 257 16. Study of Nuclear Receptor-Induced Transcription Complex Assembly and Histone Modification by Chromatin Immunoprecipitation Assays H AN MA, YONGFENG SHANG, D AVID Y. LEE, AND MICHAEL R. STALLCUP 284 Section IV. Identification of Nuclear Receptor Target Genes and Effectors 17. Serial Analysis of Gene Expression and Gene Trapping to Identify Nuclear Receptor Target Genes H ANA KOUTNIKOVA, ELISABETH FAYARD, J U ¨ RGEN LEHMANN, AND JOHAN AUWERX 299 18. Expression Cloning of Receptor Ligand Transporters P AUL A. DAWSON AND ANN L. CRADDOCK 322 19. Nuclear Receptor Target Gene Discovery Using High-Throughput Chromatin Immunoprecipi- tation J OSE ´ E LAGANIE ` RE, G ENEVIE ` VE DEBLOIS, AND VINCENT GIGUE ` RE 339 20. RNA Gel Shift Assays for Analysis of Hormone Control of mRNA Stability R OBIN E. DODSON, K ATHRYN M. GOOLSBY, MARIA ACENA-NAGEL, CHENGJIAN MAO, AND DAVID J. SHAPIRO 350 21. Expression Cloning of Ligand Biosynthetic Enzymes S HIGEAKI KATO AND KEN-ICHI TAKEYAMA 361 vi TABLE OF CONTENTS Section V. Use of Animal Models to Study Nuclear Receptor Function 22. Targeted Conditional Somatic Mutagenesis in the Mouse: Temporally-Controlled Knock Out of Retinoid Receptors in Epidermal Keratinocytes D ANIEL METZGER, ARUP KUMAR INDRA,MEI LI, B ENOIT CHAPELLIER, CE ´ CILE CALLEJA, NORBERT B. GHYSELINCK, AND PIERRE CHAMBON 379 23. Analysis of Small Molecule Metabolism in Zebrafish S HIU-YING HO, MICHAEL PACK, AND STEVEN A. FARBER 408 24. Analysis of Nuclear Receptor Function in the Mouse Auditory System M ATTHEW W. KELLEY, P AMELA J. LANFORD, IWAN JONES,LORI AMMA, LILY NG, AND DOUGLAS FORREST 426 25. Analysis of Estrogen Receptor Expression in Tissues M ARGARET WARNER, LING WANG, Z HANG WEIHUA, GUOJUN CHENG, HIDEKI SAKAGUCHI, SHIGEHIRA SAJI, S TEFAN NILSSON, T HOMAS KIESSELBACH, AND JAN-A ˚ KE GUSTAFSSON 448 26. In Vivo and In Vitro Reporter Systems for Studying Nuclear Receptor and Ligand Activities ALEXANDER MATA DE URQUIZA AND THOMAS PERLMANN 463 27. Methods to Characterize Drosophila Nuclear Receptor Activation and Function In Vivo T ATIANA KOZLOVA AND CARL S. THUMMEL 475 A UTHOR INDEX 491 S UBJECT INDEX 525 TABLE OF CONTENTS vii Contributors to Volume 364 Article numbers are in parentheses following the names of contributors. Affiliations listed are current. MARIA ACENA-NAGEL (20), Department of Neurology, University of Colorado Health Sciences Center, Denver, Colorado 80262 L ORI AMMA (24), Department of Human Genetics, Mount Sinai School of Medicine, New York, New York 10029 J OHAN AUWERX (17), Institut de Ge ´ ne ´ tique et de Biologie Mole ´ culaire et Cellulaire (IGBMC), CNRS/INSERM/Universite ´ Louis Pasteur, B.P. 163, F-67404 Illkirch, France B RUCE BLUMBERG (1), Department of Developmental and Cell Biology, University of California, Irvine, California 92697-2300 C E ´ CILE CALLEJA (22), Institut de Ge ´ ne ´ tique et de Biologie Mole ´ culaire et Cellulaire, CNRS/INSERM/ULP, Colle ` ge de France, BP 10142, 67404 Illkirch Cedex, France P IERRE CHAMBON (22), Institut de Ge ´ ne ´ tique et de Biologie Mole ´ culaire et Cellulaire, CNRS/INSERM/ULP, Colle ` ge de France, BP 10142, 67404 Illkirch Cedex, France C HING-YI CHANG (7), Department of Pharma- cology and Cancer Biology, Duke Univer- sity Medical Center, Durham, North Carolina 27710 B ENOIT CHAPELLIER (22), Institut de Ge ´ ne ´ tique et de Biologie Mole ´ culaire et Cellulaire, CNRS/INSERM/ULP, Colle ` ge de France, BP 10142, 67404 Illkirch Cedex, France G UOJUN CHENG (25), Med. Nutr., Karolinska Institute, Huddinge University Hospital, Novum, Huddinge, S-14186, Sweden H ELEN CHO (12), Howard Hughes Medical Institute, Gene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037 A NN L. CRADDOCK (18), Departments of Internal Medicine and Pathology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston- Salem, North Carolina 27157 P AUL A. DAWSON (18), Departments of Internal Medicine and Pathology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina 27157 G ENEVIE ` VE DEBLOIS (19), Molecular Oncology Group, McGill University Health Centre, Montre ´ al, Que ´ bec, Canada H3A 1A1 U LF DICZFALUSY (2), Karolinska Institutet, Department of Medical Laboratory Sciences and Technology, Division of Clinical Chemistry, Huddinge University Hospital C1.74, SE-141 86 Huddinge, Sweden R OBIN E. DODSON (20), Department of Biochemistry, University of Illinois, Urbana, Illinois 61801 R ONALD M. EVANS (12), Howard Hughes Medical Institute, Gene Expression Labora- tory, The Salk Institute for Biological Studies, La Jolla, California 92037 S TEVEN A. FARBER (23), Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson Univer- sity, Philadelphia, Pennsylvania 19107 ELISABETH FAYARD (17), Institut de Ge ´ ne ´ tique et de Biologie Mole ´ culaire et Cellulaire (IGBMC), CNRS/INSERM/Universite ´ Louis Pasteur, B.P. 163, F-67404 Illkirch, France D OUGLAS FORREST (24), Department of Human Genetics, Mount Sinai School of Medicine, New York, New York 10029 T IMOTHY R. GEISTLINGER (13), Departments of Pharmaceutical Chemistry and Cellular and Molecular Pharmacology, University of California San Francisco, California 94143- 0446 N ORBERT B. GHYSELINCK (22), Institut de Ge ´ ne ´ tique et de Biologie Mole ´ culaire et Cellulaire, CNRS/INSERM/ULP, Colle ` ge de France, BP 10142, 67404 Illkirch Cedex, France ix VINCENT GIGUE ` RE (19), Molecular Oncology Group, McGill University Health Centre, and Departments of Biochemistry, Medicine and Oncology, McGill University, Montre ´ al, Que ´ bec, Canada H3A 1A1 K ATHRYN M. GOOLSBY (20), Department of Biochemistry, University of Illinois, Urbana, Illinois 61801 F ELIX GRU ¨ N (1), Department of Developmen- tal and Cell Biology, University of Califor- nia, Irvine, California 92697-2300 M ATTHEW G. GUENTHER (14), Division of Endocrinology, Diabetes, and Metabolism, Departments of Medicine and Genetics, and The Penn Diabetes Center, University of Pennsylvania Medical Center, Philadel- phia, Pennsylvania 19104 J AN-A ˚ KE GUSTAFSSON (25), Med. Nutr., Karolinska Institute, Huddinge University Hospital, Novum, Huddinge, S-14186, Sweden R. K IPLIN GUY (13), Departments of Pharma- ceutical Chemistry and Cellular and Molecular Pharmacology, University of California San Francisco, California 94143- 0446 B IN HE (8), Laboratories for Reproductive Biology and Department of Pediatrics, and the Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-7500 S HIU-YING HO (23), Department of Micro- biology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 HUEY-JING HUANG (7), Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710 A RUP KUMAR INDRA (22), Institut de Ge ´ ne ´ t- ique et de Biologie Mole ´ culaire et Cellulaire, CNRS/INSERM/ULP, Colle ` ge de France, BP 10142, 67404 Illkirch Cedex, France M ICHELLE JANSEN (7), Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710 S TACEY A. JONES (4), Nuclear Receptor Func- tional Analysis, High Throughput Biology, GlaxoSmithKline, Research Triangle Park, North Carolina 27709-3398 I WAN JONES (24), Department of Human Genetics, Mount Sinai School of Medicine, New York, New York 10029 K IMON C. KANELAKIS (10), Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0632 SHIGEAKI KATO (21), The Institute of Mole- cular and Cellular Biosciences, The Uni- versity of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-0032, Japan J OHN A. KATZENELLENBOGEN (3), Depart- ment of Chemistry, University of Illinois, Urbana, Illinois 61801-3792 M ATTHEW W. KELLEY (24), Section on Developmental Neuroscience, National Institute on Deafness and Other Commu- nication Disorders, National Institutes of Health, 5 Research Court, Rockville, Maryland 20850 T HOMAS KIESSELBACH (25), Med. Nutr., Karolinska Institute, Huddinge University Hospital, Novum, Huddinge, S-14186, Sweden S TEVEN A. KLIEWER (4), Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas, 75390 H ANA KOUTNIKOVA (17), CareX S.A., F-67000 Strasbourg, France TATIANA KOZLOVA (27), Howard Hughes Medical Institute, Department of Human Genetics, University of Utah, Salt Lake City, Utah 84112-5331 J OSE ´ E LAGANIE ` RE (19), Molecular Oncology Group, McGill University Health Centre, and Department of Biochemistry, McGill University, Montre ´ al, Que ´ bec, Canada H3A 1A1 P AMELA J. LANFORD (24), Section on Devel- opmental Neuroscience, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, 5 Research Court, Rockville, Maryland 20850 E LIZABETH LANGLEY (8), Institute for Bio- medical Research, National University of Mexico, Mexico City, Mexico x CONTRIBUTORS VINCENT LAUDET (6), Laboratoire de Biologie Mole ´ culaire et Cellulaire, UMR CNRS 5665, Ecole Normale Supe ´ rieure de Lyon, 46 alle ´ e d’Italie, 69364 Lyon Cedex 07, France M ITCHELL A. LAZAR (14), Division of Endo- crinology, Diabetes, and Metabolism, Departments of Medicine and Genetics, and The Penn Diabetes Center, University of Pennsylvania Medical Center, Philadel- phia, Pennsylvania 19104 Y OON-KWANG LEE (9), Department of Mole- cular and Cellular Biology, Baylor College of Medicine, One BaylorPlaza,Houston,Texas 77030 D AVID Y. LEE (16), Department of Biochem- istry and Molecular Biology, University of Southern California, Los Angeles, California 90089 J U ¨ RGEN LEHMANN (17), CareX S.A., F-67000 Strasbourg, France MEI LI (22), Institut de Ge ´ ne ´ tique et de Biologie Mole ´ culaire et Cellulaire, CNRS/INSERM/ ULP, Colle ` ge de France, BP 10142, 67404 Illkirch Cedex, France E RIK G. LUND (2), Merck Research Labora- tories, RY80W-250, Rahway, New Jersey 07065 H AN MA (16), Inflammatory and Viral Dis- eases Unit, Roche Bioscience, Palo Alto, California 94304 S OHAIL MALIK (15), Laboratory of Biochem- istry and Molecular Biology, Rockefeller University, New York, New York 10021 C HENGJIAN MAO (20), Department of Bio- chemistry, University of Illinois, Urbana, Illinois 61801 A LEXANDER MATA DE URQUIZA (26), Ludwig Institute for Cancer Research, Stockholm Branch, S-171 77 Stockholm, Sweden D ONALD P. MCDONNELL (7), Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710 D ANIEL METZGER (22), Institut de Ge ´ ne ´ tique et de Biologie Mole ´ culaire et Cellulaire, CNRS/INSERM/ULP, Colle ` ge de France, BP 10142, 67404 Illkirch Cedex, France D AVID D. MOORE (9), Department of Mole- cular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030 R AMESH NARAYANAN (11), Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030 L ILY NG (24), Department of Human Genetics, Mount Sinai School of Medicine, New York, New York 10029 N GOC-HA NGUYEN (5), Departments of Phar- maceutical Chemistry and Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143-2280 S TEFAN NILSSON (25), Med. Nutr., Karolinska Institute, Huddinge University Hospital, Novum, Huddinge, S-14186, Sweden J OHN D. NORRIS (7), Department of Pharma- cology and Cancer Biology, Duke Univer- sity Medical Center, Durham, North Carolina 27710 MICHAEL PACK (23), Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 D EREK J. PARKS (4), Systems Research, GlaxoSmithKline, Research Triangle Park, North Carolina, 27709-3398 T HOMAS PERLMANN (26), Ludwig Institute for Cancer Research, Stockholm Branch, S-171 77 Stockholm, Sweden W ILLIAM B. PRATT (10), Department of Phar- macology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0632 M ARC ROBINSON-RECHAVI (6), Laboratoire de Biologie Mole ´ culaire et Cellulaire, UMR CNRS 5665, Ecole Normale Supe ´ rieure de Lyon, 46 alle ´ e d’Italie, 69364 Lyon Cedex 07, France R OBERT G. ROEDER (15), Laboratory of Bio- chemistry and Molecular Biology, Rocke- feller University, New York, New York 10021 B RIAN G. ROWAN (11), Department of Bio- chemistry and Molecular Biology, Medical College of Ohio, Toledo, Ohio 43614 S HIGEHIRA SAJI (25), Med. Nutr., Karolinska Institute, Huddinge University Hospital, Novum, Huddinge, S-14186, Sweden H IDEKI SAKAGUCHI (25), Med. Nutr., Kar- olinska Institute, Huddinge University Hos- pital, Novum, Huddinge, S-14186, Sweden CONTRIBUTORS xi THOMAS S. SCANLAN (5), Departments of Pharmaceutical Chemistry and Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143- 2280 Y ONGFENG SHANG (16), Department of Biochemistry and Molecular Biology, Peking University Health Science Center, Beijing 100083, People’s Republic of China D AVID J. SHAPIRO (20), Department of Bio- chemistry, University of Illinois, Urbana, Illinois 61801 M ICHAEL R. STALLCUP (16), Department of Pathology, University of Southern California, Los Angeles, California 90089- 9092 K EN-ICHI TAKEYAMA (21), The Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-0032, Japan A NOBEL TAMRAZI (3), Department of Chem- istry, University of Illinois, Urbana, Illinois 61801-3792 C ARL S. THUMMEL (27), Howard Hughes Medical Institute, Department of Human Genetics, University of Utah, Salt Lake City, Utah 84112-5331 L ING WANG (25), Med. Nutr., Karolinska Institute, Huddinge University Hospital, Novum, Huddinge, S-14186, Sweden M ARGARET WARNER (25), Med. Nutr., Karolinska Institute, Huddinge University Hospital, Novum, Huddinge, S-14186, Sweden N ANCY L. WEIGEL (11), Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030 Z HANG WEIHUA (25), Med. Nutr., Karolinska Institute, Huddinge University Hospital, Novum, Huddinge, S-14186, Sweden E LIZABETH M. WILSON (8), Laboratories for Reproductive Biology and Department of Pediatrics, and the Department of Biochem- istry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-7500 W EI XU (12), Howard Hughes Medical Insti- tute, Gene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037 H IKARI A. I. YOSHIHARA (5), Departments of Pharmaceutical Chemistry and Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143-2280 xii CONTRIBUTORS Section I Analysis of Nuclear Receptor Ligands [...]... and lipid homeostasis Those receptors lacking known endogenous ligands are termed as ‘‘orphan receptors. ’’ The identification of natural ligands for this class of nuclear receptors is an important goal in understanding their biology Much progress has been made in recent years identifying ligands for orphan receptors (reviewed in Refs 1–4) It is thought that nearly all nuclear receptors evolved from an... they may be classified as either being endocrine receptors, adopted orphan receptors, or orphan receptors. 2 The first group consists of well-characterized endocrine hormone receptors such as the vitamin A and D receptors, and the steroid and thyroid hormone receptors These proteins typically have very high-affinity specific ligands The second group consists of receptors for dietary or endogenous ligands present... they may be classified as either being endocrine receptors, adopted orphan receptors, or orphan receptors. 2 The first group consists of well-characterized endocrine hormone receptors such as the vitamin A and D receptors, and the steroid and thyroid hormone receptors These proteins typically have very high-affinity specific ligands The second group consists of receptors for dietary or endogenous ligands present... J Mangelsdorf, Orphan nuclear receptors as eLiXiRs and FiXeRs of sterol metabolism, J Biol Chem 17, 17 (2001) 2 J J Repa and D J Mangelsdorf, The role of orphan nuclear receptors in the regulation of cholesterol homeostasis, Annu Rev Cell Dev Biol 16, 459–481 (2000) 3 T M Willson, S A Jones, J T Moore, and S A Kliewer, Chemical genomics: functional analysis of orphan nuclear receptors in the regulation... exemplified by the PPAR// receptors, which bind fatty acids and their derivatives, the LXR/b receptors, which bind oxysterols, and FXR, which interacts with bile acids The third group consists of receptors without identified ligands, and it is possible that many receptors of this class actually do not have endogenous ligands With the identification of ligands for many different nuclear receptors, a pattern... the nuclear receptor family.1 It is now clear that members of this class of proteins serve as important signal transducers, responding to the appearance of signaling molecules with an increase or decrease in the cellular expression of target genes This behavior is typified by the classic steroid hormone receptors, which were among the first nuclear receptors to be identified and characterized Nuclear receptors. .. the nuclear receptor family.1 It is now clear that members of this class of proteins serve as important signal transducers, responding to the appearance of signaling molecules with an increase or decrease in the cellular expression of target genes This behavior is typified by the classic steroid hormone receptors, which were among the first nuclear receptors to be identified and characterized Nuclear receptors. .. exemplified by the PPAR// receptors, which bind fatty acids and their derivatives, the LXR/b receptors, which bind oxysterols, and FXR, which interacts with bile acids The third group consists of receptors without identified ligands, and it is possible that many receptors of this class actually do not have endogenous ligands With the identification of ligands for many different nuclear receptors, a pattern... novel nuclear receptor ligands is based on the key principle of activity-guided purification followed by structure determination Prior knowledge of an activity’s relation to known nuclear receptor ligands is therefore not pertinent Following sample preparation, extracts are fractionated by suitable HPLC methods and tested for their ability to modulate transcriptional activity of specific nuclear receptors. .. transactivation in small-scale formats A particular benefit of the modular structure of nuclear receptors is the ability to construct chimeras that retain the specific ligand-dependent nature of a receptor, but facilitate high-throughput screening by utilizing a common reporter construct Chimeras between the yeast GAL4 DBD and nuclear receptor LBDs have proven invaluable for developing such assays Reporter . Analysis of Nuclear Receptors 6. Bioinformatics of Nuclear Receptors M ARC ROBINSON-RECHAVI AND VINCENT LAUDET 95 7. Application of Random Peptide Phage Display to the Study of Nuclear Hormone Receptors C HING-YI. lipid homeostasis. Those receptors lacking known endogenous ligands are termed as ‘‘orphan receptors. ’’ The identification of natural ligands for this class of nuclear receptors is an important. Mangelsdorf, Orphan nuclear receptors as eLiXiRs and FiXeRs of sterol metabolism, J. Biol. Chem. 17, 17 (2001). 2 J. J. Repa and D. J. Mangelsdorf, The role of orphan nuclear receptors in the regulation