CHARACTERIZATION OF THE LOCUST RETINOID X RECEPTORS by Shaun M. Nowickyj A thesis submitted to the Department of Biology in conformity with the requirements for the degree of Master of Science Queen’s University Kingston, Ontario, Canada (December, 2007) Copyright © Shaun M. Nowickyj, 2007 ii Abstract The retinoid X receptor (RXR) participates in a multitude of nuclear receptor signaling pathways and is induced by its highly sought-after cognate ligand, 9-cis -retinoic acid (9- cis -RA). In flies and moths, molting is mediated by the ecdysone receptor that consists of a heterodimer comprising the ecdysone receptor monomer (EcR) and the invertebrate RXR homolog ultraspiracle (USP); the latter, however, is believed to have diverged from its RXR origin. From the more evolutionarily-primitive insect Locusta migratoria (Lm), long and short RXR transcripts (LmRXR-L and LmRXR-S, respectively) were detected during embryogenesis. This thesis reports the immunochemical detection of RXR cross- reactive material throughout Locusta embryogenesis, suggesting that the protein may have another role besides ecdysone signaling. Thus, the RXR isoforms were cloned for recombinant expression and purification in order to demonstrate retinoid specificity. Both isoforms bound 9-cis -RA and all-trans -RA with high affinity. Binding was further corroborated by the identification of endogenous retinoids during embryogenesis. Embryos were first subjected to modified “Bligh and Dyer” as well as solid phase extractions to circumvent oil precipitation that rendered whole homogenates unsuitable for retinoid assay and detection. The RA-inducible Cyp26A1-promoter reporter cell line identified the presence of endogenous RAs (5.4 nM) from insect embryo extracts. Finally, high pressure liquid chromatography followed by mass spectroscopy (HPLC/MS) confirmed the identity of all-trans -RA and the more abundant 9-cis -RA (1.3 nM). These findings suggest a functional role for 9-cis -RA in the invertebrate embryo and favour signaling through the combination of 9-cis -RA and RXR in evolutionarily early RA-driven animal development. iii Co-Authorship The following researchers contributed to the success of the work described in this thesis and are gratefully acknowledged with co-authorship. Chapter 2 Don Cameron and Dr. M. Petkovich (supervisor) provided the Cyp26A1-promoter reporter cell line, performed luminometer analysis and provided technical advice. Dr. James V. Chithalen and Dr. G. Jones (supervisor) helped develop the Locusta RA extraction protocol, performed HPLC/MS analysis and provided considerable advice. Dr. Michael G. Tyshenko assisted in generating anti-LmRXR and anti-hRXR antibodies and provided technical advice. Dr. Gerard R. Wyatt provided advice and assistance for the radio-ligand binding assays and in handling Locusta . iv Acknowledgements I would like to express my sincerest thanks to my supervisor Dr. Virginia Walker for her support, guidance and friendship. Her teachings, advice and conversations of science and life have helped me immensely and will be remembered. I would also like thank my committee members, Dr. Hawryshyn and Dr. Mulligan, for time taken to provide advice, critical comments and assistance in completing this thesis. Dr. Jones, Dr. Chithalen, Dr. Petkovich and Don Cameron are thanked for many helpful retinoid conversations. I truly enjoyed my research experience in their respective labs and learned a great deal from everyone in Botterell Hall. Dr. Wyatt is thanked for his advice, teachings and numerous fruitful conversations. I am grateful for the help of Dr. Tyshenko and for teaching me to balance work and fitness. Thank you to Dr. Ko for answering all last minute questions big and small. This experience will be forever memorable because of the support and friendship of past and present Bug Group members: Joslynn Affleck, Alan Brown, Raimond Gordienko, Emily Huva, Mike Kuiper, Niraj Kumar, Katerina Neumann, Wensheng Qin, Mike Tyshenko, Sandra Wilson, Suzy Wu, and Shutang Zhou. The Department of Biology’s staff is thanked for their excellent technical and administrative assistance. The Cornwall Community Hospital and Cornwall Public Library are kindly thanked for their encouragement and support. To Lynn, Stephan and Charles for making me smile everyday. v To Zachary McGlynn, Darryl Knight, Chris Boulton, Rich Lyon and Morris Lin. Your friendship means the world to me - thanks for taking care of me all these years. To Alissa Lunney whose constant support and encouragement guided me through this thesis - I can not thank you enough. Thank you to my family who has supported me from the start. For my mom, dad and grandparents to whom this thesis means the most. vi Table of Contents Abstract ...............................................................................................................................ii Co-Authorship....................................................................................................................iii Acknowledgements ............................................................................................................ iv Table of Contents ............................................................................................................... vi List of Abbreviations..........................................................................................................ix List of Tables......................................................................................................................xi List of Figures ...................................................................................................................xii CHAPTER 1: Introduction and Literature Review The Migratory Locust.......................................................................................................... 1 Early Evidence of Insect Hormones.................................................................................... 2 Insect Endocrine Structures and Molting............................................................................ 5 JH Structure and Synthesis.................................................................................................. 6 Modes of JH Action ............................................................................................................ 8 The Nuclear Receptors........................................................................................................ 9 Nuclear Receptor Domains ............................................................................................... 10 The Nuclear Mechanisms of Molting ............................................................................... 12 Vitamin A.......................................................................................................................... 13 The Retinoids .................................................................................................................... 14 9-cis -Retinoic Acid ........................................................................................................... 15 Research Objectives .......................................................................................................... 16 vii Literature Cited ................................................................................................................. 18 CHAPTER 2: Locust retinoid X receptors: 9-cis -retinoic acid in embryos from a primitive insect Abstract ............................................................................................................................. 28 Introduction ....................................................................................................................... 30 Results............................................................................................................................... 31 Ligand Binding Assays ............................................................................................... 32 Retinoids in Locust Embryos ...................................................................................... 33 Discussion ......................................................................................................................... 35 Locust RXR Characterization ..................................................................................... 35 Ligand Binding and Retinoid Identification ............................................................... 36 Materials and Methods...................................................................................................... 40 Animals ....................................................................................................................... 40 Plasmids ...................................................................................................................... 40 Protein Expression and Purification............................................................................ 42 Antibody Preparation .................................................................................................. 43 Western Blotting ......................................................................................................... 43 Ligand-Binding Assays............................................................................................... 44 Luciferase Assays........................................................................................................ 45 Embryo Retinoid Extraction.......................................................................................