The cloning, expression and partial characterization of a human delta opioid receptor Item Type text; Dissertation-Reproduction (electronic) Authors Fang, Lei. Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 29/09/2021 11:52:35 Link to Item http://hdl.handle.net/10150/186784 INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. 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Ann Arbor, MI48106 THE CLONING, EXPRESSION AND PARTIAL CHARACTERIZATION OF A HUMAN DELTA OPIOID RECEPTOR by Lei Fang A Dissertation Submitted to the Faculty of the COMMITTEE ON PHARMACOLOGY AND TOXICOLOGY (GRADUATE) In Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA 1994 2 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE As members of the Final Examination Committee, we certify that we have read the dissertation prepared by Lei Fang ----------~--------------------------- entitled The Cloning, Expression and Partial Characterization of a Human Delta Opioid Receptor and recommend that it be accepted as fulfilling the dissertation Doctor of Philosophy Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation 3 STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. SIGNED: ---=.fl----.l.Et--*----f"-l---- 4 ACKNOWLEDGMENTS I would like to thank my advisor Dr. Henry I. Yamamura for all his supervision, patience and encouragement throughout my entire graduate training. I am particularly indebted to Dr. Richard J. Knapp for his advice, criticism and enthusiasm. I would also like to thank my committee members: Dr. William R. Roeske, Dr. Frank Porreca, Dr. Thomas P. Davis and Dr. Hugh E. Laird for their valuable insight and direction in my graduate training. I would also like to thank everybody who works in Dr. Yamamura's laboratory. Particularly Ewa Malatynska, Xiaoping Li, Eva Varga, Robert Horvath, Giovanna Santoro and other graduate students for their moral and technical support and Hayes Karla for her secretarial assistance. Thanks are also given to Dr. Josephine Lai for her friendship and suggestions, and Ms. Sue Waite for the help. My special thanks are given to my parents, my aunt and my boyfriend for their deep love and tireless support and understanding. 5 TABLE OF CONTENTS Page LIST OF FIGURES .......................................................................... 9 LIST OF TABLES ............................................................................ 11 ABSTRACT ....................................................................................... 12 INTRODUCTION ............................................................................ 14 Background ................. ................................................................. 14 Opiates and Their Pharmacological Effects .............................. 14 Multiple Opioid Receptors & Endogenous Opioid Peptides . ....... ...................... ........... ........... 15 Mechanisms of Opioid Receptor Function .................... ............ 20 Delta Opioid Receptors & Selective Ligands .. 1... ...... ...... ... ..................... ... ......................... 25 Subtypes of the Delta Opioid Receptors ................................... 31 Molecular Cloning of the Opioid Receptors ............................... 37 Research Hypotheses ................................................................... 39 Explanation of Dissertation Format ............................................ 41 MATERIALS & METHODS ........................................................... 42 Cell Lines ....................................................................................... 42 Chemicals & Opioid Compounds ................................... .............. 42 Molecular Cloning Techniques ..................................................... 43 Messenger RN'A Isolation ......................................................... 39 Double Stranded cDNA Synthesis .... ..................... .................... 44 6 TABLE OF CONTENTS (Continued) Page Preparation of Delta Opioid Receptor cDNA Probes Using the Polymerase Chain Reaction (PCR) ............................. 46 Preparation of Radio labeled ([32p]) cDNA Probes .................. 47 Filter Replica Hybridization Screening of Human cDNA Libraries ........................................................ 49 Dot Blot Hybridization Analysis of cDNA Clones ..................... 53 Sequence Analysis .................................................................... 53 Reconstruction of a Human Delta Opioid Receptor (hDOR) Open Reading Frame ................................................. 55 Expression & Partial Characterization of the Reassembled Human cDNA Clone ........................................... 56 PCR Amplification of the Full Length Human Delta Opioid Receptor Open Reading Frame .. ................................... 58 COS-7 Cell Membrane Preparations ......................................... 59 RadioIigand Binding Assays .......................................................... 59 Tissue Dependence of Specific Binding .................................... 59 Saturation Studies .................................................................... 60 Inhibition Studies ..... ........................................................ ....... 61 Data Analysis .......................................................................... 62 RESULTS ....................................................................................... 63 Synthesis of Hybridization Probes ......................................... 63 Hybridization Screening of Human cDNA Libraries ............... 66 Reconstruction of a Human Delta Opioid Receptor (hDOR) ORF .......................................................... 76 Transient Expression and Partial Characterization of the Reassembled hDOR .................................................... 78 DNA and Amino Acid Sequences of the Reassembled hDOR (78x44) .................................................. 79 PCR Amplification of a Full Length hDOR ORF ............................................................................ 80 7 TABLE OF CONTENTS (Continued) Page DISCUSSION ................................................................................ 94 Cloning of a Reassembled hDOR ........................................... 94 Evidence that the Reassembled hDOR ORF Represents a Naturally Occurring hDOR ............................... 96 Sequence Comparison ofhDOR to the mDOR and rDOR ....... 97 Pharmacological Comparison ofhDOR to the mDOR and rDOR ..................................................................... 99 Delta Opioid Receptor Subtypes ............................................... 102 CONCLUSION ...............................................................................