FACTORS INFLUENCING OPIOID RECEPTOR SIGNALING TO ADENYLYL CYCLASE by Erica Sawyer Levitt A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Pharmacology) in The University of Michigan 2010 Doctoral Committee: Professor John R. Traynor, Chair Professor Henry I. Mosberg Associate Professor Jeffrey R. Martens Associate Professor Roger K. Sunahara © Erica Sawyer Levitt 2010 To my husband Trevor and my mother Barbara In memory of my father Steve ii Acknowledgements Firstly, I would like to thank my mentor Dr. John Traynor for taking the time to teach me the skills required to become a successful scientist. I appreciate his patience and guidance in learning how to think critically, prepare manuscripts, write grants and deliver presentations. I am very grateful for the true interest he has taken in preparing me for the next steps of my career. Overall, I thank him for providing a challenging and rewarding graduate school experience. I would like to thank Dr. Jeffrey Martens for providing guidance scientifically as a collaborator and thesis committee member, as well as for the special effort he has taken to help me in my career. I would also like to acknowledge Dr. Henry Mosberg and Dr. Roger Sunahara for their helpful advice and guidance. To the members of the Traynor lab, present and former, thank you for assistance scientifically and for creating a collaborative work environment. Specifically, I would like to thank Kelly Bosse, Faye Bradbury, Mary Clark, Alexander Delgado, Emily Jutkiewicz, Lauren Purington, Jennifer Thomson and Qin Wang. I would like to thank the undergraduates that have contributed to the work in this thesis, Alexander Harris, Tony McClafferty, Christine Zelnik and Jason Chen. Special thanks to those in the department who have provided assistance during my graduate career: the Jeffery Martens laboratory, especially Paul Jenkins for help with confocal imaging, lipid raft techniques and reagents; the Paul Hollenberg lab, especially Natasha Snider for providing cannabinoid ligands and advice; the Richard Neubig laboratory, including Sue Wade and Levi Blazer for providing adrenergic ligands and iii advice; the Department of Pharmacology administrative staff, especially Eileen Ferguson and Anna Taylor. I would like to recognize support and training received from the Pharmacological Sciences Training Program and the University of Michigan Substance Abuse Research Center Training Grant. I also acknowledge funding from the National Institutes of Health National Institute of Drug Abuse including a Ruth Kirschstein pre-doctoral NRSA. Finally, I would like to thank those outside of science who have supported me. My husband Trevor has been wonderful and I cannot thank him enough for the continuous encouragement and support he has provided. I would like to thank my mom for always believing in me and providing an environment that fostered my interest in learning. Last, but not least, I would like to thank my family and friends for keeping me balanced and helping me accomplish some of my other life goals, such as travel, staying active and keeping a light-hearted personality. I have made some great friends at Michigan, all of whom I am very grateful for and will miss very much. iv Table of Contents Dedication ........................................................................................................................... ii Acknowledgements ............................................................................................................ iii List of Figures ................................................................................................................... vii List of Tables ..................................................................................................................... ix List of Abbreviations .......................................................................................................... x Chapter I.............................................................................................................................. 1 General Introduction ........................................................................................................... 1 Opioid pharmacology...................................................................................................... 1 Opioid receptor signaling ................................................................................................ 4 Receptor coupling models............................................................................................... 6 Plasma membrane composition and organization........................................................... 9 Cholesterol and/or membrane microdomains in opioid receptor signaling .................. 15 Hypothesis and Aims .................................................................................................... 23 Chapter II .......................................................................................................................... 28 Differential effect of membrane cholesterol removal on MOR and DOR: A parallel comparison of acute and chronic signaling to adenylyl cyclase ....................................... 28 Summary ....................................................................................................................... 28 Introduction ................................................................................................................... 29 Materials and Methods .................................................................................................. 31 Results ........................................................................................................................... 36 v Discussion ..................................................................................................................... 47 Chapter III ......................................................................................................................... 63 Gi/o-coupled receptors compete for signaling to adenylyl cyclase in SH-SY5Y cells and reduce opioid withdrawal-mediated cAMP overshoot ..................................................... 63 Summary ....................................................................................................................... 63 Introduction ................................................................................................................... 64 Materials and Methods .................................................................................................. 65 Results ........................................................................................................................... 69 Discussion ..................................................................................................................... 75 Chapter IV ......................................................................................................................... 91 Discussion and Future Directions ..................................................................................... 91 Future directions related to compartmentalization ....................................................... 92 Future directions related to cholesterol/membrane microdomains ............................... 94 Overall summary and significance .............................................................................. 100 References ....................................................................................................................... 104 vi List of Figures Figure 1.1: Schematic of opioid receptor signaling .......................................................... 27 Figure 2.1: MOR, but not DOR, agonist-mediated G protein activation is altered following MβCD treatment. .............................................................................................. 54 Figure 2.2: Cholesterol removal by MβCD induces low-affinity binding in MOR, but not DOR, expressing cells. ...................................................................................................... 55 Figure 2.3: MOR, but not DOR, agonist-mediated inhibition of adenylyl cyclase is altered following MβCD treatment. .................................................................................. 56 Figure 2.4: MβCD treatment blocks MOR, but not DOR, agonist-mediated adenylyl cyclase sensitization. ......................................................................................................... 57 Figure 2.5: Effects of MβCD on MOR signaling are due to removal of cholesterol from the membrane. ................................................................................................................... 58 Figure 2.6: Alterations in MOR signaling are not due to changes in receptor number. .. 59 Figure 2.7: Effect of cytoskeleton inhibitors on MOR-mediated adenylyl cyclase sensitization....................................................................................................................... 60 Figure 2.8: Localization of MOR, but not DOR, in caveolin-enriched fractions, in addition to transferrin receptor-enriched fractions. .......................................................... 61 Figure 2.9: DOR is not co-localized with cholera toxin B (CTB) subunit patches. ........ 62 Figure 3.1: Gi/o-coupled receptors endogenously expressed in SH-SY5Y cells share a common pool of AC.........................................................................................................