Agonist-selective regulation of the mu opioid receptor by βarrestins DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Chad Edward Groer Graduate Program in Integrated Biomedical Science Program The Ohio State University 2010 Dissertation Committee: Professor Laura M. Bohn. Co-Advisor Professor Wolfgang Sadée, Co-Advisor Professor John Oberdick Professor Lane Wallace Copyright by Chad Edward Groer 2010 ABSTRACT Morphine and other opiates mediate their effects through activation of the mu opioid receptor (MOR). Activation of the MOR results in recruitment of regulatory proteins, arrestins, that can regulate how this receptor signals. In vivo studies suggest that disruption of βarrestin-mediated MOR regulation may enhance opiate-induced antinociception and reduce tolerance and certain unwanted side effects. Therefore, by understanding the cellular mechanisms by which this receptor is regulated, the development of analgesics which preserve the beneficial effects of opiates while eliminating unwanted side effects may be possible. In this dissertation we test the hypothesis that MOR agonists can bias MOR-arrestin interactions, and that arrestin recruitment profiles, in turn, may determine cellular responses evoked by these agonists. In the first data portion of this dissertation, we characterize several novel MOR agonists that are unable to promote βarrestin recruitment. Herkinorin is a moderately selective agonist at the MOR, based on the structure of a natural product, Salvinorin A. We find that herkinorin promotes very little MOR phosphorylation, does not recruit βarrestins, and does not induce receptor internalization in transfected cells. Herkinorin is unable to induce βarrestin recruitment or MOR internalization under conditions that facilitate receptor ii phosphorylation and subsequent arrestin recruitment with other agonists. We also evaluated several derivatives of herkinorin with similar βarrestin recruitment and MOR internalization profiles. Therefore, herkinorin and its derivatives may be a promising step toward recapitulating morphine’s effects in βarr2-KO mice, which have been used to demonstrate that MOR activation without recruiting βarrestin2 may be therapeutically useful, by producing analgesia with reduced side effects. In the second data portion of this dissertation, we evaluate the interaction and functional consequences of MOR regulation by βarrestin1 and βarrestin2, in response to the classical agonists, DAMGO and morphine. Using both qualitative (microscopy) and quantitative (cell surface biotinylation and BRET) approaches, we have confirmed that DAMGO can induce robust interactions between the MOR and both βarrestins. Morphine, however, selectively promotes interactions with βarrestin2. Additionally, the agonist specific βarrestin interactions are required for internalization of the MOR. Finally, we show that βarrestin1 is required for agonist-induced MOR ubiquitination, such that only DAMGO, and not morphine, is able to promote MOR ubiquitination. Taken together, these data suggest that MOR regulation is highly dependent on the complement of proteins available to interact with the MOR, and that the nature of the ligand can determine how the MOR is regulated by the available proteins. Therefore, the development of biased ligands for the MOR should focus activation of the MOR, but circumventing βarrestin-mediated iii regulation. These concepts may be critical to consider in the development of opiate compounds designed to retain analgesic efficacy, while reducing the occurrence of unwanted side effects. iv DEDICATION This dissertation is dedicated to: my parents, Ed and Lana Groer and my brother, sister-in-law, and niece, Eric, Trish, and Autumn Groer. v ACKNOWLEDGMENTS First of all, I would like to thank my advisor, Laura M. Bohn, Ph.D., for her guidance, resources, and patience during my graduate studies. In addition to providing all resources that needed to perform my dissertation research, she provided invaluable advice, guidance, and encouragement regarding my project, without which, I would have not succeeded. Next, I would like to thank all members of our lab, both past and present. Kirsten Raehal (formerly grad student, now post-doc) and Cullen Schmid (grad student) have been with me in Laura’s lab since the beginning. Both of these women have helped me countless times with technical and writing issues and have helped me think critically about my project. Both Kirsten and Cullen are great colleagues and great friends. John Streicher (post-doc) helped with technical issues and dissertation writing. Alex Jaeger (undergrad) helped develop the BRET cell lines. Bob Moyer, Ph.D., Lori Hudson, and Sarah Teich are former members of the lab who have also helped me tremendously, as well. I would like to thank my committee members for their time and for their suggestions concerning my dissertation work: Wolfgang Sadee, Dr.rer.nat (co- vi advisor), John Oberdick, Ph.D., and Lane Wallace, Ph.D. I would also like to thank Gopi Tejwani, Ph.D. for serving on my candidacy exam committee with only days notice to replace one member who could not serve due to health problems. I would like to thank Tom Prisinzano, Ph.D. at the University of Kansas (formerly Univ. of Iowa) and his lab members Kevin Tidgewell, PhD. and Kim Lovell for synthesizing herkinorin and the derivatives. Thanks to Robert Lefkowitz, M.D. at Duke University for providing all four MEF lines. I would like to thank Dr. Allen Yates, Dr. Virginia Sanders, and Dr. Tom Boyd for their work in directing the IBGP program. I would like to thank all administrative assistants that have made my life much less hectic, including Christine Kerr, Sherry Ring, Gina Pace, Amy Lahmers, Kelly Dillon, Elaine Wakely, Mary Krosky, and Lynn Wylie. This work was funded by NIDA: DA14600 (LMB), DA18860 (LMB), DA18151 (TEP). vii VITA March 28, 1982 . ……………….. ... Born – Pittsburgh, PA June 2000....…………..…………….Graduated from Quigley Catholic High School Baden, PA 2002-2004....................................................... Undergraduate Research Assistant Dept. of Molecular and Cellular Biochemistry College of Medicine The Ohio State University Columbus, OH June 2004. …......……………………. .. .. B.S. in Pharmaceutical Sciences Magna Cum Laude, Graduated with Distinction and Honors College of Pharmacy The Ohio State University Columbus, OH June 2004-Present……………...……………….…………..……….Ph.D. Candidate Integrated Biomedical Science Graduate Program College of Medicine The Ohio State University Columbus, OH March 2009-Present………………………..…………… External Graduate Student Scripps Florida Jupiter, FL viii Publications Tidgewell K, Groer CE, Harding WW, Lozama A, Schmidt M, Marquam A, Hiemstra J, Partilla JS, Dersch CM, Rothman RB, Bohn LM, and Prisinzano TE. Herkinorin Analogues with Differential β-Arrestin-2 Interactions. J. Med. Chem. 2008 Apr 24;51(8):2421-31. Groer CE, Tidgewell K, Moyer RA, Harding WW, Rothman RB, Prisinzano TE, and Bohn LM. An opioid agonist that does not induce mu-opioid receptor-- arrestin interactions or receptor internalization. Mol Pharmacol. 2007 Feb;71(2):549-57. Fields of Study Major Field: Integrated Biomedical Science Program Emphasis in Molecular Pharmacology, Pharmacogenomics, and Therapeutics ix Table of Contents Abstract ................................................................................................................ii Dedication ............................................................................................................ v Acknowledgments ................................................................................................vi Vita ..................................................................................................................... viii List of Tables ...................................................................................................... xiii List of Figures ..................................................................................................... xiv CHAPTER 1 Introduction .................................................................................. 1 1.1 Clinical Aspects of Opioids ......................................................................... 1 1.2 Opioid Receptors ....................................................................................... 2 1.3 Classical Regulation of G Protein-Coupled Receptors ............................... 3 1.4 Functional Selectivity.................................................................................. 7 1.5 Agonist-Directed Regulation of the MOR- In Vitro Evidence ...................... 8 1.5.1 MOR Phosphorylation .......................................................................... 9 1.5.2 βarrestin Recruitment ......................................................................... 10 1.5.3 Internalization ..................................................................................... 13 1.5.4 Desensitization ................................................................................... 14 x 1.5.5 Agonist-Directed MOR Signaling ....................................................... 16 1.6 Functional Selectivity at the Mu Opioid Receptor- In Vivo Relevance ...... 17 1.7 Hypothesis and Overview of Chapters 2 and 3 ........................................ 19 1.8 Figures ..................................................................................................... 22 CHAPTER 2