Differential regulation of serotonin 2A receptor responsiveness by agonist- directed interactions with arrestin2 DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Cullen Laura Schmid, B.S. Neuroscience Graduate Studies Program The Ohio State University 2011 Dissertation Committee: Laura M. Bohn, Co-advisor Georgia A. Bishop, Co-advisor Candice C. Askwith Wolfgang Sadee Copyright by Cullen Laura Schmid 2011 Abstract The G protein-coupled, serotonin 2A (5-HT2A) receptor is a major drug target for the treatment of a number of mental health disorders, including schizophrenia, anxiety and depression. In addition to modulating several of the physiological effects of the neurotransmitter serotonin, activation of the 5-HT2A receptor mediates the psychotomimetic effects of serotonergic hallucinogenic drugs, such as lysergic acid diethylamide (LSD), 2,5-dimethoxy-4-iodoamphetamine (DOI) and 5-methoxy-N,N- dimethyltryptamine (5-MeO-DMT). Though hallucinogens are agonists at the 5-HT2A receptor, not all 5-HT2A receptor agonists induce hallucinations in humans, including the endogenous ligand serotonin. Therefore, the activation of the 5-HT2A receptor can result in different biological responses depending upon the chemical nature of the ligand, a concept that has been referred to as “functional selectivity.” One way in which ligands can induce differential signaling at GPCRs is through interactions with arrestins, which can act to dampen or facilitate receptor signaling cascades or mediate the internalization of receptors into intracellular vesicles. The overarching hypothesis of this dissertation is that the interaction between the regulatory protein, arrestin2, and the 5-HT2A receptor is a critical point in the divergence of agonist-directed 5-HT2A receptor responsiveness. Using mice lacking arrestin2, we evaluate 5-HT2A receptor trafficking and signaling in vivo for serotonin, the hallucinogenic agonists DOI and 5-MeO-DMT and the endogenous, hallucinogenic metabolite of serotonin, N-methylserotonin. We find that ii arrestin2 mediates 5-HT2A receptor trafficking in primary neuronal cultures and can facilitate 5-HT2A receptor-mediated signaling cascades in the mouse frontal cortex, although its role is entirely dependent upon the agonist acting at the receptor. Serotonin requires arrestins to internalize the 5-HT2A receptor and to scaffold the signaling kinases Akt and Src to the receptor. The formation of this receptor scaffold results in an increase in Akt activity, which is disrupted in the absence of arrestin2, while the reintroduction of arrestin2 into primary cortical neurons rescues serotonin-induced phosphorylation of Akt. Moreover, the disruption of these cellular events, either by the absence of arrestin2 or by inhibiting the kinases, results in the inability of serotonin to induce the head twitch response in mice, which is a behavioral model of 5-HT2A receptor activation in the mouse frontal cortex. In contrast, DOI maintains its ability to internalize the 5-HT2A receptor in the absence of arrestin2 and DOI, 5-MeO-DMT and N- methylserotonin do not activate arrestin2-mediated signaling cascades in mouse embryonic fibrobalsts, primary cortical neurons or the mouse frontal cortex. The activation of the head twitch response by these hallucinogenic agonists is not disrupted in arrestin2-knockout mice or in the presence of inhibitors to Akt. Collectively, these studies advance our understanding of the mechanism through which 5-HT2A receptor activation by different agonists can lead to distinct regulatory and signaling pathways in vivo. These studies suggest that agonist-directed 5-HT2A receptor regulation bifurcates based on interactions with arrestin2. Moreover, the elucidation of these signaling pathways could have far reaching implications for the treatment of those neuropsychiatric disorders which have been associated with the disregulation of the 5- HT2A receptor. iii Dedication This dissertation is dedicated to my family. The twitch of the head is telling, shows variance in signaling. „tonin activates, arrestin migrates, not the N-methyltryptamines. -Steve Schmid iv Acknowledgments I am much in debt to a number of individuals who having guided and supported me throughout my graduate school training. First and foremost, I must thank my thesis advisor, Dr. Laura M. Bohn. I am extremely grateful to have had her as a mentor throughout this process. She has guided every aspect of this dissertation and I am thankful for the incredible amount of time, support, encouragement, insight and guidance that she has given me over the years. I am much indebted to Dr. Bohn for all of the opportunities she has provided me with, including allowing me to present at national meetings, to pursue external funding for my project and to publish my findings in scientific journals. I am enormously grateful for her investment in me and for the incredible scientific training with which she has provided me. I would also like to thank the members of my thesis committee, Dr. Georgia Bishop, Dr. Candice Askwith and Dr. Wolfgang Sadee, for their time, guidance and patience throughout my graduate training. Special thanks to Dr. Bishop for serving as my co- advisor following Dr. Bohn‟s departure from Ohio State. I am also grateful to Dr. Askwith for allowing me to complete a ten week rotation in her laboratory, during which time I learned how to culture primary cortical neurons under her guidance. v I am also incredibly grateful to the past and present members of Dr. Bohn‟s laboratory. I would like to thank Kirsten Raehal and Chad Groer for their scientific insights into my project and technical expertise. I would like to thank Lori Hudson for her assistance in maintaining the mouse colony and managing the laboratory at Ohio State. I would also like to thank Kim Lovell, John Streicher and Robert Moyer for their intellectual contributions to this work. Most of all I would like to thank all of these individuals for their constant support, encouragement and friendship. They kept me going when I wanted to quit, celebrated with me when I succeeded and made coming to work every day enjoyable. Additionally, I would also like to thank the faculty and staff at both research institutions where this dissertation work was completed. Namely, Keri Bantz, Cheryl Ring, Gina Alderson, the faculty members of the Neuroscience Graduate Studies Program and the vivarium staff at The Ohio State University. I would also like to thank Mary Krosky and the vivarium staff at The Scripps Research Institute. Finally, I would like to acknowledge my friends and family for their constant love and support throughout my doctoral training. None of this would have been possible without them. vi Vita June 2001 .................................................... Valedictorian Wooster High School Wooster, Ohio Summer of 2004 ........................................... Research Intern Department of Medicine Pennsylvania State University College of Medicine Hershey, Pennsylvania May 2005 ..................................................... B.S. Biochemistry, Summa Cum Laude Messiah College Grantham, Pennsylvania September 2005 to present .......................... Graduate Research Associate Neuroscience Graduate Studies Program The Ohio State University College of Medicine Columbus, Ohio 2005 to 2006 ............................................... University Fellow The Ohio State University Columbus, Ohio March 2009 to present ................................ External Graduate Student Departments of Molecular Therapeutics and Neuroscience The Scripps Research Institute Jupiter, Florida vii Publications Schmid CL, Bohn LM (2010) Serotonin, but not N-methyltryptamines, activates the serotonin 2A receptor via a arrestin2/Src/Akt signaling complex in vivo. Journal of Neuroscience 30:13513-13524. Bohn LM, Schmid CL (2010) Serotonin receptor signaling and regulation via arrestins. Critical Reviews in Biochemistry and Molecular Biology 45:555-566. Fei G, Raehal K, Liu S, Qu MH, Sun X, Wang GD, Wang XY, Xia Y, Schmid CL, Bohn LM, Wood JD (2010) Lubiprostone reverses the inhibitory action of morphine on intestinal secretion in guinea pig and mouse. Journal of Pharmacology and Experimental Therapeutics 334:333-340. Schmid CL, Bohn LM (2009) Physiological and pharmacological implications of arrestin regulation. Pharmacology and Therapeutics 121:285-293. Raehal KM, Schmid CL, Medvedev IO, Gainetdinov RR, Premont RT, Bohn LM (2009) Morphine-induced physiological and behavioral responses in mice lacking G protein-coupled receptor kinase 6. Drug and Alcohol Dependence 104:187-196. Schmid CL, Raehal KM, Bohn LM (2008) Agonist-directed signaling of the serotonin 2A receptor depends on arrestin2 interactions in vivo. Proceedings of the National Academy of Sciences of the United States of America 105:1079-1084. Kenney SP, Lochmann TL, Schmid CL, Parent LJ (2008) Intermolecular interactions between retroviral Gag proteins in the nucleus. Journal of Virology 82:683-691. Fields of Study Major Field: Neuroscience Graduate Studies Program viii Table of Contents Abstract ............................................................................................................................. ii Dedication ........................................................................................................................ iv Acknowledgments ............................................................................................................