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Distribution Agreement In presenting this thesis or dissertation as a partial fulfillment of the requirements for an advanced degree from Emory University, I hereby grant to Emory University and its agents the non-exclusive license to archive, make accessible, and display my thesis or dissertation in whole or in part in all forms of media, now or hereafter known, including display on the world wide web. I understand that I may select some access restrictions as part of the online submission of this thesis or dissertation. I retain all ownership rights to the copyright of the thesis or dissertation. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. Signature: _____________________________ ______________ Ryan H. Purcell Date Activation and regulation of the brain-expressed adhesion G protein-coupled receptors ADGRB1/BAI1 and ADGRB2/BAI2: Implications for human disease By Ryan Herndon Purcell Doctor of Philosophy Graduate Division of Biological and Biomedical Science Neuroscience Program _________________________________________ Randy A. Hall Advisor _________________________________________ Gary J. Bassell Committee Member _________________________________________ John R. Hepler Committee Member _________________________________________ Thomas Kukar Committee Member _________________________________________ Erwin G. Van Meir Committee Member Accepted: _________________________________________ Lisa A. Tedesco, Ph.D. Dean of the James T. Laney School of Graduate Studies ___________________ Date Activation and regulation of the brain-expressed adhesion G protein-coupled receptors ADGRB1/BAI1 and ADGRB2/BAI2: Implications for human disease By Ryan Herndon Purcell B.A., Johns Hopkins University, 2009 Advisor: Randy A. Hall, Ph.D. An abstract of A dissertation submitted to the Faculty of the James T. Laney School of Graduate Studies of Emory University In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Graduate Division of Biological and Biomedical Science Neuroscience Program Abstract Activation and regulation of the brain-expressed adhesion G protein-coupled receptors ADGRB1/BAI1 and ADGRB2/BAI2: Implications for human disease By Ryan Herndon Purcell The adhesion G protein-coupled receptors (GPCRs) are the second-largest family of GPCRs in the human genome. Most adhesion GPCRs are considered to be orphan receptors with few widely-accepted natural ligands. However, the unique architecture and common domain structures of these receptors invites the hypothesis that they may have a common activation mechanism. While the activation and signaling mechanisms of these receptors largely remain enigmatic, even less is understood in terms of how these proteins are regulated. The first third of this dissertation focuses on progress in understanding the activation mechanisms of the adhesion GPCR BAI1/ADGRB1 (B1). We found that removing almost the entire extracellular amino terminus of the receptor has no negative effect on the signaling activity of the receptor, with the heavily truncated receptor being recognized by β-arrestins and G proteins as an active receptor conformation. Thus, contrary to the tethered cryptic agonist model that has been proposed based on studies of several other aGPCRs, activation of B1 does not appear to require a tethered cryptic agonist. Next, we applied similar methods to study a human disease-associated mutation in the closely-related receptor BAI2/ADGRB2 (B2). This mutation was found in an adult female patient who has suffered from a degenerative neuromuscular condition since adolescence. We found that this arginine to tryptophan (R1465W) substitution in the intracellular C terminus of B2 significantly increases the receptor’s signaling output, but only in the active form of the receptor that has a truncated N-terminus (B2DNT). This mutation also increases receptor surface expression, and our results suggest two mechanisms underlying these differences: increased flexibility of G protein coupling and disruption in the receptor’s interaction with the endocytic protein endophilin A1. In the final third of this dissertation, studies are presented on regulation of B1 signaling by C-terminal determinants. We found that deletion of the B1 proline-rich region drastically biases the receptor’s signaling activity by eliminating G protein- dependent signaling to SRF-luciferase and greatly potentiating activity to NFAT. Finally, we report on the identification of three mutations in B1 that were discovered in individuals with schizophrenia. One of these mutations is in a similar location as the B2- R1465W, is also an ArgàTrp substitution, and greatly increases receptor activity. Taken together, these studies provide a deeper understanding of two adhesion GPCRs in terms of their signaling and regulation. Moreover, the findings described here provide a basis to connect the activity of these receptors to human diseases and set the stage for the eventual targeting of these receptors by therapeutics. Activation and regulation of the brain-expressed adhesion G protein-coupled receptors ADGRB1/BAI1 and ADGRB2/BAI2: Implications for human disease By Ryan Herndon Purcell B.A., Johns Hopkins University, 2009 Advisor: Randy A. Hall, Ph.D. A dissertation submitted to the Faculty of the James T. Laney School of Graduate Studies of Emory University In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Graduate Division of Biological and Biomedical Science Neuroscience Program Acknowledgments This work is dedicated to Riley Steiner, a source of inspiration and a steadfast and loving supporter the entire way. Special thanks to Dr. Randy Hall for unwavering optimism, exceptional ideas, and tireless mentoring. To laboratory colleagues Dr. Jason Stephenson, Zahra Nassiri-Toosi, Ayush Kishore, Michelle Giddens, Brilee Coleman and Dr. Sharon Owino for extra hands and insightful discussions. To committee members Dr. Gary Bassell, Dr. John Hepler, Dr. Tom Kukar, and Dr. Erwin Van Meir for expert advice and for guiding this work to completion. To Dr. Karen Rommelfanger, Dr. Paul Root Wolpe, Dr. Gillian Hue, Dr. John Banja, and the editorial team of AJOB Neuroscience for providing opportunities and encouraging scholarship in neuroethics. Additional thanks to Dr. David Weinshenker and Dr. Andrew Escayg for early career mentoring. Thanks to Dr. Kellie Tamashiro and Dr. Timothy Moran for igniting my interest in research. Sincere thank you to Bill and Marsha Purcell for encouraging intellectual curiosity and the pursuit of my passions. Additional acknowledgments to Dr. Malu Tansey, Dr. David Weinshenker, Dr. Victor Faundez, and Gary Longstreet for exceptional leadership of the Emory neuroscience graduate program. Final thank you to Dr. Kristian Blaich, Dr. David Saunders, Lance Harden, Dougie Coffed, and Jay Holder for helping to keep me on track. Table of Contents CHAPTER 1. INTRODUCTION ................................................................................................. 1 1.1 CELL SURFACE RECEPTORS .................................................................................................... 2 1.2 KEY FEATURES OF GPCRS ..................................................................................................... 7 1.3 REGULATION OF GPCR SIGNALING ACTIVITY ....................................................................... 8 1.4 MUTATIONS IN GPCRS CAN CAUSE HUMAN DISEASES ....................................................... 15 1.5 ADHESION GPCR FAMILY ................................................................................................... 16 1.5.1 Adhesion GPCR structure ............................................................................................ 20 1.6 ADHESION GPCR ACTIVATION MECHANISMS ..................................................................... 25 1.6.1 Adhesion GPCR Signaling Pathways and Ligands ...................................................... 32 1.6.2 Adhesion GPCR Ligands .............................................................................................. 36 1.7 ADHESION GPCRS IN DISEASE ............................................................................................ 39 1.7.1 Neuropsychiatric disorders .......................................................................................... 39 1.7.2 Cancer .......................................................................................................................... 42 1.7.3 Immune, Cardiac, and Pulmonary ............................................................................... 45 1.8 THE BAI SUB-FAMILY OF ADHESION GPCRS ...................................................................... 48 1.9 RESEARCH AIMS ................................................................................................................... 51 CHAPTER 2. ADGRB1 ACTIVATION – IS THERE A TETHERED AGONIST? ............ 54 2.1 INTRODUCTION .................................................................................................................... 55 2.2 EXPERIMENTAL PROCEDURES .............................................................................................. 58 2.2.1 Cell culture ................................................................................................................... 58 2.2.2 DNA Constructs ........................................................................................................... 58 2.2.3 Western Blot
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