Purdue University Purdue e-Pubs Open Access Dissertations Theses and Dissertations Fall 2013 Novel Modulation of Adenylyl Cyclase Type 2 Jason Michael Conley Purdue University Follow this and additional works at: https://docs.lib.purdue.edu/open_access_dissertations Part of the Medicinal-Pharmaceutical Chemistry Commons Recommended Citation Conley, Jason Michael, "Novel Modulation of Adenylyl Cyclase Type 2" (2013). Open Access Dissertations. 211. https://docs.lib.purdue.edu/open_access_dissertations/211 This document has been made available through Purdue e-Pubs, a service of the Purdue University Libraries. Please contact [email protected] for additional information. Graduate School ETD Form 9 (Revised 12/07) PURDUE UNIVERSITY GRADUATE SCHOOL Thesis/Dissertation Acceptance This is to certify that the thesis/dissertation prepared By Jason Michael Conley Entitled NOVEL MODULATION OF ADENYLYL CYCLASE TYPE 2 Doctor of Philosophy For the degree of Is approved by the final examining committee: Val Watts Chair Gregory Hockerman Ryan Drenan Donald Ready To the best of my knowledge and as understood by the student in the Research Integrity and Copyright Disclaimer (Graduate School Form 20), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy on Integrity in Research” and the use of copyrighted material. Approved by Major Professor(s): ____________________________________Val Watts ____________________________________ Approved by: Jean-Christophe Rochet 08/16/2013 Head of the Graduate Program Date i NOVEL MODULATION OF ADENYLYL CYCLASE TYPE 2 A Dissertation Submitted to the Faculty of Purdue University by Jason Michael Conley In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2013 Purdue University West Lafayette, Indiana ii For my parents iii ACKNOWLEDGEMENTS I am very grateful for the mentorship of Dr. Val Watts. His advice and support has fostered my continued growth as a scientist and I am sure that much of what I have learned from Val will also serve me well outside of the laboratory setting. I would also like to thank Dr. Greg Hockerman, Dr. Don Ready, and Dr. Dave Nichols for providing helpful feedback as thesis advisory committee members and Dr. Ryan Drenan for his participation as a thesis examination committee member. Scientific research is a highly collaborative and cumulative process. As such, the research reported in the present dissertation would not have been possible without the contributions of numerous former members of the Watts laboratory. Their studies created a solid foundation to build upon. I also thank Dr. Ben Chemel and Dr. Pierre-Alexandre Vidi, as they kindly provided a great deal of assistance with nearly all aspects of laboratory research during my early years of graduate school. Furthermore, their work ethics and expertise set high standards for me to work toward. I thank Dr. John McCorvy for his friendship and many thought-provoking conversations about our research. I thank Dr. Karin Ejendal, as she often provided a different perspective and also set a great example for both a mature and positive approach to research. I also appreciate iv the friendship of Elisabeth Garland-Kuntz and her hard work on several shared laboratory projects. I thank Tarsis Brust Fernandes, Monica Soto-Velasquez, and each of my colleagues that were acknowledged above for contributing to a fun and stimulating laboratory environment. Finally, I thank my best friend and the love of my life, Suzanne Elliott. The love, inspiration, and support that she provided at home facilitated my achievements in the laboratory. v TABLE OF CONTENTS Page ABSTRACT ........................................................................................................ viii CHAPTER 1. INTRODUCTION ............................................................................ 1 1.1 The foundation of second messenger signaling: A historical perspective .. 1 1.2 Adenylyl cyclase topology and catalytic mechanism .................................. 5 1.3 Overview of cAMP signaling: Adenylyl cyclase as a signal integrator within the cascades that link extracellular stimuli and the resulting cellular responses ......................................................................................................... 8 1.4 Adenylyl cyclase signaling properties: Contributions to cAMP signaling specificity ........................................................................................................ 15 1.4.1 G protein-coupled receptor signaling ................................................ 15 1.4.1.1 G protein activation cycle .......................................................... 15 1.4.1.2 Effector modulation .................................................................... 18 1.4.1.3 Modulation of G protein cycle by accessory proteins ................. 21 1.4.2 Regulatory properties of adenylyl cyclase isoforms .......................... 22 1.4.2.1 Group I ACs ............................................................................... 22 1.4.2.2 Group II ACs .............................................................................. 24 1.4.2.3 Group III ACs ............................................................................. 28 1.4.2.4 Group IV AC .............................................................................. 30 1.4.3 Adenylyl cyclase localization: Tissue distribution, compartmentalization, and signalosomes.................................................... 32 1.5 Heterologous sensitization of adenylyl cyclase ........................................ 37 1.6 Adenylyl cyclase isoforms in physiology and disease .............................. 41 1.6.1 Adenylyl cyclase function in peripheral tissues ................................. 42 1.6.2 Adenylyl cyclase function in the central nervous system .................. 44 1.7 Small molecule modulation of adenylyl cyclase ....................................... 46 1.8 Scope of the work .................................................................................... 52 vi Page CHAPTER 2. DIFFERENTIAL EFFECTS OF AGS3 EXPRESSION ON D2L DOPAMINE RECEPTOR-MEDIATED ADENYLYL CYCLASE SIGNALING ...... 54 2.1 Introduction .............................................................................................. 54 2.2 Materials and methods............................................................................. 57 2.2.1 Materials ........................................................................................... 57 2.2.2 Cell culture and stable cell line generation ....................................... 58 2.2.3 Cyclic AMP accumulation assay ....................................................... 59 2.2.4 Cyclic AMP quantification ................................................................. 60 2.2.5 Data analysis .................................................................................... 60 2.3 Results and discussion ............................................................................ 61 2.3.1 Characterization of HEK293 stable cell lines .................................... 61 2.3.2 Modulation of acute D2LDR-mediated AC1 and AC2 signaling by AGS3 expression ........................................................................................ 67 2.3.3 Desensitization of D2LDR-mediated AC1 and AC2 signaling ............ 70 2.3.4 Effects of AGS3 expression on heterologous sensitization of AC1 and AC2 .......................................................................................................... 75 2.3.5 Significance of AGS3-regulated AC signaling to drugs of abuse ...... 79 CHAPTER 3. HETEROLOGOUS SENSITIZATION OF ADENYLYL CYCLASE 2 IS DEPENDENT ON G PROTEIN BETA-GAMMA SUBUNIT SIGNALING ........ 81 3.1 Introduction .............................................................................................. 81 3.2 Materials and Methods............................................................................. 83 3.2.1 Materials ........................................................................................... 83 3.2.2 Cell culture and transfection ............................................................. 83 3.2.3 Cyclic AMP accumulation assay ....................................................... 84 3.2.4 Cyclic AMP quantification ................................................................. 85 3.3 Results ..................................................................................................... 85 3.4 Discussion ............................................................................................... 93 vii Page CHAPTER 4. DEVELOPMENT OF A HIGH-THROUGHPUT SCREENING PARADIGM FOR THE DISCOVERY OF SMALL MOLECULE MODULATORS OF ADENYLYL CYCLASE: IDENTIFICATION OF AN ADENYLYL CYCLASE 2 INHIBITOR ......................................................................................................... 97 4.1 Introduction .............................................................................................. 97 4.2 Materials and methods............................................................................. 99 4.2.1 Materials ........................................................................................... 99 4.2.2 Stable cell line generation and cell culture conditions..................... 100 4.2.3 Cisbio HTRF cAMP assay .............................................................. 101 4.2.4 Screening conditions ...................................................................... 102 4.2.5 3H-cAMP