Identifying signaling differences between GPCR-induced growth factor receptor transactivation and direct ligand activation by Azita Kouchmeshky A thesis presented to the University of Waterloo in fulfillment of the thesis requirement for the degree of Master of Science in Pharmacy Waterloo, Ontario, Canada, 2014 ©Azita Kouchmeshky 2014 AUTHOR'S DECLARATION I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including any required final revisions, as accepted by my examiners. I understand that my thesis may be made electronically available to the public. ii Abstract Growth factor receptors have significant effects on various normal function of body such as cell proliferation, differentiation and apoptosis. They are also involved in neuronal function and dysfunction, cardiovascular diseases, and malignancies. Recently, multiple G protein- coupled receptors (GPCRs) have been shown to transactivate receptor tyrosine kinases (RTKs). Since both classes of receptors have complicated downstream cascades individually, understanding the signaling differences between GPCR-induced growth factor receptor transactivation and direct ligand activation is an important challenge. To clarifying this phenomenon we investigated the phosphorylation profile and downstream effectors of ligand-activated vs. transactivated PDGFβ receptors. Dopamine receptors (one of the receptors of the GPCRs family) were used to compare the PDGFβ receptor phosphorylation and activity during direct activation and transactivation. Dose-response and time-course data between these two stimuli were evaluated. Furthermore, the phosphorylation site profiles and the intracellular signaling pathways of PDGFβ receptor after direct activation and transactivation were examined. In addition, possible synergic effects between transactivation and direct activation were explored. The results of this project showed that the phosphorylation profile and downstream effectors of ligand activated receptors versus transactivated receptors are different. Our data indicated that transactivation-induced pathways are more involved in survival and proliferation effects compared to ligand activation. This research answered basic questions about transactivation phenomena and proposes that these transactivation pathways could be exploited as a therapeutic approach for neurodegenerative diseases. iii Acknowledgements I wish to thank my supervisor Professor Michael Beazely for his countless hours of supervising, supporting, reflecting, reading, encouraging, and giving useful advice during my Master degree that made the completion of this project an enjoyable experience. Special thanks to Professors Praveen Nekkar and Paul Spagnuolo my committee members and Professor Jamie Joseph my committee chairman who were more than generous with their expertise and precious time. I would like to acknowledge and thank Janne Janke from writing center for her helpful advice and friendly attitude during tutoring and correcting my writing in different stages of my Master thesis. I would like to thank CIHR training program in bridging scientific domain for drug safety and effectiveness (DSECT program) for funding and supporting this research. Finally, special thanks to my friends in the School of Pharmacy and in Canada, and to the faculty and staff of the School of Pharmacy University of Waterloo for their continued support. iv Dedication A special feeling of gratitude to my loving mother, Lili, who has given me dreams to look forward to and love to support my journey in every aspect of my life To my father, Ali Reza, for being so supportive To my loving brother, Babak, who never left me alone and for being very special & To my special aunt and uncle, Parvin & Richard & To all my family and friends in Iran, Europe, and the U.S.A. v Table of Contents AUTHOR'S DECLARATION ................................................................................................. ii Abstract ................................................................................................................................... iii Acknowledgements ................................................................................................................ iv Dedication ................................................................................................................................ v Table of Contents ................................................................................................................... vi List of Figures ........................................................................................................................ viii List of Tables ........................................................................................................................... ix Abbreviations ............................................................................................................................ x Chapter 1 Introduction........................................................................................................... 1 1.1 Cell-to-cell communication ............................................................................................. 1 1.2 G-protein-coupled receptors (GPCRs) ............................................................................ 2 1.2.1 Dopamine and dopamine receptors .......................................................................... 5 1.2.2 Serotonin and serotonin receptors ........................................................................... 6 1.3 Receptor tyrosine kinases (RTKs)................................................................................... 6 1.3.1 Platelet-derived growth factors and their receptors ................................................ 7 1.4 Transactivation .............................................................................................................. 31 1.5 Rationale ....................................................................................................................... 33 1.6 Objectives ...................................................................................................................... 34 Chapter 2 Materials and Methods....................................................................................... 35 2.1 Materials ........................................................................................................................ 35 2.2 Methods ......................................................................................................................... 36 2.2.1 Treatment, cell lysis, protein assay ........................................................................ 37 2.2.2 Western blotting ...................................................................................................... 40 2.2.3 Statistical analysis .................................................................................................. 46 Chapter 3 Results .................................................................................................................. 47 3.1 Comparison between transactivation of PDGFβ receptors by various agonists of GPCRs and direct activation of PDGFβ receptors ........................................................ 47 3.2 Time-course for quinpirole transactivation of the PDGFβ receptor ............................. 49 3.3 Raclopride blocks transactivation of PDGFβ receptor by quinpirole ........................... 50 3.4 Dose-response curve for quinpirole transactivation ...................................................... 52 3.5 Does ligand-activation versus transactivation of the PDGFβ receptor produce different tyrosine phosphorylation profiles .................................................................................. 54 3.6 Infra-additive effects between direct activation by PDGF-BB and transactivation by serotonin ........................................................................................................................ 57 3.7 Downstream effectors activation by quinpirole and PDGF-BB .................................... 59 Chapter 4 Discussion ............................................................................................................ 68 4.1 Comparison between transactivation and direct activation ........................................... 68 4.1.1 Phosphorylation of PDGFβ receptors by transactivation at tyrosine residues ..... 69 4.1.2 Infra-additive effects between serotonin receptors and PDGFβ receptors ............ 73 4.1.3 MAPK/ERK pathway and transactivation .............................................................. 74 4.1.4 PI3-kinase/Akt pathway and transactivation .......................................................... 77 4.1.5 PLCγ1 pathway and transactivation ...................................................................... 82 vi 4.1.6 Other important results .......................................................................................... 83 4.1.7 Potential physiological relevance of transactivation to neurodegenerative diseases and psychiatric disorders .......................................................................... 83 4.2 Semantics: distinguishing receptor transactivation & gene transactivation .................. 85 4.3 Fundamental questions (and potential answers) about transactivation ......................... 86 1.Why do cells have two similar pathways to regulate and activate RTK receptors? .. 86 2.Why do cells in the body require transactivation at all since