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Arrestin Interactions with Rhodopsin in the Squid Visual System by Kelly Arrestin Interactions with Rhodopsin in the Squid Visual System By Kelly Ann Robinson A thesis submitted in conformity with the requirements for the degree of M.Sc. Graduate Department of Pharmacology and Toxicology University of Toronto © Copyright by Kelly Ann Robinson (2015) Arrestin Interactions with Rhodopsin in the Squid Visual System Kelly Ann Robinson A thesis submitted in conformity with the requirements for the degree of M.Sc. Graduate Department of Pharmacology and Toxicology, University of Toronto, 2015 Abstract Light activation of squid rhodopsin results in stimulation of the Gq signalling cascade. Activated rhodopsin (metarhodopsin) is a target for squid arrestin and squid rhodopsin kinase which are involved in the inactivation of metarhodopsin. The aim of this project is to characterize the interaction between squid rhodopsin and arrestin, and the role of phosphorylation on their interactions. We determined the affinity of arrestin for metarhodopsin to be 32nM. Two mutations to the polar core did not decrease the affinity of arrestin for metarhodopsin, suggesting a difference in basal structure of squid arrestin compared to other arrestins. Serine392 and Serine397 in the C-terminus of squid arrestin were phosphorylated by squid rhodopsin kinase. Arrestin phosphorylation decreased the affinity of arrestin binding to metarhodopsin, while metarhodopsin phosphorylation increased the dissociation of the two proteins. Further studies are required to identify mechanisms of metarhodopsin and arrestin dephosphorylation in the squid visual system. ii Acknowledgements I am extremely grateful for the opportunity that Dr. Jane Mitchell provided me by taking me on as a Master’s student. I pursued my Master’s in hopes of getting hands on experience and learning something new, and I can say that I have definitely accomplished both of these over the past two years. I am extremely grateful for the guidance and support that Dr. Mitchell provided as my supervisor; under her guidance I’ve discovered an entirely new area of pharmacology, that if you told me 10 years ago I would be studying the squid visual system—I wouldn’t have believed it. I would also like to thank Dr. Kim Sugamori, who has taught me many of the methods that I have used in this project. I’ve learned so much having you as a mentor and I greatly appreciate all of your help and all of your support! Dr. Abhishek Bandyopadhyay has also greatly contributed to the methods and knowledge that I will be taking with me from this project. Your passion for this project, and the squid visual system was very motivational. I am also thankful to Dr. Oliver Ernst for his collaboration with this project and for allowing me to produce and purify my recombinant proteins in his laboratory. I am also thankful for the continued help and support of my fellow lab members, Ariana Dela Cruz, Lucia Zhang, and Seanna Yoon, as well as the members of Dr. Ernst’s laboratory. Lastly, I am grateful to the Department of Pharmacology and Toxicology and NSERC for the funding which allowed me to perform this project. iii Table of Contents Abstract ........................................................................................................................................... ii Acknowledgements ........................................................................................................................ iii Table of Contents ........................................................................................................................... iv List of Tables ................................................................................................................................ vii List of Figures .............................................................................................................................. viii List of Abbreviations: .................................................................................................................... xi 1. Introduction ............................................................................................................................. 1 1.1. Structure of Squid Eyes ....................................................................................................... 1 1.2. Activation Pathway of the Visual System ........................................................................... 2 1.2.1. Rhodopsin......................................................................................................................... 5 1.2.2. G proteins ......................................................................................................................... 8 1.2.3. Phospholipase C (PLC) .................................................................................................. 12 1.3. Inactivation Pathway of the Visual System ....................................................................... 14 1.3.1. Squid Rhodopsin Kinase (SQRK) .................................................................................. 16 1.4. Arrestin .............................................................................................................................. 18 1.4.1. Arrestin Structure ........................................................................................................... 19 1.5. Key Binding Elements in Arrestin ..................................................................................... 20 1.5.1. Polar Core Residues ....................................................................................................... 21 1.5.2. 3- Element Interaction .................................................................................................... 22 1.5.3. N- and C- Domain Residues........................................................................................... 23 1.6. Models of Arrestin Binding ............................................................................................... 25 1.6.1. Clam-Shell Model .......................................................................................................... 25 1.6.2. Multi-Site Interaction Model .......................................................................................... 26 1.6.3. One Arrestin to Two Rhodopsin Binding Model ........................................................... 29 1.7. Invertebrate Arrestin .......................................................................................................... 31 1.8. Arrestin Phosphorylation ................................................................................................... 33 1.8.1. Role of Arrestin Phosphorylation ................................................................................... 35 1.9. Rationale ............................................................................................................................ 37 1.10. Research Goals ............................................................................................................... 37 1.11. Hypotheses ..................................................................................................................... 38 iv 2. Materials and Methods .......................................................................................................... 39 2.1. Materials ............................................................................................................................ 39 2.2. Purification of native squid visual proteins ....................................................................... 39 2.2.1. Rhodopsin....................................................................................................................... 40 2.2.2. Arrestin, SQRK and PLC ............................................................................................... 40 2.3. cDNA Preparation .............................................................................................................. 41 2.3.1. Preparation of XL1 Competent Cells ............................................................................. 42 2.4. Mutagenesis of the Phosphorylation Sites and of the Polar Core of Squid Arrestin ......... 43 2.5. Production and Preparation of Recombinant Squid Arrestin ............................................. 45 2.6. Arrestin Binding Assays .................................................................................................... 46 2.7. Phosphorylation Assays ..................................................................................................... 47 2.8. Dissociation Assays ........................................................................................................... 48 2.9. Additional Methods ........................................................................................................... 48 2.10. Data Analysis ................................................................................................................. 49 3. Results ................................................................................................................................... 49 3.1. Purification of Native Proteins of the Squid Visual System .............................................. 49 3.2. Purification of Recombinant, Strep-Tagged Squid Arrestin Proteins ................................ 55 3.3. Rhodopsin-Arrestin Association Assays ........................................................................... 58 3.4. Effect of Mutations to the Polar Core on Arrestin-Rhodopsin Binding ............................ 62 3.5.
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