Novel insights into the interaction of the Calcium Sensing Receptor with the Receptor Activity Modifying Proteins PhD Aditya Jwalant Desai Department of Human Metabolism November 2012 Acknowledgements: It is very easy to name all the people who helped me get through this, though it is equally difficult to thank them adequately. First and foremost, I would like to extend my deepest gratitude to my supervisor, Prof.Tim Skerry for giving me an opportunity to work with him. Without his constant support, encouragement, optimism and teaching; this thesis would not have been possible. I thank him for believing in me especially during the “downs” of the project. He taught me that scientific research is the disinterested reporting of observations, which I shall always follow. I would like to thank Dr.Gareth Richards from the bottom of my heart for perusing my thesis, for being an excellent mentor and a very good friend. I thank him for being supportive after my project got changed near the end of the first year and helping me in framing the current project. He made me understand science clearly and simply by ignoring my endless mistakes and has played an instrumental role in my training. I am also deeply grateful to Dr.Dave Roberts for his invaluable and enduring training, guidance and support. He taught me techniques like molecular cloning, FRET and SPA used in this project. I also thank him for kindly providing me with the constructs used in this project. The discussions, encouragement and critiques made by Gareth and Dave were of immense significance to the progress of this work. I would like to thank Dr. Peter Grabowski for his excellent suggestions and insights during my PhD. I thank Dr.Stephen Brown from the RNAi facility for letting me use the plate-reader for Ca2+ imaging. I thank Dr.Colin Gray for helping me with microscopy. I am grateful to Dr.Paul Heath and Dr.Clive Buckle for allowing me to use the Nanodrop machine. I would like to thank Lan Zhu for being a great student and efficiently performing the GPRC6A work during her MSc project under my supervision. My cordial Thanks to Brindha, Susana and Suruchi for a friendly support in and out of the lab. I would like to thank my friends Karan, Marc, Ning, Ruchika, Akash, Ankita, Nick, Hannah, Robin, Shikha and Yogi for the ‘good-times’ and making life in Sheffield really enjoyable. I would also like to thank my friends in India- Kunal, Heeral, Hiral and the rest of the troop for always being there for me. Last but by no way the least, I am deeply indebted to my family and Baba who have fostered me lovingly and encouraged me in all my endeavors. They have always been my strength. I dedicate my thesis to my parents for their unending love and support. 1 Abstract: Calcium (Ca2+) homeostasis is a tightly controlled mechanism by which adequate Ca2+ levels are maintained in the body. A G-protein coupled receptor known as the Calcium Sensing Receptor (CaSR) plays crucial role in Ca2+ homeostasis by sensing minute changes in extracellular Ca2+ and modulating the secretion of calciotropic hormones. It was shown that a group of accessory proteins known as Receptor Activity Modifying Proteins (RAMPs), specifically RAMP1 and 3 are responsible for cell-surface trafficking of the CaSR. Based on this, it was hypothesised that CaSR and RAMPs traffic to the cell-surface as high-order oligomers. FRET-based stoichiometry revealed equal abilities of RAMP1 and 3 to chaperone CaSR to the cell surface, even though RAMP3 interacted more efficiently. Furthermore, a higher fraction of RAMP3 than RAMP1 was observed in the CaSR-complex on the cell-surface, suggesting a higher-order oligomer. Next, it was hypothesised that CaSR and RAMPs associate in an endogenous expression system and that RAMPs play a role in CaSR signalling. In medullary thyroid carcinoma TT cells, an attenuation of CaSR signalling by RAMP1 knock-down suggested an association between them. Also, blocking of RAMP1 using antibodies significantly attenuated CaSR-mediated signalling in these cells; consequently demonstrating its role in CaSR signalling. Finally it was hypothesised that RAMP expression is regulated by agents of Ca2+ homeostasis. However, no changes in RAMP mRNA expression were observed upon treatment with Ca2+ and calcitriol in TT cells; and with Ca2+ in human osteosarcoma cells. Furthermore, it was shown using FRET that RAMP1 associated and caused cell-surface trafficking of GPRC6A, a GPCR closely related to the CaSR. Taken together, it is demonstrated for the first time that RAMP plays a role in CaSR signalling and further research could establish the importance of RAMPs in Ca2+ homeostasis and subsequently as important drug targets against the pathologies of the CaSR. 2 Table of Contents Acknowledgements: ....................................................................................................................... 1 Abstract: .......................................................................................................................................... 2 List of figures ................................................................................................................................... 9 List of tables .................................................................................................................................. 12 List of Abbreviations ..................................................................................................................... 13 CHAPTER 1: GENERAL INTRODUCTION ........................................................................................................... 18 1.1. Ca2+ homeostasis: .............................................................................................................. 19 1.2. G-protein coupled receptors (GPCRs): ............................................................................... 21 1.3. CaSR and its role in Ca2+ homeostasis: .............................................................................. 25 1.3.1. Parathyroid cells: ......................................................................................................... 26 1.3.2. Thyroid: ........................................................................................................................ 26 1.3.3. Kidney: ......................................................................................................................... 26 1.3.4. Bone: ............................................................................................................................ 27 1.3.5. Gut: .............................................................................................................................. 29 1.4. Pathological mutations and knock-out models of CaSR: ................................................... 30 1.5. Ligands of CaSR: ................................................................................................................. 32 1.5.1. Cations: ........................................................................................................................ 32 1.5.2. Polyamines:.................................................................................................................. 33 1.5.3. Aminoglycoside antibiotics: ........................................................................................ 33 1.5.4. Amino-acids and polypeptides: ................................................................................... 34 1.5.5. Phenylalkylamine derivatives ...................................................................................... 35 1.6. Structure-functional relationships of the CaSR: ................................................................ 36 1.6.1. The extracellular domain (ECD): .................................................................................. 36 1.6.2. The trans-membrane domain:..................................................................................... 39 1.6.3. The C-terminal domain: ............................................................................................... 41 1.6.4. Homo-dimerization of CaSR: ....................................................................................... 43 1.7. Signalling of CaSR: .............................................................................................................. 45 1.7.1. Pleiotropic G-protein coupling and activation of phospholipases: ............................. 45 1.7.2. Protein Kinases: ........................................................................................................... 45 3 1.8. Interaction of CaSR with other receptors and proteins: ................................................... 46 1.8.1. Family C GPCRs: ........................................................................................................... 46 1.8.2. Caveolae: ..................................................................................................................... 47 1.8.3. Filamin A: ..................................................................................................................... 48 1.8.4. Receptor Activity Modifying Proteins (RAMPs): .......................................................... 48 1.9. GPRC6A and its role in physiology: .................................................................................... 50 1.10. RAMPs and their interacting partners: .......................................................................... 53 1.11. Structure of RAMPs: ......................................................................................................