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The functional regulation of kisspeptin receptor by calmodulin and Ca2+/calmodulin-dependent protein kinase II Abdirahman M Jama BSc (Hons), MSc Medical Research Council, Centre for Reproductive Health 47 Little France Crescent Edinburgh EH16 4TH This thesis is submitted to the University of Edinburgh for the degree of Doctor of Philosophy 2014 Doctor of Philosophy – The University of Edinburgh – 2014 Table of Contents Declaration............................................................................................................................... i Publications ............................................................................................................................ ii Acknowledgements ............................................................................................................... iii Abstract .................................................................................................................................. iv List of Figures .......................................................................................................................... v List of Tables ........................................................................................................................ vii Abbreviation ........................................................................................................................ viii 1. Introduction ........................................................................................................................ 1 1.1. Overview ....................................................................................................................... 1 1.2. G protein coupled receptors ........................................................................................... 1 1.2.1. Trafficking to the cell surface ................................................................. 1 1.2.2. Overview of GPCR structure and function ............................................. 4 1.2.2.1. The ternary complex model .................................................................. 13 1.2.3. Desensitization and endocytosis ........................................................... 15 1.2.4. The Receptosome: GPCR interacting proteins (GIPs) ......................... 17 1.3. The kisspeptin system .................................................................................................. 26 1.3.1. RFamides and their cognate receptors ................................................. 26 1.3.2. Kisspeptins and KISS1R ....................................................................... 30 1.3.3. KISS1R Agonists ................................................................................... 35 1.3.4. The role of kisspeptin in reproduction and puberty .............................. 36 1.3.5. The role of kisspeptin and KISS1R in pathology .................................. 40 1.3.6. Cell-line models of GnRH neurons. ...................................................... 43 1.4. Ca2+/calmodulin ........................................................................................................... 44 1.4.1 Structural features of calmodulin ......................................................... 45 1.4.2. Calmodulin conformational states ........................................................ 46 1.4.3. Calmodulin binding to GPCRs ............................................................. 49 1.5. αCaMKII ..................................................................................................................... 51 1.5.1. Structure and regulation ....................................................................... 51 1.5.2. T286 autophosphorylation .................................................................... 53 1.5.3. T305/306 autophosphorylation ............................................................. 53 1.5.4. Other posttranslational modifications .................................................. 54 1.5.5. αCaMKII binding GPCRs..................................................................... 54 1.6. Aims and Objectives ................................................................................................... 55 2. Materials and Methods ............................................................................................... 56 2.1. Introduction ................................................................................................................. 56 2.2. Peptides and Proteins ................................................................................................... 56 2.3. Transforming of competent cells ................................................................................. 56 2.4. Preparation of DNA plasmids ...................................................................................... 57 2.5. Preparation of glycerol stocks ..................................................................................... 57 2.6. Agarose gel electrophoresis ......................................................................................... 58 2.7. KISS1R cDNA victors and Site-directed mutagenesis ................................................ 58 2.8. Co-immunoprecipitation .............................................................................................. 58 2.9. Expression and purification of calmodulin .................................................................. 59 2.10. Determination of protein concentration ....................................................................... 61 2.11. Site-directed fluorescence labelling of calmodulin ..................................................... 61 2.12. Reverse phase HPLC ................................................................................................... 62 2.13. Calmodulin-binding experiments ................................................................................ 62 2.14. Free Ca2+ calculation ................................................................................................... 64 2.15. Steady state kinase activity assay ................................................................................ 65 2.16. ADP calibration ........................................................................................................... 68 2.17. Calculation of CaMKII specific activity ...................................................................... 68 2.18. Cryopreservation and resurrection of cell-lines ........................................................... 69 2.19. Cell culture .................................................................................................................. 69 2.20. Confocal laser scanning microscopy ........................................................................... 70 2.21. Transient transfection by electroporation .................................................................... 70 2.22. KP-10 iodination and whole cell competitive radioligand binding assay .................... 71 2.23. Radioimmunoassay ...................................................................................................... 72 2.24. Measurement of inositol phosphate turnover............................................................... 72 2.25. Preparation of cell extracts .......................................................................................... 73 2.26. Western blotting .......................................................................................................... 74 2.27 Software and data analysis ........................................................................................... 75 3. The physical association of calmodulin with kisspeptin receptor ................................ 76 3.1 Introduction ................................................................................................................ 77 3.2. Aim and hypothesis ..................................................................................................... 78 3.3. Results ......................................................................................................................... 79 3.3.1. Ca2+/CaM binding to KISS1R intracellular loops ........................................ 79 3.3.2. Ca2+ and KP-10 dependent CaM binding to KISS1R ................................... 94 3.4 Discussion .................................................................................................................. 105 4. The functional relevance of calmodulin binding to kisspeptin receptor ................... 110 4.1. Introduction ............................................................................................................... 111