Characterisation of Calcium-Sensing Receptor Extracellular pH Sensitivity and Intracellular Signal Integration A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy (PhD) in the Faculty of Life Sciences. 2013 Katherine Louisa Campion CONTENTS PAGE LIST OF FIGURES AND TABLES 9 ABBREVIATIONS 13 ABSTRACT 16 DECLARATION 17 COPYRIGHT STATEMENT 18 ACKNOWLEDGEMENTS 19 CHAPTER 1 INTRODUCTION 1.1 Extracellular calcium homeostasis 21 1.1.1 Discovery of the parathyroid gland and its role in calcium homeostasis 21 1.1.2 Discovery of the calcium-sensing receptor its role in calcium homeostasis 22 1.2 Calcium-sensing receptor structure 24 1.2.1 Extracellular domain 24 1.2.2 Venus flytrap and calcium binding domains 24 1.2.3 Glycosylation and the ECD 25 1.2.4 Dimerisation 26 1.2.5 Transmembrane domain 27 1.2.6 TMD extracellular loops 27 1.2.7 TMD Intracellular loops 28 1.2.8 Intracellular domain 29 1.3 Calcium-sensing receptor tissue expression 30 2 1.3.1 Parathyroid gland 31 1.3.2 Kidney 32 1.3.3 Bone 35 1.3.4 Gastrointestinal system 35 1.3.5 Central and peripheral nervous system 36 1.3.6 Blood vessels 37 1.3.7 Developing lung 38 1.3.8 Other tissues 38 1.4 Calcium-sensing receptor pharmacology 38 1.4.1 Orthosteric ligands 38 1.4.2 CaR allosteric modulators – aromatic amino acids 39 1.4.3 Allosteric CaR activators – Calcimimetics 40 1.4.4 Allosteric CaR inhibitors – Calcilytics 41 1.4.5 Effect of extracellular pH on CaR 41 1.5 Diseases of the calcium-sensing receptor and parathyroid gland 46 1.5.1 Loss-of-function CaR mutations 46 1.5.2 Gain-of-function CaR mutations 48 1.5.3 Primary hyperparathyroidism 48 1.5.4 Secondary hyperparathyroidism 49 1.6 Calcium-sensing receptor signalling 49 1.6.1 Phospholipase activation 50 1.6.1.1 Phospholipase C 50 1.6.1.2 Phospholipases A 2 and D 50 1.6.2 Protein kinase regulation 51 3 1.6.2.1 Protein Kinase C 52 1.6.2.2 Protein Kinase A 53 1.6.3 Mitogen-activated protein kinases 54 1.6.3.1 Extracellular-regulated protein kinase 54 1.6.3.2 p38 MAP kinase 55 1.6.3.3 C-Jun N-terminal kinase 55 1.6.4 Calcium-sensing receptor interacting proteins 55 1.6.4.1 Beta arrestin 55 1.6.4.2 Calmodulin 56 1.6.4.3 Potassium channels 57 1.6.4.4 14-3-3 proteins 57 1.6.4.5 Other interactions 58 1.7 Summary 58 1.8 Aims and objectives 59 CHAPTER 2 MATERIALS AND METHODS 60 2.1 Cell culture 61 2.2 Bovine parathyroid cell isolation and culture 61 2.3 Transient cell transfection 61 2.4 Preparation of cell lysates 62 2.5 Immunoblotting 62 2.6 Intracellular calcium imaging 63 2.7 Site-Directed Mutagenesis 63 2.7.1 Mutation primers 64 2.7.2 Sequencing primers 66 4 2.8 Microarray 67 2.9 ERK Assay 68 2.10 Protein references 68 2.11 Statistical analysis 68 CHAPTER 3: MODULATION OF CALCIUM-SENSING RECEPTOR ACTIVITY BY EXTRACELLULAR PH 69 3.1 Introduction 70 3.2 Materials and Methods 70 3.3 Effect of albumin addition on extracellular pH sensitivity in CaR-HEK cells and bovine parathyroid cells 70 3.4 Determination of the contribution of extracellular histidine residues to calcium-sensing receptor pH o-sensitivity 74 3.4.1 Confirmation of each CaR histidine mutant’s base sequence protein expression in HEK-293 cells. 74 3.4.2 Characterisation of the extracellular calcium sensitivity of the CaR histidine mutants expressed abundantly in HEK-293 cells. 75 3.4.3 Characterisation of the extracellular calcium sensitivity of the CaR histidine mutants CaR H41V and CaR H595V 92 3.4.4 Determination of the extracellular pH sensitivity of the CaR histidine mutants that respond to modest extracellular calcium concentrations. 95 3.4.5 Determination of the extracellular pH sensitivity of the CaR histidine mutants CaR H41V and CaR H595V . 110 3.4.6 Characterisation of the extracellular calcium and pH sensitivity of the CaR double histidine mutant, CaRH429V/H495V. 113 3.4.7 Characterisation of the extracellular calcium and pH C482S sensitivity of the free cysteine mutant, CaR . 117 5 3.4.8 Summary 120 3.5 Discussion 121 3.5.1 The effect of altered extracellular pH on the CaR in a heterologous expression system 121 3.5.2 Potential effect of altered calcium binding to albumin following changes in pH o 123 3.5.3 Potential contribution of extracellular histidine residues to CaR pH o sensitivity 124 3.5.4 Extracellular pH sensitivity exhibited by the mGluRs 126 3.5.5 pH sensitivity of non-elemental agonists. 127 3.5.6 pH o sensitivity of CaR mutants 128 3.5.7 Physiological relevance of extracellular pH sensing 128 3.5.8 Summary 131 CHAPTER 4: CALCIUM-SENSING RECEPTOR INTRACELLULAR SIGNALLING INTEGRATION 132 4.1 Introduction 133 4.2 Materials and Methods 133 4.3 Effect of increasing intracellular cAMP on CaR signalling in a heterologous expression system 133 4.3.1 Effect of forskolin addition during CaR-induced 2+ Ca i mobilisation 133 2+ 4.3.2 Effect of forskolin on CaR-induced Ca i mobilisation 2+ at sub-threshold Ca o concentrations 134 2+ 4.3.3 Effect of 1,9-dideoxyforksolin on CaR-induced Ca i 2+ mobilisation at subthreshold Ca o concentrations 136 2+ 4.3.4 Effect of forskolin on CaR-induced Ca i mobilisation 2+ at low Ca o concentration 138 6 2+ 4.3.5 Effect of forskolin on CaR-induced Ca i mobilisation as a function of concentration 138 2+ 4.3.6 Effect of forskolin on CaR-induced Ca i mobilisation as a function of extracellular calcium concentration 139 2+ 4.3.7 Effect of IBMX on CaR-induced Ca i mobilisation at 2+ subthreshold Ca o concentrations 141 4.3.8 Effect of forskolin cotreatment on CaR-induced ERK phosphorylation 142 4.3.9 Effect of chronic phorbol ester pretreatment on 2+ 2+ Ca o–induced Ca i mobilisation 144 4.3.10 Effect of pertussis toxin pretreatment on 2+ 2+ Ca o–induced Ca i mobilisation 146 2+ 4.3.11 Effect of isoproterenol on CaR-induced Ca i 2+ mobilisation at subthreshold Ca o concentrations 149 4.4 Investigation of the role of protein kinase A in CaR signalling 153 4.4.1 Detection of protein kinase A activity using a motif-specific anti-PKA phospho-substrate antibody 153 4.4.2 Effect of protein kinase A inhibition 2+ and forksolin cotreatment on CaR-induced Ca i mobilisation 2+ at moderate Ca o concentration 155 4.4.3 Effect of protein kinase A inhibition 2+ and forksolin cotreatment on CaR-induced Ca i mobilisation 2+ at threshold Ca o concentration 158 4.4.4 Effect of PKA- and EPAC-selective cAMP analogues 2+ 2+ on Ca i mobilisation at moderate Ca o concentration 159 4.4.5 Characterisation of the CaR Ser-899 and Ser-900 mutants 162 2+ 2+ 4.4.6 Effect of forskolin on Ca o–induced Ca i mobilisation in CaR mutants lacking the PKA sites Ser-899 and Ser-900. 167 7 4.5 Discussion 169 4.5.1 The interaction between use of HEK-293 cells as a suitable model of CaR signalling 169 4.5.2 The involvement of cAMP and PKC signalling in parathyroid physiology 170 4.5.3 Effect of cAMP and CaR on the activation threshold 2+ for CaR-mediated Ca i mobilisation 171 4.5.4 Identification of possible cyclic AMP targets in lowering 2+ the agonist threshold for CaR-induced Ca i mobilisation 172 4.5.5 Effect of pertussis toxin on CaR- 2+ mediated Ca i mobilisation 172 4.5.6 Effect of endogenous stimulation of cAMP levels and the potential physiological significance of this effect. 174 2+ 4.5.7 Effect of PKA inhibition on Ca o induced 2+ Ca i mobilisation 177 4.5.8 Functional consequence of mutating the PKA consensus sites in CaR. 178 4.5.9 Possible mechanism of cAMP influence in CaR signalling 180 4.5.10 Relevance of CaR S899A functional data for the concept of agonist-driven insertional signalling 181 4.5.11 Summary 184 CHAPTER 5: GENERAL DISCUSSION. 185 5.1 General discussion 186 5.2 Conclusions 188 REFERENCES 189 WORD COUNT 50,670 8 LIST OF FIGURES AND TABLES Figure 1.1. Schematic overview of extracellular free-ionised calcium homeostasis. 31 Figure 1.2. Schematic of mineral reabsorption in the cortical thick ascending limb. 34 2+ Figure 1.3. Effect of pH o increase on CaR-induced Ca i mobilisation in CaR-HEK cells. 43 2+ Figure 1.4. Effect of pH o increase on CaR-induced Ca i mobilisation in bovine parathyroid chief cells 44 Table 1. CaR extracellular histidine conservation. 45 Figure 1.5. PyMOL model of the extracellular histidines, free- cysteine and putative Ca 2+ binding regions 46 Figure 1.6. Schematic of CaR signalling 50 Table 2. Summary of thermal cycler protocol. 64 Figure 3.1. Effect of pH o on bovine parathyroid cells in the presence of 5% (w/v) albumin 72 Figure 3.2. Effect of pH o on CaR-HEK in the presence of 5% (w/v) albumin 73 Table 3.1. EC 50 values and relative maximal responses for the CaR histidine mutants versus wild-type.