The Effect of μ-Opioid Receptor Polymorphisms on Receptor Signalling Systems Alisa Knapman (BMedSci Hons) Faculty of Human Science Australian School of Advanced Medicine Macquarie University 2014 A thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy Table of Contents Chapter 1: Introduction 1 1.1 Opioid Receptors 2 1.1.1 MOP 4 1.1.2 DOPr 5 1.1.3 KOPr 5 1.1.4 NOPr 6 1.2 Opioid receptors are G protein coupled receptors 7 1.3 MOPr Structure and Signalling 8 1.4 MOPr Effectors 9 1.4.1 MOPr inhibition of adenylyl cyclase 9 1.4.2 MOPr activation of potassium channels 11 1.4.3 MOPr inhibition of calcium channels 12 1.4.4 MOPr release of intracellular calcium 13 1.4.5 MOPr and the MAP kinase cascade 14 1.4.6 Other MOPr effectors 15 1.4.7 MOPr desensitisation and endocytosis 16 1.5 MOPr Ligands 18 1.5.1 Endogenous MOPr ligands 18 1.5.2 Exogenous MOPr ligands 18 1.5.3 Ligand affinity and efficacy 19 1.6 Ligand-biased signalling at MOPr 20 1.7 OPRM1 Polymorphisms 22 1.8 MOPr variants and differential signalling 25 i Chapter 2: Review Article 27 “Cellular signalling of non-synonymous single nucleotide polymorphisms of the human μ-opioid receptor” Hypotheses and Aims 77 Methods - Overview 78 Chapter 3: Research Article 79 “A real-time, fluorescence-based assay for measuring μ-opioid receptor modulation of adenylyl cyclase activity in Chinese hamster ovary cells” Chapter 4: Research Article 107 “A continuous, fluorescence-based assay of μ-opioid receptor activation in AtT-20 cells” Results - Overview 135 Chapter 5: Research Article 137 “ Buprenorphine signalling is compromised at the N40D polymorphism of the human μ-opioid receptor in vitro” Chapter 6: Research Article 185 “ The A6V polymorphism of the human μ-opioid receptor negatively impacts signalling of morphine and endogenous opioids in vitro” Chapter 7: Research Article (in preparation) 233 “Mu-opioid receptor polymorphisms differentially affect receptor signalling via adenylyl cyclase inhibition and ERK1/2 phosphorylation” ii Chapter 8: General Discussion and Summary 263 References 275 Appendix: Book Chapter 329 “Fluorescence-Based, High-Throughput Assays for µ-Opioid Receptor Activation using Membrane Potential Sensitive Dye” iii List of Figures 2.1 Naturally occurring non-synonymous OPRM1 variants 35 3.1 Stimulating adenylyl cyclase hyperpolarises CHO cells 87 3.2 Opioids inhibit forskolin stimulated membrane hyperpolarisation 89 in CHO cells 3.3 Protein kinase A activators mimic the effects of forskolin 93 3.4 Forskolin induced hyperpolarisation is dependent on extracellular 95 K concentration 4.1 Example traces of fluorescent signal in the membrane potential assay 114 4.2 Trace of fluorescent signal illustrating reversal of DAMGO stimulated 117 decrease in signal by MOPr antagonist naloxone 4.3 DAMGO and morphine stimulated hyperpolarisation is PTX sensitive 120 4.4 Decreasing [K]Ex increases maximum change in fluorescent signal 120 4.5 Nigericin causes maximum membrane hyperpolarisation 121 4.6 DAMGO, morphine, buprenorphine and pentazocine concentration- 121 response curves for GIRK activation 4.7 Desensitisation of MOPr signalling in AtT-20 cells 123 5.1 Saturation binding curve of [3H]DAMGO in intact CHO-MOPr cells 149 5.2 DAMGO inhibits adenylyl cyclase and activates ERK1/2 in CHO cells 153 expressing MOPr-WT and MOPr-N40D 5.3 Endogenous opioids inhibit adenylyl cyclase and activate ERK1/2 in 155 CHO cells expressing MOPr-WT or MOPr-N40D 5.4 Buprenorphine inhibits adenylyl cyclase and activates ERK1/2 less 157 effectively in CHO cells expressing MOPr-N40D 5.5 Fentanyl, oxycodone and methadone inhibit adenylyl cyclase and activate 159 ERK1/2 in CHO cells expressing MOPr-WT or MOPr-N40D 5.6 DAMGO causes membrane hyperpolarisation in AtT-20 cells expressing 164 MOPr-WT 5.7 Buprenorphine is less potent for GIRK activation in AtT-20 cells 167 expressing MOPr-N40D 6.1 Saturation binding curve of [3H]DAMGO in intact CHO-MOPr-WT 196 and CHO-MOPr-A6V cell 6.2 DAMGO inhibits adenylyl cyclase and activates ERK1/2 in CHO cells 199 expressing MOPr-WT and MOPr-A6V iv 6.3 Morphine, buprenorphine and pentazocine inhibition of adenylyl cyclase 203 and activation of ERK1/2 is compromised in CHO cells expressing MOPr-A6V 6.4 Endogenous opioid inhibition of adenylyl cyclase and activation of 207 ERK1/2 is significantly affected by the A6V variant in CHO cells expressing MOPr 6.5 Fentanyl, methadone and oxycodone inhibition of adenylyl cyclase 211 and activation of ERK1/2 is significantly affected by the A6V variant in CHO cells expressing MOPr 6.6 DAMGO causes membrane hyperpolarisation in AtT-20 cells expressing 213 MOPr-WT 6.7 Morphine activates GIRK less effectively in AtT20 cells expressing 215 MOPr-A6V 7.1 DAMGO, morphine, buprenorphine and pentazocine inhibit adenylyl 245 cyclase and activate ERK1/2 in CHO cells expressing MOPr-WT or MOPr variants 7.2 Endogenous opioids inhibit adenylyl cyclase and activate ERK1/2 247 in CHO cells expressing MOPr-WT or MOPr variants 7.3 Methadone, fentanyl and oxycodone inhibit adenylyl cyclase and activate ERK1/2 in CHO cells expressing MOPr-WT or MOPr variants 251 7.4 High concentrations of methadone and fentanyl cause membrane 252 depolarisation in CHO-MOPr-L85I cells v List of Tables 2.1 Summary of non-synonymous MOPr variants 38 2.2 Summary of key findings about MOPr SNP signalling 48 - 51 4.1 Potencies and efficacies of the range of structurally distinct 119 opioid ligands tested using the membrane potential assay 5.1 Summary of opioid efficacy and potency in assays of AC 160 inhibition in CHO cells expressing MOPr-WT and MOPr-N40D 5.2 Summary of opioid efficacy and potency in assays of ERK1/2 161 phosphorylation in CHO cells expressing MOPr-WT and MOPr-N40D 5.3 Summary of opioid efficacy and potency in assays of GIRK 168 activation in in CHO cells expressing MOPr-WT and MOPr-N40D 6.1 Summary of opioid efficacy and potency in assays of AC 200 inhibition in CHO cells expressing MOPr-WT and MOPr-A6V 6.2 Summary of opioid efficacy and potency in assays of ERK1/2 208 phosphorylation in CHO cells expressing MOPr-WT and MOPr-A6V 6.3 Summary of opioid efficacy and potency in assays of GIRK 216 activation in in CHO cells expressing MOPr-WT and MOPr-A6V 7.1 Surface receptor expression and DAMGO Kd for MOPr 242 variants in CHO cells 7.2 Forskolin and PMA responses in MOPr variants 242 7.3 Summary of opioid efficacy and potency in assays of AC 254 inhibition in CHO cells expressing MOPr-WT and MOPr variants 7.4 Summary of opioid efficacy and potency in assays of ERK1/2 255 phosphorylation in CHO cells expressing MOPr-WT and MOPr variants vi Abbreviations 4-AP 4-aminopyridine aa amino acid AC adenylyl cyclase ACTH adrenocorticotropic hormone Akt protein kinase B β-CNA β-chlornaltrexamine β-FNA β-funal trexamine BSA bovine serum albumin CaM calmodulin cAMP cyclic adenosine monophosphate CaMKII Ca2+/calmodulin-dependent protein kinase II CHO Chinese Hamster Ovary CRC concentration-response curve CRE cAMP response element DAMGO [D-Ala2, N-MePhe4, Gly-ol]-enkephalin DMEM Dulbecco’s Modified Eagle Medium DOPr δ-opioid receptor DPDPE [2-Dpenicillamin, 5-Dpenicillamin]-enkephalin ECL extracellular loop EGFR epidermal growth factor receptor ELISA enzyme-linked immunosorbent assay EM1 endomorphin 1 EM2 endomorphin 2 EPAC exchange signal activated by cAMP vii ERK extracellular signal regulated kinase FSK forskolin GIRK G protein gated, inwardly rectifying potassium channel GLR3 glycine receptor α3 GPCR G protein couple receptors GRK G protein coupled receptor kinase HBSS Hanks balanced salt solution hMOPr human µ-opioid receptor HPA hypothalamic-pituitary-adrenal ICa voltage gated Ca channels ICL intracellular loop IP3 inositol (1,4,5) triphosphate JNK Jun N-terminal kinase KO knock-out KOPr κ-opioid receptor M-6-G morphine-6-glucuronide MAPK mitogen activated protein kinase MOPr µ-opioid receptor mRNA messenger ribonucleic acid N/OFQ nociceptin/orphanin FQ NMDA N-methyl-d-aspartate NOPr nociceptin/orphanin FQ receptor NR1 NMDA receptor 1 OPRD1 opioid receptor δ 1 viii OPRK1 opioid receptor κ 1 OPRL1 opioid receptor-like 1 OPRM1 opioid receptor mu 1 p38MAPK p38 mitogen activated kinase PAG periaqueductal grey pDyn prodynorphin pENK proenkephalin pERK phosphorylated ERK1/2 PI3K phosphatidylinositol-3 kinase PKA protein kinase A PKC protein kinase C POMC proopiomelanocortin PLC phospholipase C PTX pertussis toxin SNP single nucleotide polymorphism STAT5 signal transducer and activator of transcription 5 TEA tetraethylammonium TM transmembrane TRPV1 transient receptor potential cation channel, subfamily V, member 1 ix x Summary There is significant variation in individual response to opioid drugs, one cause of which is likely to be polymorphisms on the opioid receptors themselves. The μ-opioid receptor (MOPr) is the primary site of action for most analgesic opioids. Previous studies have identified a number of naturally occurring single nucleotide polymorphisms (SNPs) in the coding region of MOPr. The A118G SNP (N40D), present at allelic frequencies ranging from 10 – 50%, has been associated with diverse phenotypic effects as well as differences in receptor signalling in vitro, with little consistency between studies. Few studies have examined the consequences of other MOPr polymorphisms on receptor function, or potential ligand and pathway specific effects. In this study, the relative potency and efficacy of a range of clinically important and/or structurally distinct opioid ligands were assessed in Chinese hamster ovary (CHO) cells and mouse pituitary neuroblastoma (AtT- 20) cells stably transfected with human wild type MOPr and 5 naturally occurring MOPr variants, N40D, A6V, L85I, R260H and R265H.