The Structure and Function of V1b Vasopressin Receptors Yukie Goto A thesis submitted to the University of Birmingham for the degree of Doctor of Philosophy School of Biosciences University of Birmingham Edgbaston, Birmingham B15 2TT, United Kingdom September 2009 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Acknowledgement Firstly I would like to thank my supervisor Professor Mark Wheatley for giving me this opportunity to participate in his research, and for his support and guidance throughout my study. I would also like to thank those who have worked in his research group: in particular John for providing us with molecular models of vasopressin receptors; Alex, Mattew, Denise, Cymone and Amelia, for equipping me with the laboratory techniques required for carrying out this study; and Rachel and Richard for their companies and supports in the research group for last few years. I am truly grateful to Dr. David Poyner and Prof. Ian Martin for their inspiring teachings on this subject area in my undergraduate years. My gratitude goes to Rosemary for her conscientious hard work in looking after laboratories, equipments, and students; and to Eva, David, Karthik, Prof. Wharton, Bob, Pei, Tim, Umbrene, Yiu Pin, Raul, Martin and Mohammed for their companies in the laboratories and in the office; and also to Eric for his support in general outside the university. I am also thankful to Dr. Dove and Dr. Ladds for reading manuscripts and giving me advices on corrections. I would like to acknowledge Dr. Jeremy Presland, Dr. Grant Wishart, Dr. Mark Craighead and their co-workers in Organon Laboratory, a part of Schering-Plough Research Institute (Newhouse, Scotland) for collaboration, intellectual input, and all the supports provided. Finally, I am truly grateful to BBSRC and Schering-Plough for financial and material supports given to this project. i Abstract The V1b vasopressin receptor (V1bR) is a receptor for a neurohypophysial nonapeptide 8 [arginine ] vasopressin (AVP). V1bR is a G-protein coupled receptor (GPCR) belonging to the Family A GPCR superfamily. The structures of seven α-helical transmembrane domains of this family members can be predicted based on the crystal structure of bovine rhodopsin (bRho) and human β2 adrenergic receptor (β2AR) obtained by the X-ray diffraction techniques. This study aimed to identify amino acid residues which participate in ligand binding of the V1bR by site- directed mutagenesis with the aid of molecular models of vasopressin receptors based on the crystal structure of bRho. The V1bR is a potential drug target in treating stress-related conditions such as depression, anxiety and post-traumatic stress disorders. Since it is the latest subtype identified among the mammalian neurohypophysial hormone receptors, it remains as the least studied subtype. A closely related subtype V1a receptor (V1aR) has been studied in far more detail for its potential of being a drug target in treating cardiac conditions and epilepsy. Hence, effective means of studying the V1bR can be accomplished by exploring the information already available on the V1aR and thereby defining the differences and similarities existing between the two. Detailed subtype comparisons are also fundamental for designing subtype selective drugs for effective therapy with fewer side-effects. This project was designed also to elucidate amino acid residues which determine selectivity of ligands for the V1bR over the V1aR. This study demonstrated that the charged residues Glu1.35 and Arg3.26, polar residues Gln2.61 and 5.38 2.60 Tyr , and cyclic residue Pro are crucial in AVP binding to the V1bR. These residues are all ii located at the extracellular facing surface of transmembrane domains (TMs). Also at the vicinity, charged residues Arg1.27 and Asp2.65, and aromatic Trp2.64 are shown to be important components of the AVP binding cavity. The study demonstrated that two residues Trp6.48 and Phe6.51 located at the TM6 are required for high affinity binding of non-peptide antagonists to the V1bR. 7.35 The reciprocal mutagenesis between V1bR and V1aR residues revealed that Phe located at the exofacial surface of TM7 is a key residue required for a high affinity binding of a 7.39 non-peptide antagonist with selectivity for the V1bR over V1aR. Also on the TM7, Met was shown to be involved in a high affinity binding of an antagonist with selectivity for the V1bR 5.39 5.42 over V1aR. Two residues on the top of TM5, Leu and Thr were also shown to participate in V1bR-selective binding of non-peptide antagonists. From the perspectives of drug development, the variants of V1bR were studied in two streams: firstly to characterise pharmacological properties of single nucleotide polymorphism (SNP) variants of human V1bR; and secondly to determine differences in TM architectures between rat and human V1bRs. The study showed that a SNP variant of the V1bR with a residue substitution of Gly to Arg at position 191 is more readily expressed on the cell-surface in comparison to the wild-type. The variant was also found to generate InsP-InsP3 accumulation effectively in response to AVP stimulation. The mutagenesis involving introduction of specific rat V1bR residues into human V1bR revealed that there is a slight difference in the local environment of TM4 between the V1bRs found in the two species. iii Abbreviations In the production of this thesis, the abbreviations recommended by the Journal of Biological Chemistry (http://www.jbc.org/misc/ifora.shtml) have been applied. In addition, the following abbreviations were used: A1AR A1 Adenosine receptor A2AR A2 Adenosine receptor αAR α adrenergic receptor AC Adenylyl cyclase ACTH Adrenocorticotropin AIMAH ACTH-independent macronodular adrenal hyperplasia AKA A-kinase anchoring protein ARF6 ADP-ribosylation factor 6 ARNO ADP-ribosylation factor nucleotide site opener AT1AR Angiotensin receptor type 1A AVP [Arginine8]vasopressin AVT [Arginine8]vasotocin β1AR β1 adrenergic receptor β2AR β2 adrenergic receptor bRho Bovine rhodopsin BSA Bovine serum albumin BNST Bed nucleus of the stria terminalis BRET Bioluminscence energy transfer 8 CA Cyclic antagonist: [d(CH2)5Tyr(Me)Arg ]vasopressin CA2 Cornu Ammonis 2 CaM Calmodulin CAM Constitutively active mutant cAMP Cyclic AMP CCKBR Cholecystokinin B receptor iv CCR5 C-C chemokine receptor type 5 CD8 Cluster of differentiation 8 CHO Chinese hamster ovary cell-line CNS Central nervous system CP Carboxy-terminal of glycopeptide CRF Corticotrophin releasing factor CRFR1α Corticotrophin releasing factor receptor 1α CRLR Calcitonin receptor-like receptor DAG 1, 2-diacylglycerol dAVP [deamino-Cys1, Arg8]vasopressin d[Cha4]AVP [deamino-Cys1, Cyclohyxylalanine4, Arg8]vasopressin dDAVP [deamino-Cys1, D-Arg8]vasopressin DI Diabetes insipidus dVDAVP [deamino-Cys1, Val4, D-Arg8]vasopressin DMEM Dulbecco’s modified Eagle’s medium DMSO Di-methyl-sulfoxide EGFR Epidermal growth factor receptor ECL Extracellular loop ELISA Enzyme-linked immunosorbent assay ERK Extracellular signal regulated kinase FBS Foetal bovine serums FRET Fluorescence energy transfer FSH Follicle-stimulating hormone G-protein Guanine-nucleotide-binding protein GABA Gamma-aminobutyric acid GIT G protein-coupled receptor kinase-interactor GPCR G-protein-coupled receptor GDP Guanosine diphosphate GEF Guanine nucleotide exchange factor GRK G-protein-coupled receptor kinase v GSK Glycogen synthase kinase GTP Guanosine triphosphate H2R H2 Histamine receptor H8 Helix 8 HA Haemagglutinin HEK Human embryonic kidney HPA Hypothalamic-pituitary adrenal 5-HT 5-hydroxytryptamine InsP Inositol monophosphate InsP3 Inositol 1,4,5-trisphosphate ICL Intracellular loop 2 8 LA Linear antagonist: [PhACD-Tyr(Me) Arg Tyr-(NH2)]-vasopressin LH Luteinising hormone MA Medial amygdaloid nucleus mAChR Muscarinic acetylcholine receptor MAPK Mitogen activated protein kinase MCS Multiple cloning sequence MD Molecular dynamic mGluR Metabotropic glutamate receptor MLCK Myosin light chain kinase MLCP Myosin light chain phosphatase μOR μ opioid receptor NDI Nephrogenic diabetes insipidus NF-AT Nuclear factor of activated T-cells NMR Nuclear magnetic resonance spectroscopy NP Neurophysin OT Oxytocin OTR Oxytocin receptor PDL Poly-D lysine PDZ PSD-95-discs-large-ZO-1 vi PEI Poly-ethylenimine PKC Protein kinase C PLC Phospholipase C PTSD Post-traumatic stress disorders PVN Paraventricular nucleus RAMP Receptor activity modifying protein RGS Regulator of G-protein signalling RMSD Root mean square deviations RTK Receptor tyrosin kinase SCN Suprachiasmatic nucleus SEM Standard errors of means SH3 Src homology 3 SON Supraoptic nucleus SNP Single nucleotide polymorphism TM Transmembrane domain TSH Thyrotropin stimulating hormone V1aR V1a vasopressin receptor V1bR V1b vasopressin receptor V2R V2 vasopressin receptor VTR [Arginine8]vasotocin receptor Wt Wild-type vii Contents Acknowledgements ................................................................................................................... i Abstruct