Identification and characterization of Adenosine A2A heteromers in the CNS Identificació i caracterització d’heteròmers d’Adenosina A2A al SNC Marc Brugarolas Campillos ADVERTIMENT. La consulta d’aquesta tesi queda condicionada a l’acceptació de les següents condicions d'ús: La difusió d’aquesta tesi per mitjà del servei TDX (www.tdx.cat) i a través del Dipòsit Digital de la UB (diposit.ub.edu) ha estat autoritzada pels titulars dels drets de propietat intel·lectual únicament per a usos privats emmarcats en activitats d’investigació i docència. No s’autoritza la seva reproducció amb finalitats de lucre ni la seva difusió i posada a disposició des d’un lloc aliè al servei TDX ni al Dipòsit Digital de la UB. No s’autoritza la presentació del seu contingut en una finestra o marc aliè a TDX o al Dipòsit Digital de la UB (framing). Aquesta reserva de drets afecta tant al resum de presentació de la tesi com als seus continguts. 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On having consulted this thesis you’re accepting the following use conditions: Spreading this thesis by the TDX (www.tdx.cat) service and by the UB Digital Repository (diposit.ub.edu) has been authorized by the titular of the intellectual property rights only for private uses placed in investigation and teaching activities. Reproduction with lucrative aims is not authorized nor its spreading and availability from a site foreign to the TDX service or to the UB Digital Repository. Introducing its content in a window or frame foreign to the TDX service or to the UB Digital Repository is not authorized (framing). Those rights affect to the presentation summary of the thesis as well as to its contents. In the using or citation of parts of the thesis it’s obliged to indicate the name of the author. FACULTAT DE BIOLOGIA DEPARTAMENT DE BIOQUÍMICA I BIOLOGIA MOLECULAR Identification and characterization of Adenosine A2A heteromers in the CNS. Identificació i caracterització d’heteròmers d’Adenosina A2A al SNC. Memòria presentada pel Llicenciat en Biologia Marc Brugarolas Campillos per a optar al grau de Doctor per la Universitat de Barcelona. Aquesta tesi s’ha adscrit al Departament de Bioquímica i Biologia Molecular de la Universitat de Barcelona, dins del programa de doctorat de Biomedicina. El treball experimental i la redacció de la present memoria han estat realitzats per Marc Brugarolas Campillos sota la direcció del Dr. Rafael Franco Fernández i del Dr. Vicent Casadó Burillo. Dr. Rafael Franco Fernández Dr. Vicent Casadó Burillo Marc Brugarolas Campillos Barcelona, setembre del 2013. 2 INDEX I. Introduction 1. G protein coupled receptors …………………………………………….………17 1.1. GPCR function and structure ………………………………...…………18 1.2. GPCR classification ……………………………………………………..22 1.3. Signaling pathways ………………………………………….…………..25 1.4. GPCR interacting proteins ………………………………….…………..27 2. GPCR oligomerization …………………………………………………...……..29 2.1. GPCR oligomerization evidence …………………………………...……29 2.2. Architecture of the heteromers …………………………….…………….30 2.3. Techniques used to identify GPCR dimers …………………………..….33 2.3.1. Ligand binding in oligomeric receptors ……………………...38 2.4. GPCR heteromerization: functional consequences ……………..………..42 3. Dopamine receptors ………………………………………………………...…..47 3.1. Dopamine receptor characteristics …………………………...………….48 3.2. Dopamine D2 receptor …………………………………………..………51 3.3. Basal ganglia and dopaminergic circuitry ……………………..…………53 4. Adenosine receptor …………………………………………………………..….57 4.1. Adenosine ……………………………………………………….………60 4.2. Adenosine A2A receptors …………………………………………………63 4.3. A2A receptor heteromers ……………………………………...………….68 4.3.1. Postsynaptic A2A receptor heteromers ………………………..68 4.3.2. Presynaptic A2A receptor heteromers ……………….………..73 4.4. Adenosine receptor in Huntington’s disease ……………………………..75 4.4.1. Huntington’s disease and huntingtin ………………………..75 4.4.2. Adenosine A2A receptors in HD ……………………………..81 3 4.4.3. A2A receptor antagonists in HD treatment …………..………84 5. Cannabinoid receptors ………………………………………………………….86 5.1. CB1 receptors ………………………………………………………….86 5.2. CB1 receptor heteromers ………………………………………………90 5.3. CB1 receptors in Huntington’s disease …………………………………92 II. Aims ………………………………………………………………....……………...99 III. Methods …………………………………………………………………………...103 IV. Results and discussion ……………………………........…………………………133 Chapter 1.1: A1R-A2AR heteromers coupled to Gs and Gi/o proteins modulate GABA transport into astrocytes …………………………….133 Chapter 1.2: Adenosine A1R and A2AR heteromers form dynamic but stable tetrameric complexes with two different G proteins ……………..….155 Chapter 2: Agonist and antagonist allosteric interactions between receptors in the A2AR-D2R heteromer ………………………………………….175 Chapter 3: Striatal pre- and postsynaptic profile of adenosine A2AR antagonists ………………………………………………………………193 Chapter 4: Pharmacological and functional characterization of A2AR-CB1R heteromer ………………………………………………………....219 4 Chapter 5: Compound screening of different A2AR antagonists in stable CHO cell lines expressing A2AR, A1R-A2AR, A2AR-D2R or A2AR-CB1R heteromers ………………………………………………………..235 V. Conclusions ………………………………………………………….……………239 VI. Bibliography ………………………………………………………………………245 VII. Resum en català ……………………………………………….………………….301 5 6 LIST OF ABBREVIATIONS [3H]: 3H-labeled [35S]: 35S-labelled 2-AG: 2-Arachidonoylglycerol 5-HT2BR: 5- hydroxytryptamine (serotonin) receptor 2B A1R: Adenosine A1 receptor A2AR: Adenosine A2A receptor A374 A2A R: Adenosine receptor A2A with a mutation in the position 374 of a serine to an alanine. A3R: Adenosine A3 receptor AC: Adenylyl cyclase ADA: Adenosine deaminase ADP: Adenosine diphosphate Ala: Alanine α-MEM: α-Modification of minimum essential medium. AMP: Adenosine monophosphate AMPA: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ANOVA: Analysis of variance ATP: Adenosine triphosphate B50: Concentration competing 50% of radioligand binding BAY-59-3074: 3-[2-Cyano-3-(trifluoromethyl) phenoxy] phenyl 4, 4, 4-trifluoro-1- butanesulfonic acid ester BCA: Bicinchoninic acid assay BDNF: Brain-derived neurotrophic factor BiLFC: Bimolecular fluorescence complementation 7 BRET: Bioluminescence resonance energy transfer BSA: Bovine serum albumin CADO: 2-Chloro-adenosine Ca2+: Calcium cAMP: Cyclic adenosine monophosphate CB1R: Cannabinoid CB1 receptor CCPA: 2-Chloro-N-cyclopentyladenosine cDNA: Cyclic deoxyribonucleic acid CDS: Cellular dielectric spectroscopy CGS 21680: 4-[2-[[6-Amino-9-(N-ethyl-β-D-ribofuranuronamidosyl)-9H-purin- yl]amino]ethyl]benzenepropanoic acid hydrochloride CHDI: Curing Huntington’s disease initiative CHO: Chinese hamster ovary cells ChTx: Cholera toxin cm: Centimeter CNS: Central nervous system CP 55,940: (-)-cis-3-[2-Hydroxy-4-(1,1-dimethyl-heptyl)phenyl]-trans-4-(3- hydroxypropyl)cyclohexanol CPA: (N6-Cyclopentyladenosine) CREB: cAMP response element-binding protein cRluc: C-terminus renilla luciferase Cy3: Cyanine 3 cYFP: C-terminus yellow fluorescent protein D2R: Dopamine D2 receptor DAG: Diacylglycerol DARPP-32: Dopamine- and cyclic AMP-regulated phosphoprotein of 32 kDa 8 DAT: Dopamine transporter DCB: Dimer cooperativity index for the competing ligand B ddH2O: Double distilled water Δ9-THC: Δ9-tetrahydrocannabinol DGL: Diacylglycerol lipase DMEM: Dulbeco’s modified Eagle’s medium DMSO: Dimethyl-sulfoxyde DNA: Deoxyribonucleic acid DPCPX: 8-Cyclophenyl-1,3-dipropylxantine DTT: Dithiothreitol ECL: Extracellular EDTA: Ethylenediaminetetraacetic acid eGFP: Enhanced green fluorescent protein Elk1: ETS domain-containing protein 1 EMCCD: Electron multiplying charge-coupled device EMG: Electromyographic ER: Endoplasmic reticulum ERK: Extracellular signal-regulated kinases ETS: E-twenty six transcription factor EYFP: Enhanced yellow fluorescent protein FAD: Flavin adenine dinucleotide FAAH: Fatty acid amide hydrolase FBS: Foetal Bovine Serum Fig.: Figure FITC: Fluorescein isothiocyanate FRAP: Fluorescence recovery after photobleaching 9 FRET: Förster resonance energy transfer FSH: Follicle-stimulating hormone GABA: Gamma-aminobutyric acid GABAergic: Gamma-Aminobutyric-acidergic GAT: Gamma-aminobutyric acid transporter GDP: Guanosine-5’-diphosphate GEF: Guanine nucleotide exchange factor GFAP: Glial fibrillary acidic protein GFP: Green fluorescent protein GPCR: G-protein coupled receptor Gpe: Globus pallidus external segment Gpi: Globus pallidus internal segment GRK: G-protein receptor kinase GTP: Guanosine-5’-triphosphate GTP-γ-S: Guanosine-5’-O-gamma-thiophosphate H: Hour HBSS: Hank’s balanced salt solution HD: Huntington’s disease HEPES: 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic