The four cysteine residues in the second extracellular loop of the human adenosine A receptor: role in ligand binding and receptor function Anke C. Schiedel, Sonja Hinz, Dominik Thimm, Farag Sherbiny, Thomas Borrmann, Astrid Maaß, Christa E. Müller To cite this version: Anke C. Schiedel, Sonja Hinz, Dominik Thimm, Farag Sherbiny, Thomas Borrmann, et al.. The four cysteine residues in the second extracellular loop of the human adenosine A receptor: role in ligand binding and receptor function. Biochemical Pharmacology, Elsevier, 2011, 82 (4), pp.389. 10.1016/j.bcp.2011.05.008. hal-00718035 HAL Id: hal-00718035 https://hal.archives-ouvertes.fr/hal-00718035 Submitted on 16 Jul 2012 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Accepted Manuscript Title: The four cysteine residues in the second extracellular loop of the human adenosine A2B receptor: role in ligand binding and receptor function Authors: Anke C. Schiedel, Sonja Hinz, Dominik Thimm, Farag Sherbiny, Thomas Borrmann, Astrid Maaß, Christa E. Muller¨ PII: S0006-2952(11)00301-7 DOI: doi:10.1016/j.bcp.2011.05.008 Reference: BCP 10900 To appear in: BCP Received date: 18-2-2011 Revised date: 9-5-2011 Accepted date: 11-5-2011 Please cite this article as: Schiedel AC, Hinz S, Thimm D, Sherbiny F, Borrmann T, Maaß A, Muller¨ CE, The four cysteine residues in the second extracellular loop of the human adenosine A2B receptor: role in ligand binding and receptor function, Biochemical Pharmacology (2010), doi:10.1016/j.bcp.2011.05.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 1 2 3 4 The four cysteine residues in the second extracellular loop of the human 5 6 7 adenosine A2B receptor: role in ligand binding and receptor function 8 9 10 11 a, a a b 12 Anke C. Schiedel *, Sonja Hinz , Dominik Thimm , Farag Sherbiny , Thomas 13 a b a 14 Borrmann , Astrid Maaß , and Christa E. Müller 15 16 17 18 a 19 PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University 20 21 of Bonn, An der Immenburg 4, D-53121 Bonn, Germany 22 23 b 24 Fraunhofer Institute SCAI, Schloss Birlinghoven, 53754 Sankt Augustin, Germany 25 26 *corresponding author: [email protected], phone ++49 (228) 736457; fax ++49 27 28 29 (228) 732567; An der Immenburg 4, D-53121 Bonn, Germany 30 31 [email protected], [email protected], [email protected], 32 33 [email protected] , [email protected], 34 35 36 [email protected] 37 38 39 40 41 category: (5) Inflammation and Immunopharmacology 42 43 44 45 46 47 48 49 50 Accepted Manuscript 51 52 53 54 55 56 57 58 59 60 61 62 1 63 64 Page 1 of 56 65 1 2 3 4 Abstract 5 6 7 8 9 The adenosine A2B receptor is of considerable interest as a new drug target for the 10 11 12 treatment of asthma, inflammatory diseases, pain, and cancer. In the present study we 13 14 investigated the role of the cysteine residues in the extracellular loop 2 (ECL2) of the 15 16 receptor, which is particularly cysteine-rich, by a combination of mutagenesis, molecular 17 18 19 modeling, chemical and pharmacological experiments. Pretreatment of CHO cells 20 21 recombinantly expressing the human A2B receptor with dithiothreitol led to a 74-fold 22 23 24 increase in the EC50 value of the agonist NECA in cyclic AMP accumulation. In the 25 3.25 45.50 26 C78 S and the C171 S mutants high-affinity binding of the A2B antagonist 27 28 3 29 radioligand [ H]PSB-603 was abolished and agonists were virtually inactive in cAMP 30 31 assays. This indicates that the C3.25-C45.50 disulfide bond, which is highly conserved 32 33 in GPCRs, is also important for binding and function of A receptors. In contrast, the 34 2B 35 45.45 45.46 45.45 45.46 36 C166 S and the C167 S mutants as well as the C166 S-C167 S double 37 38 mutant behaved like the wild-type receptor, while in the C15445.33S mutant significant, 39 40 41 although more subtle effects on cAMP accumulation were observed - decrease (BAY60- 42 43 6583) or increase (NECA) - depending on the structure of the investigated agonist. In 44 45 46 contrast to the X-ray structure of the closely related A2A receptor, which showed four 47 48 disulfide bonds, the present data indicate that in the A2B receptor only the C3.25-C45.50 49 Accepted Manuscript 50 disulfide bond is essential for ligand binding and receptor activation. Thus, the cysteine 51 52 53 residues in the ECL2 of the A2B receptor not involved in stabilization of the receptor 54 55 structure may have other functions. 56 57 58 59 60 Keywords 61 62 2 63 64 Page 2 of 56 65 1 2 3 4 5 6 7 Adenosine A2B receptor; disulfide bonds; extracellular loop 2; mutagenesis; PSB-603 8 9 10 11 12 1 Introduction 13 14 15 16 Adenosine A receptors belong to the large group of purinergic G protein-coupled 17 2B 18 19 receptors (GPCRs), which comprise P2 (P2Y and P2X, nucleotide-activated ) and P1 20 21 (adenosine) receptors [1]. Brunschweiger and Müller [2] proposed to add P0 (adenine) 22 23 24 receptors as a third class to the group of purinergic receptors. The P1 or adenosine 25 26 receptor (AR) family consists of four subtypes, A1, A2A, A2B and A3 [3]. A1 and A3 27 28 29 receptors are coupled to Gi type G proteins, leading to the inhibition of the adenylate 30 31 cyclase upon receptor activation, while A2A and A2B receptors are mainly coupled to Gs 32 33 proteins resulting in an increase in intracellular cAMP concentrations via stimulation of 34 35 36 adenylate cyclase [4]. In several cell systems, such as HEK-293 and HMC-1 mast cells 37 38 A2B receptors are additionally coupled to phospholipase C via Gq proteins, and are 39 40 2+ 41 thereby linked to intracellular Ca release [5-6]. In the human leukemia cell line Jurkat 42 43 T, A2B-mediated calcium mobilization independent of inositol-1,4,5-trisphosphate was 44 45 46 observed [7]. Coupling of the A2B receptor to the MAPK cascade via ERK1/2 has been 47 48 described for recombinant CHO cells overexpressing human A2B receptors and for mast 49 Accepted Manuscript 50 cells, showing an involvement in proliferation, differentiation and apoptosis [4, 8]. 51 52 53 Furthermore a link of A2B receptor signaling to the arachidonic acid signal transduction 54 55 pathway via phospholipase A and cyclooxygenase activation leading to vasoconstriction 56 57 58 in smooth muscle cells has been described [9]. 59 60 61 62 3 63 64 Page 3 of 56 65 1 2 3 4 Among the four AR subtypes A2B has been the least well characterized receptor, mainly 5 6 7 due to the lack of suitable, specific ligands [10]. Meanwhile highly selective A2B 8 3 9 antagonists have been developed and an A2B-specific antagonist radioligand, [ H]PSB- 10 11 12 603 (for structure see figure 1), with high potency and specificity across species, 13 14 including rodents and humans, has recently become available [11]. As for agonists, 15 16 besides the nucleoside derivative NECA [12], which is non-selective, and related 17 18 19 adenosine derivatives, the first highly selective A2B agonist BAY60-6583 [13], a non- 20 21 nucleosidic compound, has been developed (structures are shown in supplemental 22 23 24 figure 1). 25 26 In many tissues, A2B receptors are considered low affinity receptors with typically low 27 28 29 expression levels [14]. Therefore, adenosine concentrations typically have to reach 30 31 micromolecular levels to activate natively expressed A2B receptors, which occurs under 32 33 pathological conditions, such as hypoxia, ischemia, inflammation or massive cell death 34 35 36 [15-16]. While their distribution is ubiquitous, A2B receptors are found at higher densities 37 38 mainly in the large intestine, in mast cells, hematopoietic cells, and in the brain, mainly 39 40 41 in astrocytes [6, 14, 17-18]. Upregulation has been found in several cancer cell lines 42 43 [19]. A2B receptors are thought to be involved in a number of diseases and the first 44 45 46 antagonist is now being evaluated in clinical trials for the treatment of asthma and 47 48 chronic obstructive pulmonary disease [10]. Other potential indications include secretory 49 Accepted Manuscript 50 diarrhea associated with inflammation, Alzheimer’s disease, inflammatory diseases, 51 52 53 pain, cancer, type II diabetes, and diabetic retinopathy [20]. Thus, A2B receptors 54 55 represent important new drug targets. 56 57 58 To fully understand interactions of the human A2B receptor with its ligands, agonists and 59 60 antagonists, it is of major importance to gain knowledge about the structure of the 61 62 4 63 64 Page 4 of 56 65 1 2 3 4 receptor, the amino acid residues involved in ligand binding, and to determine the 5 6 7 receptor's 3D structure, which in turn can then be used for the development of new 8 9 ligands [21-22].