Elsevier

Biochemical Pharmacology

Volume 82, Issue 4, 15 August 2011, Pages 389-399
Biochemical Pharmacology

The four cysteine residues in the second extracellular loop of the human adenosine A2B receptor: Role in ligand binding and receptor function

https://doi.org/10.1016/j.bcp.2011.05.008Get rights and content

Abstract

The adenosine A2B receptor is of considerable interest as a new drug target for the treatment of asthma, inflammatory diseases, pain, and cancer. In the present study we investigated the role of the cysteine residues in the extracellular loop 2 (ECL2) of the receptor, which is particularly cysteine-rich, by a combination of mutagenesis, molecular modeling, chemical and pharmacological experiments. Pretreatment of CHO cells recombinantly expressing the human A2B receptor with dithiothreitol led to a 74-fold increase in the EC50 value of the agonist NECA in cyclic AMP accumulation. In the C783.25S and the C17145.50S mutant high-affinity binding of the A2B antagonist radioligand [3H]PSB-603 was abolished and agonists were virtually inactive in cAMP assays. This indicates that the C3.25–C45.50 disulfide bond, which is highly conserved in GPCRs, is also important for binding and function of A2B receptors. In contrast, the C16645.45S and the C16745.46S mutant as well as the C16645.45S–C16745.46S double mutant behaved like the wild-type receptor, while in the C15445.33S mutant significant, although more subtle effects on cAMP accumulation were observed – decrease (BAY60-6583) or increase (NECA) – depending on the structure of the investigated agonist. In contrast to the X-ray structure of the closely related A2A receptor, which showed four disulfide bonds, the present data indicate that in the A2B receptor only the C3.25–C45.50 disulfide bond is essential for ligand binding and receptor activation. Thus, the cysteine residues in the ECL2 of the A2B receptor not involved in stabilization of the receptor structure may have other functions.

Graphical abstract

Adenosine A2B receptors form only one disulfide bond between the conserved cysteine residues C783.25 and C17145.50; the other three cysteines in the loop are not involved in disulfide bond formation.

  1. Download : Download full-size image

Introduction

Adenosine A2B receptors belong to the large group of purinergic G protein-coupled receptors (GPCRs), which comprise P2 (P2Y and P2X, nucleotide-activated) and P1 (adenosine) receptors [1]. Brunschweiger and Müller [2] proposed to add P0 (adenine) receptors as a third class to the group of purinergic receptors. The P1 or adenosine receptor (AR) family consists of four subtypes, A1, A2A, A2B and A3 [3]. A1 and A3 receptors are coupled to Gi type G proteins, leading to the inhibition of the adenylate cyclase upon receptor activation, while A2A and A2B receptors are mainly coupled to Gs proteins resulting in an increase in intracellular cAMP concentrations via stimulation of adenylate cyclase [4]. In several cell systems, such as HEK-293 and HMC-1 mast cells A2B receptors are additionally coupled to phospholipase C via Gq proteins, and are thereby linked to intracellular Ca2+ release [5], [6]. In the human leukemia cell line Jurkat T, A2B-mediated calcium mobilization independent of inositol-1,4,5-trisphosphate was observed [7]. Coupling of the A2B receptor to the MAPK cascade via ERK1/2 has been described for recombinant CHO cells overexpressing human A2B receptors and for mast cells, showing an involvement in proliferation, differentiation and apoptosis [4], [8]. Furthermore a link of A2B receptor signaling to the arachidonic acid signal transduction pathway via phospholipase A and cyclooxygenase activation leading to vasoconstriction in smooth muscle cells has been described [9].

Among the four AR subtypes A2B has been the least well characterized receptor, mainly due to the lack of suitable, specific ligands [10]. Meanwhile highly selective A2B antagonists have been developed and an A2B-specific antagonist radioligand, [3H]PSB-603 (for structure see Supplemental Figure 1), with high potency and specificity across species, including rodents and humans, has recently become available [11]. As for agonists, besides the nucleoside derivative NECA [12], which is non-selective, and related adenosine derivatives, the first highly selective A2B agonist BAY60-6583 [13], a non-nucleosidic compound, has been developed (structures are shown in Supplemental Figure 1).

In many tissues, A2B receptors are considered low affinity receptors with mostly low expression levels [14]. Therefore, adenosine concentrations typically have to reach micromolar levels to activate natively expressed A2B receptors, which occurs under pathological conditions, such as hypoxia, ischemia, inflammation or massive cell death [15], [16]. While their distribution is ubiquitous, A2B receptors are found at higher densities mainly in the large intestine, in mast cells, hematopoietic cells, and in the brain, particularly in astrocytes [6], [14], [17], [18]. Upregulation has been found in several cancer cell lines [19]. A2B receptors are thought to be involved in a number of diseases and the first antagonist is now being evaluated in clinical trials for the treatment of asthma and chronic obstructive pulmonary disease [10]. Other potential indications include secretory diarrhea associated with inflammation, Alzheimer's disease, inflammatory diseases, pain, cancer, type II diabetes, and diabetic retinopathy [20]. Thus, A2B receptors represent important new drug targets.

To fully understand interactions of the human A2B receptor with its ligands, agonists and antagonists, it is of major importance to gain knowledge about the structure of the receptor, the amino acid residues involved in ligand binding, and to determine the receptor's 3D structure, which in turn can then be used for the development of new ligands [21], [22]. Except for a few mutagenesis studies [16], [23], [24], [25] and homology models [26], [27], [28], the most recent one based on the X-ray structure of the closely related A2A receptor [29], not much structural information about the A2B receptor is available.

A common feature of most GPCRs is the existence of a highly conserved disulfide bond between C3.25 (Ballesteros Weinstein nomenclature [30]) at the extracellular site of transmembrane domain 3 (TMD3) and cysteine residue C45.50 [31] in the extracellular loop 2 (ECL2) located between TMD4 and TMD5 [26], [27], [32]. Recently de Graaf et al. [31] undertook a molecular modeling project and aligned ECL2 sequences of 365 human GPCRs. More than 92% of the investigated receptors showed the conserved disulfide bond.

The A2B receptor possesses the longest ECL2 of all four adenosine receptor subtypes, with four cysteine residues – the highest number found in any GPCR – of which three (C154, C167, C171) are homologous to the three (C146, C159, C166) found in the A2A receptor (see Fig. 1, Fig. 2). Those four cysteine residues are conserved in the known mammalian A2B receptor orthologs. Therefore, the goal of the present study was to investigate the role of the cysteine residues in the cysteine-rich ECL2 of the A2B receptor with respect to disulfide bond formation, ligand binding, and receptor activation.

Section snippets

Material and methods

All chemicals were obtained from Roth (Karlsruhe, Germany) or Applichem (Darmstadt, Germany) unless otherwise noted.The Radioligand [3H]PSB-603 were obtained from Quotient Bioresearch (Cardiff, UK) by custom-labeling.

Comparison of extracellular loops 1 and 2 of A2A and A2B receptors

The A2A receptor is the adenosine receptor subtype, which is most closely related to the A2B receptor. Sequence analysis of the human A2A and A2B receptors show an overall identity of 58% and a similarity of 73%. The most conserved residues are found within the transmembrane domains. By comparing the extracellular loops 1 and 2, which show 44% and 34% identity, and 56% and 46% similarity, respectively, one can find extremely high degrees of homology when comparing the residues close to the

Discussion

The extracellular loops ECL1 [25], ECL2 [31], and ECL3 [42] of GPCRs belonging to the rhodopsin family have been found to contribute considerably to receptor function [46]. However, extracellular loops differ widely in length, sequence, and structure between different GPCRs and even between closely related receptor subtypes [31]. Cysteine residues and disulfide bonds present in the extracellular domains of GPCRs have been reported to play important roles in ligand binding, receptor stability,

Acknowledgements

A.C.S., D.T., and C.E.M. are supported by the state of NRW (NRW International Research Graduate School BIOTECH-PHARMA). T.B. was supported by a stipend provided by the Bischöfliche Studienförderung Cusanuswerk. We would like to thank Susan Jean Johns for upgrading the TOPO2 program to fulfill our needs for more colors in the topology model. We are also grateful to Dr. Thomas Krahn, Bayer Healthcare (Germany) for providing BAY60-6583.

References (63)

  • Z. Ding et al.

    Inactivation of the human P2Y12 receptor by thiol reagents requires interaction with both extracellular cysteine residues, Cys17 and Cys270

    Blood

    (2003)
  • L.A. Rubenstein et al.

    Molecular dynamics of a biophysical model for beta2-adrenergic and G protein-coupled receptor activation

    J Mol Graph Model

    (2006)
  • R.W. Mercier et al.

    hCB2 ligand-interaction landscape: cysteine residues critical to biarylpyrazole antagonist binding motif and receptor modulation

    Chem Biol

    (2010)
  • W.A. Goddard et al.

    Predicted 3D structures for adenosine receptors bound to ligands: comparison to the crystal structure

    J Struct Biol

    (2010)
  • S. Barrondo et al.

    Allosteric modulation of 5-HT(1A) receptors by zinc: binding studies

    Neuropharmacology

    (2009)
  • C.E. Elling et al.

    Metal ion site engineering indicates a global toggle switch model for seven-transmembrane receptor activation

    J Biol Chem

    (2006)
  • J.M. Klco et al.

    C5a receptor oligomerization. I. Disulfide trapping reveals oligomers and potential contact surfaces in a G protein-coupled receptor

    J Biol Chem

    (2003)
  • M. Berthouze et al.

    Two transmembrane Cys residues are involved in 5-HT4 receptor dimerization

    Biochem Biophys Res Commun

    (2007)
  • G. Burnstock

    Purine and pyrimidine receptors

    Cell Mol Life Sci

    (2007)
  • A. Brunschweiger et al.

    P2 receptors activated by uracil nucleotides—an update

    Curr Med Chem

    (2006)
  • B.B. Fredholm et al.

    Nomenclature and classification of adenosine receptors – an update

    Pharmacol Rev

    (2011)
  • J. Linden et al.

    Characterization of human A(2B) adenosine receptors: radioligand binding, western blotting, and coupling to G(q) in human embryonic kidney 293 cells and HMC-1 mast cells

    Mol Pharmacol

    (1999)
  • I. Feoktistov et al.

    Adenosine A2B receptors

    Pharmacol Rev

    (1997)
  • M. Mirabet et al.

    Calcium mobilization in Jurkat cells via A2b adenosine receptors

    Br J Pharmacol

    (1997)
  • G. Pearson et al.

    Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions

    Endocr Rev

    (2001)
  • M.V. Donoso et al.

    A2B adenosine receptor mediates human chorionic vasoconstriction and signals through arachidonic acid cascade

    Am J Physiol Heart Circ Physiol

    (2005)
  • C.E. Müller et al.

    Recent developments in adenosine receptor ligands and their potential as novel drugs

    Biochim Biophys Acta

    (2010)
  • T. Borrmann et al.

    1-Alkyl-8-(piperazine-1-sulfonyl)phenylxanthines: development and characterization of adenosine A2B receptor antagonists and a new radioligand with subnanomolar affinity and subtype specificity

    J Med Chem

    (2009)
  • L. Yan et al.

    Adenosine receptor agonists: from basic medicinal chemistry to clinical development

    Expert Opin Emerg Drugs

    (2003)
  • P.G. Baraldi et al.

    Recent improvements in the development of A(2B) adenosine receptor agonists

    Purinergic Signal

    (2008)
  • B.B. Fredholm

    Adenosine, an endogenous distress signal, modulates tissue damage and repair

    Cell Death Differ

    (2007)
  • Cited by (0)

    View full text