The second extracellular loop of GPCRs determines subtype-selectivity and controls efficacy as evidenced by loop exchange study at A2 adenosine receptors

doi:10.1016/j.bcp.2013.03.005

Biochemical Pharmacology

Volume 85, Issue 9, 1 May 2013, Pages 1317-1329

https://doi.org/10.1016/j.bcp.2013.03.005 Get rights and content

Abstract

The second extracellular loop (EL2) of G protein-coupled receptors (GPCRs), which represent important drug targets, may be involved in ligand recognition and receptor activation. We studied the closely related adenosine receptor (AR) subtypes A_2A and A_2B by exchanging the complete EL2 of the human A_2BAR for the EL2 of the A_2AAR. Furthermore, single amino acid residues (Asp148^45.27, Ser149^45.28, Thr151^45.30, Glu164^45.43, Ser165^45.44, and Val169^45.48) in the EL2 of the A_2BAR were exchanged for alanine. The single mutations did not lead to any major effects, except for the T151A mutant, at which NECA showed considerably increased efficacy. The loop exchange entailed significant effects: The A_2A-selective agonist CGS21680, while being completely inactive at A_2BARs, showed high affinity for the mutant A_2B(EL2-A_2A)AR, and was able to fully activate the receptor. Most strikingly, all agonists investigated (adenosine, NECA, BAY60-6583, CGS21680) showed strongly increased efficacies at the mutant A_2B(EL2-A_2A) as compared to the wt AR. Thus, the EL2 of the A_2BAR appears to have multiple functions: besides its involvement in ligand binding and subtype selectivity it modulates agonist-bound receptor conformations thereby controlling signalling efficacy. This role of the EL2 is likely to extend to other members of the GPCR family, and the EL2 of GPCRs appears to be an attractive target structure for drugs.

Graphical abstract

Introduction

G protein-coupled receptors (GPCRs) are a major class of targets for drugs on the market as well as for those presently in development. More than one third of current therapeutics are directed towards GPCRs [1]. GPCRs are divided into six different receptor classes (A to F) among which class A is the largest one comprising all rhodopsin-like GPCRs. In recent years many class A GPCRs have been co-crystallized with a variety of ligands ranging from inverse agonists/antagonists to full agonists [2], [3], [4]. Furthermore, the crystal structure of ternary complexes consisting of the agonist-bound β₂ adrenergic receptor and G_s protein, and of rhodopsin bound to a fragment of the G_α protein have been described revealing the conformational changes during receptor activation [5], [6]. Each crystal structure provides a snapshot of a specific conformational stage along the activation process of the receptor. By comparing the X-ray structures of GPCRs from different families global patterns of conformational changes leading to receptor activation have emerged [2], [3], [7], [8], [9], [10]. X-ray structures also provide insights into the respective binding pockets and allow the optimization of homology models that are highly useful for virtual screening [11], [12], [13]. This is particularly true for homology models of closely related receptor subtypes. One example is the A_2B adenosine receptor (A_2BAR) model [14], [15], which has been constructed based on X-ray structures of the closely related A_2AAR [16], [17].

Adenosine receptors (ARs) are members of class A GPCRs, subdivided into four distinct subtypes: A₁, A_2A, A_2B, and A₃. A₁ and A₃ ARs are G_i-coupled receptors whereas A_2A and A_2B ARs are coupled to G_s proteins thereby mediating an increase in intracellular cAMP levels. Furthermore, coupling of the A₁, A_2B, and A₃ARs to G_q has been found in several cell types [18].

Due to their ubiquitous distribution throughout the body, ARs are involved in many physiological and pathophysiological processes. Ligands for ARs are therefore of considerable interest as novel drugs [18], [19], [20]. The A_2AAR subtype mediates antiinflammatory and immunosuppressive effects [21]. A_2AAR antagonists are currently in advanced clinical trials for Parkinson's disease [22]; furthermore, they may be active in Alzheimer's disease, and for the treatment of addiction and depression [18], [19], [20], [23]. For the A_2BAR subtype, antagonists are being developed and evaluated for the treatment of asthma and bronchial inflammation [24]. Further potential indications include cancer, diabetes, (neuro) inflammation, and Alzheimer's disease [23], [25], [26].

The endogenous ligand adenosine (1, Fig. 1) and the vast majority of its derivatives, e.g. NECA (2) and CGS21680 (3), show considerably higher affinity for the human A_2AAR than for the A_2BAR. The reason for this and the amino acids involved are yet unknown. Sequence analysis of the human A_2A and A_2B ARs show an overall identity of 58% and a similarity of 73%, whereas the EL1 and especially the EL2 are less conserved exhibiting 44% and 34% identity, and 56% and 46% similarity, respectively [14], [20].

In many GPCRs the EL2 is not well conserved regarding length, amino acid composition, and number of disulfide bonds. Nevertheless it participates in ligand recognition and binding as evidenced by crystal structures [27], [28]. Meanwhile several X-ray structures of the human A_2AAR are available, including inactive, antagonist-bound conformations (ZM241385, 3EML [16]; XAC, 3REY and caffeine, 3RFM [29]), intermediate states, which are NECA (2YDV, [30]) and adenosine-bound (2YDO, [30]), respectively, and the fully activated UK-432097-bound conformation (3QAK, [17]). The resolutions of the crystal structures are in the same range for all structures with 2.6 Å for the inactive and NECA-bound structure, 2.7 Å for the fully active structure 3QAK, and 3.0 Å for the adenosine-bound structure, and 3.6 Å for the caffeine-bound structure 3RFM [16], [17], [29], [30]. Only the most recent structure of the A_2AAR in complex with ZM241385 (4EIY), shows a significantly higher resolution of 1.8 Å [31]. In this structure several ordered water clusters, a bound sodium ion, as well as lipids and cholesterol molecules are resolved. Except for the two transition-state structures 2YDV and 2YDO [30] and the inactive structure 4EIY [31], where the extracellular loops, especially EL2, are completely resolved, all other structures show very low loop resolution or incomplete loops. From the EL2 only the part close to TM5, containing the cysteine residues involved in disulfide bonds and the stabilizing β-sheet, which is also the most conserved part of the EL2, are resolved in all structures of the A_2AAR, except for 3VG9, where the β-sheet is missing [16], [17], [29], [30], [31], [32], [33].

Since each crystal structure only provides one single, fixed receptor conformation, and active conformations and loop structures are in most cases poorly resolved, mutagenesis studies are still the method of choice to investigate the role and the involvement of particular amino acids or whole loop structures. Furthermore, solving X-ray structures for all subtypes of a particular GPCR subfamily may be too costly and not practical [3], and for the A_2BAR no X-ray structure is available.

In the present study we investigated the role of the EL2 of the A_2BAR by exchanging the whole loop by the EL2 of the closely related A_2AAR. Moreover, we investigated the role of single amino acids in the EL2 by exchanging them for alanine. Finally we performed homology modelling studies of the A_2BAR based on recently published X-ray structural data of the A_2AAR. Our main goal was to find explanations (i) for the relatively low affinity and potency of adenosine and its derivatives at A_2BARs in comparison to the closely related A_2AAR subtype, and about subtype-selectivity for A_2A versus A_2B ARs, and (ii) to get more insight into the roles of the EL2 in GPCR activation by comparing two closely related receptors, which differ considerably in their EL2 length and sequence. By a complete loop exchange approach of closely related GPCRs we learned that the EL2 is not only involved in ligand recognition and receptor subtype selectivity, but beyond that, it plays an important role in receptor activation and ligand efficacy.

Section snippets

Methods

Chemicals were obtained from Roth (Karlsruhe, Germany) or Applichem (Darmstadt, Germany) unless otherwise noted. All enzymes and competent bacteria were obtained from New England Biolabs (Frankfurt, Germany), primers, the vector pUC19, cell culture reagents and supplements, including antibiotics, were obtained from Life Technologies - Invitrogen (Darmstadt, Germany). Radioligands were obtained from Amersham - GE Healthcare (Frankfurt, Germany).

Cell culture

Chinese hamster ovary (CHO) cells were cultured at 37 °C and 5% CO₂ in DMEM-F12 supplemented with 10 % FCS, 100 U/ml penicillin G, 100 μg/ml streptomycin, 1% ultraglutamine and 200 μg/ml geneticin (G418). After transfection, G418 (800 μg/ml) was added for two weeks. GP⁺envAM12 packaging cells were maintained in HXM media containing DMEM supplemented with 10 % FCS, 100 U/ml penicillin G, 100 μg/ml streptomycin, 1 % ultraglutamine, 15 μg/ml hypoxanthine, 250 μg/ml xanthine, 25 μg/ml mycophenolic acid and

cAMP assays

CHO cells were seeded into a 24-well plate at a density of 200,000 cells/well 24 h before performing the cAMP assay. Cells were washed with Hank's Balanced Salt Solution (HBSS; 20 mM HEPES, 135 mM NaCl, 5.5 mM glucose, 5.4 mM KCl, 4.2 mM NaHCO₃, 1.25 mM CaCl₂, 1 mM MgCl₂, 0.8 mM MgSO₄, 0.44 mM KH₂PO₄ and 0.34 mM Na₂HPO₄, pH adjusted to 7.3) with 1 U/ml of adenosine deaminase (ADA, Sigma–Aldrich, Taufkirchen, Germany). Then the cells were incubated in 300 μl HBSS at 37 °C and 5 % CO₂ for 2 h. ADA was omitted

Data analysis

All data were analyzed by GraphPad Prism version 4.02 for Windows, GraphPad Software, San Diego California USA [38]. For curve fitting standard equations offered by Prism for one-site competition and two-site fits were used. The one-site fit was taken for the calculation, since the two-site fit did not lead to meaningful results. Levels of significance were determined using a two-tailed t-test.

The second extracellular loop of the human A₂ adenosine receptors

By comparing the EL2 of the human AR subtypes A_2A and A_2B, which show an overall similarity of 46% and an identity of 34% one can find high degrees of similarity at the region close to TM5, especially between the β-sheet and TM5 (see Fig. 2, Fig. 3). The β-sheet is found in all A_2AAR X-ray structures [16], [17], [29], [30], where it is in anti-parallel conformation with a second β-sheet in EL1. Both sheets are predicted to be present in the human A_2BAR as well [14], being probably stabilized by

Radioligand binding

Radioligand binding assays were performed using membrane preparations of cells recombinantly expressing either the wt or the mutant A_2BARs. Homologous competition assays using unlabeled PSB-603 versus [³H]PSB-603 as a radioligand allowed to estimate the expression levels of the wt and mutant receptors as well as the calculation of the K_D values (see Table 1). The expression of the wt A_2A and A_2BARs was similar (A_2A, 390 ± 167 fmol/mg protein; A_2B, 271 ± 85 fmol/mg protein). The levels of most of the A

Discussion

G protein-coupled receptors are major targets in drug discovery. Information on their structure and function is therefore not only of interest in basic science but, in addition, has important practical implications in the development of better drugs. Besides the orthosteric binding site, which is occupied by the physiological agonist and by many drugs, evidence has recently accumulated that the extracellular region of the GPCR receptor proteins are directly involved in ligand recognition and

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¹: Authors contributed equally.

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The second extracellular loop of GPCRs determines subtype-selectivity and controls efficacy as evidenced by loop exchange study at A2 adenosine receptors

Abstract

Graphical abstract

Introduction

Section snippets

Methods

Cell culture

cAMP assays

Data analysis

The second extracellular loop of the human A2 adenosine receptors

Radioligand binding

Discussion

Trends Pharmacol Sci

Trends Pharmacol Sci

Cell

Trends Pharmacol Sci

Adv Pharmacol

Biochem Pharmacol

Biochim Biophys Acta

Adv Pharmacol

Trends Pharmacol Sci

Structure

Gene

Anal Biochem

Meth Neurosci

Trends Pharmacol Sci

Pharmacol Ther

J Biol Chem

Biochem Pharmacol

The year in G protein-coupled receptor research

Mol Endocrinol

New insights for drug design from the X-ray crystallographic structures of GPCRs

Mol Pharmacol

A new era of GPCR structural and chemical biology

Nat Chem Biol

Crystal structure of the β2 adrenergic receptor-Gs protein complex

Nature

Crystal structure of metarhodopsin II

Nature

The GPCR Network: a large-scale collaboration to determine human GPCR structure and function

Nat Rev Drug Discov

Structure-based discovery of novel chemotypes for adenosine A2A receptor antagonists

J Med Chem

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J Med Chem

Homology modelling of the human adenosine A2B receptor based on X-ray structures of bovine rhodopsin, the β2 adrenergic receptor and the human adenosine A2A receptor

J Comput Aided Mol Des

The second extracellular loop of GPCRs determines subtype-selectivity and controls efficacy as evidenced by loop exchange study at A₂ adenosine receptors

The second extracellular loop of the human A₂ adenosine receptors

Crystal structure of the β₂ adrenergic receptor-G_s protein complex

Structure-based discovery of novel chemotypes for adenosine A_2A receptor antagonists

Structure-based discovery of A_2A adenosine receptor ligands

Homology modelling of the human adenosine A_2B receptor based on X-ray structures of bovine rhodopsin, the β₂ adrenergic receptor and the human adenosine A_2A receptor