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

Abstract

The adenosine A(2B) 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 A(2B) receptor with dithiothreitol led to a 74-fold increase in the EC(50) value of the agonist NECA in cyclic AMP accumulation. In the C78(3.25)S and the C171(45.50)S mutant high-affinity binding of the A(2B) antagonist radioligand [(3)H]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 A(2B) receptors. In contrast, the C166(45.45)S and the C167(45.46)S mutant as well as the C166(45.45)S-C167(45.46)S double mutant behaved like the wild-type receptor, while in the C154(45.33)S 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 A(2A) receptor, which showed four disulfide bonds, the present data indicate that in the A(2B) 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 A(2B) receptor not involved in stabilization of the receptor structure may have other functions.

BAY60-6583 Acts as a Partial Agonist at Adenosine A2B Receptors

“…In antagonist radioligand binding studies, BAY60-6583 showed high affinity for human (K i 212 nM) A 2B ARs. The nonselective A 2B agonist NECA displayed a 5-to 11-fold lower affinity than BAY60-6583 in binding studies versus the antagonist radioligand [ 3 H]PSB-603 (Schiedel et al, 2011).…”

The rat adenine receptor: pharmacological characterization and mutagenesis studies to investigate its putative ligand binding site

“…We have now experimentally supported the assumption that probably no disulfide bond is formed, by measuring ligand binding and receptor activation (by cAMP accumulation studies) without and after preincubation with the disulfide-reducing agent DTT. In contrast to many other GPCRs that require an intact extracellular disulfide bond for proper functioning [16,48,52], the rAdeR did not show any difference, neither in adenine binding, nor in adenine-induced adenylate cyclase inhibition (Fig. 10) after incubation with DTT to reduce accessible disulfide bonds.…”

“…AJ311952) used for the mutagenesis studies was cloned into the pUC19 vector and subcloned into the pFastBac1 vector (Invitrogen, Karlsruhe) after mutagenesis. Site-directed mutagenesis was performed as previously described [16]. In brief, complementary primers with the desired base exchanges were designed.…”

Characterization of new G protein-coupled adenine receptors in mouse and hamster

“…In the past when searching for a suitable expression system to characterize adenine receptors, we discovered that there is an endogenous high affinity binding site for adenine in CHO K1 cells [24]. We concluded that there may be an ortholog in the Chinese hamster genome as well, whose gene product might be responsible for [ 3 H]adenine binding in CHO K1 cells.…”

“…* For both cell lines, the medium was supplemented with 10 % fetal calf serum, 100 U penicillin, and 100 μg/ml streptomycin. 1321N1 astrocytoma cells stably transfected with the mAde1R, and CHO K1 cells stably transfected with cAdeR were generated using a retroviral expression system as previously described [24]. In brief, GP + env AM12 packaging cells were transfected with retroviral vectors containing adenine receptor cDNA.…”