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.