The four cysteine residues in the second extracellular loop of the human adenosine A₂B receptor: Role in ligand binding and receptor function
2011
Schiedel, Anke C. | Hinz, Sonja | Thimm, Dominik | Sherbiny, Farag | Borrmann, Thomas | Maaß, Astrid | Müller, Christa E.
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.
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