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

Anke C. Schiedel (1) , Sonja Hinz (1) , Dominik Thimm (1) , Farag Sherbiny (2) , Thomas Borrmann (1) , Astrid Maass (2) , Christa E. Müller (1)

1 Pharmaceutical Institute
2 Fraunhofer SCAI - Fraunhofer Institute for Algorithms and Scientific Computing

The adenosine A 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 receptor with dithiothreitol led to a 74-fold increase in the EC value of the agonist NECA in cyclic AMP accumulation. In the C78S and the C171S mutants high-affinity binding of the A antagonist radioligand [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 receptors. In contrast, the C166S and the C167S mutants as well as the C166S-C167S double mutant behaved like the wild-type receptor, while in the C154S 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 receptor, which showed four disulfide bonds, the present data indicate that in the A 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 receptor not involved in stabilization of the receptor structure may have other functions.

Mots clés: Adenosine A receptor; disulfide bonds; extracellular loop 2; mutagenesis; PSB-603

Domaines: Pharmacologie

Anke C. Schiedel, Sonja Hinz, Dominik Thimm, Farag Sherbiny, Thomas Borrmann, et al.. The four cysteine residues in the second extracellular loop of the human adenosine A receptor: role in ligand binding and receptor function. Biochemical Pharmacology, 2011, 82 (4), pp.389. ⟨10.1016/j.bcp.2011.05.008⟩. ⟨hal-00718035⟩