Real-time allosteric modulation at human and rat adenosine A1 receptor

Adenosine receptors (A1, A2A, A2B, A3) are highly distributed throughout the human body. PD 81,723 (PD) is a positive allosteric modulator (PAM) which specifically enhances the response of the adenosine A1 receptor (A1AR) induced by an agonist. Research on subtype selective PAMs has a high potential for diminishing side effects due to a reduced demand for agonist concentration. To observe positive allosteric modulation an experimental procedure was established using fluorescence resonance energy transfer (FRET)-based A1AR sensors to monitor conformational change of the receptor in real-time and in single living cells. This approach enabled the investigation of allosteric modulation in combination with the endogenous ligand adenosine (Ado), in contrast to radioligand binding assays. The affinity of PD as well as cooperativity was successfully determined for human and rat A1AR occupied by Ado. Positive allosteric regulation on A1AR is most often characterized by the prolongation of agonist dissociation from the receptor. Here the pure unbinding kinetics of PD from the Ado-occupied-receptor were observed for the first time. Promising point mutations were introduced in the second extracellular loop, the putative allosteric binding site, and their influence on affinity, cooperativity and kinetics of PD was investigated. Taken together, human and rat A1AR FRET-sensors allowed real-time recording of allosteric enhancement of the endogenous ligand adenosine under physiological conditions. This work provides affinity (EC50) of PD for human and rat A1AR, provides evidence for probe dependency of PD with NECA, reveals the mechanism of prolonged dissociation of Ado in the presence of PD and indicates the importance of residue at site 162 located in the second extracellular loop for the kinetic behavior of PD.