Effect of a toggle switch mutation in TM6 of the human adenosine A₃ receptor on Gi protein-dependent signalling and Gi-independent receptor internalization.
Bottom Line: We set out to characterize the effect of this mutation on the efficacy of two agonists at multiple signalling pathways downstream of the adenosine A₃ receptor.Mutation of W6.48F therefore resulted in differential effects on agonist efficacy, and introduced signalling pathway bias for HEMADO at the adenosine A3 receptor.Investigation of the pharmacology of the W6.48F mutant of the adenosine A₃ receptor confirms that this region is important in forming the active conformation of the receptor for stimulating a number of different signalling pathways and that mutations in this residue can lead to changes in agonist efficacy and signalling bias.
Affiliation: Institute of Cell Signalling, School of Life Sciences, The University of Nottingham, Nottingham, UK.Show MeSH
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Mentions: By introducing the W243F mutation to the A3 receptor, it is possible that ligand-binding site has been sufficiently altered to prevent the binding of HEMADO to the receptor. To confirm that HEMADO was still capable of binding to the A3 W243F-YFP receptor, we investigated the affinity of NECA and HEMADO at both A3-YFP and A3 W243F-YFP receptors using a previously characterized fluorescence-based competition binding assay, using an xanthine amine congener-based fluorescent antagonist, CA200645 (Stoddart et al., 2012). Initially, we established that the fluorescent antagonist used in the competition binding assay, CA200645, itself retained affinity at both A3-YFP and A3 W243F-YFP. This was achieved by measuring the shift in NECA internalization concentration-response curves at both A3-YFP and A3 W243F-YFP in the presence of 25 nM CA200645 (Supporting Information Figure S2). Subsequent Gaddum analysis revealed the pKD for CA200645 was 7.82 ± 0.13 at the A3-YFP receptor and 7.81 ± 0.05 at the A3 W243F-YFP receptor. A3-YFP and A3 W243F-YFP cells were treated with increasing concentrations of NECA or HEMADO in the presence of 25 nM CA200645, automated confocal images were obtained on the Ultra confocal plate reader and total image intensities were determined. As shown in Figure 4A, a concentration-dependent decrease in CA200645 binding was observed with both NECA and HEMADO in both cell lines. Calculation of pKi values from the resulting IC50 values showed that the affinity of both NECA and HEMADO was similar at both wild-type and mutant receptors (Table 1). To confirm that HEMADO was acting as an antagonist at the A3 W243F-YFP receptor (compared with its agonist properties at the A3-YFP receptor), A3 W243F-YFP-expressing cells were pretreated with 10 μM HEMADO before the addition of increasing concentrations of NECA. Under these conditions, the ability of NECA to cause internalization of the A3 W243F-YFP receptor was virtually abolished (Figure 5).
Affiliation: Institute of Cell Signalling, School of Life Sciences, The University of Nottingham, Nottingham, UK.