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: To investigate the time- and concentration-dependence of A3-YFP and A3 W243F-YFP internalization to both agonists, quantification of internalized receptor was carried out using a MD ImageXpress Ultra confocal plate reader. This high-content screening based approach automatically collects images from each well of a 96-well plate and quantifies the accumulation of receptor in granules within the cells (predominantly in the perinuclear compartment) using a mathematical algorithm (Kilpatrick et al., 2010; Watson et al., 2012). This enabled full concentration-response curves to both NECA and HEMADO to be generated for A3-YFP and A3 W243F-YFP (Figure 2). NECA stimulated receptor internalization with similar potency in the A3 W243F-YFP- and A3-YFP-expressing cells (Figure 2A, Table 1). In contrast, HEMADO stimulated internalization with a higher potency than NECA at A3-YFP but did not stimulate any significant internalization of the mutant A3 W243F-YFP receptor at concentrations up to 10 μM. These experiments were performed at a single time point (45 min); therefore, to confirm that the observed results were not due to slower kinetics of the HEMADO-mediated response, the time course of internalization was determined for both agonists. HEMADO-mediated internalization of the wild-type A3-YFP was indeed significantly slower than that observed with NECA (HEMADO t1/2 = 18.0 ± 4.8 min vs. NECA t1/2 = 8.5 ± 0.8 min, P < 0.05, unpaired Student's t-test), but at the A3 W243F-YFP receptor, no accumulation of internalized receptor could be detected even after 2 h stimulation with 10 μM HEMADO (Figure 2C,D) and visual inspection of the images showed no change in receptor distribution (data not shown). The difference in the kinetics of NECA- and HEMADO-mediated internalization of A3-YFP suggests the involvement of different downstream signalling pathways to stimulate receptor internalization and to test this, cells expressing A3-YFP or A3 W243F-YFP were treated with PTx for 16 h before stimulation to prevent coupling to Gi/o G-proteins. Pretreatment of A3-YFP- or A3 W243F-YFP-expressing cells with PTx resulted in no change in the potency of NECA (pEC50 values in the presence of PTx of 5.58 ± 0.31 and 6.29 ± 0.11 for A3-YFP and A3 W243F-YFP, respectively; n = 4, P > 0.05, Student's unpaired t-test vs. pEC50 values in the absence of PTx) (Figure 3). In addition, PTx treatment did not alter the distribution of either receptor before or after agonist treatment (data not shown). The influence of PTx treatment on the concentration-response characteristics of HEMADO-mediated internalization of the wild-type A3-YFP receptor is shown in Figure 3. In contrast to NECA-mediated A3-YFP receptor internalization, pretreatment with PTx significantly reduced the extent of HEMADO-stimulated internalization. There was a pronounced decrease in the maximal levels of internalization in the presence of PTx (30 ± 6% reduction vs. 10 μM NECA internalization, n = 5) but no significant change in the potency of the agonist (pEC50 = 6.55 ± 0.21, n = 5).
Affiliation: Institute of Cell Signalling, School of Life Sciences, The University of Nottingham, Nottingham, UK.