Rate of β-arrestin recruitment to the β2 adrenoceptor is governed by agonist efficacy

Elizabeth Rosethorne , Steven J Charlton. Novartis Institutes for Biomedical Research, Horsham, United Kingdom.

Efficacy is a relative scale that describes the strength or efficiency of receptor activation by an agonist. Efficacy is typically thought of as influencing the maximal response elicited in a system (intrinsic activity), but in kinetic terms, a more efficient agonist might also be expected to lead to a increased rate of receptor signalling. We have investigated the time-course of β-arrestin recruitment to the β2 adrenoceptor (β2 AR) upon stimulation by 5 agonists of varying relative efficacy, to determine whether efficacy influences the rate as well as the maximal level of recruitment. The PathHunter™ β-arrestin assay (DiscoverX) uses enzyme fragment complementation to measure recruitment of β-arrestin to a GPCR after activation. PathHunter™ CHO-K1 β2 AR/ β-arrestin cells were seeded overnight in opaque 384-well CulturPlates ( PerkinElmer, UK) at 3,000 cells/well. Cells were stimulated with a range of β2 AR agonists (adrenaline, isoprenaline, formoterol, salbutamol and salmeterol) for 90 min, the Flash detection reagent added and luminescence read on the LeadSeeker (GE Healthcare, UK) after a 1 min incubation. Time course studies revealed that β-arrestin was recruited to the β2AR after as little as 5 min, with maximal levels obtained after 2 h. At 2 h, both EC50 values and intrinsic activity obtained using this assay format were comparable to those observed previously using cAMP accumulation as a readout for receptor activity (Battram et al., 2006). We investigated whether intrinsic activity and potency changed with time. EC50 values for β-arrestin recruitment were determined over a 6 h time-course (see table for data from 15 min and 2 h time points). Potency was lower at 15 min than 2 h for all of the agonists tested, although the time taken to reach full potency varied for each agonist. Despite this, rank order of potency did not change with increasing stimulation time. The time taken to reach maximal response was also different for each agonist and this correlated well with intrinsic activity (r2 = 0.87). In conclusion, both the rate and maximal degree of β-arrestin recruitment to the β2 AR is governed by the efficacy of the agonist. This means that EC50 estimates from rapid time points are likely to underestimate the potency of low efficacy agonists to a greater degree than high efficacy agonists.

Table 1 I.A. = intrinsic activity.

Battram, C., Charlton, SJ., Cuenoud, B., et al., (2006) JPET 317:762-770.