Kinetic investigations into G protein activation and biased agonism at the dopamine D2 receptor

[Speaker] Alastair C Keen:1,2
[Co-author] David A Sykes:2, Steven J Charlton:2, J Robert Lane:1
1:Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Australia, 2:School of Life Sciences, Queen's Medical Centre, University of Nottingham, UK

Background-The dopamine D2 receptor (D2R) is a therapeutic target for the treatment of several neuropsychiatric disorders. The D2R can couple to all members of the heterotrimeric Galpha-i/o/z protein family but the physiological importance of coupling to distinct G protein subtypes remains unclear. It has been demonstrated that slow-dissociating agonists display time-dependent biased agonism between endpoints at different levels in the D2R signalling cascade(1). The aim of this study was to explore biased agonism at the level of coupling to different G proteins and how this might be influenced by agonist binding kinetics.
Methods-D2R expressing FLPIN HEK293 cells were stimulated by a panel of agonists with varied intrinsic efficacy and binding kinetics. Galpha-i/o/z activation was measured in live cells by BRET-based biosensors which detect dissociation of the Gbeta-gamma subunit upon activation of the G protein(2). Biased agonism was quantified by analysing concentration response curves of agonists at different timepoints using an operational model of agonism(3).
Results-The D2R showed a coupling preference for GoA/B and Gz over Gi1/2/3 as indicated by the relative potency of high efficacy agonists. Time-dependent biased agonism was observed for some agonists relative to ropinirole primarily between Gi1/2/3 and Gz activation. Interestingly, all agonists, independent of their receptor binding kinetics, displayed a large increase in potency over time for Gz activation while the potency of agonists at all other G proteins either did not change, or changed in manner consistent with their dissociation rate from the D2R. We provide evidence that the large increase in potency over time at Gz is dependent on a slow rate of GTP hydrolysis.
Conclusion-We observed no correlation between association or dissociation rate of selected agonists and their efficacy to activate different G proteins. Subtle but statistically significant bias was detected between Gz and Gi1/2/3 activation for some agonists, and this pattern of bias changes over time. Finally, we have provided evidence that rates of G protein activation/deactivation in combination with ligand binding kinetics may underlie some observations of biased agonism.
1.Klein-Herenbrink, C. et al. (2016)Nat. Commun. 7,10842
2.Hollins, B. et al. (2009)Cell. Signal. 21,1015-1021
3.Kenakin, T. et al. (2012)ACS Chem. Neurosci. 3,193-203
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