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Effect of Gβ5 on dopamine-mediated activation of D2R-coupled G protein signaling as measured by a fast kinetic BRET assay.

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posted on 2014-08-27, 02:52 authored by J. Christopher Octeau, Joseph M. Schrader, Ikuo Masuho, Meenakshi Sharma, Christopher Aiudi, Ching-Kang Chen, Abraham Kovoor, Jeremy Celver

A. Average BRET dose-repsonse curve elicited by the application of 10 pM - 10 µM concentrations of dopamine to cells expressing only D2R (black trace) and cells transiently coexpressing low (dark grey) and high (light grey) levels of Gβ5. D2R stimulation by dopamine application leads to the dissociation of the G protein heterotrimer into Gβγ-Venus and GTP-bound Gαo subunits. Free Gβγ-Venus interacts with masGRK3ct-NanoLuc to produce a BRET signal. The black trace is from HEK293 cells that did not coexpress Gβ5 and the dark and light grey traces are from HEK293 cells transiently coexpressing two different levels (l, for low and h, for high) of Gβ5 (mean ± SEM; n = 4). B. Quantification of the maximal amplitude (Emax) of the BRET signal elicited by the application of dopamine in the cells described above. The Emax only significantly differed between D2R and D2R + Gβ5 (h) (*p<0.01, ANOVA followed by Tukey’s post-hoc test). C. Quantification of the average EC50 derived for the dopamine mediated activation of D2R in the cells described in D. The response at each dose is expressed as a percent of the average maximal response (n = 4). The EC50 (in nM) for D2R (black bar), D2R + Gβ5 (l, dark grey bar), and D2R + Gβ5 (h, light grey bar) was 19.8±0.825, 21.3±0.863, and 25.4±0.431 (mean ± SEM), respectively. The EC50 only significantly differed between D2R and D2R + Gβ5 (h) (*p<0.01, ANOVA followed by Tukey’s post-hoc test). D. Averaged traces (± SEM) of changes in the BRET signal (ΔBRET or the BRET response) over time obtained from HEK293 cells transfected with cDNA for D2R, Gαo, Venus-Gβγ, masGRK3ct-NanoLuc and treated sequentially with dopamine (10 nM) and haloperidol (100 µM). E. Quantification of the deactivation kinetics of the dopamine-elicited BRET response after application of the D2R antagonist, haloperidol (100 µM).