Fig. 6: Impact of GABAB-FLNA interactions on Gi protein signaling.
From: Filamin A organizes γ‑aminobutyric acid type B receptors at the plasma membrane
a Scheme of the FRET sensor34 used to monitor Gi protein activation in real-time. This consists of a single plasmid encoding Gαi fused to the fluorescent donor mTurquoise2 (mTq2), Gβ1, and Gγ2 fused to the fluorescent acceptor cp173Venus (cpVenus). A decrease in FRET is indicative of Gi protein activation. b Representative FRET measurement of Gi protein activation and deactivation in response to transient GABA stimulation (100 µM). c Comparison of the kinetics of Gi protein deactivation from experiments as in b in the presence of FLNA17-18 or FLNA19-20. Data are mean ± SEM. n = 22 and 30 cells (FLNA17-18 and FLNA19-20, respectively) examined over three independent experiments. d Corresponding estimated time constants (τ) for Gi protein deactivation. Data are mean ± SEM. n = 22 and 30 cells (FLNA17-18 and FLNA19-20, respectively) examined over three independent experiments. e Top, representative images of GABAB1 (magenta) over-expressed in M2 cells (FLNA−) or A7 cells (FLNA+) together with Lifeact-GFP (green). Scale bars, 5 µm. Bottom, corresponding MCC analyses. Data are mean ± SEM. n = 16 and 16 cells (M2 and A7 cells, respectively) examined over two independent experiments. f Comparison of the kinetics of Gi protein deactivation in M2 and A7 cells after a transient stimulation with GABA as in b. Data are mean ± SEM. n = 23 and 20 cells (M2 and A7 cells, respectively) examined over two independent experiments. g Corresponding estimated time constants (τ) for Gi protein deactivation. Data are mean ± SEM. n = 23 and 20 cells (M2 and A7 cells, respectively) examined over two independent experiments. h Schematic summary of the results. The interactions of GABAB1 with FLNA tethers GABAB receptors to the actin cytoskeleton, controlling their localization on the plasma membrane. In addition, interaction of either full length FLNA or FLNA19-20 with GABAB1 leads to prolonged Gi signaling after transient GABA stimulation, presumably via an allosteric mechanism. This effect is lost in M2 cells lacking FLNA. *p < 0.05, **p < 0.01, ****p < 0.0001 by two-tailed unpaired Welch’s t-test. Source data are provided as a Source Data file.
