Fig. 2: Mechano-activation of GABA_B receptor requires integrin β₃ interaction.

a IP₁ production in HEK293 cells transfected with either control siRNA or integrin β₃ siRNA, along with the expressing of vector and G_qi9, or GABA_B receptor and G_qi9, under either adhesion or suspension conditions. Data are present as mean ± s.e.m. from five biologically independent experiments and analyzed using unpaired t test (two-tailed) to determine significance. ***P < 0.001, not significant (ns) > 0.05. b Co-immunoprecipitation of GABA_B receptor and integrin β₃ in HEK293 cells transfected with GABA_B receptor constructs (Snap-tagged GB1 and GB2) using anti-integrin β₃ antibody, under basal condition. Only GB1 in cell surface was labeled and visualized using an impermeable Snap fluorescent substrate. c–f Co-immunoprecipitation of GABA_B receptor and integrin β₃ in HEK293 cells transfected with GABA_B receptor constructs (HA-tagged GB1 and GB2) using anti-HA antibody, under conditions including suspension or adhesion (c), without (control) or with shear stress (c), Blebbistatin (50 μM, 30 min) treatment (d), RGDS (10 μM, 12 h) treatment (e), or CGP54626 (50 μM, 30 min) treatment (f). Blots are representative from at least three biologically independent experiments (b, n = 4; c, suspension or adhesion, n = 3; control or with shear stress, n = 4; d, n = 3; e, n = 3; f, n = 4). The amount of integrin β₃ immunoprecipitated by IgG or HA antibody are present as mean ± s.e.m. in (e) and analyzed using unpaired t test (two-tailed) to determine significance. **P < 0.01. g Schematic representation of the BRET assay detecting direct interaction between GABA_B receptor and integrin β₃. Rluc was fused in C-terminal of GB1 or GB2 subunit (GB1^Rluc or GB2^Rluc) as luminescence donor. Venus was fused in C-terminal of integrin β₃ or integrin α_V subunit (integrin β₃^Venus or integrin α_V^Venus) as fluorescence acceptor. h, i Interaction of GABA_B receptor and integrin β₃ interaction between GB1 and integrin β₃ or GB1 and integrin α_V (h), or GB2 and integrin β₃ (i) detected using BRET titration assay. The BRET between mGlu2 with Rluc fused in the C-terminal and 5HT2a receptor with Venus fused in the C-terminal was measured as positive control. The BRET between GB1^Rluc and Venus was measured as negative control. Data were analyzed by nonlinear regression on a pooled data set from at least three biologically independent experiments (upper to lower, h, left, n = 16, 10, 12; right, n = 5, 5; i, n = 9, 9) each performed in triplicates, fitting with 1-site binding model in GraphPad Prism 8.