Journal: Nature Communications

Article Title: GABA-independent activation of GABA B receptor by mechanical forces

doi: 10.1038/s41467-025-64811-2

Figure Lengend Snippet: a Schematic illustration depicting the modulation of GABA B receptor activity by ligands (e.g. agonists: GABA and baclofen; antagonist: CGP54626 ). b Schematic representation of the experiments applying traction force and shear stress to cells in this study, encompassing conditions such as cell suspension or adhesion; PDL or FN coating; and shear stress treatment. No GABA was added in these experiments. c IP 1 production in HEK293 cells transfected with vector or GABA B receptor, along with G qi9 chimera in the corresponding conditions: suspension or adhesion (left) ; PDL or FN coating (middle) , without (control) or with shear stress (15 dyn/cm 2 , 15 min) (right) . Data are present as mean ± s.e.m. from at least three biologically independent experiments (from left to right , n =4, 5, 5, respectively), each performed in triplicate and analyzed using an unpaired t-test (two-tailed) to determine significance. *** P < 0.001, * P < 0.05, not significant (ns) > 0.05. d Real-time recording of intracellular Ca 2+ release in HEK293 cells expressing GABA B receptor and G qi9 . Left: Schematic presentation of shear stress loading device and real-time recording of Ca 2+ response in single cells. Right: Real-time Ca 2+ signal measurement. After the recording of basal state of Ca 2+ release for 50 seconds, cells were subjected to shear stress for 100 seconds. Shear stress was then halted for 150 seconds, after which baclofen was injected and the Ca 2+ signal was measured for another 200 seconds. Data are present as mean ± s.e.m. from 85 cells recorded. e Traction force and GABA-induced G i protein activation in HEK293 cells measured by optimized G i protein BRET sensors. Schematic presentation of Gi protein activation measurement by BRET assay based on G αi1 -Nluc, Venus-G γ2 and endogenous G β rearrangement ( left ). Traction force- ( middle ) and GABA- ( right ) induced G i protein activation in HEK293 cells transfected with vector or GABA B receptor together with G i protein sensor. Cells were treated under suspension or adhesion conditions. The traction force-induced net BRET ( middle ) was calculated by the BRET ratio obtained under suspension conditions, subtracting the BRET ratio in adhesion conditions. PBS or GABA-induced net BRET ( right ) was calculated by the BRET ratio before PBS or GABA treatment, subtracting the BRET ratio after PBS or GABA treatment. Data are present as mean ± s.e.m. from five biologically independent experiments, and analyzed using an unpaired t-test (two-tailed) to determine significance. **** P < 0.0001, ** P < 0.01.

Article Snippet: HEK293 cells were stained with 4′6-diamidino-2-phenylindole (DAPI) for 30 min. Then cells were washed with PBS and mounted with FluorSave reagent (AR1109, Boster Biological Technology Co. Ltd., Wuhan, China) for fluorescent imaging.

Techniques: Activity Assay, Shear, Suspension, Transfection, Plasmid Preparation, Control, Two Tailed Test, Expressing, Injection, Activation Assay, Bioluminescence Resonance Energy Transfer

Journal: Nature Communications

Article Title: GABA-independent activation of GABA B receptor by mechanical forces

doi: 10.1038/s41467-025-64811-2

Figure Lengend Snippet: a IP 1 production in HEK293 cells transfected with either control siRNA or integrin β 3 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 β 3 in HEK293 cells transfected with GABA B receptor constructs (Snap-tagged GB1 and GB2) using anti-integrin β 3 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 β 3 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 β 3 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 β 3 . 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 β 3 or integrin α V subunit (integrin β 3 Venus or integrin α V Venus ) as fluorescence acceptor. h , i Interaction of GABA B receptor and integrin β 3 interaction between GB1 and integrin β 3 or GB1 and integrin α V ( h ), or GB2 and integrin β 3 ( 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.

Article Snippet: HEK293 cells were stained with 4′6-diamidino-2-phenylindole (DAPI) for 30 min. Then cells were washed with PBS and mounted with FluorSave reagent (AR1109, Boster Biological Technology Co. Ltd., Wuhan, China) for fluorescent imaging.

Techniques: Transfection, Control, Expressing, Plasmid Preparation, Suspension, Two Tailed Test, Immunoprecipitation, Construct, Labeling, Shear, Bioluminescence Resonance Energy Transfer, Fluorescence, Titration, Positive Control, Negative Control, Binding Assay

Journal: Nature Communications

Article Title: GABA-independent activation of GABA B receptor by mechanical forces

doi: 10.1038/s41467-025-64811-2

Figure Lengend Snippet: a –c Left: IP 1 production in HEK293 cells transfected with the indicated constructs, along with G qi9 under either suspension or adhesion conditions: ( a ): vector, GABA B receptor (GB1 and GB2), GB1 only or GB2 only; ( b ): vector, GABA B receptor, GABA B -ΔG (mutant with L685 in GB2 substituted with Phenylalanine that fails to couple G protein), or GABA B -ΔB (mutant with the residues S276 and E465 in GB1 mutated to Analine that fails to bind GABA); ( c ): vector or GABA B receptor, treated with or without antagonist CGP54626 (50 μM, 30 min). Data are present as mean ± s.e.m. from at least three biologically independent experiments (from left to right, a , n = 4, 4, 4, 4; b, n = 6, 6, 4, 4; c, n = 5, 3, 6, 4) each performed in triplicates, and analyzed using unpaired t test (two-tailed) to determine significance. *** p < 0.001, * p < 0.05, not significant (ns) > 0.05. Right: Model of the traction force-induced GABA B receptor activation through the closure of GB1 VFT -induced LB2 GB1 and LB2 GB2 in contact. The traction force-activated GABA B receptor requires both GB1 and GB2, and relies on GB2 for G protein coupling. Whereas the traction force-activated GABA B receptor is independent of GABA binding, preventing VFT GB1 closure by GABA B receptor antagonist CGP54626 , abolishes traction force-induced GABA B receptor activation, indicating that the closure of GB1 VFT -induced LB2 GB1 and LB2 GB2 in contact is important for traction force-induced GABA B receptor activation.

Article Snippet: HEK293 cells were stained with 4′6-diamidino-2-phenylindole (DAPI) for 30 min. Then cells were washed with PBS and mounted with FluorSave reagent (AR1109, Boster Biological Technology Co. Ltd., Wuhan, China) for fluorescent imaging.

Techniques: Transfection, Construct, Suspension, Plasmid Preparation, Mutagenesis, Two Tailed Test, Activation Assay, Binding Assay

Journal: Nature Communications

Article Title: GABA-independent activation of GABA B receptor by mechanical forces

doi: 10.1038/s41467-025-64811-2

Figure Lengend Snippet: a Schematic representation of GABA B -ΔVFT truncation, in which GB1 subunit lacks of the VFT domain (GB1-ΔVFT), but retains the ability to be activated by positive allosteric modulator Rac BHFF (yellow square). Co-immunoprecipitation experiments were performed using anti-HA antibody targeting to the HA tag, which is fused in the N-terminal of GB1 or GB1-ΔVFT. b Co-immunoprecipitation of GABA B receptor or GABA B -ΔVFT and integrin β 3 using anti-HA antibody. Blots are from one representative of five biologically independent experiments. c IP 1 production in cells transfected with GABA B receptor, or GABA B -ΔVFT, along with G qi9 under suspension or adhesion conditions. Data are present as mean ± s.e.m. from four biologically independent experiments each performed in triplicates and analyzed using unpaired t test (two-tailed) to determine significance. * P < 0.05, not significant (ns) > 0.05. d Real-time recording of intracellular Ca 2+ release in HEK293 cells expressing GABA B -ΔVFT and G qi9 . After recording the basal state of Ca 2+ release for 50 seconds, cells were subjected to shear stress for 100 seconds. Shear stress was then halted for 150 seconds, after which Rac BHFF was added for 200 seconds. Data are present as mean ± s.e.m. from 135 cells recorded.

Article Snippet: HEK293 cells were stained with 4′6-diamidino-2-phenylindole (DAPI) for 30 min. Then cells were washed with PBS and mounted with FluorSave reagent (AR1109, Boster Biological Technology Co. Ltd., Wuhan, China) for fluorescent imaging.

Techniques: Immunoprecipitation, Transfection, Suspension, Two Tailed Test, Expressing, Shear

Journal: Nature Communications

Article Title: GABA-independent activation of GABA B receptor by mechanical forces

doi: 10.1038/s41467-025-64811-2

Figure Lengend Snippet: a 3D model of the VFT of GB1 and GB2. N-glycosylation mutations (M1-7) are highlighted in LB2 of GB1 VFT. b Detailed presentation of the mutants of M1-7 and 5 A. c Schematic representation of GABA B -M1-7 and GABA B -5A. d IP 1 production in HEK293 cells transfected with GABA B receptor WT, or GABA B receptor mutants M1-7 along with G qi9 under suspension or adhesion conditions. Data are present as mean ± s.e.m. from at least three biologically independent experiments (from left to right: n = 6, 6, 6, 5, 6, 5, 5, 4, 5) each performed in triplicates and analyzed using paired t test (two-tailed) to determine significance. ** P < 0.01, * P < 0.05, not significant (ns) > 0.05. e Intracellular calcium release induced by different dose of GABA in HEK293 cells transfected with GABA B receptor WT and G qi9 , or GABA B -5A and G qi9 . Data are presented as mean ± s.e.m. from three biologically independent experiments each performed in triplicates. f IP 1 production in HEK293 cells transfected with GABA B receptor WT and G qi9 , or GABA B -5A and G qi9 under suspension or adhesion conditions. Data are mean ± s.e.m. from four biologically independent experiments each performed in triplicates and analyzed using unpaired t test (two-tailed) to determine significance. * P < 0.05, not significant (ns) > 0.05. g Real-time recording of intracellular Ca 2+ release in HEK293 cells expressing GABA B -5A and G qi9 . After recording the basal state of Ca 2+ release for 50 seconds, cells were subjected to shear stress for 100 seconds. Shear stress was then halted for 150 seconds, after which baclofen was added and the Ca 2+ signal was measured for another 200 seconds. Data are present as mean ± s.e.m. from 111 cells recorded.

Article Snippet: HEK293 cells were stained with 4′6-diamidino-2-phenylindole (DAPI) for 30 min. Then cells were washed with PBS and mounted with FluorSave reagent (AR1109, Boster Biological Technology Co. Ltd., Wuhan, China) for fluorescent imaging.

Techniques: Glycoproteomics, Transfection, Suspension, Two Tailed Test, Expressing, Shear

Journal: Nature Communications

Article Title: GABA-independent activation of GABA B receptor by mechanical forces

doi: 10.1038/s41467-025-64811-2

Figure Lengend Snippet: a Schematic representation of the active state of GABA B receptor, while the TM6s of GB1 and GB2 are rearranged towards each other. b Highlight of the cysteine substitutions (red balls) in the position 6.56 of TM6 in GB1 and GB2 in its active state structure (PDB: 7EB2). c Cross-linking of cell surface GABA B receptor between the two cysteines in GB1 6.56 and GB2 6.56 with CuP treatment along with no shear stress (control), shear stress treatment (15 dyn/cm 2 , 30 min) or GABA treatment (100 μΜ, 30 min). d , e Cross-linking of cell surface GABA B receptor dimer between the two cysteines in GB1 6.56 and GB2 6.56 encompassing three conditions: without CuP treatment, with CuP treatment, with both CuP and GABA treatment, under PDL or FN-treated condition. MW, molecular weight. Blots in ( c , d ) are one representative experiment of five biologically independent experiments. The bars show the percentage of GB1-GB2 dimer, which are relative to total amount of GB1 including GB1-GB2 dimer and GB1 monomer in each lane. Data are present as mean ± s.e.m. from five biologically independent experiments respectively in ( c , d ) and analyzed using paired t test (two-tailed) to determine significance. * P < 0.05, ** P < 0.01. f , g Dose response curve and E max analysis of GABA-induced IP 1 production in HEK293 cells transfected with GABA B receptor and G qi9 under suspension or adhesion conditions ( f ), or under PDL or FN-treatment ( g ). Data are present as mean ± s.e.m. from five and three biologically independent experiments each performed in triplicates in ( f ) and ( g ) respectively. E max are analyzed using paired t test (two-tailed) to determine significance. * p < 0.05.

Article Snippet: HEK293 cells were stained with 4′6-diamidino-2-phenylindole (DAPI) for 30 min. Then cells were washed with PBS and mounted with FluorSave reagent (AR1109, Boster Biological Technology Co. Ltd., Wuhan, China) for fluorescent imaging.

Techniques: Shear, Control, Molecular Weight, Two Tailed Test, Transfection, Suspension