Extended Data Fig. 2: Mechanical-force-induced separation between the α and β subunit of ADGRG2, and CFTR current increase via ADGRG2.

a, The whole cell Cl^- current elicited by voltage steps between −100 mV and +100 mV in HEK293 cells overexpressing either CFTR and ADGRG2(FL), or CFTR and ADGRG2-AA, or CFTR alone, after stimulation with mechano-forces (cells were treated with shaking at 60 rpm for 2 h as indicated by previous literature). Note that the nonciliated cells of efferent ductules enriched with ADGRG2 expression constantly withstood the pressure of fluids. We therefore examined the CFTR current in HEK293 cells overexpressing ADGRG2 and CFTR after stimulation with mechanical forces using a protocol from research on other aGPCRs. b, Corresponding I-V curves of whole-cell Cl^- current as shown in a. Values are the mean ± s.e.m. of six independent experiments (n = 6). c, Average current densities (pA/pF) recorded at 100 mV as shown in b. Values are the mean ± s.e.m. of six independent experiments (n = 6). ***P < 0.001; *P < 0.05; n.s., no significant difference. HEK293 cells transfected with CFTR and ADGRG2(FL), or CFTR and ADGRG2-AA, or CFTR alone treated with or without shaking were compared. All data were analysed by two-sided one-way ANOVA with Tukey’s test. (P = 0.4908, <0.0001, 0.0215 from left to right). d, Box plots showed the increased current densities (pA/pF) recorded at 100 mV after mechano-forces administration as shown in b. In box plots, whiskers indicate the minimum and maximum values, the bounds of the box are 25th and 75th percentiles of the data, and the centre line indicates the mean. Values are the mean ± s.e.m. of six independent experiments (n = 6). Note that the CFTR current was increased after stimulation with mechanical forces, with a significantly larger increase in the wild-type ADGRG2 group than in the ADGRG2-AA group. ***P < 0.001; mechano-forces-stimulated increased current densities in CFTR and ADGRG2(FL) overexpressed cells compared with that in CFTR overexpressed cells. ^###P < 0.001; mechano-forces-stimulated increased current densities in CFTR and ADGRG2-AA overexpressed cells compared with that in CFTR and ADGRG2(FL) overexpressed cells. All data were analysed by two-sided one-way ANOVA with Tukey’s test. (P <0.0001, <0.0001 from left to right). e, The auto-cleavage ratio of the ADGRG2 after application of mechano-force examined by western blot, as depicted in Extended Data Fig. 1e. Full gel scans are shown. Cells were transfected with wild-type or auto-proteolysis deficient ADGRG2 (ADGRG2-H597A/T599A (AA)) with Flag and GFP tags fused at their N- and C-terminus, respectively. Whole-cell lysates were analysed by western blot using anti-Flag (α subunit) or anti-GFP (β subunit) antibody. Western blot membranes were co-stained against the Flag (ADGRG2-α subunit, left panel) and GFP (ADGRG2 β subunit, middle panel). Cleaved ADGRG2 α subunit bands (~130 kDa), cleaved ADGRG2 β subunit bands (~60 kDa), and ADGRG2(FL) uncleaved bands (~180 kDa) or uncleavable ADGRG2-AA bands (~180 kDa) are highlighted with blue, red, and green arrows, respectively. Representative blots from at least three independent experiments (n=3) are shown. The percentage of auto-cleaved ADGRG2 (right panel) were obtained by dividing of the average densitometry of ADGRG2 β subunit bands with the average densitometry of the ADGRG2-AA bands. Values are mean ± s.e.m. from three independent experiments (n = 3). Notably, the sum of the average densitometries of the ADGRG2 β subunit bands and ADGRG2(FL) uncleaved bands were equal to the densitometry of the ADGRG2-AA bands. f, The co-immunoprecipitation analysis of the association between the α and β subunit of ADGRG2 after application of mechano-force, as depicted in Extended Data Fig. 1g. Full gel scans are shown. Representative blots from at least three independent experiments (n = 3) are shown. β subunit of ADGRG2 were immune-precipitated from whole-cell lysates using anti-GFP antibody covalently linked sepharose beads. The immune-precipitants were analysed by western blot and simultaneously co-stained with an antibody against the Flag (α subunit, left panel) and GFP (β subunit, middle panel). Cleaved ADGRG2 α subunit bands (~130 kDa), cleaved ADGRG2 β subunit bands (~60 kDa), ADGRG2(FL) uncleaved bands (~180 kDa) or uncleavable ADGRG2-AA bands (~180 kDa) are highlighted with blue, red, and green arrows, respectively. The association ratio of α and β subunit of ADGRG2 (right panel) were obtained by dividing of the densitometry of α subunit bands with the β subunit bands, using the densitometry of the ADGRG2-AA bands for normalization (Because we have subjected the same amount of the ADGRG2-AA/GAIN-ADGRG4-β-AA for western blot. The separation ratio between the α and β subunits were calculated by subtraction of 100% with the association α/β ratio.) Values are mean ± s.e.m. from three independent experiments (n = 3). Mechanical force stimulation accelerated the separation of the ADGRG2-β subunit from its α subunit because no detectable α subunit was found to be associated with the ADGRG2-β subunit by co-IP (cells were treated with shaking at 60 rpm for 2 h according to previous literature). We speculated that the mechanical force-induced separation ratio between the α and β subunits was 100%. Note that the mechanical forces in our experimental conditions probably accelerated the separation of the ADGRG2-β subunit from its α subunit because no detectable α subunit was found to be associated with the ADGRG2-β subunit by co-IP.