Extended Data Fig. 8: Cryo-EM data processing of NOX-6-7-GPR132-Gi1 complex and effects of residues mutations of GPR132 in response to NOX-6-7 stimulation.

(a) BRET ratio changes curves in HEK293 cells overexpressing GPR132 in response to stimulations with NOX-6-7, NOX-6-1, dxd-190904-1, xgf-1-10 or NOX-6-13. Data are from three independent experiment (n = 3). (b) Relative mRNA levels of Il-1β, Tnf-α, Il-6 and Ccl2 in CD11c⁺Ly6C⁻F4/80^low-labeled islet-resident macrophages isolated from WT mice pretreated with or without 100 ng/ml PTX and in response to stimulations with 1 μM 9(S)-HODE, NOX-6-7 or vehicle for 24h. Approximately 6*10⁴ CD11c⁺Ly6C⁻F4/80^low-labeled islet-resident macrophages isolated from 18–24 WT mice (1*10⁴ CD11c⁺ Ly6C⁻F4/80^low cells/group), Data are from six independent experiments (n = 6). (from left to right, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001). (c) Phagocytosis of CD11c⁺Ly6C⁻F4/80^low-labeled islet-resident macrophages isolated from WT mice pretreated with or without 100ng/ml PTX in response to stimulations with 1 μM 9(S)-HODE, NOX-6-7 or vehicle for 24h. Approximately 6*10⁴ CD11c⁺Ly6C⁻F4/80^low-labeled islet-resident macrophages isolated from 18–24 WT mice (1*10⁴ CD11c⁺ Ly6C⁻F4/80^low cells/group), Data are from six independent experiments (n = 6). (from left to right, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001). (d) Representative size-exclusion chromatography profile (left panel) and SDS-PAGE Coomassie blue staining of the peak fraction for purification (right panel) of the NOX-6-7-GPR132-G_i1 complex. (e, g) The cryo-EM images and data processing of NOX-6-7-GPR132-G_i1 complex. In total, 520250 particles were selected to construct EM density maps of the NOX-6-7-GPR132-Gi1 complex at an overall resolution of 3.0 Å. Cryo-EM micrograph (Scale bar: 50 nm) and reference-free two-dimensional class averages (Scale bar: 10 nm) of the NOX-6-7-GPR132-G_i1 complex (E). Workflow chart of cryo-EM data processing for the NOX-6-7-GPR132-G_i1 complex (F). The Gold-standard Fourier shell correlation (FSC) curves showing an overall resolution at 3.04Å for the NOX-6-7-GPR132-G_i1 complex (G). Cryo-EM map colored based on local resolution (in Extended Data Fig. 10f) for NOX-6-7-GPR132-G_i1 complex (right panel) (F). (h) Cryo-EM density map of the NOX-6-7-GPR132-G_i1 complex. NOX-6-7 is shown in blue, GPR132 in green, Gα_i1 in yellow, Gβ in cyan, Gγ in pink and scFv16 in grey. (i) Effects of alanine scanning mutations of key residues in the ligand binding pocket of GPR132 in response to NOX-6-7 stimulation. The heatmap is colored according to the values of ΔpEC₅₀ and Emax (ΔpEC₅₀ = pEC₅₀ of mutant-pEC₅₀ of the wild type); nd, signal not detectable. (j) Effects of key residues allelic mutations that effect the ligand-binding pockets of GPR132 on other lipid-binding GPCRs in response to NOX-6-7 stimulation. The heatmap is colored according to the value of ΔpEC₅₀ and Emax (ΔpEC₅₀ = pEC₅₀ of mutant-pEC₅₀ of wild type); nd, signal not detectable. (B-C) ***P < 0.001, CD11c⁺Ly6C⁻F4/80^low-labeled islet-resident macrophages isolated from WT mice treated with NOX-6-7 or 9(S)-HODE compared with those treated with vehicle. ^###P < 0.001, CD11c⁺Ly6C⁻F4/80^low-labeled islet-resident macrophages isolated from WT mice pretreated with 100 ng/ml PTX compared with those treated with vehicle in response to the stimulation with NOX-6-7. ^$$$P < 0.001, CD11c⁺Ly6C⁻F4/80^low-labeled islet-resident macrophages isolated from WT mice pretreated with 100 ng/ml PTX compared with those treated with vehicle in response to the stimulation with 9(S)-HODE. The bars indicate the mean ± SEM values. Data were statistically analyzed using two-way ANOVA with Dunnett’s post hoc test.