Journal: The Journal of Neuroscience

Article Title: A Novel CCK Receptor GPR173 Mediates Potentiation of GABAergic Inhibition

doi: 10.1523/JNEUROSCI.2035-22.2023

Figure Lengend Snippet: CCK8s could trigger intracellular Ca 2+ mobilization through GPR173 in CHO-GPR173 cells. A , Schematic illustration of Ca 2+ imaging assay in CHO-GPCR cell lines. B , Dose-dependent Ca 2+ responses of CHO-CCK1R (left; EC 50 = 1.72 ± 1.11 n m ) and CHO-CCK2R (right; EC 50 = 4.64 ± 2.37 n m ) cells provoked by CCK8s. C , EC 50 curve of CCK8s for the CHO-GPR173 cell line (EC 50 = 3.23 ± 1.94 n m ). D , Dose-dependent Ca 2+ response of CHO-GPCR cells provoked by the CCK2R agonist CCK4. E , Dose-dependent Ca 2+ response of CHO-GPCR cells provoked by the CCK1R-specific agonist A-71 623. F , Schematic illustration of knockdown of GPR173 expression in CHO-GPR173 cells using an shRNA of Gpr173. G , 200 n m CCK8s provoked the Ca 2+ response in the scramble or shRNA-infected CHO-GPR173 cells and Gpr173 mRNA expression level in the scramble or shRNA-infected CHO-GPR173 cells. H , Ca 2+ signal responses of CHO-GPR173 cells treated with 10 μ m PNX or 200 n m CCK8s. Relative fluorescence unit (RFU) is defined as the ratio of RFU before and 30 s after adding CCK8s/HHBS (unpaired Student's t test, * p < 0.05,**** p < 0.0001, N.S., not significant). I , Illustration of the β-arrestin1/2 recruitment assay design. J , Dose-dependent response of CCK8s-induced β-arrestin recruitment to CCK1Rs determined by the β-arrestin2 recruitment and β-arrestin1/2 recruitment assays. The two control groups were without GPCR plasmid transfection in HTLA cells. K , L , Dose-dependent response of CCK8s-induced β-arrestin recruitment to CCK2Rs ( K ) and GPR173 ( L ) determined by the β-arrestin2 recruitment and β-arrestin1/2 recruitment assays (at least n = 3 independent experiments; one-way ANOVA with Tukey's post hoc test or unpaired Student's t test, **** p < 0.0001). Data are expressed as mean ± SEM.

Article Snippet: We incubated them for 10–12 h in a mixture of calcium phosphate transfection reagent [31.25 μl 2 m CaCl 2 solution, 250 μl 2× HBS (0.05 mol/l HEPES, 0.012 mol/l D-(+)-Glucose, 0.28 mol/l NaCl, 0.023 mol/l KCl, 0.0015 mol/l Na 2 HPO 4 , and 218.75 μl H 2 O)] and 1 ml fresh medium with 4 μg pLVX-puro-GPCR plasmid (Public Protein/Plasmid Library), 3 μg pSPAX2 plasmid (catalog #12260, Addgene), and 1.2 μg pMD2.G plasmid (catalog #12 259, Addgene).

Techniques: Imaging, Knockdown, Expressing, shRNA, Infection, Fluorescence, Control, Plasmid Preparation, Transfection