Fig 2: Bioinformatics and histology screening for a novel CCK receptor. A, Percentage identity correlation matrix of orphan receptors with CCK1R/2R. B, Left, Illustration of mDlx-DIO-ChR2-mCherry injected into the AC of CCK-Cre mice. Middle, Immunochemistry staining images of candidate receptors in CCK-GABAergic synapses. Scale bars: 5 µm. Expanded images are from the white rectangles (top right). The white arrows indicate the colocalization of GPR173 with CCK-GABAergic synapses. Scale bars: 1 µm. C, Bar chart shows numbers of overlay dots [bottom right, mCherry+, Syp (synaptophysin)+, and GPCR+, per mm2; n = 3 independent experiments; one-way ANOVA with Tukey's post hoc test, ****p < 0.0001]. D, Confocal images of Flag-GPR173-transfected HEK293T cells colabeled with anti-GPR173, anti-Flag, and DAPI. Scale bar, 10 µm. Quantitative analysis of colocalization of Flag and GPR173 across the dotted line (labeled in confocal images). The bar chart depicts the percentage of colabeled cells positive for Flag and GPR173 from Flag-GPR173–transfected HEK293T cells stained with anti-GPR173, anti-Flag, and DAPI (n = 21 cells). E, Super-resolution image of colocalized GPR173 with CCK-GABAergic synapses, GABAAR with CCK-GABAergic synapses, and GABAAR and GPR173 (left). Scale bar, 1 µm. The values for PCC and MOC indicate the colocalization possibility of mCherry with GPR173, mCherry with GABAAR, and GABAAR with GPR173. F, Immunochemistry images of costaining of GPR173 with CaMKII (top) or GAD67 (bottom) in C57BL/6J mice. Scale bar, 50 µm. Group data from colocalization analysis of GPR173 with CaMKII (n = 11 sections, N = 3 mice) or GAD 67 (n = 10 sections, N = 3 mice). G, Immunochemistry images for colocalized GPR173 with CCK-GABAergic synapses contacting pyramidal cells and box plot showing numbers of colocalized GPR173 and CCK-GABAergic contacts per pyramidal cell (n = 49 neurons, 5.69 ± 0.38). Scale bar, 5 µm. Data are expressed as mean ± SEM.

Fig 3: Known CCK receptors do not mediate CCK signaling in CCK-INs. A, Schematic drawings show rAAV9-mDlx-DIO-ChR2-mCherry-WPRE-pA (mDlx-DIO-ChR2-mCherry) and AAV9-mDlx-Cre-WPRE-pA (mDlx-Cre) injected into CCK1R/2R-KO or CCK-KO mice. B, AAV9-infected mCherry neurons colabeled with GAD67 (left, CCK1R/2R-KO mouse; right, CCK-KO mouse). Scale bars: 50 and 10 µm, respectively. Right, Bar chart shows the percentage of mCherry neurons colabeled with GAD67 in CCK1R/2R-KO and CCK-KO mice. C, Schematic drawings show in vitro patch-clamp recordings from pyramidal neurons while delivering a laser pulse to activate infected GABAergic neurons in CCK-KO and CCK1R/2R-KO mice. Neuronal responses to a step current confirmed that the recorded neurons were excitatory. D, Traces of IPSCs in response to the laser pulse in CCK1R/2R-KO (top) and CCK-KO (bottom) mice before and after HFLS of infected GABAergic neurons. E, Sampled neuronal responses to HFLS in CCK1R/2R-KO and CCK-KO mice. F, Time course of normalized IPSCs before and after (for 25 min) HFLS in CCK-KO (black, n = 9 neurons; one-way ANOVA with Tukey's post hoc test, N.S., not significant) and CCK1R/2R-KO mice (green, n = 9 neurons; one-way ANOVA with Tukey's post hoc test, ****p < 0.0001; two-way RM ANOVA with Tukey's post hoc test, ####p < 0.0001). G, Schematic illustration of the setup for in vivo recordings with laser stimulation in the AC and a natural AS (a noise burst; SOM, PV neuron, PC) in CCK1R/2R-KO (left) and CCK-KO (right) mice. Bottom, Example waveforms from the two groups. H, Normalized LFP response amplitudes to the AS with a preceding laser pulse before and after HFLS in CCK1R/2R-KO (green, n = 7 recording sites, N = 5 mice) and CCK-KO (black, n = 8 recording sites, N = 6 mice; two-way RM ANOVA with Tukey's post hoc test, ****p < 0.0001) mice. Data are expressed as mean ± SEM.

Fig 4: CCK8s could trigger intracellular Ca2+ mobilization through GPR173 in CHO-GPR173 cells. A, Schematic illustration of Ca2+ imaging assay in CHO-GPCR cell lines. B, Dose-dependent Ca2+ responses of CHO-CCK1R (left; EC50 = 1.72 ± 1.11 nm) and CHO-CCK2R (right; EC50 = 4.64 ± 2.37 nm) cells provoked by CCK8s. C, EC50 curve of CCK8s for the CHO-GPR173 cell line (EC50 = 3.23 ± 1.94 nm). D, Dose-dependent Ca2+ response of CHO-GPCR cells provoked by the CCK2R agonist CCK4. E, Dose-dependent Ca2+ 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 nm CCK8s provoked the Ca2+ 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, Ca2+ signal responses of CHO-GPR173 cells treated with 10 µm PNX or 200 nm 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 ß-arrestin1/2 recruitment assay design. J, Dose-dependent response of CCK8s-induced ß-arrestin recruitment to CCK1Rs determined by the ß-arrestin2 recruitment and ß-arrestin1/2 recruitment assays. The two control groups were without GPCR plasmid transfection in HTLA cells. K, L, Dose-dependent response of CCK8s-induced ß-arrestin recruitment to CCK2Rs (K) and GPR173 (L) determined by the ß-arrestin2 recruitment and ß-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.