Figure 2. Adgrl3 CTF signals through Gq and G13.

Screen of Adgrl3 signaling in the major G protein signaling pathways utilizing a CRISPR knockout cell line (HEKΔ7) and a panel of gene expression assays. (a-c) Assay controls showing that the Gα_s-coupled β₂AR signals in CRE only when Gα_s is reintroduced (a) and that ETA signals in NFκB only when Gα_q (b) or Gα₁₃ (c) is reintroduced. In (a) an unpaired two-tailed t-test was used to determine statistical significance between the No Gα and Gα_s conditions; in (b,c) One-way ANOVA was employed with Tukey’s multiple-comparison post-hoc test (b, ****p<0.0001). (d-f) Gene expression signals for Adgrl3 constructs FL, CTF, and Δ5-CTF. (d) CRE with Gα_s (e) NFκB with Gα_q (f) NFκB with Gα₁₃. Each Gα protein species was reintroduced at an optimized cDNA concentrations (Supplementary Fig. 5). In (d-f) One-way ANOVA with Tukey’s multiple-comparison post-hoc test was performed to determine statistical significance between the FL, CTF and Δ5-CTF conditions (ns p>0.05, ***p<0.001, ****p<0.0001). In panels (a-f) the baseline signal of empty vector was subtracted to show receptor-dependent luminescence (lumi). The full screen for Adgr3 in HEKΔ7 is shown in Extended Data Fig. 3. (g-i) ADGRL3 N-terminal dissociation induced by urea enhances G13 and Gq activation. Mock and urea-treated ADGRL3 membranes or empty High-Five membranes were reconstituted with purified Gα_s (g) Gα_q (h) Gα₁₃ (i) and Gβ₁Gγ₂ heterodimer and receptor-stimulated [³⁵S]-GTPγS binding kinetics were measured^15,51,52. In panels (a-f) bars are presented as mean ±SEM from (a) n=4 (b-c) n=3 (d) n=4 (e-f) n=5 independent experimental replicates. In panels (g-i) data are from one representative experiment performed 3 times. Error bars mean ±SD from three technical replicates. See Supplementary Data for the full set of p-values.