Extended Data Fig. 3: Functional comparison between hNTSR1(FL) and hNTSR1(∆ICL3).
From: Conformational transitions of a neurotensin receptor 1–Gi1 complex
a, FSEC-TS74 for hNTSR1(FL)–Gi1 (left) and hNTSR1(∆ICL3)–Gi1 (right). Each profile is a representative of two independent experiments. Only about 50% of the hNTSR1(FL)–Gi1 complex survives after a 45 °C incubation for 10 min, whereas over 90% of the hNTSR1(FL)–Gi1 complex survives after the same heat stress. b, GTPase-Glo assay39 of hNTSR1(FL) and hNTSR1(∆ICL3). Sample sizes for both hNTSR1(FL) and hNTSR1(∆ICL3) are 3. The intrinsic GTP hydrolysis activities of Gi1 heterotrimer and Gq heterotrimer are enhanced by hNTSR1. The guanine-nucleotide exchange factor activities of hNTSR1(FL) and hNTSR1(∆ICL3) proteins are equally potent. Symbols and bars represent individual value and mean of a single experiment performed in triplicate. c, Cell-surface expression level. HEK293 cells transiently expressing a Flag-epitope-tagged NTSR1 construct were analysed by flow cytometry. Sample sizes are shown in parentheses. Centre lines and error bars represent mean and s.e.m. of the indicated experiments. One-way analysis of variance (ANOVA) with Dunnett’s post hoc test was used to assess statistical analyses (ANOVA P value = 0.90, not significantly different (NS) among the four samples). d–g, NanoBiT G-protein dissociation assay. Concentration–response curves for G-protein dissociation signals (d, top) and their summary (d, bottom), for hNTSR1(FL) and hNTSR1(∆ICL3). Symbols and error bars represent mean and s.e.m. of indicated independent numbers of experiments, each performed in duplicate. e, Heat map of NanoBiT G-protein dissociation signals for hNTSR1(∆ICL3) (10 μM JMV449), β2AR (10 μM isoproterenol) and μOR (10 μM DAMGO). Mean values of test GPCR-specific signal-changes (differences in NanoBiT-G protein dissociation signal between test GPCR-transfected cells and mock-transfected cells) are shown. Sample sizes for Gs, Gi1, Go, Gq and G13 are as follows: 5, 5, 5, 5 and 5 (hNTSR1), 6, 5, 3, 3 and 3 (β2AR) and 7, 7, 6, 5 and 5 (μOR). Unlike β2AR and μOR, the NTSR1 agonist (JMV449) causes a signal decrease for all G-proteins (e), which suggest that all G proteins can be recognized and activated by hNTSR1, and dissociated into Gα and Gβγ subunits. f, The summary of NanoBiT G-protein dissociation assay of wild-type hNTSR1 and the hNTSR1(S93A/L94A/R294A/H373A) mutant for full-length constructs. Concentration–response curves are shown in Fig. 5b. We used an unpaired t-test with correction for multiple comparisons using the Holm–Sidak method. NS, not significantly different from wild type; **P < 0.01. g, NanoBiT G-protein dissociation assay of wild-type hNTSR1 and the hNTSR1(S93A/L94A/R294A/H373A) mutant for ∆ICL3 constructs. Concentration–response curves of Gs, Gi1, Go, Gq and G13 signalling (top), and the summary of the assay result (bottom). Symbols and error bars (top) represent mean and s.e.m. of indicated independent numbers of experiments (bottom), each performed in duplicate. We used an unpaired t-test with correction for multiple comparisons using the Holm–Sidak method. NS, not significantly different from wild type; *P < 0.05, **P < 0.01, ***P < 0.001.
