Abstract
Gαs serves as the prototypical signal transducer for G-protein-coupled receptors (GPCRs) and is the heterotrimeric G protein most frequently mutated in cancer. The classical view of the plasma membrane as the only cellular location where GPCR signal transduction occurs has been challenged by evidence suggesting that Gs also signals from intracellular compartments. However, progress on this topic has stalled because of insufficient approaches with adequate spatiotemporal resolution. Here we describe genetically encoded probes and cell-penetrating compounds that block the effector-binding site of active Gαs in cells to prevent signal propagation at discrete subcellular locations, at user-specified times and across diverse experimental conditions. Using these tools, we show direct evidence of Gαs-mediated signaling on intracellular organelles, unique spatiotemporal features of signaling by Gαs oncomutants and specific regulation of physiologically relevant responses in cardiac or immune cells. These findings pave the way to harnessing the spatiotemporal modulation of Gs signaling and its untapped therapeutic potential.
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Data availability
The data supporting the findings of this study are available within the paper and its Supplementary Information. Source data are provided with this paper.
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Acknowledgements
This project was primarily funded by National Institutes of Health (NIH) grants R35GM156286 and R21NS127065 (to M.G-M). Work by R.I. was funded by NIH grant R35GM133521. Work by X.V. was funded by NIH grants R01HL124392 and NIH R01DE033519. Research by J.-P.V. was funded by NIH grants R01DK116780 and R21-DE032478, as well as by the US Department of Veterans Affairs (VA) under grant I01BX006371. S.S. was supported by NIH fellowship F31DK136222. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or the VA.
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J.Z. and M.G.-M. designed the majority of the experiments. J.Z. conducted and analyzed the majority of the experiments. A.L., M.M. and M.G.-M. contributed to designing, conducting and analyzing some of the experiments with purified proteins. R.J., E.G., A.W. and A.S. contributed to designing, conducting and analyzing some of the cell-based experiments involving BRET and/or gene expression analysis. S.S. and J.-P.V. designed, conducted and analyzed experiments to assess PTH signaling by FRET. T.-Y.L. and R.I. designed, conducted and analyzed experiments in cardiomyocytes. J.Z., N.C. and X.V. designed, conducted and analyzed experiments in T cells. J.Z., X.V., R.I., J.-P.V. and M.G-M. designed experiments and analyzed data. J.Z. and M.G-M. wrote the paper with input from all authors. M.G-M. conceptualized and supervised the project.
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Boston University has filed a provisional patent (US Patent application no. 63/932,172) on behalf of M.G.-M. related to peptides described in this manuscript. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 GαsBP1 and GαsBP2 binding to different Gα subunits in cells.
(A) Diagram showing the design of the BRET assay to measure binding of Nluc-fused peptides to active (QL mutant) or inactive (WT) Gα subunits fused to YFP. (B) GαsBP1 and GαsBP2 specifically bind to the active form of Gαs. BRET measurements were carried out in HEK293T cells co-expressing Nluc-fused plasma membrane anchored GαsBP1 (mas-GαsBP1) or GαsBP2 (mas-GαsBP2) with either Venus-fused Gαs wild-type (WT) or the constitutively active mutant Q227L (QL). Results are presented as the ratio between luminescence at 535 nm and 450 nm (“BRET ratio (535/450 nm)”). Mean ± S.E.M. (n = 3 independent experiments). (C) Diagram depicting the investigation of G protein specificity for GαsBP1 and GαsBP2 (D-H) GαsBP2 does not bind to any of the Gα subunits tested, whereas GαsBP1 binds to Gαi3. BRET measurements were carried out in HEK293T cells co-expressing Nluc-fused mas-GαsBP1, mas-GαsBP2, mas-KB1753 (in D, E and F), mas-GRKRH (in G), or mas-PRGRH (in H), with either wild-type (WT) or constitutively active (QL) mutants of Gαi3 (in D), Gαo (in E), Gαz (in F), Gαq (in G), or Gα13 (in H) fused to a YFP, as indicated. Results are presented as the ratio between luminescence at 535 nm and 450 nm (“BRET ratio (535/450 nm)”). Mean ± S.E.M. (n = 3-6 independent experiments).
Extended Data Fig. 2 GαsBP2 accelerates the re-association of Gβγ with Gαs after GPCR signaling termination.
(A) mem-GαsBP2 does not affect Gαs-Gβγ association prior to GPCR stimulation, but accelerates Gβγ binding to Gαs after GPCR signal termination. Diagram on the left corresponds to the BRET-based assay to measure free Gβγ and the mechanism investigated with GαsBP2 probes. Graphs in the middle correspond to BRET measurements in HEK293T cells co-expressing a BRET-based biosensor for free Gβγ and different amounts of mem-GαsBP2 WT or mem-GαsBP2 W11A, as indicated. Changes in BRET before β2AR stimulation compared to the condition without GαsBP2 (“ΔBRET (no GαsBP2)”) are shown in the graphs on the left, and real-time changes in BRET upon isoproterenol (agonist)/ alprenolol (antagonist) treatment compared to the unstimulated baseline (“ΔBRET (baseline)”) are shown in the graphs on the right. Mean ± S.E.M. (n = 4-5 independent experiments). Immunoblots on the right correspond to representative results of experiments confirming the expression of mem-GαsBP2 and BRET sensor components. (B) mem-GαsBP2 does not affect the efficacy or potency of isoproterenol on Gβγ responses. Diagram on the left corresponds to the BRET-based assay to measure free Gβγ and the mechanism investigated with GαsBP2 probes. Graphs correspond to BRET measurements in HEK293T cells co-expressing a BRET-based biosensor for free Gβγ and the indicated amounts of mem-GαsBP2 were stimulated with the indicated concentrations of isoproterenol. The changes in BRET upon isoproterenol stimulation compared to the unstimulated condition (“ΔBRET·103 (no agonist)”) are shown on the left graph, the maximal response (“maximum ΔBRET ·103”) is shown on the middle graph, and the negative logarithm of half-maximal effective concentration (pEC50) is shown on the right graph. Mean ± S.E.M. (n = 3 independent experiments). ns, p > 0.05 by one-way ANOVA corrected for multiple comparisons (Dunnett). (C) cyto-GαsBP2 accelerates Gβγ binding to Gαs after GPCR signal termination, whereas the GαsBP2 W11A mutant has no effect. Diagram on the left corresponds to the BRET-based assay to measure free Gβγ and the mechanism investigated with GαsBP2 probes Real-time changes in BRET compared to the unstimulated baseline (“ΔBRET (baseline)”) were measured in HEK293T cells expressing a BRET-based biosensor for free Gβγ co-transfected with 500 ng of plasmid DNA for cyto-GαsBP2 WT, mem-GαsBP2 WT, or their corresponding W11A mutants, upon isoproterenol (agonist)/ alprenolol (antagonist) treatment. Mean ± S.E.M. (n = 4 independent experiments). Immunoblots on the right correspond to representative results of experiments confirming the expression of GαsBP2 constructs and BRET sensor components.
Extended Data Fig. 3 Temporally resolved inhibition of Gαs signaling in cells via chemically controlled GαsBP2 probes.
(A) Rapamycin-induced recruitment of GαsBP2 to membranes blocks GPCR-stimulated AC activity. Diagram on the left depicts the recruitment of a GαsBP2 probe to the plasma membrane upon rapamycin stimulation to block Gαs-AC signaling. Graphs with kinetic traces in the middle correspond to luminescence-based cAMP measurements in HEK293T cells expressing FKBP-GαsBP2 and membrane-anchored FRB, or mem-GαsBP2 upon stimulation with isoproterenol in the presence or absence of rapamycin pre-treatment, as indicated. The bar graph corresponds to the quantification of the area under the curve (AUC) from the kinetic response curves. Mean ± S.E.M. (n = 4 independent experiments). ns, p > 0.05, ***p < 0.005 by paired, two-tailed Student’s t-test. Immunoblots on the right correspond to representative results of experiments confirming the expression of GαsBP2 constructs, membrane-anchored FRB, and G proteins. (B) Acute, rapamycin-induced recruitment of GαsBP2 to membranes rapidly inhibits the kinetics GPCR-stimulated AC activity. Experiments were carried out as in (A), except that rapamycin was added at different times relative to the addition of isoproterenol, as indicated by the color coding and the diagram on the left. Grey traces correspond to the isoproterenol stimulated control with no addition of rapamycin. Mean ± S.E.M. (n = 3 independent experiments). (C) Abscisic acid (ABA)-induced recruitment of GαsBP2 to membranes blocks GPCR-stimulated AC activity. Diagram on the left the recruitment of a GαsBP2 probe to the plasma membrane upon ABA stimulation to block Gαs-AC signaling. Graphs with kinetic traces in the middle correspond to luminescence-based cAMP measurements in HEK293T cells expressing PYLcs-GαsBP2 and membrane-anchored ABIcs, or mem-GαsBP2 upon stimulation with isoproterenol in the presence or absence of ABA pre-treatment, as indicated. The bar graph corresponds to the quantification of the area under the curve (AUC) from the kinetic response curves. Mean ± S.E.M. (n = 3 independent experiments). ns, for p > 0.05, **p < 0.01, by paired, two-tailed Student’s t-test. Immunoblots on the right correspond to representative results of experiments confirming the expression of GαsBP2 constructs, membrane-anchored ABIcs, and G proteins.
Extended Data Fig. 4 endo-GαsBP2 has a modest effect of cAMP responses, but efficiently blocks the ERK phosphorylation.
(A) endo-GαsBP2 minimally reduces GPCR-stimulated AC activity in cells, whereas mem-GαsBP2 efficiently blocks it. Diagram on the top left corresponds to the mechanism of cAMP regulation investigated with GαsBP2 probes. Graphs with kinetic traces on the top right correspond to luminescence-based cAMP measurements in HEK293T cells expressing 100 ng mem-GαsBP2 or 250 ng endo-GαsBP2 stimulated with isoproterenol as indicated. Mean ± S.E.M. (n = 4 independent experiments). The bar graph on the bottom left corresponds to the quantification of the area under the curve (AUC) from the kinetic response curves. Mean ± S.E.M. (n = 4 independent experiments); ns, p > 0.05, **p < 0.01 by one-way ANOVA corrected for multiple comparisons (Dunnett). Immunoblots on the bottom right correspond to representative results of experiments confirming the expression of GαsBP2 constructs. The kinetic curve of the cAMP response from cells expressing mem-GαsBP2 is reproduced from Fig. 2e. (B) Endo-GαsBP2 efficiently blocks the GPCR-stimulated ERK phosphorylation. Diagram on the left corresponds the mechanism of ERK1/2 regulation investigated with GαsBP2 probes. ERK1/2 phosphorylation was detected in HEK293T cells expressing endo-GαsBP2 or mem-GαsBP2 were stimulated (or not) with 10 µM isoproterenol for 5 min, as indicated. A representative immunoblotting result is shown in the middle and the quantification of ERK1/2 phosphorylation is shown on the bar graph on the right. Mean ± S.E.M. (n = 5 independent experiments). *p < 0.05 by one-way ANOVA corrected for multiple comparisons (Dunnett).
Extended Data Fig. 5 Inhibition of isoproterenol-induced transcriptional responses by different GαsBP2 constructs.
(A) Design of different plasma membrane-anchored GαsBP2 constructs. HEK293T cells transfected with GαsBP2-mem constructs were stained as indicated and visualized by confocal fluorescence microscopy. Scale bars = 10 µm. (B) GαsBP2-mem blocks the isoproterenol-stimulated cAMP response as efficiently as mem-GαsBP2. Diagram on the top left corresponds the mechanism of cAMP regulation investigated with GαsBP2 probes. Graphs with kinetic traces on the bottom correspond to luminescence-based cAMP measurements in HEK293T cells expressing the indicated constructs. Mean ± S.E.M. (n = 3 independent experiments). Immunoblots on the top right correspond to representative results of experiments confirming the expression of GαsBP2 constructs. (C) GαsBP2-mem blocks the isoproterenol-induced gene transcription as efficiently as mem-GαsBP2. Bar graphs on the bottom correspond to relative mRNA levels of PCK1 or FOS in HEK293T cells transfected with GαsBP2-mem or mem-GαsBP2 in the presence or absence of isoproterenol stimulation, as indicated. Mean ± S.E.M. (n = 4 independent experiments); ns, p > 0.05, *p < 0.05, by one-way ANOVA corrected for multiple comparisons (Dunnett). (D) Diagram on the top shows two potential scenarios of Gαs-mediated regulation of gene expression triggered by β2AR. Bar graphs on the bottom correspond to relative mRNA levels of PCK1, FOS, or DUSP1 in HEK293T cells transfected with mem-GαsBP2, endo-GαsBP2, or both constructs simultaneously in the presence or absence of isoproterenol stimulation, as indicated. Mean ± S.E.M. (n = 4 independent experiments); ns, p > 0.05, *p < 0.05, **p < 0.01 by one-way ANOVA corrected for multiple comparisons (Dunnett).
Extended Data Fig. 6 GαsBP2 does not have GAP-like activity on constitutively active Gαs, but promotes active sequestration into an inactive complex with Gβγ at the plasma membrane.
(A) GαsBP2 does not accelerate GTP hydrolysis by Gαs R201C. Single-turnover GTPase activity was measured with [γ-33P]GTP-loaded Gαs R201C in the absence (Gαs only, 1% DMSO, grey) or presence GαsBP2 (orange) by quantifying the release of 33P[Pi] at 21 °C. Mean ± S.E.M. (n = 3 independent experiments). (B) Gβγ remains dissociated from Gαs R201C after GPCR signal termination both in the presence and absence of mem-GαsBP2. Diagram on the top left corresponds to the BRET-based assay to measure free Gβγ and the mechanism investigated with GαsBP2 probes. Graphs on the left correspond to real-time changes in BRET relative to unstimulated cells expressing Gαs WT alone (“ΔBRET (Gαs WT)”) in HEK293T cells co-expressing a BRET-based biosensor for free Gβγ and Gαs R201C (top graph) or Gαs WT (bottom graph), with or without mem-GαsBP2 expression. After isoproterenol stimulation, cells were treated with alprenolol or buffer as indicated. Mean ± S.E.M. (n = 4 independent experiments). Immunoblots on the bottom left right correspond to representative results of experiments confirming the expression of mem-GαsBP2, G proteins, and BRET sensor components. (C) mem-GαsBP2 promotes the association of Gαs Q227L with Gβγ. Diagram on the left corresponds to the BRET-based assay to measure free Gβγ and the mechanism investigated with GαsBP2 probes. Graphs in the middle correspond to BRET measurements in HEK293T cells co-expressing a BRET-based biosensor for free Gβγ, Gαs Q227L, and different amounts of mem-GαsBP2 WT (orange) or mem-GαsBP2 W11A (blue) as indicated. Open circles correspond to controls in which Gαs Q227L was replaced by Gαs WT in the presence of mem-GαsBP2 WT. Changes in BRET (ΔBRET) relative to cells expressing Gαs WT in the absence of mem-GαsBP2 (“ΔBRET (Gαs WT)”) are shown in the graphs. Mean ± S.E.M. (n = 4 independent experiments). Immunoblots on the left right correspond to representative results of experiments confirming the expression of mem-GαsBP2, G proteins, and BRET sensor components. (D) mem-GαsBP2, but not cyto-GαsBP2, recruits Gαs Q227L to the plasma membrane. Diagram on the left corresponds to the BRET-based assay to measure the association of Gαs with membranes. Graphs in the middle correspond to BRET measurements in HEK293T cells co-expressing a plasma membrane-anchored nanoluciferase (mas-Nluc) and Venus-fused Gαs WT or Q227L in the absence or presence of mem-GαsBP2 or cyto-GαsBP2. Changes in BRET relative to cells expressing Gαs WT alone (“ΔBRET·103 (Gαs WT)”) are shown in the graph. Mean ± S.E.M. (n = 3 independent experiments). Immunoblots on the left right correspond to representative results of experiments confirming the expression of GαsBP2 constructs, G proteins variants, and mas-Nluc. (E) As opposed to mem-GαsBP2, cyto-GαsBP2 does not promote the association of Gαs Q227L with Gβγ. Graphs on the left correspond to BRET measurements in HEK293T cells co-expressing a BRET-based biosensor for free Gβγ, Gαs WT or Q227L, and the indicated GαsBP2 constructs. Changes in BRET (ΔBRET) relative to cells expressing Gαs WT in the absence of mem-GαsBP2 (“ΔBRET (Gαs WT)”) are shown in the graph. Mean ± S.E.M. (n = 4 independent experiments). Immunoblots on the left right correspond to representative results of experiments confirming the expression of GαsBP2 constructs, G proteins, and BRET sensor components. (F) Proposed model for the mechanism by which mem-GαsBP2 sequesters Gαs Q227L into an inactive complex with Gβγ. Upon recruitment of Gαs Q227L from the cytosol to the plasma membrane by mem-GαsBP2 (red), but not cyto-GαsBP2 (green), concentration of the G protein favors its association with membrane-resident Gβγ.
Extended Data Fig. 7 Gβγ favors the association of Gαs with membranes and mem-GαsBP2 prevents the cytosolic translocation of active Gαs upon GPCR stimulation.
(A) Gβγ facilitates the association of Gαs with the plasma membrane. Diagram on the left corresponds to the BRET-based assay to measure the association of Gαs with membranes. Graphs in the middle correspond BRET measurements in HEK293T cells co-expressing plasma membrane anchored Venus (Venus-KRas), nanoluciferase-fused Gαs (Gαs-Nluc), and different amounts of Gβγ, as indicated, in the presence or absence of 100 nM isoproterenol stimulation as indicated. Results are presented as the ratio between luminescence at 535 nm and 450 nm (“BRET ratio (535/450 nm)”). Mean ± S.E.M. (n = 4 independent experiments). Immunoblots on the left right correspond to representative results of experiments confirming the expression of Venus-KRas, Gαs-Nluc, and Gβγ. (B) mem-GαsBP2 prevents the translocation of Gαs from membranes to the cytosol upon the GPCR stimulation. Diagram on the left corresponds to the BRET-based assay to measure the association of Gαs with membranes. Graphs in the middle correspond BRET measurements in HEK293T cells co-expressing plasma membrane anchored Venus (Venus-KRas), nanoluciferase fused Gαs (Gαs-Nluc), and mem-GαsBP2 WT or mem-GαsBP2 W11A, as indicated. Changes in BRET compared to the unstimulated baseline (“ΔBRET·103 (baseline)”) are shown in the kinetic curves on the right graph, whereas the bar graph on the right corresponds to the quantification of ΔBRET before or after isoproterenol stimulation for 10 min. Mean ± S.E.M. (n = 4 independent experiments). Immunoblots on the left right correspond to representative results of experiments confirming the expression of Venus-KRas, Gαs-Nluc, and mem-GαsBP2.
Extended Data Fig. 8 Structure-guided, rational design of a cell penetrating GαsBP2 derivative to block Gαs signaling.
(A) GαsBP2 fused to the cell penetrating TAT sequence (TAT-GαsBP2) partially blocks GPCR-stimulated AC activity in cells. Diagram on the left displays the sequence of TAT-GαsBP2 and the mechanism of GPCR-Gs-AC signaling investigated with it. Graphs with kinetic traces in the middle correspond to luminescence-based cAMP measurements in HEK293T cells pre-treated with DMSO (1%, “no GαsBP2”, gray), or TAT-GαsBP2 WT (100 µM, orange) or TAT-GαsBP2 W11A mutant (100 µM, blue) before stimulation with isoproterenol. Mean ± S.E.M. (n = 4-6 independent experiments). Graph on the right correspond to luminescence-based cAMP measurements in HEK293T cells pre-treated the indicated concentrations of TAT-GαsBP2 WT (orange) or TAT-GαsBP2 W11A mutant (blue) before stimulation with isoproterenol (100 nM). Mean ± S.E.M. (n = 3 independent experiments). (B) Systematic identification of structural determinants of GαsBP2 required for Gαs binding. Diagram on the top corresponds to the cell-based BRET assay to measure binding between GαsBP2 and Gαs. Bar graph on the bottom corresponds to BRET measurements in HEK293T cells co-expressing Nluc-fused mem-GαsBP2 (WT or the indicated mutants) with either Venus-fused Gαs WT or Q227L. Relative Gαs binding was calculated using as reference the BRET values for Nluc-fused mem-GαsBP2 co-expressed with Gαs WT (as the minimum, 0%) or with Gαs Q226L (as the maximum, 100%). Mean ± S.E.M. (n = 3 independent experiments). (C) Mapping of the structural determinants of GαsBP2 within the binding interface with Gαs. The structural model of GαsBP2 (orange) bound to Gαs (purple) was generated using ColabFold. Yellow stars indicate amino acid positions of GαsBP2 that result in complete loss of binding in panel B upon mutation, which dock onto the α3/SwII groove of active Gαs. A potential electrostatic hindrance between GαsBP2 S9 and Gαs I235 (red dashed circle), and the positions connected through a hydrocarbon “staple” (gray) in panel D are shown in the bottom left. (D) Optimization of GαsBP2 peptides to efficiently block Gαs-stimulated AC activity in cells. Sequences on the left display amino acids and modification features of TAT-GαsBP2 peptide derivatives; S9A mutation (purple) and hydrocarbon “staple” modification (green) linking positions E10 to S17 (E10^S17). Graph with kinetic traces in the middle correspond to luminescence-based cAMP measurements in HEK293T cells pre-treated with DMSO (1%, “no GαsBP2”, gray), TAT-GαsBP2 S9A (100 µM, purple) or TAT-GαsBP2 S9A E10^S17 (100 µM, green) before stimulation with isoproterenol. Mean ± S.E.M. (n = 3-4 independent experiments). Graphs with concentration-dependence curves on the right correspond to luminescence-based cAMP measurements in untransfected HEK293T cells pre-treated the indicated concentrations of TAT-GαsBP2 WT (orange), GαsBP2 S9A (purple), or TAT-GαsBP2 S9A E10^S17 (green) before stimulation with isoproterenol (100 nM), or in cells transfected with Gαs R201C. Mean ± S.E.M. (n = 3, independent experiments). The half-maximal inhibitory concentration (IC50) was determined from curve fits.
Extended Data Fig. 9 α-sintide blocks GPCR-mediated activation of Gαs, but not of Gαi3, Gαq, or Gα13.
(A) Diagram depicting the G protein dependent effects investigated with α -sintide (B) α-sintide specifically inhibits Gαs activation. Graphs with kinetic traces on the top correspond to BRET measurements in HEK293T cells co-expressing the indicated combination of GPCR and ONE-GO biosensor after preincubation with 100 µM α-sintide WT (orange), 100 µM α-sintide W11A (green), or 1% (v:v) DMSO (grey) for 2 hrs. Data is presented as real-time changes in BRET relative to the unstimulated baseline (“ΔBRET·103 (baseline)”). Bar graphs on the bottom display the maximal changes in BRET after agonist stimulation relative to the unstimulated baseline (“Max ΔBRET·103”). Mean ± S.E.M. (n = 3-4 independent experiments). (C) α-sintide has no effect on the basal activity of any of the G proteins investigated. BRET was measured in HEK293T cells as in (A) but without stimulation and results are represented as the ratio between luminescence at 535 nm and 450 nm (“BRET ratio (535/450 nm)”). Mean ± S.E.M. (n = 3-4 independent experiments).
Extended Data Fig. 10 α-sintide improves cytotoxic cytokine production by chronically activated CD8+ T cells.
Diagram on the left represent the experimental design to assess the effect of α-sintide on T cells. Chronically activated human CD8+ T cells were treated with 30 µM of α-sintide WT (orange) or α-sintide W11 A (blue) for 1 day before analysis. Graphs in the middle correspond to the percentage of CD8+ T cells positive for IL-2 and IFNγ (IL-2+/ IFNγ+, top graph), or for IL-2 and TNFα (IL-2+/ TNFα+, bottom graph) quantified by flow cytometry. Bar graphs are mean ± S.E.M. (n = 3 independent experiments), ns, p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA corrected for multiple comparisons (Dunnett). Pseudocolor plots on the right display the average distribution of CD8+ T cells across all replicates. The flow cytometry plots corresponding to the DMSO conditions are reproduced from Fig. 6d because these were the same controls for experiments presented in the two figures.
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Zhao, J., Luebbers, A., Savransky, S. et al. Inhibitory probes for spatiotemporal analysis of Gαs protein signaling. Nat Chem Biol (2026). https://doi.org/10.1038/s41589-025-02138-1
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DOI: https://doi.org/10.1038/s41589-025-02138-1
