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.

References

Hauser, A. S., Attwood, M. M., Rask-Andersen, M., Schioth, H. B. & Gloriam, D. E. Trends in GPCR drug discovery: new agents, targets and indications. Nat. Rev. Drug Discovery 16, 829–842 (2017).

Article CAS PubMed Google Scholar 1.

Ross, E. M. & Gilman, A. G. Reconstitution of catecholamine-sensitive adenylate cyclase activity: interactions of solubilized components with receptor-replete membranes. Proc. Natl Acad. Sci. USA 74, 3715–3719 (1977).

Article CAS PubMed PubMed Central Google Scholar 1.

Ross, E. M., Howlett, A. C., Ferguson, K. M. & Gilman, A. G. Reconstitution of hormone-sensitive adenylate cyclase activity with resolved components of the enzyme. J. Biol. Chem. 253, 6401–6412 (1978).

Article CAS PubMed Google Scholar 1.

Wu, V. et al. Illuminating the Onco-GPCRome: novel G protein-coupled receptor-driven oncocrine networks and targets for cancer immunotherapy. J. Biol. Chem. 294, 11062–11086 (2019).

Article CAS PubMed PubMed Central Google Scholar 1.

Patra, K. C. et al. Mutant GNAS drives pancreatic tumourigenesis by inducing PKA-mediated SIK suppression and reprogramming lipid metabolism. Nat. Cell Biol. 20, 811–822 (2018).

Article CAS PubMed PubMed Central Google Scholar 1.

Ideno, N. et al. GNASR201C induces pancreatic cystic neoplasms in mice that express activated KRAS by inhibiting YAP1 signaling. Gastroenterology 155, 1593–1607 e1512 (2018).

Article CAS PubMed Google Scholar 1.

Ramms, D. J. et al. Gαs–protein kinase A (PKA) pathway signalopathies: the emerging genetic landscape and therapeutic potential of human diseases driven by aberrant Gαs–PKA signaling. Pharmacol. Rev. 73, 155–197 (2021).

Article CAS PubMed Google Scholar 1.

Wu, V. H. et al. The GPCR–Gα–PKA signaling axis promotes T cell dysfunction and cancer immunotherapy failure. Nat. Immunol. 24, 1318–1330 (2023).

Article CAS PubMed PubMed Central Google Scholar 1.

Zhu, J. et al. Tumour immune rejection triggered by activation of α2-adrenergic receptors. Nature 618, 607–615 (2023).

Article CAS PubMed Google Scholar 1.

Irannejad, R. & von Zastrow, M. GPCR signaling along the endocytic pathway. Curr. Opin. Cell Biol. 27, 109–116 (2014).

Article CAS PubMed Google Scholar 1.

Bock, A., Irannejad, R. & Scott, J. D. cAMP signaling: a remarkably regional affair. Trends Biochem. Sci. 49, 305–317 (2024).

Article CAS PubMed PubMed Central Google Scholar 1.

Calebiro, D., Miljus, T. & O’Brien, S. Endomembrane GPCR signaling: 15 years on, the quest continues. Trends Biochem. Sci. 50, 46–60 (2025).

Article CAS PubMed Google Scholar 1.

Ferrandon, S. et al. Sustained cyclic AMP production by parathyroid hormone receptor endocytosis. Nat. Chem. Biol. 5, 734–742 (2009).

Article CAS PubMed PubMed Central Google Scholar 1.

Calebiro, D. et al. Persistent cAMP-signals triggered by internalized G-protein-coupled receptors. PLoS Biol. 7, e1000172 (2009).

Article PubMed PubMed Central Google Scholar 1.

Irannejad, R. et al. Conformational biosensors reveal GPCR signalling from endosomes. Nature 495, 534–538 (2013).

Article CAS PubMed Google Scholar 1.

Sutkeviciute, I. & Vilardaga, J. P. Structural insights into emergent signaling modes of G protein-coupled receptors. J. Biol. Chem. 295, 11626–11642 (2020).

Article CAS PubMed PubMed Central Google Scholar 1.

Irannejad, R., Tsvetanova, N. G., Lobingier, B. T. & von Zastrow, M. Effects of endocytosis on receptor-mediated signaling. Curr. Opin. Cell Biol. 35, 137–143 (2015).

Article CAS PubMed PubMed Central Google Scholar 1.

Kwon, Y. et al. Non-canonical β-adrenergic activation of ERK at endosomes. Nature 611, 173–179 (2022).

Article CAS PubMed PubMed Central Google Scholar 1.

Stoeber, M. et al. A genetically encoded biosensor reveals location bias of opioid drug action. Neuron 98, 963–976 (2018).

Article CAS PubMed PubMed Central Google Scholar 1.

Wright, S. C. et al. BRET-based effector membrane translocation assay monitors GPCR-promoted and endocytosis-mediated Gq activation at early endosomes. Proc. Natl Acad. Sci. USA 118, e2025846118 (2021).

Article CAS PubMed PubMed Central Google Scholar 1.

Eiger, D. S. et al. Location bias contributes to functionally selective responses of biased CXCR3 agonists. Nat. Commun. 13, 5846 (2022).

Article CAS PubMed PubMed Central Google Scholar 1.

Tsvetanova, N. G. & von Zastrow, M. Spatial encoding of cyclic AMP signaling specificity by GPCR endocytosis. Nat. Chem. Biol. 10, 1061–1065 (2014).

Article CAS PubMed PubMed Central Google Scholar 1.

Godbole, A., Lyga, S., Lohse, M. J. & Calebiro, D. Internalized TSH receptors en route to the TGN induce local Gs-protein signaling and gene transcription. Nat. Commun. 8, 443 (2017).

Article PubMed PubMed Central Google Scholar 1.

Klauer, M. J., Willette, B. K. A. & Tsvetanova, N. G. Functional diversification of cell signaling by GPCR localization. J. Biol. Chem. 300, 105668 (2024).

Article CAS PubMed PubMed Central Google Scholar 1.

Willette, B. K. A., Zhang, J. F., Zhang, J. & Tsvetanova, N. G. Endosome positioning coordinates spatially selective GPCR signaling. Nat. Chem. Biol. 20, 151–161 (2024).

Article CAS PubMed Google Scholar 1.

Lin, T. Y. et al. Cardiac contraction and relaxation are regulated by distinct subcellular cAMP pools. Nat. Chem. Biol. 20, 62–73 (2024).

Article CAS PubMed Google Scholar 1.

Irannejad, R. et al. Functional selectivity of GPCR-directed drug action through location bias. Nat. Chem. Biol. 13, 799–806 (2017).

Article CAS PubMed PubMed Central Google Scholar 1.

White, A. D. et al. Ca2+ allostery in PTH-receptor signaling. Proc. Natl Acad. Sci. USA 116, 3294–3299 (2019).

Article CAS PubMed PubMed Central Google Scholar 1.

White, A. D. et al. Spatial bias in cAMP generation determines biological responses to PTH type 1 receptor activation. Sci. Signal. 14, eabc5944 (2021).

Article CAS PubMed PubMed Central Google Scholar 1.

Wedegaertner, P. B., Bourne, H. R. & von Zastrow, M. Activation-induced subcellular redistribution of Gs alpha. Mol. Biol. Cell 7, 1225–1233 (1996).

Article CAS PubMed PubMed Central Google Scholar 1.

Jang, W., Senarath, K., Feinberg, G., Lu, S. & Lambert, N. A. Visualization of endogenous G proteins on endosomes and other organelles. eLife 13, RP97033 (2024).

Article CAS PubMed PubMed Central Google Scholar 1.

Martin, B. R. & Lambert, N. A. Activated G protein Gαs samples multiple endomembrane compartments. J. Biol. Chem. 291, 20295–20302 (2016).

Article CAS PubMed PubMed Central Google Scholar 1.

Pizzoni, A., Zhang, X. & Altschuler, D. L. From membrane to nucleus: a three-wave hypothesis of cAMP signaling. J. Biol. Chem. 300, 105497 (2024).

Article CAS PubMed Google Scholar 1.

Thiyagarajan, M. M. et al. Activation-induced subcellular redistribution of Gαs is dependent upon its unique N-terminus. Biochemistry 41, 9470–9484 (2002).

Article CAS PubMed Google Scholar 1.

Lazar, A. M. et al. G protein-regulated endocytic trafficking of adenylyl cyclase type 9. eLife 9, e58039 (2020).

Article CAS PubMed PubMed Central Google Scholar 1.

Thomsen, A. R. B. et al. GPCR–G protein–β-arrestin super-complex mediates sustained G protein signaling. Cell 166, 907–919 (2016).

Article CAS PubMed PubMed Central Google Scholar 1.

Krumins, A. M. & Gilman, A. G. Targeted knockdown of G protein subunits selectively prevents receptor-mediated modulation of effectors and reveals complex changes in non-targeted signaling proteins. J. Biol. Chem. 281, 10250–10262 (2006).

Article CAS PubMed Google Scholar 1.

Burghi, V. et al. Gαs is dispensable for β-arrestin coupling but dictates GRK selectivity and is predominant for gene expression regulation by β2-adrenergic receptor. J. Biol. Chem. 299, 105293 (2023).

Article CAS PubMed PubMed Central Google Scholar 1.

Takasaki, J. et al. A novel Gαq/11-selective inhibitor. J. Biol. Chem. 279, 47438–47445 (2004).

Article CAS PubMed Google Scholar 1.

Schrage, R. et al. The experimental power of FR900359 to study Gq-regulated biological processes. Nat. Commun. 6, 10156 (2015).

Article CAS PubMed Google Scholar 1.

Kostenis, E., Pfeil, E. M. & Annala, S. Heterotrimeric Gq proteins as therapeutic targets? J. Biol. Chem. 295, 5206–5215 (2020).

Article CAS PubMed PubMed Central Google Scholar 1.

Pfeil, E. M. et al. Heterotrimeric G protein subunit Gαq is a master switch for Gβγ-mediated calcium mobilization by Gi-coupled GPCRs. Mol. Cell 80, 940–954 (2020).

Article CAS PubMed Google Scholar 1.

Dai, S. A. et al. State-selective modulation of heterotrimeric Gαs signaling with macrocyclic peptides. Cell 185, 3950–3965 (2022).

Article CAS PubMed PubMed Central Google Scholar 1.

Janicot, R. et al. Direct interrogation of context-dependent GPCR activity with a universal biosensor platform. Cell 187, 1527–1546 (2024).

Article CAS PubMed PubMed Central Google Scholar 1.

Berman, D. M., Wilkie, T. M. & Gilman, A. G. GAIP and RGS4 are GTPase-activating proteins for the Gi subfamily of G protein α subunits. Cell 86, 445–452 (1996).

Article CAS PubMed Google Scholar 1.

Tesmer, J. J., Berman, D. M., Gilman, A. G. & Sprang, S. R. Structure of RGS4 bound to AlF4-activated Giα1: stabilization of the transition state for GTP hydrolysis. Cell 89, 251–261 (1997).

Article CAS PubMed Google Scholar 1.

Waldo, G. L. et al. Kinetic scaffolding mediated by a phospholipase C-β and Gq signaling complex. Science 330, 974–980 (2010).

Article CAS PubMed PubMed Central Google Scholar 1.

Luebbers, A. et al. Dissecting the molecular basis for the modulation of neurotransmitter GPCR signaling by GINIP. Structure 32, 47–59 (2024).

Article CAS PubMed Google Scholar 1.

Garcia-Marcos, M. Heterotrimeric G protein signaling without GPCRs: the Gα-binding-and-activating (GBA) motif. J. Biol. Chem. 300, 105756 (2024).

Article CAS PubMed PubMed Central Google Scholar 1.

Kimple, R. J., Kimple, M. E., Betts, L., Sondek, J. & Siderovski, D. P. Structural determinants for GoLoco-induced inhibition of nucleotide release by Gα subunits. Nature 416, 878–881 (2002).

Article CAS PubMed Google Scholar 1.

Tesmer, J. J., Sunahara, R. K., Gilman, A. G. & Sprang, S. R. Crystal structure of the catalytic domains of adenylyl cyclase in a complex with Gsα·GTPγS. Science 278, 1907–1916 (1997).

Article CAS PubMed Google Scholar 1.

Hollins, B., Kuravi, S., Digby, G. J. & Lambert, N. A. The C-terminus of GRK3 indicates rapid dissociation of G protein heterotrimers. Cell. Signal. 21, 1015–1021 (2009).

Article CAS PubMed PubMed Central Google Scholar 1.

Masuho, I. et al. Distinct profiles of functional discrimination among G proteins determine the actions of G protein-coupled receptors. Sci Signal 8, ra123 (2015).

Article PubMed PubMed Central Google Scholar 1.

Masuho, I. et al. A global map of G protein signaling regulation by RGS proteins. Cell 183, 503–521 (2020).

Article CAS PubMed PubMed Central Google Scholar 1.

DiGiacomo, V. et al. Probing the mutational landscape of regulators of G protein signaling proteins in cancer. Sci. Signal. 13, eaax8620 (2020).

Article CAS PubMed PubMed Central Google Scholar 1.

Lambert, N. A. et al. Regulators of G-protein signaling accelerate GPCR signaling kinetics and govern sensitivity solely by accelerating GTPase activity. Proc. Natl Acad. Sci. USA 107, 7066–7071 (2010).

Article CAS PubMed PubMed Central Google Scholar 1.

Inoue, T., Heo, W. D., Grimley, J. S., Wandless, T. J. & Meyer, T. An inducible translocation strategy to rapidly activate and inhibit small GTPase signaling pathways. Nat. Methods 2, 415–418 (2005).

Article CAS PubMed PubMed Central Google Scholar 1.

Cullum, S. A., Veprintsev, D. B. & Hill, S. J. Kinetic analysis of endogenous β2-adrenoceptor-mediated cAMP GloSensor responses in HEK293 cells. Br. J. Pharmacol. 180, 1304–1315 (2023).

Article CAS PubMed Google Scholar 1.

Edwards, S. R. & Wandless, T. J. The rapamycin-binding domain of the protein kinase mammalian target of rapamycin is a destabilizing domain. J. Biol. Chem. 282, 13395–13401 (2007).

Article CAS PubMed Google Scholar 1.

Liang, F. S., Ho, W. Q. & Crabtree, G. R. Engineering the ABA plant stress pathway for regulation of induced proximity. Sci. Signal. 4, rs2 (2011).

Article PubMed PubMed Central Google Scholar 1.

Vilardaga, J. P. et al. Molecular mechanisms of PTH/PTHrP class B GPCR signaling and pharmacological implications. Endocr. Rev. 44, 474–491 (2023).

Article PubMed Google Scholar 1.

Wedegaertner, P. B. & Bourne, H. R. Activation and depalmitoylation of Gsα. Cell 77, 1063–1070 (1994).

Article CAS PubMed Google Scholar 1.

Hu, Q. & Shokat, K. M. Disease-causing mutations in the G protein Gαs subvert the roles of GDP and GTP. Cell 173, 1254–1264 e1211 (2018).

Article CAS PubMed PubMed Central Google Scholar 1.

Digby, G. J., Lober, R. M., Sethi, P. R. & Lambert, N. A. Some G protein heterotrimers physically dissociate in living cells. Proc. Natl Acad. Sci. USA 103, 17789–17794 (2006).

Article CAS PubMed PubMed Central [Google Scholar](http://scholar.google.com/scholar_lookup?&title=Some%20G%20protein%20heterotrimers%20physically%20dissociate%20in%20living%20cells&journal=Proc.%20Natl%20Acad.%20Sci.%20USA&doi=10.1073%2Fpnas.0607116103&volume=103&pages=17789-17794&publication_year=2006&author=Digby%2CGJ&author=Lober%2CRM&autho