1 Gilman, A. G. G proteins: transducers of receptor-generated signals. Annu. Rev. Biochem. 56, 615–649 (1987).

2 Oldham, W. M. & Hamm, H. E. Heterotrimeric G protein activation by G-protein-coupled receptors. Nat. Rev. Mol. Cell Biol. 9, 60–71 (2008).

3 Komolov, K. E. & Benovic, J. L. G protein-coupled receptor kinases: past, present and future. Cell. Signal. 41, 17–24 (2018).

4 Homan, K. T. & Tesmer, J. J. G. Structural insights into G protein-coupled receptor kinase function. Curr. Opin. Cell Biol. 27, 25–31 (2014).

5 Peterson, Y. K. & Luttrell, L. M. The diverse roles of arrestin scaffolds in G protein–coupled receptor signaling. Pharmacol. Rev. 69, 256 (2017).

6 Alvarez-Curto, E. et al. Targeted elimination of G proteins and arrestins defines their specific contributions to both intensity and duration of G protein-coupled receptor signalling. J. Biol. Chem. 291, 27147–27159 (2016).

7 Eichel, K., Jullie, D. & von Zastrow, M. β-Arrestin drives MAP kinase signalling from clathrin-coated structures after GPCR dissociation. Nat. Cell Biol. 18, 303–310 (2016).

8 Grundmann, M. et al. Lack of β-arrestin signaling in the absence of active G proteins. Nat. Commun. 9, 341 (2018).

9 O'Hayre, M. et al. Genetic evidence that β-arrestins are dispensable for the initiation of β2 adrenergic receptor signaling to ERK. Sci. Signal. 10, eaal3395 (2017).

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

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

12 Santos, R. et al. A comprehensive map of molecular drug targets. Nat. Rev. Drug Discov. 16, 19 (2016).

13 Fishman, M. C. & Porter, J. A. Pharmaceuticals: a new grammar for drug discovery. Nature 437, 491–493 (2005).

14 Van Raamsdonk, C. D. et al. Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi. Nature 457, 599–602 (2009). This paper identifies constitutively activating mutations of G α q at high prevalence in uveal melanoma and identifies G α q as a potential therapeutic target for the treatment of uveal melanoma.

15 Van Raamsdonk, C. D. et al. Mutations in GNA11 in uveal melanoma. N. Engl. J. Med. 363, 2191–2199 (2010).

16 Kenakin, T. Signaling bias in drug discovery. Expert Opin. Drug Discov. 12, 321–333 (2017).

17 Smith, J. S., Lefkowitz, R. J. & Rajagopal, S. Biased signalling: from simple switches to allosteric microprocessors. Nat. Rev. Drug Discov. 17, 243–260 (2018).

18 Bohn, L. M. et al. Enhanced morphine analgesia in mice lacking β-arrestin 2. Science 286, 2495–2498 (1999). These authors demonstrate that genetic deletion of β-arrestin 2 enhances the analgesic potency of morphine. This and subsequent work provide the basis for the idea of development of G protein-biased opioid analgesics.

19 Bohn, L. M., Gainetdinov, R. R., Lin, F. T., Lefkowitz, R. J. & Caron, M. G. μ-Opioid receptor desensitization by β arrestin 2 determines morphine tolerance but not dependence. Nature 408, 720–723 (2000).

20 Schmid, C. L. et al. Bias factor and therapeutic window correlate to predict safer opioid analgesics. Cell 171, 1165–1175 (2017).

21 Koblish, M. et al. TRV0109101, a G protein-biased agonist of the μ-opioid receptor, does not promote opioid-induced mechanical allodynia following chronic administration. J. Pharmacol. Exp. Ther. 362, 254–262 (2017).

22 Singla, N. et al. A randomized, phase IIb study investigating oliceridine (TRV130), a novel μ receptor G protein pathway selective (μ-GPS) modulator, for the management of moderate to severe acute pain following abdominoplasty. J. Pain Res. 10, 2413–2424 (2017).

23 Simon, M. I., Strathmann, M. P. & Gautam, N. Diversity of G proteins in signal transduction. Science 252, 802–808 (1991).

24 Robishaw, J. D. & Berlot, C. H. Translating G protein subunit diversity into functional specificity. Curr. Opin. Cell Biol. 16, 206–209 (2004).

25 Oldham, W. M. & Hamm, E. Structural basis of function in heterotrimeric G proteins. Q. Rev. Biophys. 39, 117–166 (2006).

26 Khan, S. M. et al. The expanding roles of Gβγ subunits in G protein-coupled receptor signaling and drug action. Pharmacol. Rev. 65, 545–577 (2013).

27 Hepler, J. R. & Gilman, A. G. G proteins. Trends Biochem. Sci. 17, 383–387 (1992).

28 Northup, J. K. et al. Purification of the regulatory component of adenylate cyclase. Proc. Natl Acad. Sci. USA 77, 6516–6520 (1980).

29 Smrcka, A. V., Hepler, J. R., Brown, K. O. & Sternweis, P. C. Regulation of polyphosphoinositide-specific phospholipase C activity by purified Gq. Science 251, 804–807 (1991).

30 Hart, M. J. et al. Direct stimulation of the guanine nucleotide exchange activity of p115 RhoGEF by Gα13. Science 280, 2112–2114 (1998).

31 Taussig, R., Iniguez-Lluhi, J. A. & Gilman, A. G. Inhibition of adenylyl cyclase by Giα. Science 261, 218–221 (1993).

32 Logothetis, D. E., Kurachi, Y., Galper, J., Neer, E. J. & Clapham, D. E. The βγ subunits of GTP-binding proteins activate the muscarinic K+ channel in heart. Nature 325, 321–326 (1987).

33 Camps, M. et al. Isozyme-selective stimulation of phospholipase Cβ2 by G protein βγ subunits. Nature 360, 684–686 (1992).

34 Stephens, L. et al. A novel phosphoinositide 3 kinase activity in myeloid-derived cells is activated by G protein βγ subunits. Cell 77, 83–93 (1994).

35 Smrcka, A. V. G protein βγ subunits: central mediators of G protein-coupled receptor signaling. Cell. Mol. Life Sci. 65, 2191–2214 (2008).

36 Sunahara, R. K., Dessauer, C. W. & Gilman, A. G. Complexity and diversity of mammalian adenylyl cyclases. Ann. Rev. Pharmacol. Toxicol. 36, 461–480 (1996).

37 Hohenegger, M. et al. Gsα-selective G protein antagonists. Proc. Natl Acad. Sci. USA 95, 346–351 (1998).

38 Sternweis, P. C. & Robishaw, J. D. Isolation of two proteins with high affinity for guanine nucleotides from membranes of bovine brain. J. Biol. Chem. 259, 13806–13813 (1984).

39 Bokoch, G. M., Katada, T., Northup, J. K., Ui, M. & Gilman, A. G. Purification and properties of the inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. J. Biol. Chem. 259, 3560–3567 (1984).

40 Katada, T., Bokoch, G. M., Northup, J. K., Ui, M. & Gilman, A. G. The inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. Properties and function of the purified protein. J. Biol. Chem. 259, 3568–3577 (1984).

41 Codina, J. et al. Pertussis toxin substrate, the putative N i component of adenylyl cyclases, in an αβ heterodimer regulated by guanine nucleotide and magnesium. Proc. Natl Acad. Sci. USA 80, 4276–4280 (1983).

42 Strathmann, M., Wilkie, T. M. & Simon, M. I. Diversity of the G protein family: sequences from five additional α subunits in the mouse. Proc. Natl Acad. Sci. USA 86, 7407–7409 (1989).

43 Linder, M. E., Ewald, D. A., Miller, R. J. & Gilman, A. G. Purification and characterization of G oα and three types of G iα after expression in Escherichia coli. J. Biol. Chem. 265, 8243–8251 (1990).

44 Solis, G. P. et al. Golgi-resident Gαo promotes protrusive membrane dynamics. Cell 170, 939–955 (2017).

45 Casey, P. J., Fong, H. K. W., Simon, M. I. & Gilman, A. G. Gz, a guanine nucleotide-binding protein with unique biochemical properties. J. Biol. Chem. 265, 2383–2390 (1990).

46 Murayama, T. & Ui, M. Loss of the inhibitory function of the guanine nucleotide regulatory component of adenylate cyclase due to its ADP ribosylation by islet-activating protein, pertussis toxin, in adipocyte membranes. J. Biol. Chem. 258, 3319–3326 (1983).

47 Lee, C. H., Park, D., Wu, D., Rhee, S. G. & Simon, M. I. Members of the Gαq subunit gene family activate phospholipase Cβ isozymes. J. Biol. Chem. 267, 16044–16047 (1992).

48 Taylor, S. J., Chae, H. Z., Rhee, S. G. & Exton, J. H. Activation of the β1 isozyme of phospholipase C by α subunits of the G q class of G proteins. Nature 350, 516–518 (1991).

49 Rhee, S. G. Regulation of phospho-specific phospholipase C. Annu. Rev. Biochem 70, 281–312 (2001).

50 Singer, W. D., Brown, H. A. & Sternweis, P. C. Regulation of eukaryotic phosphatidylinositol-specific phospholipase C and phospholipase D. Ann. Rev. Biochem. 66, 475–509 (1997).

51 Kadamur, G. & Ross, E. M. Mammalian phospholipase C. Annu. Rev. Physiol. 75, 127–154 (2013).

52 Harden, T. K., Waldo, G. L., Hicks, S. N. & Sondek, J. Mechanism of activation and inactivation of Gq/phospholipase C β signaling nodes. Chem. Rev. 111, 6120–6129 (2011).

53 Wettschureck, N. & Offermanns, S. Mammalian G proteins and their cell type specific functions. Physiol. Rev. 85, 1159–1204 (2005).

54 Chagin, A. S. et al. G Protein stimulatory subunit alpha and Gq/11α G proteins are both required to maintain quiescent stem-like chondrocytes. Nat. Commun. 5, 3673 (2014).

55 Wang, S. et al. P2Y(2) and Gq/G(1)(1) control blood pressure by mediating endothelial mechanotransduction. J. Clin. Invest. 125, 3077–3086 (2015).

56 Sivaraj, K. K. et al. Endothelial Gαq/11 is required for VEGF-induced vascular permeability and angiogenesis. Cardiovasc. Res. 108, 171–180 (2015).

57 John, A. E. et al. Loss of epithelial Gq and G11 signaling inhibits TGFbeta production but promotes IL 33 mediated macrophage polarization and emphysema. Sci. Signal. 9, ra104 (2016).

58 Wilkie, T. M., Scherly, P. A., Strathmann, M. P., Slepak, V. Z. & Simon, M. I. Characterization of G protein α subunits in the G q class: expression in murine tissues and in stromal and hematopoietic cell lines. Proc. Natl Acad. Sci. USA 88, 10049–10053 (1991).

59 Hepler, J. R. et al. Purification from Sf9 cells and characterization of recombinant Gq α and G11 α. Activation of purified phospholipase C isozymes by G α subunits. J. Biol. Chem. 268, 14367–14375 (1993).

60 Kozasa, T. et al. Purification and characterization of recombinant G16 α from Sf9 cells: activation of purified phospholipase C isozymes by G protein α subunits. Proc. Natl Acad. Sci. USA 90, 9176–9180 (1993).

61 Wirotanseng, L. N., Kuner, R. & Tappe-Theodor, A. Gq rather than G11 preferentially mediates nociceptor sensitization. Mol. Pain 9, 54 (2013).

62 Aittaleb, M., Boguth, C. A. & Tesmer, J. J. G. Structure and function of heterotrimeric G protein-regulated Rho guanine nucleotide exchange factors. Mol. Pharmacol. 77, 111–125 (2010).

63 Momotani, K. et al. p63RhoGEF couples Gα(q/11)-mediated signaling to Ca2+ sensitization of vascular smooth muscle contractility. Circ. Res. 109, 993–1002 (2011).

64 Momotani, K. & Somlyo, A. V. p63RhoGEF: a new switch for G(q)-mediated activation of smooth muscle. Trends Cardiovasc. Med. 22, 122–127 (2012).

65 Vaque, J. P. et al. A genome-wide RNAi screen reveals a Trio-regulated Rho GTPase circuitry transducing mitogenic signals initiated by G protein-coupled receptors. Mol. Cell 49, 94–108 (2013).

66 Sanchez-Fernandez, G. et al. Gαq signalling: the new and the old. Cell. Signal. 26, 833–848 (2014).

67 Garcia-Hoz, C. et al. G α(q) acts as an adaptor protein in protein kinase C ζ (PKCζ)-mediated ERK5 activation by G protein-coupled receptors (GPCR). J. Biol. Chem. 285, 13480–13489 (2010).

68 Garcia-Hoz, C. et al. Protein kinase C (PKC)ζ mediated Gαq stimulation of ERK5 protein pathway in cardiomyocytes and cardiac fibroblasts. J. Biol. Chem. 287, 7792–7802 (2012).

69 Schrage, R. et al. The experimental power of FR900359 to study Gq regulated biological processes. Nat. Commun. 6, 10156 (2015). This comprehensive analysis reviews the specificity and efficacy of FR900359 as an inhibitor of Gα q in vitro and in cell models.

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

71 Strathmann, M. P. & Simon, M. I. Gα12 and Gα13 subunits define a fourth class of G protein α subunits. Proc. Natl Acad. Sci. USA 88, 5582–5586 (1991).

72 Kozasa, T. et al. p115 RhoGEF, a GTPase activating protein for Gα12 and Gα13. Science 280, 2109–2111 (1998).

73 Davis, T. L., Bonacci, T. M., Sprang, S. R. & Smrcka, A. V. Structural and molecular characterization of a preferred protein interaction surface on G protein βγ subunits. Biochemistry 44, 10593–10604 (2005).

74 Ford, C. E. et al. Molecular basis for interactions of G protein βγ subunits with effectors. Science 280, 1271–1274 (1998).

75 Lin, Y. & Smrcka, A. V. Understanding molecular recognition by G protein βγ subunits on the path to pharmacological targeting. Mol. Pharmacol. 80, 551–557 (2011).

76 Scott, J. K. et al. Evidence that a protein-protein interaction 'hot spot' on heterotrimeric G protein βγ subunits is used for recognition of a subclass of effectors. EMBO J. 20, 767–776 (2001).

77 Cabrera, J. L., de Freitas, F., Satpaev, D. K. & Slepak, V. Z. Identification of the Gβ5-RGS7 protein complex in the retina. Biochem. Biophys. Res. Commun. 249, 898–902 (1998).

78 Witherow, D. S. et al. Complexes of the G protein subunit gβ5 with the regulators of G protein signaling RGS7 and RGS9. Characterization in native tissues and in transfected cells. J. Biol. Chem. 275, 24872–24880 (2000).

79 Snow, B. E. et al. A G protein γ subunit-like domain shared between RGS11 and other RGS proteins specifies binding to Gβ5 subunits. Proc. Natl Acad. Sci. USA 95, 13307–13312 (1998).

80 Ueda, N. et al. G protein βγ subunits. Simplified purification and properties of novel isoforms. J. Biol. Chem. 269, 4388–4395 (1994).

81 Wickman, K. D. et al. Recombinant G-protein βγ subunits activate the muscarinic-gated atrial potassium channel. Nature 368, 255–257 (1994).

82 Diverse-Pierluissi, M. et al. Selective coupling of G protein βγ complexes to inhibition of Ca2+ channels. J. Biol. Chem. 275, 28380–28385 (2000).

83 Mayeenuddin, L. H., McIntire, W. E. & Garrison, J. C. Differential sensitivity of P-Rex1 to isoforms of G protein βγ dimers. J. Biol. Chem. 281, 1913–1920 (2006).

84 Gibson, S. K. & Gilman, A. G. Giα and Gβ subunits both define selectivity of G protein activation by α2-adrenergic receptors. Proc. Natl Acad. Sci. USA 103, 212–217 (2006).

85 Lindorfer, M. A. et al. Differential activity of the G protein β5 γ2 subunit at receptors and effectors. J. Biol. Chem. 273, 34429–34436 (1998).

86 Liang, Y. L. et al. Phase-plate cryo-EM structure of a class B GPCR-G-protein complex. Nature 546, 118–123 (2017).

87 Liang, Y. L. et al. Phase-plate cryo-EM structure of a biased agonist-bound human GLP-1 receptor-Gs complex. Nature 555, 121–125 (2018).

88 Zhang, Y. et al. Cryo-EM structure of the activated GLP-1 receptor in complex with a G protein. Nature 546, 248–253 (2017).

89 Kleuss, C., Scher¸bl, H., Hescheler, J., Schultz, G. & Wittig, B. Selectivity in signal transduction determined by γ subunits of heterotrimeric G proteins. Science 259, 832–834 (1993).

90 Wang, Q., Mullah, B., Hansen, C., Asundi, J. & Robishaw, J. D. Ribozyme-mediated suppression of the G Protein γ 7 subunit suggests a role in hormone regulation of adenylylcyclase activity. J. Biol. Chem. 272, 26040–26048 (1997).

91 Schwindinger, W. F. et al. Loss of G protein γ7 alters behavior and reduces striatal α(olf) level and cAMP production. J. Biol. Chem. 278, 6575–6579 (2003).

92 Schwindinger, W. F. et al. Mice with deficiency of G protein γ3 are lean and have seizures. Mol. Cell. Biol. 24, 7758–7768 (2004).

93 Schwindinger, W. F. et al. Synergistic roles for G-protein γ3 and γ7 subtypes in seizure susceptibility as revealed in double knock-out mice. J. Biol. Chem. 287, 7121–7133 (2012).

94 Cho, J. H., Saini, D. K., Karunarathne, W. K., Kalyanaraman, V. & Gautam, N. Alteration of Golgi structure in senescent cells and its regulation by a G protein γ subunit. Cell. Signal. 23, 785–793 (2011).

95 O'Neill, P. R., Karunarathne, W. K. A., Kalyanaraman, V., Silvius, J. R. & Gautam, N. G-Protein signaling leverages subunit-dependent membrane affinity to differentially control βγ translocation to intracellular membranes. Proc. Natl Acad. Sci. USA 109, E3568–E3577 (2012).

96 Ajith Karunarathne, W. K., O'Neill, P. R., Martinez-Espinosa, P. L., Kalyanaraman, V. & Gautam, N. All G protein βγ complexes are capable of translocation on receptor activation. Biochem. Biophys. Res. Commun. 421, 605–611 (2012).

97 Lambright, D. G. et al. The 2.0 Å crystal structure of a heterotrimeric G protein. Nature 379, 311–319 (1996).

98 Sondek, J., Bohm, A., Lambright, D. G., Hamm, H. E. & Sigler, P. B. Crystal structure of a G-protein βγ dimer at 2.1Å resolution. Nature 379, 369–374 (1996).

99 Clapham, D. E. & Neer, E. J. G protein βγ subunits. Annu. Rev. Pharmacol. Toxicol. 37, 167–203 (1997).

100 Pitcher, J. A. et al. Role of βγ subunits of G proteins in targeting the β-adrenergic receptor kinase to membrane-bound receptors. Science 257, 1264–1267 (1992).

101 Koch, W. J., Hawes, B. E., Inglese, J., Luttrell, L. M. & Lefkowitz, R. J. Cellular expression of the carboxyl terminus of a G protein-coupled receptor kinase attenuates G βγ-mediated signaling. J. Biol. Chem. 269, 6193–6197 (1994).

102 Koch, W. J. et al. Cardiac function in mice overexpressing the β-adrenergic receptor kinase or a βARK inhibitor. Science 268, 1350–1353 (1995). This study shows that cardiac expression of a protein that binds to Gβγ improves cardiac function, which is evidence that targeting Gβγ could be useful in the treatment of heart failure.

103 Rockman, H. A. et al. Expression of a β-adrenergic receptor kinase 1 inhibitor prevents the development of myocardial failure in gene-targeted mice. Proc. Natl Acad. Sci. USA 95, 7000–7005 (1998).

104 Eckhart, A. D., Ozaki, T., Tevaearai, H., Rockman, H. A. & Koch, W. J. Vascular-targeted overexpression of G protein-coupled receptor kinase-2 in transgenic mice attenuates β-adrenergic receptor signaling and increases resting blood pressure. Mol. Pharmacol. 61, 749–758 (2002).

105 Bonacci, T. M. et al. Differential targeting of Gβγ-subunit signaling with small molecules. Science 312, 443–446 (2006). This study identifies prototypical small-molecule inhibitors of Gβγ subunit signalling. In this work, the concept of selective blockage of Gβγ downstream signalling by small molecules is introduced.

106 Taniguchi, M. et al. YM-254890, a novel platelet aggregation inhibitor produced by Chromobacterium sp. QS3666. J. Antibiot. (Tokyo) 56, 358–363 (2003).

107 Kawasaki, T. et al. Antithrombotic and thrombolytic efficacy of YM-254890, a G q/11 inhibitor, in a rat model of arterial thrombosis. Thromb. Haemost. 90, 406–413 (2003). This study demonstrates the utility of small-molecule Gα q inhibition in the treatment of thrombosis.

108 Kawasaki, T. et al. Pharmacological properties of YM-254890, a specific Gαq/11 inhibitor, on thrombosis and neointima formation in mice. Thromb. Haemost. 94, 184–192 (2005).

109 Nishimura, A. et al. Structural basis for the specific inhibition of heterotrimeric Gq protein by a small molecule. Proc. Natl Acad. Sci. USA 107, 13666–13671 (2010). These investigators solve the X-ray crystal co-structure of YM-254890 bound to Gα q , which provides a mechanism of action and a potential starting point for the development of Gα subunit-selective inhibitors.

110 Wall, M. A. et al. The structure of the G protein heterotrimer Giα1β1γ2 . Cell 83, 1047–1058 (1995).

111 Lambright, D. G., Noel, J. P., Hamm, H. E. & Sigler, P. B. Structural determinants for activation of the α-subunit of a heterotrimeric G protein. Nature 369, 621–628 (1994).

112 Rasmussen, S. G. F. et al. Crystal structure of the β2 adrenergic receptor-Gs protein complex. Nature 477, 549–555 (2011).

113 Dror, R. O. et al. Structural basis for nucleotide exchange in heterotrimeric G proteins. Science 348, 1361–1365 (2015).

114 Charpentier, T. H. et al. Potent and selective peptide-based inhibition of the G protein Gαq. J. Biol. Chem. 291, 25608–25616 (2016).

115 Coughlin, S. R. Protease-activated receptors in hemostasis, thrombosis and vascular biology. J. Thromb. Haemost. 3, 1800–1814 (2005).

116 Leon, C. et al. Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y(1) receptor-null mice. J. Clin. Invest. 104, 1731–1737 (1999).

117 Gachet, C. P2Y(12) receptors in platelets and other hematopoietic and non-hematopoietic cells. Purinerg. Signal 8, 609–619 (2012).

118 Carr, R. 3rd et al. Interdicting Gq activation in airway disease by receptor-dependent and receptor-independent mechanisms. Mol. Pharmacol. 89, 94–104 (2016).

119 Matthey, M. et al. Targeted inhibition of Gq signaling induces airway relaxation in mouse models of asthma. Sci. Transl Med. 9, eaag2288 (2017). This thorough study demonstrates the utility of FR900359 in the treatment of asthma. The authors use an aerosol-based approach to selectively deliver the compound to the lungs, thereby avoiding the global effects of Gα q inhibition.

120 Gao, Z. G. & Jacobson, K. A. On the selectivity of the Gαq inhibitor UBO-QIC: A comparison with the Gαi inhibitor pertussis toxin. Biochem. Pharmacol. 107, 59–66 (2016).

121 Ohta, H., Okajima, F. & Ui, M. Inhibition by islet-activating protein of a chemotactic peptide-induced early breakdown of inositol phospholipids and Ca2+ mobilization in guinea pig neutrophils. J. Biol. Chem. 260, 15771–15780 (1985).

122 Baldassare, J. J., Henderson, P. A. & Fisher, G. J. Plasma membrane associated phospholipase C from human platelets: synergistic stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis by thrombin and guanosine 5′-O-(3-thiotriphosphate). Biochemistry 28, 56–60 (1989).

123 Portilla, D., Morrissey, J. & Morrison, A. R. Bradykinin-activated membrane-associated phospholipase C in Madin-Darby canine kidney cells. J. Clin. Invest. 81, 1896–1902 (1988).

124 Smrcka, A. V. & Sternweis, P. C. Regulation of purified subtypes of phosphatidylinositol specific phospholipase Cβ by G protein α and βγ subunits. J. Biol. Chem. 268, 9667–9674 (1993).

125 Boyer, J. L., Waldo, G. L. & Harden, T. K. βγ-Subunit activation of G-protein-regulated phospholipase C. J. Biol. Chem. 267, 25451–25456 (1992).

126 Philip, F., Kadamur, G., Silos, R. G. l., Woodson, J. & Ross, E. M. Synergistic activation of phospholipase C-β3 by Gαq and Gβγ describes a simple two-state coincidence detector. Curr. Biol. 20, 1327–1335 (2010).

127 Rebres, R. A. et al. Synergistic Ca2+ responses by Gβγ and Gαq-coupled G-protein-coupled receptors require a single PLCβ isoform that is sensitive to both Gβγ and Gαq. J. Biol. Chem. 286, 942–951 (2011).

128 Stehno-Bittel, L., Krapivinsky, G., Krapivinsky, L., Perez-Terzic, C. & Clapham, D. E. The G protein βγ subunit transduces the muscarinic receptor signal for Ca2+ release in Xenopus oocytes. J. Biol. Chem. 270, 30068–30074 (1995).

129 Maruko, T. et al. Involvement of the βγ subunits of G proteins in the cAMP response induced by stimulation of the histamine H1 receptor. Naunyn Schmiedebergs Arch. Pharmacol. 372, 153–159 (2005).

130 Rensing, D. T., Uppal, S., Blumer, K. J. & Moeller, K. D. Toward the selective inhibition of G proteins: total synthesis of a simplified YM-254890 analog. Org. Lett. 17, 2270–2273 (2015).

131 Xiong, X. F. et al. Total synthesis and structure-activity relationship studies of a series of selective G protein inhibitors. Nat. Chem. 8, 1035–1041 (2016). This paper synthesizes a series of compounds related to FR900359 demonstrating total synthesis of these natural products and a possible route to designing new selective inhibitors of Gα subunits.

132 Zhang, H. et al. Structure-activity relationship studies of the cyclic depsipeptide natural product YM-254890, targeting the Gq protein. ChemMedChem 12, 830–834 (2017).

133 Iaccarino, G., Smithwick, L. A., Lefkowitz, R. J. & Koch, W. J. Targeting Gβγ signaling in arterial vascular smooth muscle proliferation: a novel strategy to limit restenosis. Proc. Natl Acad. Sci. USA 96, 3945–3950 (1999).

134 Iaccarino, G. & Koch, W. J. Transgenic mice targeting the heart unveil G protein-coupled receptor kinases as therapeutic targets. Assay. Drug Dev. Technol. 1, 347–355 (2003).

135 Rengo, G., Lymperopoulos, A., Leosco, D. & Koch, W. J. GRK2 as a novel gene therapy target in heart failure. J. Mol. Cell Cardiol. 50, 785–792 (2011).

136 Bookout, A. L. et al. Targeting Gβγ signaling to inhibit prostate tumor formation and growth. J. Biol. Chem. 278, 37569–37573 (2003).

137 Lymperopoulos, A., Rengo, G., Funakoshi, H., Eckhart, A. D. & Koch, W. J. Adrenal GRK2 upregulation mediates sympathetic overdrive in heart failure. Nat. Med. 13, 315 (2007).

138 Siuda, E. R., Carr, R. 3rd, Rominger, D. H. & Violin, J. D. Biased μ-opioid receptor ligands: a promising new generation of pain therapeutics. Curr. Opin. Pharmacol. 32, 77–84 (2017).

139 Gulati, S. et al. Targeting G protein-coupled receptor signaling at the G protein level with a selective nanobody inhibitor. Nat. Commun. 9, 1996 (2018).

140 Lehmann, D. M., Seneviratne, A. M. P. B. & Smrcka, A. V. Small molecule disruption of G protein βγ subunit signaling inhibits neutrophil chemotaxis and inflammation. Mol. Pharmacol. 73, 410–418 (2008).

141 Neer, E. J., Schmidt, C. J., Nambudripad, R. & Smith, T. F. The ancient regulatory-protein family of WD-repeat proteins. Nature 371, 297–300 (1994).

142 Arkin, M. R. & Wells, J. A. Small-molecule inhibitors of protein-protein interactions: progressing towards the dream. Nat. Rev. Drug Discov. 3, 301–317 (2004).

143 Bolshan, Y. et al. Synthesis, optimization, and evaluation of novel small molecules as antagonists of WDR5-MLL interaction. ACS Med. Chem. Lett. 4, 353–357 (2013).

144 Grebien, F. et al. Pharmacological targeting of the Wdr5-MLL interaction in C/EBPα N-terminal leukemia. Nat. Chem. Biol. 11, 571–578 (2015). This paper describes the development and the structural basis for binding of a therapeutically relevant compound that binds to a WD40 repeat protein to block protein–protein interactions, which suggests that development of novel Gβγ inhibitors that bind with high affinity is possible.

145 Schapira, M., Tyers, M., Torrent, M. & Arrowsmith, C. H. WD40 repeat domain proteins: a novel target class? Nat. Rev. Drug Discov. 16, 773–786 (2017).

146 Seneviratne, A. M., Burroughs, M., Giralt, E. & Smrcka, A. V. Direct-reversible binding of small molecules to G protein βγ subunits. Biochim. Biophys. Acta 1814, 1210–1218 (2011).

147 Tonge, P. J. Drug–target kinetics in drug discovery. ACS Chem. Neurosci. 9, 29–39 (2018).

148 Bignante, E. A. et al. APP/Go protein Gβγ-complex signaling mediates Aβ degeneration and cognitive impairment in Alzheimer's disease models. Neurobiol. Aging 64, 44–57 (2018).

149 Sanz, G. et al. Gallein, a Gβγ subunit signalling inhibitor, inhibits metastatic spread of tumour cells expressing OR51E2 and exposed to its odorant ligand. BMC Res. Notes 10, 541 (2017).

150 Jensen, D. D. et al. Protein kinase D and Gβγ subunits mediate agonist-evoked translocation of protease-activated receptor-2 from the golgi apparatus to the plasma membrane. J. Biol. Chem. 291, 11285–11299 (2016).

151 Gautam, J. et al. 4-Hydroxynonenal-induced GPR109A (HCA2 receptor) activation elicits bipolar responses, Gαi -mediated anti-inflammatory effects and Gβγ -mediated cell death. Br. J. Pharmacol. 175, 2581–2598 (2018).

152 Kajimoto, T. et al. Involvement of Gβγ subunits of Gi protein coupled with S1P receptor on multivesicular endosomes in F-actin formation and cargo sorting into exosomes. J. Biol. Chem. 293, 245–253 (2018).

153 Wells, C. A. et al. Gβγ inhibits exocytosis via interaction with critical residues on soluble N-ethylmaleimide-sensitive factor attachment protein-25. Mol. Pharmacol. 82, 1136–1149 (2012).

154 Stephens, L. R. et al. The Gβγ sensitivity of a PI3K is dependent upon a tightly associated adaptor, 101. Cell 89, 105–114 (1997).

155 Tesmer, V. M., Kawano, T., Shankaranarayanan, A., Kozasa, T. & Tesmer, J. J. G. Snapshot of activated G proteins at the membrane: the Gαq-GRK2-Gβγ complex. Science 310, 1686–1690 (2005).

156 Whorton, M. R. & MacKinnon, R. X-Ray structure of the mammalian GIRK2-βγ G-protein complex. Nature 498, 190–197 (2013). These authors solve the X-ray structure of the Gβγ subunit bound to one of its effector molecules, GIRK. This structure reveals why small molecules that bind to the top of Gβγ do not inhibit GIRK channel regulation.

157 Gaudet, R., Bohm, A. & Sigler, P. B. Crystal structure at 2.4 angstroms resolution of the complex of transducin βγ and its regulator, phosducin. Cell 87, 577–588 (1996).

158 Mathews, J. L., Smrcka, A. V. & Bidlack, J. M. A. Novel Gβγ subunit inhibitor selectively modulates μ-opioid-dependent antinociception and attenuates acute morphine-induced antinociceptive tolerance and dependence. J. Neurosci. 28, 12183–12189 (2008). This study shows that small-molecule inhibition of Gβγ potentiates opioid analgesia and prevents the development of acute tolerance and dependence, which suggests this as a strategy for improving the safety of opioid analgesics.

159 Bourinet, E., Soong, T. W., Stea, A. & Snutch, T. P. Determinants of the G protein-dependent opioid modulation of neuronal calcium channels. Proc. Natl Acad. Sci. USA 93, 1486–1491 (1996).

160 Ikeda, S. R. Voltage-dependent modulation of N-type calcium channels by G-protein βγ subunits. Nature 380, 255–258 (1996).

161 Kamal, F. A. et al. Simultaneous adrenal and cardiac GPCR-Gβγ inhibition halts heart failure progression. J. Am. Coll. Cardiol. 63, 2549–2557 (2014).

162 Yoon, E. J., Gerachshenko, T., Spiegelberg, B. D., Alford, S. & Hamm, H. E. Gβγ interferes with Ca2+-dependent binding of synaptotagmin to the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Mol. Pharmacol. 72, 1210–1219 (2007).

163 Araldi, D., Ferrari, L. F. & Levine, J. D. Repeated μ-opioid exposure induces a novel form of the hyperalgesic priming model for transition to chronic pain. J. Neurosci. 35, 12502–12517 (2015).

164 Bianchi, E., Norcini, M., Smrcka, A. & Ghelardini, C. Supraspinal Gβγ-dependent stimulation of PLCβ originating from G inhibitory protein-μ opioid receptor-coupling is necessary for morphine induced acute hyperalgesia. J. Neurochem. 111, 171–180 (2009).

165 Joseph, E. K., Bogen, O., Alessandri-Haber, N. & Levine, J. D. PLC-β3 signals upstream of PKC ε in acute and chronic inflammatory hyperalgesia. Pain 132, 67–73 (2007).

166 Xie, W. et al. Genetic alteration of phospholipase Cβ3 expression modulates behavioral and cellular responses to μ opioids. Proc. Natl Acad. Sci. USA 96, 10385–10390 (1999). This study demonstrates that deletion of PLCβ3 in mice potentiates morphine-dependent opioid analgesia, which suggests a mechanism by which Gβγ inhibitors potentiate opioid analgesia through blockade of Gβγ-dependent regulation of PLCβ3.

167 Park, D., Jhon, D. Y., Lee, C. W., Lee, K. H. & Goo Rhee, S. Activation of phospholipase C isozymes by G protein βγ subunits. J. Biol. Chem. 268, 4573–4576 (1993).

168 Hoot, M. R. et al. Inhibition of Gβγ-subunit signaling potentiates morphine-induced antinociception but not respiratory depression, constipation, locomotion, and reward. Behav. Pharmacol. 24, 144–152 (2013).

169 Xu, J. et al. Divergent signals and cytoskeletal assemblies regulate self-organizing polarity in neutrophils. Cell 114, 201–214 (2003).

170 Surve, C. R., To, J. Y., Malik, S., Kim, M. & Smrcka, A. V. Dynamic regulation of neutrophil polarity and migration by the heterotrimeric G protein subunits Gαi-GTP and Gβγ. Sci. Signal. 9, ra22 (2016).

171 Rangel-Moreno, J. et al. Inhibition of G protein βγ subunit signaling abrogates nephritis in lupus-prone mice. Arthritis Rheumatol. 68, 2244–2256 (2016). This study shows that chronic Gβγ inhibition with gallein prevents the development of lupus, which shows the utility of this approach in chronic inflammatory disease and that chronic administration of gallein is well tolerated in mice.

172 Casey, L. M. et al. Small molecule disruption of G βγ signaling inhibits the progression of heart failure. Circ. Res. 107, 532–539 (2010). These authors demonstrate that small-molecule Gβγ inhibition prevents the development of heart failure.

173 Kamal, F. A. et al. G protein-coupled receptor-G-protein βγ-subunit signaling mediates renal dysfunction and fibrosis in heart failure. J. Am. Soc. Nephrol. 28, 197–208 (2017).

174 Lorenz, K., Schmitt, J. P., Schmitteckert, E. M. & Lohse, M. J. A new type of ERK1/2 autophosphorylation causes cardiac hypertrophy. Nat. Med. 15, 75–83 (2009).

175 Zhang, L. et al. Phospholipase Cε hydrolyzes perinuclear phosphatidylinositol 4-phosphate to regulate cardiac hypertrophy. Cell 153, 216–227 (2013).

176 Malik, S. et al. G protein βγ subunits regulate cardiomyocyte hypertrophy through a perinuclear Golgi phosphatidylinositol 4-phosphate hydrolysis pathway. Mol. Biol. Cell 26, 1188–1198 (2015).

177 Travers, J. G. et al. Pharmacological and activated fibroblast targeting of Gβγ-GRK2 after myocardial ischemia attenuates heart failure progression. J. Am. Coll. Cardiol. 70, 958–971 (2017).

178 Wells, J. A. & McClendon, C. L. Reaching for high-hanging fruit in drug discovery at protein–protein interfaces. Nature 450, 1001 (2007).

179 Garcia-Olivares, J. et al. Gβγ subunit activation promotes dopamine efflux through the dopamine transporter. Mol. Psychiatry 22, 1673–1679 (2017).

180 Yost, E. A., Hynes, T. R., Hartle, C. M., Ott, B. J. & Berlot, C. H. Inhibition of G-protein βγ signaling enhances T cell receptor-stimulated interleukin 2 transcription in CD4+ T helper cells. PLOS One 10, e0116575 (2015).

181 Ouellaa-Benslama, R. et al. Identification of a GαGβγ, AKT and PKCα signalome associated with invasive growth in two genetic models of human breast cancer cell epithelial-to-mesenchymal transition. Int. J. Oncol. 41, 189–200 (2012).

182 Meens, M. J. et al. G-Protein βγ subunits in vasorelaxing and anti-endothelinergic effects of calcitonin gene-related peptide. Br. J. Pharmacol. 166, 297–308 (2012).

183 Irannejad, R. & Wedegaertner, P. B. Regulation of constitutive cargo transport from the trans-Golgi network to plasma membrane by Golgi-localized G protein betagamma subunits. J. Biol. Chem. 285, 32393–32404 (2010).

184 Kirui, J. K. et al. Gβγ signaling promotes breast cancer cell migration and invasion. J. Pharmacol. Exp. Ther. 333, 393–403 (2010).

185 Tang, X. et al. A critical role of Gbetagamma in tumorigenesis and metastasis of breast cancer. J. Biol. Chem. 286, 13244–13254 (2011).

186 Paudyal, P., Xie, Q., Vaddi, P. K., Henry, M. D. & Chen, S. Inhibiting G protein βγ signaling blocks prostate cancer progression and enhances the efficacy of paclitaxel. Oncotarget 8, 36067–36081 (2017).

187 Purcell, R. H., Toro, C., Gahl, W. A. & Hall, R. A. Hum. Mutat. 38, 1751–1760 (2017).

188 Arensdorf, A. M. et al. Sonic Hedgehog activates phospholipase A2 to enhance smoothened ciliary translocation. Cell Rep. 19, 2074–2087 (2017).

189 Gont, A., Daneshmand, M., Woulfe, J., Lavictoire, S. J. & Lorimer, I. A. PREX1 integrates G protein-coupled receptor and phosphoinositide 3-kinase signaling to promote glioblastoma invasion. Oncotarget 8, 8559–8573 (2017).