Heterotrimeric G-proteins are ubiquitous molecular switches that transduce extracellular signals into intracellular cascades of biochemical reactions to steer cellular responses (Gilman, 1987). The ON/OFF state of these switches is defined by their nucleotide binding status—GDP-bound G-proteins are OFF, whereas GTP-bound G-proteins are ON. The switching between states is primarily determined by two biochemical events. The first one is nucleotide exchange of GDP for GTP, which determines the rate of activation, and the second one is hydrolysis of GTP to GDP, which determines the rate of inactivation. While these two reactions can be carried out spontaneously by G-proteins, they are tightly regulated by the enzymatic activity of other proteins in the cellular context. The G-protein regulatory mechanism controlled by G-protein-coupled receptors (GPCRs) is the best characterized to date (Pierce et al., 2002; Weis and Kobilka, 2018). GPCRs in their active conformation are guanine nucleotide exchange factors (GEFs) that catalyze nucleotide exchange on the Gα subunit of Gα-Gβγ heterotrimers. Upon GTP loading, Gβγ disengages from Gα, leading to the formation of two active signaling species, that is, Gα-GTP and free Gβγ, that can engage their respective intracellular effectors to initiate signaling cascades. In addition to GPCR-mediated activation, G-proteins are regulated by many cytoplasmic proteins with different enzymatic activities (Sato et al., 2006; Siderovski and Willard, 2005). These include proteins that accelerate the intrinsic rate of nucleotide hydrolysis by G-proteins (GTPase accelerating proteins [GAPs]) (Ross and Wilkie, 2000), proteins that block nucleotide exchange (guanine nucleotide dissociation inhibitors [GDIs]) (Blumer et al., 2012), or even GEFs that are not GPCRs (Cismowski et al., 2000; DiGiacomo et al., 2018; Tall, 2013; Figure 1A).

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From a historical perspective, the heterotrimeric G-protein research field has relied heavily on reductionist approaches. Breaking down complex systems into defined biochemical entities that can be reconstituted and rigorously characterized in vitro has been a powerful approach to subsequently understand the mechanisms operating in cells. This approach has proven particularly successful for understanding GPCR-mediated regulation of G-proteins, a mechanism with broad biomedical implications that has been extensively characterized at the molecular level and that represents one of the most widely pursued pharmacological targets (Sriram and Insel, 2018). Similar biochemical reductionist approaches have also been useful to define the enzymatic activities of cytoplasmic regulators of G-proteins like GAPs, GDIs, and non-receptor GEFs, but have left several open questions about how these regulatory mechanisms operate in cells. For example, if G-protein signaling activity in cells is defined by the formation of Gα-GTP or free Gβγ, as each one of these species is sufficient to trigger downstream signaling, it is unclear how some GDIs and non-receptor GEFs affect G-protein signaling as a whole. For example, GDIs of the GoLoco motif family have paradoxical effects on G-proteins. On one hand, they bind to inactive, GDP-loaded Gα subunits to prevent the formation of Gα-GTP in vitro (De Vries et al., 2000; Kimple et al., 2002; Natochin et al., 2000). On the other hand, they also prevent the association of Gβγ with Gα-GDP (Ghosh et al., 2003; Webb et al., 2005). In fact, GoLoco motif GDIs were originally identified in a genetic screen for ‘activators of G-protein signaling (AGS)’ in yeast that relied on signaling readouts activated by free Gβγ (Cismowski et al., 1999). The situation with several non-receptor GEFs is similarly unclear. A group of non-receptor GEFs characterized by a Gα-binding-and-activating (GBA) motif has been shown not only to promote nucleotide exchange in vitro, but also to physically displace Gβγ from GDP-bound Gα (Aznar et al., 2015; Garcia-Marcos et al., 2009; Maziarz et al., 2018), raising the question of what is the relative contribution of each mechanism, Gα-GTP formation and Gβγ release, to downstream signaling in cells. Other non-receptor GEFs like Ric-8 proteins promote nucleotide exchange on monomeric Gα but not on Gα-Gβγ heterotrimers (Tall et al., 2003), which has led to the contentious speculation that Ric-8 proteins might not regulate directly G-protein activity in cells, but rather work primarily as folding chaperones (Chan et al., 2013; Tall et al., 2013). Yet another non-receptor GEF, AGS1 (a.k.a. Dexras1), has been shown to activate nucleotide exchange on both monomeric Gα or Gα-Gβγ heterotrimers in vitro (Cismowski et al., 2000), but how it influences G-protein signaling in cells is not understood well.

Despite these gaps in mechanistic knowledge, activation of G-proteins by cytoplasmic proteins has been proven to impact various cellular processes and its dysregulation to be linked to different pathologies. This has been made particularly evident for non-receptor GEFs of the GBA family, for which the G-protein regulatory activity can be specifically disabled through mutagenesis (Coleman et al., 2016; de Opakua et al., 2017; Garcia-Marcos et al., 2009; Garcia-Marcos et al., 2012; Maziarz et al., 2018). This surgical approach has been leveraged to establish that G-protein regulation by GBA proteins, like GIV(a.k.a. Girdin) and DAPLE, is involved in normal physiological processes (e.g., formation of the neural tube during embryonic development), or in disease (e.g., cancer metastasis or birth defects) (Aznar et al., 2015; Garcia-Marcos et al., 2015; Ghosh, 2015; Leyme et al., 2017; Marivin et al., 2019). Overall, the involvement of these cytoplasmic regulators in (patho)physiological processes make imperative a more detailed understanding of their mechanisms of action in cells.

The slow progress in understanding G-protein regulation by cytoplasmic proteins compared to their regulation by GPCRs could be due to two experimental issues. One is that the G-protein regulatory function of many GPCRs can be stimulated (and inhibited) with high precision by the simple addition of extracellular ligands, whereas this is not possible for cytoplasmic regulators of G-proteins. The second experimental issue is that approaches to directly detect G-protein activity in cells have typically relied on the detection of Gα-Gβγ dissociation instead of detecting Gα-GTP. While these two signaling events correlate well in the process of GPCR-mediated G-protein activation, this is not necessarily the case for many cytoplasmic regulators of G-proteins, making evident the need for detecting both free Gβγ and Gα-GTP formation to understand how they operate in cells. Here, a cell-based approach was developed and implemented to overcome current limitations to study the mechanisms of G-protein regulation by cytoplasmic proteins. For this, the action of individual cytoplasmic regulators on their cognate G-proteins was triggered with an exogenous small molecule, and the responses evaluated with real-time biosensors for both Gα-GTP and free Gβγ. The resulting experimental system allows to precisely modulate and detect G-protein activity, similar to what can be done biochemically with purified proteins in vitro, but in the more physiologically relevant environment of the cell. This newly developed approach allowed to pinpoint key differences between the modes of G-protein signaling regulation in cells exerted by various proteins with GEF activity, including both receptor (i.e., GPCRs) and non-receptor proteins.

This work introduces an experimental paradigm to investigate the regulation of G-proteins in cells with high precision and for a wide range of regulators beyond GPCRs. The experimental framework presented here is poised to complement other approaches that have traditionally been used to study G-protein signaling, like reductionist biochemical assays with purified proteins and genetic manipulations for more complex systems like cultured cells or whole organisms. Experiments with purified proteins allow gaining detailed insights into molecular mechanisms but can suffer from lack of physiological context, whereas genetic manipulations can reveal functionality in more physiological contexts but can fall short in delivering mechanistic detail at the molecular level. The approach presented here leverages some advantages of reductionism, in that it permits studying isolated G-protein biochemical events like nucleotide binding status and subunit dissociation upon modulation by specific protein regulators, but within the more physiological context of the cell. In this sense, it could be considered a ‘cell-based reductionist’ approach. The benefits of this are demonstrated here by the discovery that G-protein signaling modes in cells can differ greatly among proteins with similar G-protein regulatory functions in vitro, including differences between receptor and non-receptor GEFs or even among different non-receptor GEFs (see further discussion below). Also, the interplay between GPCRs and different cytoplasmic regulators in controlling G-protein activity in cells was further clarified. Like with biochemical reductionist approaches, a limitation of the studies presented here and the approach in general if considered alone is that they still rely on non-native conditions, like the use of overexpression, protein fragment fusions, or artificial means to alter the subcellular localization of proteins. It is in the context of complementarity with other well-established approaches like in vitro biochemistry and genetics that this limitation is outweighed by the additional mechanistic insights it can provide.

The in-depth characterization of the mechanisms by which non-GPCR proteins of the GBA family trigger G-protein signaling in cells exemplifies well the additional insights that can be gained through the approach presented in this work. On one hand, it was known from in vitro biochemical experiments that GBA motifs have GEF activity on Gαi proteins and that they also promote the dissociation of Gβγ from Gαi-GDP (Aznar et al., 2015; Coleman et al., 2016; de Opakua et al., 2017; Garcia-Marcos et al., 2011a; Garcia-Marcos et al., 2010; Garcia-Marcos et al., 2009; Garcia-Marcos et al., 2012; Garcia-Marcos et al., 2011c; Marivin et al., 2019; Maziarz et al., 2018). On the other hand, genetic approaches had also established that the GBA motif of some proteins, like GIV and DAPLE, controls G-protein signaling in cells or whole organisms (Aznar et al., 2015; Garcia-Marcos et al., 2011a; Garcia-Marcos et al., 2010; Garcia-Marcos et al., 2009; Garcia-Marcos et al., 2012; Landin Malt et al., 2020; Leyme et al., 2016; Leyme et al., 2017; Leyme et al., 2015; Lin et al., 2014; Lo et al., 2015; Lopez-Sanchez et al., 2014; Ma et al., 2015b; Marivin et al., 2019; Midde et al., 2015; Sasaki et al., 2015). Thus, an unresolved question so far had been the relative contribution of the two potentially overlapping mechanism of G-protein activation-mediated GBA motifs, that is, generation of Gαi-GTP and formation of free Gβγ. A key conclusion of the studies presented here (Figure 2, Figure 4) is that GIV, and most likely other GBA proteins, activates G-protein signaling in cells primarily through the formation of free Gβγ rather than through the formation of Gαi-GTP, despite its GEF activity in vitro. GIV enhances nucleotide exchange in vitro approximately 2.5- to 3-fold (Garcia-Marcos et al., 2011a; Garcia-Marcos et al., 2010; Garcia-Marcos et al., 2009; Garcia-Marcos et al., 2012), which is very similar to the approximately 3-fold enhancement mediated by AGS1 under similar conditions in vitro (Cismowski et al., 2000). In contrast, the studies described here reveal that AGS1 efficiently triggers the formation of Gαi-GTP in cells, whereas GIV does not (Figure 2). Similarly, the enhancement of nucleotide exchange on Gi by a GPCR (e.g. α2 adrenergic receptor) in vitro is 3- to 6-fold (Cerione et al., 1986; Kurose et al., 1991), which is stronger than that of GIV or AGS1 but still within the same order of magnitude. Thus, the results presented here indicate that the relative GEF activity in vitro across different G-protein activators does not correlate well with their ability to generate Gα-GTP in cells. These observations not only provide mechanistic insights into G-protein activation by GBA proteins that were not evident from experiments using other approaches, but also highlight the importance of elucidating the molecular mechanisms of G-protein regulation in a cellular context.

The conclusion that GIV promotes G-protein signaling in cells primarily through the formation of free Gβγ prompts the re-evaluation of previous signaling studies in cells. In fact, the vast majority of signaling readouts regulated by the GBA motif of GIV (or DAPLE) have been shown or are known to be controlled by Gβγ rather than by Gαi-GTP. This includes activation of the PI3K-Akt axis (Aznar et al., 2015; Bhandari et al., 2015; Garcia-Marcos et al., 2010; Garcia-Marcos et al., 2009; Garcia-Marcos et al., 2012; Gupta et al., 2016; Leyme et al., 2016; Leyme et al., 2017; Leyme et al., 2015; Ma et al., 2015a), p114RhoGEF-mediated activation of RhoA (Marivin et al., 2019), or Rac1 activation (Aznar et al., 2015). Although GBA-mediated inhibition of adenylyl cyclase (Aznar et al., 2015; Maziarz et al., 2018; Midde et al., 2015) could have been explained as an effect of Gαi-GTP, evidence presented here (Figure 3) strongly suggests that this is indeed mediated by Gβγ as well. Thus, although previous evidence has identified detectable levels of Gαi-GTP in cells upon GIV action (Lin et al., 2014; Lopez-Sanchez et al., 2014; Midde et al., 2015), these are probably much lower than those achieved upon activation of a GPCR and insufficient to drive robust signaling directly. Thus, the functional role of the weak GEF activity of proteins with a GBA motif remains to be elucidated, whereas efficient release of Gβγ subunits seems to be main mechanism by which this type of G-protein activator promotes signaling in cells.

Results shown here (Figure 5, Figure 6) also indicate that proteins with a GBA motif differ mechanistically from other regulators that promote the dissociation of Gβγ from G-protein heterotrimers, like GDIs that contain a GoLoco motif. This in turn might be important for the interplay between GBA proteins and GPCRs in regulating G-proteins. The data suggest that activation of G-proteins by GIV, which in many cases is triggered by surface receptors different from GPCRs (Garcia-Marcos et al., 2015; Leyme et al., 2015; Lopez-Sanchez et al., 2014), might operate without opposing efficient GPCR-mediated G-protein activation. In contrast, GDIs with a GoLoco motif efficiently suppress GPCR-mediated activation of G-proteins. Interestingly, the artificial system implemented here to recruit GIV to membranes mimics the recruitment of native GIV from the cytosol to membranes when it binds non-GPCR surface receptors upon ligand stimulation (Ghosh et al., 2010; Leyme et al., 2015), which might be the mechanism by which GIV action on G-proteins is controlled under native conditions (Parag-Sharma et al., 2016). Nevertheless, the role of GIV in the interplay between GPCR and non-GPCR receptors in G-protein regulation needs to be characterized in more detail in the future.

Beyond insights gained in the understanding of GBA-mediated mechanisms of G-protein signaling regulation, the present study also provides other useful information. For example, it was shown that pertussis toxin is not a generic inhibitor of all mechanisms of Gi activation or a specific inhibitor of GPCR-mediated activation of Gi proteins (Figure 2—figure supplement 1), which warranties caution in the interpretation of past and future experiments with this widely used reagent. In addition, the chemogenetic tools presented here could be easily adapted for other applications, like synthetic biology approaches to turn different modes of G-protein signaling ON and OFF.

In summary, the combination of chemogenetics and optical biosensors presented here has demonstrated the potential to become an experimental paradigm to expand how we study and understand signal transduction mechanisms mediated by heterotrimeric G-proteins.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Mikel Garcia-Marcos

   Department of Biochemistry, Boston University School of Medicine, Boston, United States

   Contribution

   Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Writing - original draft, Project administration

   For correspondence

   mgm1@bu.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9513-4826

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