Heterotrimeric G proteins transduce a vast number of important physiological signals (Gilman, 1987), most often in response to activation by G-protein-coupled receptors (GPCRs; Pierce et al., 2002). Canonical G protein signaling occurs when a cell surface GPCR is activated by an extracellular ligand, which in turn promotes activation of plasma membrane G protein heterotrimers and downstream effectors. Recently, it has become clear that GPCRs can also signal from intracellular compartments (Calebiro et al., 2010; Eichel and von Zastrow, 2018), most notably endosomes and the Golgi apparatus (Calebiro et al., 2009; Ferrandon et al., 2009; Irannejad et al., 2017; Irannejad et al., 2013; Mullershausen et al., 2009). Signaling from endosomes is often a continuation of signaling that starts at the plasma membrane and persists as (or resumes after) active receptors are endocytosed (Tsvetanova et al., 2015). Much is known about the machinery responsible for GPCR internalization, and also about the trafficking itineraries of specific receptors after endocytosis. Some receptors are efficiently sorted for recycling and are returned to the plasma membrane, whereas other receptors are rapidly degraded (Hanyaloglu and von Zastrow, 2008). It is also known that at least one isoform of the G protein effector adenylyl cyclase is actively internalized (Lazar et al., 2020).

In contrast, much less is known about how G protein heterotrimers traffic from the plasma membrane through intracellular compartments (Wedegaertner, 2012). It is not known how efficiently heterotrimers are loaded onto endocytic vesicles at the plasma membrane, how receptor activation might change this process, or what the fate of G proteins might be after endocytosis. Activation at the plasma membrane promotes heterotrimer dissociation, and the resulting loss of membrane avidity allows Gβγ dimers and some Gα subunits (most notably Gα_s) to translocate through the cytosol to sample intracellular membranes (Akgoz et al., 2004; Hynes et al., 2004; Ransnäs et al., 1989; Slepak and Hurley, 2008; Wedegaertner et al., 1996). However, these processes reverse quickly when activation ceases (Akgoz et al., 2004; Martin and Lambert, 2016), meaning that activation-dependent translocation of free Gα subunits and Gβγ dimers would be an inefficient mechanism to deliver inactive heterotrimers to intracellular membranes. G proteins have been detected on the surface of endosomes and other intracellular compartments using a variety of approaches (Hewavitharana and Wedegaertner, 2012; Irannejad et al., 2013; Scarselli and Donaldson, 2009; Wedegaertner, 2012). However, there has been no quantitative comparison of G protein distribution across identified subcellular compartments. Studies of endogenous G proteins are limited by the availability of well-validated antibodies suitable for immunostaining, and overexpression of tagged G protein subunits may lead to aberrant localization.

Here, we study the subcellular distribution of endogenous heterotrimeric G proteins in cultured cells using CRISPR-mediated gene editing, confocal imaging, and bioluminescence resonance energy transfer (BRET). We find that G proteins are abundant on membrane compartments that are functionally continuous with the plasma membrane, including early, late, and recycling endosomes. However, heterotrimer density on endocytic membranes is lower than on the plasma membrane, suggesting that G protein endocytosis is inefficient. Endocytic trafficking of G proteins is not regulated by GPCRs. Our findings are likely to have implications for GPCR signaling from endosomes, as internalized receptors are concentrated in G-protein-deficient compartments.

To study the localization of endogenous G proteins, we used gene editing to attach small peptide tags to the amino terminus of Gβ₁ subunits (GNB1) in HEK 293 cells. We chose this subunit because it is the most abundant Gβ subunit in this cell type (Cho et al., 2022), it can associate with any type of Gα or Gγ subunit (Hillenbrand et al., 2015), and it can be labeled at this position without disrupting heterotrimer formation or function. For bioluminescence experiments we added the HiBit tag (Schwinn et al., 2018) and isolated clonal ‘HiBit-β₁’ cell lines. An advantage of this approach over adding a full-length Nanoluc luciferase is that it requires coexpression of LgBit to produce a complemented luciferase. This limits luminescence to cotransfected cells and thus eliminates background from untransfected cells. For imaging experiments we added a tandem tag that included the 11^th beta strand of mNeonGreen2 (mNG2(11); Feng et al., 2017) and HiBit in cells constitutively expressing mNG2(1–10) and isolated ‘mNG-β₁’ cell lines (Figure 1A). Amplicon sequencing verified that cell lines had correctly edited GNB1 genes and SDS-PAGE revealed single proteins with apparent molecular weights consistent with edited Gβ₁ subunits (Figure 1B). BRET assays demonstrated that tagged subunits in HiBit-β₁ and mNG-β₁ cell lines formed functional heterotrimers with endogenous Gα and Gγ subunits. For example, we observed agonist-induced BRET between the D2 dopamine receptor and mNG-β₁, an interaction that requires association with endogenous Gα subunits (Figure 1C). Similarly, we observed BRET between HiBit-β₁ and the free Gβγ sensor memGRKct-Venus after activation of receptors that couple G_i/o, G_s, and G_q heterotrimers, indicating that HiBit-β₁ associated with endogenous Gα subunits from these three families (Figure 1D). We also found that cyclic AMP accumulation in response to stimulation of endogenous β adrenergic receptors was similar in edited cell lines and their unedited parent lines (Figure 1—figure supplement 1). Endogenous Gα and Gβ subunits are expressed at approximately a 1:1 ratio, and Gβ subunits are tightly associated with Gγ and inactive Gα subunits (Cho et al., 2022; Gilman, 1987; Krumins and Gilman, 2006). Moreover, proteins that bind to free Gβγ dimers are found in the cytosol of unstimulated HEK 293 cells, suggesting at most only a small population of free Gβγ in these cells (Barak et al., 1999). Therefore, we assume that almost all mNG-β₁ and HiBit-β₁ subunits in unstimulated cells are part of heterotrimers.

Figure 1 with 1 supplement see all

Download asset Open asset

Figure 1—source data 1

PDF file containing original HiBit blot shown in panel B, indicating the relevant bands.

https://cdn.elifesciences.org/articles/97033/elife-97033-fig1-data1-v1.pdf

Download elife-97033-fig1-data1-v1.pdf

Figure 1—source data 2

Original files for HiBit blot shown in panel B.

https://cdn.elifesciences.org/articles/97033/elife-97033-fig1-data2-v1.zip

Download elife-97033-fig1-data2-v1.zip

Figure 1—source data 3

Numerical data for traces shown in panels C and D.

https://cdn.elifesciences.org/articles/97033/elife-97033-fig1-data3-v1.xlsx

Download elife-97033-fig1-data3-v1.xlsx

Endogenous G proteins primarily associate with the plasma membrane and endolysosomes

Confocal imaging of mNG-β₁ cells revealed the expected bright fluorescence at the plasma membrane. Most cells also contained pleiomorphic intracellular structures and dim cytosolic fluorescence that was sufficient to suggest relative exclusion of mNG-β₁ from the nucleus. Especially notable were clusters of large vesicular structures located at the cell periphery which were later identified as lysosomes (Figure 2A; see below). Large intracellular organelles such as the endoplasmic reticulum, mitochondria, and Golgi apparatus were not evident. Unsurprisingly, our images are quite similar to those made as part of previous study that labeled Gβ₁ subunits with mNG2 (Cho et al., 2022).

Figure 2 with 3 supplements see all

Download asset Open asset

To identify the intracellular membrane compartments with mNG-β₁ fluorescence, we coexpressed a series of organelle markers tagged with red fluorescent proteins. Markers of the endoplasmic reticulum, mitochondria and medial-trans Golgi apparatus indicated that these large compartments were virtually devoid of mNG-β₁ fluorescence (Figure 2B–D, Figure 2—figure supplements 1–3). In some cells, an indistinct region of mNG-β₁ fluorescence was interleaved with leaflets of the Golgi apparatus, but line profiles suggested that this was a distinct structure (Figure 2D, Figure 2—figure supplement 3), most likely the perinuclear recycling compartment (see below).

In contrast, mNG-β₁ clearly colocalized with the marker FYVE, which binds to phosphatidylinositol-3-phosphate (PI3P) on the surface of endosomes (Figure 3A and B, Figure 3—figure supplement 1). However, mNG-β₁ fluorescence was not detected on every FYVE-positive vesicle and when present was much less intense than fluorescence of adjacent segments of the plasma membrane (Figure 3B). The median signal-to-background ratio for FYVE-positive structures was less than one-fourth that of the plasma membrane (Figure 3C). FYVE domains primarily localize to early endosomes (Hammond and Balla, 2015), so it was not surprising that similar colocalization of mNG-β₁ was observed with the early endosome marker rab5a. As was the case with FYVE, mNG-β₁ was detectable in some rab5a-positive vesicles but not others and was not as intense as the nearby plasma membrane (Figure 3A and C, Figure 3—figure supplement 2). In order to determine the fate of G proteins after endocytosis, we then examined mNG-β₁ colocalization with markers of recycling and late endosomes (Stenmark, 2009). Dim mNG-β₁ fluorescence was detected on indistinct rab11a-positive structures clustered diffusely in the vicinity of the nucleus (Figure 3A, Figure 3—figure supplement 3), which we presumptively identified as the perinuclear recycling compartment (PNRC). Similarly, mNG-β₁ colocalized extensively with vesicles labeled with rab7a, a marker of late endosomes (Figure 3A, Figure 3—figure supplement 4). Notably, mNG-β₁ fluorescence was more intense on rab7a-positive late endosomes than on FYVE- or rab5a-positive early endosomes (Figure 3C). The presence of mNG-β₁ on late endosomes suggested that some G proteins may be degraded by lysosomes. Accordingly, mNG-β₁ strongly colocalized with lysosomes marked with LysoView 633 (Figure 3A, Figure 3—figure supplement 5), long-term incubation with fluorescent dextran (Figure 3—figure supplement 5), or the lysosome marker LAMP1 (Figure 3—figure supplement 6). While some mNG fluorescence was detected in the lumen of lysosomes, much of the signal remained on the cytosolic surface of these structures, and in many instances the intensity of mNG-β₁ fluorescence on lysosomes was similar to that of the nearby plasma membrane (Figure 3A). We also detected robust mNG-β₁ signals on structures labeled with TGNP (Figure 3—figure supplement 7), a marker of the trans-Golgi network. These imaging results suggest that G proteins are likely to undergo endocytosis and enter both recycling and degradative pathways and may become more concentrated as late endosomes mature.

Figure 3 with 7 supplements see all

Download asset Open asset

Figure 3—source data 1

Numerical data for line profiles (panel B), signal/background ratios (panel C) and bystander BRET (panel D).

https://cdn.elifesciences.org/articles/97033/elife-97033-fig3-data1-v1.xlsx

Download elife-97033-fig3-data1-v1.xlsx

As an alternative approach, we performed bystander BRET experiments to map the subcellular localization of endogenous HiBit-β₁. This approach provides an unbiased index of membrane protein colocalization from large populations of cells and has the additional advantage of very high sensitivity (Lan et al., 2012). We expressed LgBit and a series of inert Venus-tagged membrane markers in HiBit-β₁ cells and observed large bystander signals at the plasma membrane, smaller bystander signals at endosomes, and very small bystander signals at the endoplasmic reticulum and mitochondria (Figure 3D). Although bystander BRET signals cannot be directly compared between different compartments, these results are generally consistent with what we observed using confocal imaging and confirm the presence of G proteins on multiple endosomal compartments.

Constitutive G protein endocytosis is inefficient

That mNG-β₁ fluorescence was less intense on endosomes than the plasma membrane suggested that G protein density may be lower on the surface of endosomes than on the plasma membrane. However, differences in fluorescence intensity could also be due to differences in the amount of membrane surface area sampled in the imaging volume. Likewise, differences in bystander BRET between compartments could be due to differences in several factors, including expression and efficiency of compartment-specific BRET acceptors. Therefore, we devised a co-labeling protocol that allowed us to compare mNG-β₁ fluorescence to the amount of newly internalized membrane imaged at endocytic vesicles, and to simultaneously make the same measurements at the plasma membrane. To stain both the plasma membrane as well as newly formed endocytic vesicle membrane, we exposed live cells to the styryl dye FM4-64, which rapidly and reversibly partitions into (but does not cross) membranes and is only fluorescent in a hydrophobic environment (Betz et al., 1996). When cells are exposed to FM4-64 at physiological temperatures the plasma membrane is stained immediately, and this is followed over the course of several minutes by the appearance of intracellular vesicles that have trapped the dye (Figure 4A, Figure 4—figure supplement 1). As an orthogonal approach we stained cells with CellMask Deep Red, a lipophilic dye that permanently stains the plasma membrane and therefore is incorporated into endocytic vesicles. Both dyes are expected to produce fluorescence signals proportional to the surface area of the membrane sampled by the imaging method, allowing us to normalize the fluorescence of individual vesicles to the nearby plasma membrane. We reasoned that if G proteins are passively incorporated into endocytic vesicles without any enrichment or exclusion, then mNG-β₁ fluorescence in each vesicle should have the same intensity relative to the plasma membrane as lipophilic dyes. After staining cells and allowing 15 min for constitutive endocytosis, we found that FM4-64 and CellMask dyes reported similar amounts of membrane surface area in endocytic vesicles (Figure 4A and B); in both cases, peak vesicle intensity was on average similar to the intensity of the plasma membrane (Figure 4C). In contrast, peak mNG-β₁ fluorescence on the same endocytic vesicles was much less intense than the plasma membrane (Figure 4A–C). There was considerable variability between individual vesicles, such that some vesicles contained no detectable mNG-β₁ fluorescence (Figure 4A, Figure 4—figure supplement 1). Using FM4-64 fluorescence as a standard for membrane surface area, we calculated that mNG-β₁ density on FM4-64-positive vesicles was 20 ± 8% (mean ± 95% CI; n=45) of the nearby plasma membrane. This result confirms that heterotrimeric G proteins are present on newly internalized membrane but also suggests that G proteins are partially excluded from endocytic vesicles. To test if other peripheral membrane proteins are similarly depleted from endocytic vesicles, we performed analogous experiments by overexpressing mNG bearing the C-terminal membrane anchor of HRas (mNG-HRas ct). We found that mNG-HRas ct was also less abundant on FM4-64-positive endocytic vesicles than on the plasma membrane, although not to the same extent as mNG-β1 (Figure 4—figure supplement 2); mNG-HRas ct density on FM4-64-positive vesicles was 64 ± 17% (mean ± 95% CI; n=78) of the nearby plasma membrane.

Figure 4 with 2 supplements see all

Download asset Open asset

Figure 4—source data 1

Numerical data for line profiles (panel C).

https://cdn.elifesciences.org/articles/97033/elife-97033-fig4-data1-v1.xlsx

Download elife-97033-fig4-data1-v1.xlsx

Receptor activation does not change G protein endocytosis

The above results suggested that constitutive endocytosis of heterotrimeric G proteins is inefficient. However, it is possible that GPCR and G protein activation could change how G proteins are loaded onto endocytic vesicles. To test this possibility, we performed similar imaging experiments with mNG-β₁ cells transfected with SNAP-tagged β₂ adrenergic receptors (SNAPf-β₂AR). This receptor is often used as a model of activity-dependent GPCR internalization (Benovic et al., 1988; von Zastrow and Kobilka, 1992) and has been shown to activate G proteins on endosomes (Bowman et al., 2016; Irannejad et al., 2013). We labeled SNAPf-β₂AR with a membrane-impermeant SNAP ligand (AF 647) at room temperature to prevent constitutive endocytosis, then incubated cells with FM4-64 and the agonist isoproterenol for 15 min at physiological temperature to promote receptor endocytosis. Confocal imaging after agonist washout revealed numerous intracellular vesicles with intense AF 647 fluorescence, consistent with robust receptor internalization (Figure 5A). Normalization and comparison to FM4-64 fluorescence indicated that SNAPf-β₂AR was enriched approximately threefold on endocytic vesicles compared to the nearby plasma membrane (Figure 5B and C), consistent with active recruitment of active receptors to clathrin-coated pits and endocytic vesicles. In contrast, mNG-β₁ fluorescence in the same vesicles was again lower than expected given the amount of membrane imaged in each vesicle (Figure 5B and C). Once again there was considerable variability between individual endocytic vesicles (Figure 5A and D). Using FM4-64 fluorescence as a standard for membrane surface area, we calculated that mNG-β₁ density on receptor-containing vesicles was 28 ± 8% (mean ±95% CI; n=91) of the nearby plasma membrane. Although this density was higher than that calculated for vesicles formed by constitutive endocytosis, the difference did not reach significance (Figure 5D). These results demonstrate that activation-dependent internalization of β₂ adrenergic receptors does not significantly promote or prevent loading of G proteins onto endocytic vesicles.

Figure 5 with 2 supplements see all

Download asset Open asset

Figure 5—source data 1

Numerical data for line profiles (panel C), intensity ratios (panel D) and bystander BRET (panel E).

https://cdn.elifesciences.org/articles/97033/elife-97033-fig5-data1-v1.xlsx

Download elife-97033-fig5-data1-v1.xlsx

These findings suggested that β₂ adrenergic receptor activation should have no impact on the abundance of heterotrimeric G proteins on endosomes. To test this idea for this and other receptors, we performed bystander BRET experiments with HiBit-β₁ cells transiently expressing β₂ adrenergic, D2 dopamine or M3 muscarinic receptors to activate G_s, G_i/o, and G_q/11 heterotrimers, respectively. With overexpressed G proteins free Gβγ dimers translocate from the plasma membrane and sample intracellular membrane compartments when dissociated from Gα subunits. Consistent with this, we observed small decreases in bystander BRET at the plasma membrane and small increases in bystander BRET at intracellular compartments during activation of GPCRs, suggesting that endogenous Gβγ subunits undergo similar translocation (Figure 5—figure supplement 1). Notably, these changes occurred at room temperature, suggesting that endocytosis was not involved, and developed over the course of minutes. The latter observation and the small magnitude of agonist-induced changes are both consistent with expression of primarily slowly-translocating endogenous Gγ subtypes in HEK 293 cells. Moreover, as shown previously for overexpressed Gβγ, the changes we observed with endogenous Gβγ were readily reversible (Figure 5—figure supplement 1), suggesting that most heterotrimers reassemble at the plasma membrane after activation ceases. In order to assess changes in heterotrimer trafficking to endosomes without interference from Gβγ translocation we incubated cells with agonist under conditions permissive for vesicular trafficking, then washed with antagonist to inactivate receptors and allow heterotrimers to reassemble prior to measuring BRET. Under these conditions no significant changes in bystander BRET were observed at any endosome compartment after 30 min of receptor stimulation (Figure 5E). Similar results were obtained when stimulation was maintained for 15 min or 5 min (Figure 5—figure supplement 2), although small increases were apparent in a few instances. Taken together these results indicate that activation of receptors and G proteins does not substantially change heterotrimer abundance on the surface of endosomes.

While the mechanisms involved in the biosynthesis, chaperoning and trafficking of nascent G protein heterotrimers are fairly well understood (Gabay et al., 2011; Marrari et al., 2007; Wedegaertner, 2012), the mechanisms that regulate the subcellular distribution of heterotrimers after delivery to the plasma membrane have not been studied as extensively. Here, we show that constitutive and activity-dependent endocytosis of G proteins is inefficient. Avoidance of endocytosis is likely to be important for maintaining a high density of heterotrimers at the plasma membrane, where much important signaling takes place. On the other hand, this limits the abundance of G proteins on the surface of endosomes. At present we can only speculate regarding the mechanism that limits G protein density on endocytic vesicles. Many endocytosis mechanisms, including clathrin-mediated endocytosis, rely on bulky coat proteins and adapters to induce membrane curvature and recruit cargo (Doherty and McMahon, 2009). One possibility is that heterotrimeric G proteins are simply excluded from nascent endocytic vesicles by steric occlusion. While large extracellular domains are known to impede endocytosis of membrane proteins (DeGroot et al., 2018), a similar relationship has not been demonstrated for intracellular domains. Our finding that the small mNG-HRas ct probe is loaded onto endocytic vesicles more efficiently than mNG-β₁-containing heterotrimers is consistent with this idea. It is noteworthy that the monomeric G proteins HRas and NRas are also less abundant on endosomes than the plasma membrane, and therefore are separated from internalized growth factor receptors (Pinilla-Macua et al., 2016; Surve et al., 2021).

Some studies using overexpressed G protein subunits have suggested that a large pool of G proteins is located on intracellular membranes, including the Golgi apparatus (Chisari et al., 2007; Saini et al., 2007; Tsutsumi et al., 2009), whereas others have indicated a distribution that is dominated by the plasma membrane (Crouthamel et al., 2008; Evanko et al., 2000; Marrari et al., 2007; Takida and Wedegaertner, 2003). A likely factor contributing to these discrepant results is the stoichiometry of overexpressed subunits, as neither Gα nor Gβγ traffic appropriately to the plasma membrane as free subunits (Wedegaertner, 2012). Our gene-editing approach presumably maintains the native subunit stoichiometry, providing a more accurate representation of native G protein distribution. Our results show that endogenous G proteins are primarily located on the plasma membrane and are present on internal membranes at substantially lower levels. We identify the specific intracellular compartments where G proteins are found and show the relative abundance of G proteins on each compartment. Nascent heterotrimers are likely formed and lipid modified on the endoplasmic reticulum and Golgi apparatus (Wedegaertner, 2012), yet few heterotrimers can be found on these compartments at any given moment, consistent with a relatively slow rate of turnover compared to forward trafficking during biosynthesis (Gabay et al., 2011). Notably, when Gβγ dimers are expressed alone they accumulate on the endoplasmic reticulum (Michaelson et al., 2002; Takida and Wedegaertner, 2003). That we detect almost no endogenous Gβγ on the endoplasmic reticulum supports our conclusion that the large majority of Gβγ in unstimulated HEK 293 cells is associated with Gα. Likewise, we found that few heterotrimers are associated with mitochondria, despite the fact that previous studies have demonstrated functional roles for G proteins on these organelles (Hewavitharana and Wedegaertner, 2012). Our results suggest that GPCR signaling from intracellular compartments will generally have to be transduced by a lower density of G proteins. Consistent with these findings, a recent large-scale study assessing protein abundance on organelles in HEK 293 cells found that Gβ₁ is enriched on the plasma membrane and lysosomes but is not significantly enriched on endosomes, the Golgi apparatus, endoplasmic reticulum, or mitochondria (Hein et al., 2023).

Fully lipid-modified heterotrimers in their inactive state are unlikely to detach from membranes at a significant rate (Shahinian and Silvius, 1995). Therefore, we infer from the presence of Gβ₁ on early, late, and recycling endosomes that heterotrimers undergo vesicle-mediated endocytosis in unstimulated cells and are not efficiently sorted to either the slow recycling pathway or the degradative pathway. Our imaging results also show that G proteins are apparently more abundant on late endosomes and lysosomes than on early endosomes, suggesting that they become concentrated as late endosomes mature. We do not know the fate of G proteins located on the surface of lysosomes. Since lysosomes may fuse with the plasma membrane under certain circumstances (Xu and Ren, 2015), it is possible that this represents a route of G protein recycling to the plasma membrane. Our results are largely consistent with the hypothesis that G proteins passively follow bulk endocytic flow of membrane and suggest that at least some G proteins are recycled to the plasma membrane. We also cannot exclude the possibility that heterotrimers traffic between membrane compartments by mechanisms other than vesicular trafficking (Saini et al., 2009). However, even if this is the case our conclusions that G proteins are internalized inefficiently and are present at lower density on most intracellular membranes are still valid.

Tagging endogenous proteins with HiBit and other luciferase fragments has proven to be useful for studying several aspects of GPCR signaling (White et al., 2020). The cell lines we developed should prove useful for answering additional questions related to G protein regulation, such as possible non-vesicular translocation due to palmitate turnover (Saini et al., 2009; Wedegaertner and Bourne, 1994), the role of ubiquitination (Dohlman and Campbell, 2019), and localization in subcompartments not studied here. In addition, it may also be possible to use these cells in combination with targeted sensors to study endogenous G protein activation in different subcellular compartments. More broadly, our results show that subcellular localization of endogenous membrane proteins can be studied in living cells by adding a HiBit tag and performing bystander BRET mapping. Applied at large scale this approach would have some advantages over fluorescent protein complementation (Cho et al., 2022), most notably the ability to localize endogenous membrane proteins that are expressed at levels that are too low to permit fluorescence microscopy.

Our study is not without limitations. Our labeling strategy was designed to interfere as little as possible with heterotrimer function, but we cannot rule out the possibility that the tags we used to visualize and track G proteins had some influence on their trafficking. By labeling Gβ₁ subunits, we cannot directly distinguish heterotrimers from free Gβγ dimers, complicating interpretation. This strategy also does not allow us to resolve heterotrimers containing different Gα subunits. It is quite possible that heterotrimers containing different Gα subunits could be subject to different trafficking mechanisms. Our conclusion that GPCR activation has no lasting effect on the subcellular distribution of G proteins rests on three representative receptors, chosen because they activate three of the four major G protein families. It is possible that other receptors will influence G protein distribution using mechanisms not shared by the receptors we studied. For example, a few receptors are thought to form relatively stable complexes with Gβγ, which could provide a mechanism of trafficking to endosomes (Thomsen et al., 2016; Wehbi et al., 2013). Finally, our study was limited to a single non-differentiated cell type. It would not be surprising to find that differentiated cells have mechanisms to regulate G protein trafficking and distribution that are not shared by the model cells we used (Calebiro et al., 2009; Kotowski et al., 2011; Lin et al., 2024; Puri et al., 2022).

In summary, here we show that heterotrimeric G proteins are more abundant on the plasma membrane than on any intracellular compartment where they are thought to be important for signaling. Our results are likely to have functional implications for signaling from intracellular compartments. Receptor-G protein coupling is thought to be rate-limited by collision and G protein abundance (Hein et al., 2005), and decreasing G protein expression is known to impair downstream signaling (Gabay et al., 2011; Schwindinger et al., 1997). Signaling from endosomes and other compartments may thus be disadvantaged by a low density of G proteins. Further studies are warranted to examine the stoichiometry of receptors, G proteins, regulators, and effectors in different subcellular compartments and how this affects signaling.

All data generated or analyzed during this study are included in the manuscript and supporting files; source data files have been provided for Figures 1, 3—5, Figure 1—figure supplement 1, Figure 4—figure supplement 2, and Figure 5—figure supplements 1 and 2.

1. 1. Akgoz M
   2. Kalyanaraman V
   3. Gautam N

   (2004) Receptor-mediated reversible translocation of the G protein betagamma complex from the plasma membrane to the Golgi complex

   The Journal of Biological Chemistry 279:51541–51544.

   https://doi.org/10.1074/jbc.M410639200

   • PubMed
   • Google Scholar
2. 1. Barak LS
   2. Warabi K
   3. Feng X
   4. Caron MG
   5. Kwatra MM

   (1999) Real-time visualization of the cellular redistribution of G protein-coupled receptor kinase 2 and beta-arrestin 2 during homologous desensitization of the substance P receptor

   The Journal of Biological Chemistry 274:7565–7569.

   https://doi.org/10.1074/jbc.274.11.7565

   • PubMed
   • Google Scholar
3. 1. Benovic JL
   2. Bouvier M
   3. Caron MG
   4. Lefkowitz RJ

   (1988) Regulation of adenylyl cyclase-coupled beta-adrenergic receptors

   Annual Review of Cell Biology 4:405–428.

   https://doi.org/10.1146/annurev.cb.04.110188.002201

   • PubMed
   • Google Scholar
4. 1. Betz WJ
   2. Mao F
   3. Smith CB

   (1996) Imaging exocytosis and endocytosis

   Current Opinion in Neurobiology 6:365–371.

   https://doi.org/10.1016/s0959-4388(96)80121-8

   • PubMed
   • Google Scholar
5. 1. Bowman SL
   2. Shiwarski DJ
   3. Puthenveedu MA

   (2016) Distinct G protein-coupled receptor recycling pathways allow spatial control of downstream G protein signaling

   The Journal of Cell Biology 214:797–806.

   https://doi.org/10.1083/jcb.201512068

   • PubMed
   • Google Scholar
6. 1. Calebiro D
   2. Nikolaev VO
   3. Gagliani MC
   4. de Filippis T
   5. Dees C
   6. Tacchetti C
   7. Persani L
   8. Lohse MJ

   (2009) Persistent cAMP-signals triggered by internalized G-protein-coupled receptors

   PLOS Biology 7:e1000172.

   https://doi.org/10.1371/journal.pbio.1000172

   • PubMed
   • Google Scholar
7. 1. Calebiro D
   2. Nikolaev VO
   3. Persani L
   4. Lohse MJ

   (2010) Signaling by internalized G-protein-coupled receptors

   Trends in Pharmacological Sciences 31:221–228.

   https://doi.org/10.1016/j.tips.2010.02.002

   • PubMed
   • Google Scholar
8. 1. Chisari M
   2. Saini DK
   3. Kalyanaraman V
   4. Gautam N

   (2007) Shuttling of G protein subunits between the plasma membrane and intracellular membranes

   The Journal of Biological Chemistry 282:24092–24098.

   https://doi.org/10.1074/jbc.M704246200

   • PubMed
   • Google Scholar
9. 1. Cho NH
   2. Cheveralls KC
   3. Brunner AD
   4. Kim K
   5. Michaelis AC
   6. Raghavan P
   7. Kobayashi H
   8. Savy L
   9. Li JY
   10. Canaj H
   11. Kim JYS
   12. Stewart EM
   13. Gnann C
   14. McCarthy F
   15. Cabrera JP
   16. Brunetti RM
   17. Chhun BB
   18. Dingle G
   19. Hein MY
   20. Huang B
   21. Mehta SB
   22. Weissman JS
   23. Gómez-Sjöberg R
   24. Itzhak DN
   25. Royer LA
   26. Mann M
   27. Leonetti MD

   (2022) Opencell: endogenous tagging for the cartography of human cellular organization

   Science 375:eabi6983.

   https://doi.org/10.1126/science.abi6983

   • PubMed
   • Google Scholar
10. 1. Clement K
    2. Rees H
    3. Canver MC
    4. Gehrke JM
    5. Farouni R
    6. Hsu JY
    7. Cole MA
    8. Liu DR
    9. Joung JK
    10. Bauer DE
    11. Pinello L

    (2019) CRISPResso2 provides accurate and rapid genome editing sequence analysis

    Nature Biotechnology 37:224–226.

    https://doi.org/10.1038/s41587-019-0032-3

    • PubMed
    • Google Scholar
11. 1. Crouthamel M
    2. Thiyagarajan MM
    3. Evanko DS
    4. Wedegaertner PB

    (2008) N-terminal polybasic motifs are required for plasma membrane localization of Galpha(s) and Galpha(q)

    Cellular Signalling 20:1900–1910.

    https://doi.org/10.1016/j.cellsig.2008.06.019

    • PubMed
    • Google Scholar
12. 1. DeGroot ACM
    2. Busch DJ
    3. Hayden CC
    4. Mihelic SA
    5. Alpar AT
    6. Behar M
    7. Stachowiak JC

    (2018) Entropic control of receptor recycling using engineered ligands

    Biophysical Journal 114:1377–1388.

    https://doi.org/10.1016/j.bpj.2018.01.036

    • PubMed
    • Google Scholar
13. 1. Doherty GJ
    2. McMahon HT

    (2009) Mechanisms of endocytosis

    Annual Review of Biochemistry 78:857–902.

    https://doi.org/10.1146/annurev.biochem.78.081307.110540

    • PubMed
    • Google Scholar
14. 1. Dohlman HG
    2. Campbell SL

    (2019) Regulation of large and small G proteins by ubiquitination

    The Journal of Biological Chemistry 294:18613–18623.

    https://doi.org/10.1074/jbc.REV119.011068

    • PubMed
    • Google Scholar
15. 1. Eichel K
    2. von Zastrow M

    (2018) Subcellular organization of GPCR signaling

    Trends in Pharmacological Sciences 39:200–208.

    https://doi.org/10.1016/j.tips.2017.11.009

    • PubMed
    • Google Scholar
16. 1. Evanko DS
    2. Thiyagarajan MM
    3. Wedegaertner PB

    (2000) Interaction with Gbetagamma is required for membrane targeting and palmitoylation of Galpha(s) and Galpha(q)

    The Journal of Biological Chemistry 275:1327–1336.

    https://doi.org/10.1074/jbc.275.2.1327

    • PubMed
    • Google Scholar
17. 1. Feng S
    2. Sekine S
    3. Pessino V
    4. Li H
    5. Leonetti MD
    6. Huang B

    (2017) Improved split fluorescent proteins for endogenous protein labeling

    Nature Communications 8:370.

    https://doi.org/10.1038/s41467-017-00494-8

    • PubMed
    • Google Scholar
18. 1. Ferrandon S
    2. Feinstein TN
    3. Castro M
    4. Wang B
    5. Bouley R
    6. Potts JT
    7. Gardella TJ
    8. Vilardaga JP

    (2009) Sustained cyclic AMP production by parathyroid hormone receptor endocytosis

    Nature Chemical Biology 5:734–742.

    https://doi.org/10.1038/nchembio.206

    • PubMed
    • Google Scholar
19. 1. Gabay M
    2. Pinter ME
    3. Wright FA
    4. Chan P
    5. Murphy AJ
    6. Valenzuela DM
    7. Yancopoulos GD
    8. Tall GG

    (2011) Ric-8 proteins are molecular chaperones that direct nascent G protein α subunit membrane association

    Science Signaling 4:ra79.

    https://doi.org/10.1126/scisignal.2002223

    • PubMed
    • Google Scholar
20. 1. Gilman AG

    (1987) G proteins: transducers of receptor-generated signals

    Annual Review of Biochemistry 56:615–649.

    https://doi.org/10.1146/annurev.bi.56.070187.003151

    • PubMed
    • Google Scholar
21. 1. Hammond GRV
    2. Balla T

    (2015) Polyphosphoinositide binding domains: Key to inositol lipid biology

    Biochimica et Biophysica Acta 1851:746–758.

    https://doi.org/10.1016/j.bbalip.2015.02.013

    • PubMed
    • Google Scholar
22. 1. Hanyaloglu AC
    2. von Zastrow M

    (2008) Regulation of GPCRs by endocytic membrane trafficking and its potential implications

    Annual Review of Pharmacology and Toxicology 48:537–568.

    https://doi.org/10.1146/annurev.pharmtox.48.113006.094830

    • PubMed
    • Google Scholar
23. 1. Hein P
    2. Frank M
    3. Hoffmann C
    4. Lohse MJ
    5. Bünemann M

    (2005) Dynamics of receptor/G protein coupling in living cells

    The EMBO Journal 24:4106–4114.

    https://doi.org/10.1038/sj.emboj.7600870

    • PubMed
    • Google Scholar
24. 1. Hein MY
    2. Peng D
    3. Todorova V
    4. McCarthy F
    5. Kim K
    6. Liu C
    7. Savy L
    8. Januel C
    9. Baltazar-Nunez R
    10. Bax S
    11. Vaid S
    12. Vangipuram M
    13. Ivanov IE
    14. Byrum JR
    15. Pradeep S
    16. Gonzalez CG
    17. Aniseia Y
    18. Wang E
    19. Creery JS
    20. McMorrow AH
    21. Burgess J
    22. Sunshine S
    23. Yeung-Levy S
    24. DeFelice BC
    25. Mehta SB
    26. Itzhak DN
    27. Elias JE
    28. Leonetti MD

    (2023) Global organelle profiling reveals subcellular localization and remodeling at proteome scale

    Cell Biology 1:e2249.

    https://doi.org/10.1101/2023.12.18.572249

    • Google Scholar
25. 1. Hewavitharana T
    2. Wedegaertner PB

    (2012) Non-canonical signaling and localizations of heterotrimeric G proteins

    Cellular Signalling 24:25–34.

    https://doi.org/10.1016/j.cellsig.2011.08.014

    • PubMed
    • Google Scholar
26. 1. Hillenbrand M
    2. Schori C
    3. Schöppe J
    4. Plückthun A

    (2015) Comprehensive analysis of heterotrimeric G-protein complex diversity and their interactions with GPCRs in solution

    PNAS 112:E1181–E1190.

    https://doi.org/10.1073/pnas.1417573112

    • PubMed
    • Google Scholar
27. 1. Hollins B
    2. Kuravi S
    3. Digby GJ
    4. Lambert NA

    (2009) The c-terminus of GRK3 indicates rapid dissociation of G protein heterotrimers

    Cellular Signalling 21:1015–1021.

    https://doi.org/10.1016/j.cellsig.2009.02.017

    • PubMed
    • Google Scholar
28. 1. Hynes TR
    2. Mervine SM
    3. Yost EA
    4. Sabo JL
    5. Berlot CH

    (2004) Live cell imaging of Gs and the beta2-adrenergic receptor demonstrates that both alphas and beta1gamma7 internalize upon stimulation and exhibit similar trafficking patterns that differ from that of the beta2-adrenergic receptor

    The Journal of Biological Chemistry 279:44101–44112.

    https://doi.org/10.1074/jbc.M405151200

    • PubMed
    • Google Scholar
29. 1. Irannejad R
    2. Tomshine JC
    3. Tomshine JR
    4. Chevalier M
    5. Mahoney JP
    6. Steyaert J
    7. Rasmussen SGF
    8. Sunahara RK
    9. El-Samad H
    10. Huang B
    11. von Zastrow M

    (2013) Conformational biosensors reveal GPCR signalling from endosomes

    Nature 495:534–538.

    https://doi.org/10.1038/nature12000

    • PubMed
    • Google Scholar
30. 1. Irannejad R
    2. Pessino V
    3. Mika D
    4. Huang B
    5. Wedegaertner PB
    6. Conti M
    7. von Zastrow M

    (2017) Functional selectivity of GPCR-directed drug action through location bias

    Nature Chemical Biology 13:799–806.

    https://doi.org/10.1038/nchembio.2389

    • PubMed
    • Google Scholar
31. 1. Kotowski SJ
    2. Hopf FW
    3. Seif T
    4. Bonci A
    5. von Zastrow M

    (2011) Endocytosis promotes rapid dopaminergic signaling

    Neuron 71:278–290.

    https://doi.org/10.1016/j.neuron.2011.05.036

    • PubMed
    • Google Scholar
32. 1. Krumins AM
    2. Gilman AG

    (2006) Targeted knockdown of G protein subunits selectively prevents receptor-mediated modulation of effectors and reveals complex changes in non-targeted signaling proteins

    The Journal of Biological Chemistry 281:10250–10262.

    https://doi.org/10.1074/jbc.M511551200

    • PubMed
    • Google Scholar
33. Book

    1. Lan TH
    2. Liu Q
    3. Li C
    4. Wu G
    5. Lambert NA

    (2012) Sensitive and High Resolution Localization and Tracking of Membrane Proteins in Live Cells with BRET

    Wiley.

    https://doi.org/10.1111/j.1600-0854.2012.01401.x

    • Google Scholar
34. 1. Lazar AM
    2. Irannejad R
    3. Baldwin TA
    4. Sundaram AB
    5. Gutkind JS
    6. Inoue A
    7. Dessauer CW
    8. Zastrow M

    (2020) G protein-regulated endocytic trafficking of adenylyl625 cyclase type

    eLife 9:9.

    https://doi.org/10.7554/eLife.58039

    • Google Scholar
35. 1. Lin TY
    2. Mai QN
    3. Zhang H
    4. Wilson E
    5. Chien HC
    6. Yee SW
    7. Giacomini KM
    8. Olgin JE
    9. Irannejad R

    (2024) Cardiac contraction and relaxation are regulated by distinct subcellular cAMP pools

    Nature Chemical Biology 20:62–73.

    https://doi.org/10.1038/s41589-023-01381-8

    • PubMed
    • Google Scholar
36. 1. Marrari Y
    2. Crouthamel M
    3. Irannejad R
    4. Wedegaertner PB

    (2007) Assembly and trafficking of heterotrimeric G proteins

    Biochemistry 46:7665–7677.

    https://doi.org/10.1021/bi700338m

    • PubMed
    • Google Scholar
37. 1. Martin BR
    2. Lambert NA

    (2016) Activated g protein gαs samples multiple endomembrane compartments

    The Journal of Biological Chemistry 291:20295–20302.

    https://doi.org/10.1074/jbc.M116.729731

    • PubMed
    • Google Scholar
38. 1. Michaelson D
    2. Ahearn I
    3. Bergo M
    4. Young S
    5. Philips M

    (2002) Membrane trafficking of heterotrimeric G proteins via the endoplasmic reticulum and Golgi

    Molecular Biology of the Cell 13:3294–3302.

    https://doi.org/10.1091/mbc.e02-02-0095

    • PubMed
    • Google Scholar
39. 1. Mullershausen F
    2. Zecri F
    3. Cetin C
    4. Billich A
    5. Guerini D
    6. Seuwen K

    (2009) Persistent signaling induced by FTY720-phosphate is mediated by internalized S1P1 receptors

    Nature Chemical Biology 5:428–434.

    https://doi.org/10.1038/nchembio.173

    • PubMed
    • Google Scholar
40. 1. Pierce KL
    2. Premont RT
    3. Lefkowitz RJ

    (2002) Seven-transmembrane receptors

    Nature Reviews. Molecular Cell Biology 3:639–650.

    https://doi.org/10.1038/nrm908

    • PubMed
    • Google Scholar
41. 1. Pinilla-Macua I
    2. Watkins SC
    3. Sorkin A

    (2016) Endocytosis separates EGF receptors from endogenous fluorescently labeled HRas and diminishes receptor signaling to MAP kinases in endosomes

    PNAS 113:2122–2127.

    https://doi.org/10.1073/pnas.1520301113

    • PubMed
    • Google Scholar
42. 1. Puri NM
    2. Romano GR
    3. Lin TY
    4. Mai QN
    5. Irannejad R

    (2022) The organic cation transporter 2 regulates dopamine D1 receptor signaling at the Golgi apparatus

    eLife 11:e75468.

    https://doi.org/10.7554/eLife.75468

    • PubMed
    • Google Scholar
43. 1. Ransnäs LA
    2. Svoboda P
    3. Jasper JR
    4. Insel PA

    (1989) Stimulation of beta-adrenergic receptors of S49 lymphoma cells redistributes the alpha subunit of the stimulatory G protein between cytosol and membranes

    PNAS 86:7900–7903.

    https://doi.org/10.1073/pnas.86.20.7900

    • PubMed
    • Google Scholar
44. 1. Saini DK
    2. Kalyanaraman V
    3. Chisari M
    4. Gautam N

    (2007) A family of G protein βγ subunits translocate reversibly from the plasma membrane to endomembranes on receptor activation

    The Journal of Biological Chemistry 282:24099–24108.

    https://doi.org/10.1074/jbc.M701191200

    • PubMed
    • Google Scholar
45. 1. Saini DK
    2. Chisari M
    3. Gautam N

    (2009) Shuttling and translocation of heterotrimeric G proteins and Ras

    Trends in Pharmacological Sciences 30:278–286.

    https://doi.org/10.1016/j.tips.2009.04.001

    • PubMed
    • Google Scholar
46. 1. Scarselli M
    2. Donaldson JG

    (2009) Constitutive internalization of G protein-coupled receptors and G proteins via clathrin-independent endocytosis

    The Journal of Biological Chemistry 284:3577–3585.

    https://doi.org/10.1074/jbc.M806819200

    • PubMed
    • Google Scholar
47. 1. Schwindinger WF
    2. Reese KJ
    3. Lawler AM
    4. Gearhart JD
    5. Levine MA

    (1997) Targeted disruption of Gnas in embryonic stem cells

    Endocrinology 138:4058–4063.

    https://doi.org/10.1210/endo.138.10.5439

    • PubMed
    • Google Scholar
48. 1. Schwinn MK
    2. Machleidt T
    3. Zimmerman K
    4. Eggers CT
    5. Dixon AS
    6. Hurst R
    7. Hall MP
    8. Encell LP
    9. Binkowski BF
    10. Wood KV

    (2018) CRISPR-mediated tagging of endogenous proteins with a luminescent peptide

    ACS Chemical Biology 13:467–474.

    https://doi.org/10.1021/acschembio.7b00549

    • PubMed
    • Google Scholar
49. 1. Shahinian S
    2. Silvius JR

    (1995) Doubly-lipid-modified protein sequence motifs exhibit long-lived anchorage to lipid bilayer membranes

    Biochemistry 34:3813–3822.

    https://doi.org/10.1021/bi00011a039

    • PubMed
    • Google Scholar
50. 1. Slepak VZ
    2. Hurley JB

    (2008) Mechanism of light-induced translocation of arrestin and transducin in photoreceptors: interaction-restricted diffusion

    IUBMB Life 60:2–9.

    https://doi.org/10.1002/iub.7

    • PubMed
    • Google Scholar
51. 1. Stenmark H

    (2009) Rab GTPases as coordinators of vesicle traffic

    Nature Reviews. Molecular Cell Biology 10:513–525.

    https://doi.org/10.1038/nrm2728

    • PubMed
    • Google Scholar
52. 1. Surve S
    2. Watkins SC
    3. Sorkin A

    (2021) EGFR-RAS-MAPK signaling is confined to the plasma membrane and associated endorecycling protrusions

    The Journal of Cell Biology 220:e202107103.

    https://doi.org/10.1083/jcb.202107103

    • PubMed
    • Google Scholar
53. 1. Takida S
    2. Wedegaertner PB

    (2003) Heterotrimer formation, together with isoprenylation, is required for plasma membrane targeting of Gbetagamma

    The Journal of Biological Chemistry 278:17284–17290.

    https://doi.org/10.1074/jbc.M213239200

    • PubMed
    • Google Scholar
54. 1. Thomsen ARB
    2. Plouffe B
    3. Cahill TJ III
    4. Shukla AK
    5. Tarrasch JT
    6. Dosey AM
    7. Kahsai AW
    8. Strachan RT
    9. Pani B
    10. Mahoney JP
    11. Huang L
    12. Breton B
    13. Heydenreich FM
    14. Sunahara RK
    15. Skiniotis G
    16. Bouvier M
    17. Lefkowitz RJ

    (2016) GPCR-G protein-β-arrestin super-complex mediates sustained g protein signaling

    Cell 166:907–919.

    https://doi.org/10.1016/j.cell.2016.07.004

    • PubMed
    • Google Scholar
55. 1. Tsutsumi R
    2. Fukata Y
    3. Noritake J
    4. Iwanaga T
    5. Perez F
    6. Fukata M

    (2009) Identification of G protein alpha subunit-palmitoylating enzyme

    Molecular and Cellular Biology 29:435–447.

    https://doi.org/10.1128/MCB.01144-08

    • PubMed
    • Google Scholar
56. 1. Tsvetanova NG
    2. Irannejad R
    3. Zastrow M

    (2015) GPCR signaling via heterotrimeric G proteins from endosomes

    Journal of Biological Chemistry 290:jbc.

    https://doi.org/10.1074/jbc.R114.617951

    • Google Scholar
57. 1. von Zastrow M
    2. Kobilka BK

    (1992) Ligand-regulated internalization and recycling of human beta 2-adrenergic receptors between the plasma membrane and endosomes containing transferrin receptors

    Journal of Biological Chemistry 267:3530–3538.

    https://doi.org/10.1016/S0021-9258(19)50762-1

    • Google Scholar
58. 1. Wedegaertner PB
    2. Bourne HR

    (1994) Activation and depalmitoylation of Gs alpha

    Cell 77:1063–1070.

    https://doi.org/10.1016/0092-8674(94)90445-6

    • PubMed
    • Google Scholar
59. 1. Wedegaertner PB
    2. Bourne HR
    3. von Zastrow M

    (1996) Activation-induced subcellular redistribution of Gs alpha

    Molecular Biology of the Cell 7:1225–1233.

    https://doi.org/10.1091/mbc.7.8.1225

    • PubMed
    • Google Scholar
60. 1. Wedegaertner PB

    (2012) G protein trafficking

    Sub-Cellular Biochemistry 63:193–223.

    https://doi.org/10.1007/978-94-007-4765-4_11

    • PubMed
    • Google Scholar
61. 1. Wehbi VL
    2. Stevenson HP
    3. Feinstein TN
    4. Calero G
    5. Romero G
    6. Vilardaga JP

    (2013) Noncanonical GPCR signaling arising from a PTH receptor-arrestin-Gβγ complex

    PNAS 110:1530–1535.

    https://doi.org/10.1073/pnas.1205756110

    • PubMed
    • Google Scholar
62. 1. White CW
    2. Caspar B
    3. Vanyai HK
    4. Pfleger KDG
    5. Hill SJ

    (2020) CRISPR-mediated protein tagging with nanoluciferase to investigate native chemokine receptor function and conformational changes

    Cell Chemical Biology 27:499–510.

    https://doi.org/10.1016/j.chembiol.2020.01.010

    • PubMed
    • Google Scholar
63. 1. Xu H
    2. Ren D

    (2015) Lysosomal physiology

    Annual Review of Physiology 77:57–80.

    https://doi.org/10.1146/annurev-physiol-021014-071649

    • PubMed
    • Google Scholar

Author details

1. Wonjo Jang

   Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, Augusta, United States

   Contribution

   Conceptualization, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1928-8978

2. Kanishka Senarath

   Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, Augusta, United States

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

3. Gavin Feinberg

   Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, Augusta, United States

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0009-0002-7065-0917

4. Sumin Lu

   Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, Augusta, United States

   Contribution

   Resources, Investigation

   Competing interests

   No competing interests declared

5. Nevin A Lambert

   Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, Augusta, United States

   Contribution

   Conceptualization, Funding acquisition, Investigation, Writing - original draft, Project administration

   For correspondence

   NELAMBERT@augusta.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7550-0921

• 1,416

  views

• 100

  downloads

• 3

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.