Conformational biosensors delineate endosomal G protein regulation by GPCRs

Domain: chemistry
Published: 2026-02-18
Editorial outcome: major_revision

Abstract
Many GPCRs trigger a second phase of G protein-coupled signaling from endosomes after signaling from the plasma membrane, necessitating GPCRs to increase the concentration of active-state G proteins on the endosome membrane. How this is achieved remains unclear. Here, we show that three Gs-coupled GPCRs – the β2-adrenergic receptor, VIP-1 receptor, and adenosine 2B receptor – each trigger a net redistribution of Gαs from the plasma membrane to endosomes at native expression levels and without requiring receptor internalization. We then show that active-state Gαs production on endosomes, in contrast, is GPCR internalization-dependent. We further identify location bias in the selectivity of GPCR coupling between Gs and Gq on endosomes relative to the plasma membrane. We propose that endosomal Gs regulation involves discrete GPCR-G protein coupling reactions, one at the plasma membrane controlling Gs concentration and another at endosomes controlling Gs activity, and that GPCR endocytosis can switch signaling selectivity between G protein classes.

Introduction
G protein-coupled receptors (GPCRs) constitute the largest family of signaling receptors and regulate nearly every physiological process. After activation by binding an agonist, GPCRs initiate signaling by coupling to cognate heterotrimeric G proteins, consisting of a Gα subunit and Gβγ subcomplex, which function collectively as key transducers of downstream signaling. This allosteric coupling reaction promotes guanine nucleotide exchange on the G protein α-subunit, resulting in GTP binding to the α-subunit that converts it from an inactive to active state. G protein classes are defined according to the identity of their α-subunit (e.g., Gs, Gi/o, Gq/11, and G12/13), with individual GPCRs differing in selectivity for coupling among G protein classes that produce distinct downstream regulatory effects1,2. The central importance of GPCR signaling in physiology, as well as its dysfunction or dysregulation in a variety of pathological states, has motivated intense interest in GPCRs as therapeutic targets, and presently over 30% of FDA-approved drugs target GPCRs3.

The importance of GPCR signaling from the plasma membrane has been recognized for many years1,4, and there is now considerable interest in the ability of GPCRs to produce distinct and additional effects from intracellular membranes4–6. Endomembrane signaling is perhaps most strongly supported from the study of Gs-coupled GPCRs4,5,7–17, but there is also significant evidence for endomembrane signaling through other G protein classes as well5,18,19. Based on first principles, such signaling fundamentally depends on the presence of active-state G proteins on the appropriate membrane1,2. However, it remains unclear how the concentration of active-state G proteins on endomembranes is regulated.

Under basal conditions, Gs is enriched on the plasma membrane and present in lower amounts on intracellular membranes20. Gs activation by coupling to a GPCR on the plasma membrane promotes dissociation of Gαs and its net intracellular redistribution, increasing the concentration of Gαs on multiple endomembrane compartments, including endosomes20–24. The production of active-state Gαs on endosomes is then thought to require a second coupling reaction occurring locally on the endosome limiting membrane4,5,10. There is considerable evidence supporting such activation (e.g8–11,16), but the presence of active-state, GTP-bound Gαs on endosomes has not been directly demonstrated. Accordingly, the subcellular location(s) of G protein activation by GPCRs, and of active-state Gαs subunit accumulation on endomembranes, remain incompletely understood.

Here, we addressed this knowledge gap by dissecting the regulation of endosomal Gαs localization and activity by GPCRs. We first verify that the prototypical β2-adrenergic receptor (β2AR) triggers a rapid intracellular redistribution of Gαs from the plasma membrane20,21,23–25. We then extend the present understanding by showing that this process is triggered by a variety of Gs-coupled GPCRs, and under physiologically relevant conditions of native or near-native levels of GPCR and G protein expression. Next, using conformational biosensors, we demonstrate sequential phases of both Gs activation and active-state Gαs accumulation, first on the plasma membrane and then on endosomes, and at endogenous levels of G protein expression. We then show that the accumulation of active-state Gαs on endosomes is specifically dependent on receptor endocytosis. Finally, we provide evidence for a type of location bias in the biochemical selectivity of endosomal G protein activation that is programmed by GPCRs in a receptor-specific manner.

Results
Gαs colocalizes with internalized receptors and Gβγ on early endosomes after GPCR activation

Gαs associates with various cellular membranes and is enriched on the plasma membrane in unstimulated cells20,23–25. It is generally thought that Gαs dissociates from the plasma membrane after its activation there and subsequently samples a variety of intracellular membrane compartments, including endosomes20,23,24,26,27, where a second round of GPCR-triggered activation occurs4,5,8,10,11. We sought to verify and further examine this fundamental signaling process in living cells. We began by imaging Gαs by confocal fluorescence microscopy, using β2AR as a model Gs-coupled GPCR that is well known to trigger intracellular redistribution of Gαs and has traditionally been used to study it20,21,23–25. We labeled Gαs by inserting EGFP into the linker region between its α-helical domain and conserved Ras-like domain, a strategy shown previously to preserve the signaling function of Gαs25, and then expressed this labeled construct in HEK293 cells stably expressing Flag-tagged β2ΑR. Consistent with previous studies21,23–25, EGFP-Gαs visibly redistributed intracellularly from the plasma membrane within several minutes after application of isoproterenol, a β2AR agonist (Iso, Fig. 1a). EGFP-Gαs was diffusely distributed in the cytoplasm and concentrated on various endomembranes, including endosomes, as indicated by colocalization with mApple-EEA1 (Supplementary Fig. 1a). We previously described Iso-induced intracellular redistribution of an epitope-tagged Gαs, but not specific localization to endomembranes, in formaldehyde-fixed cells21. Potentially explaining this, we found such fixation to incompletely preserve endomembrane association of EGFP-Gαs (Supplementary Fig. 1b).

According to the present understanding, Gs activation on endosomes would require receptors to be present in the same endosome membrane2,4–6. We tested this in our system by imaging EGFP-Gαs and internalized β2ARs. Confocal microscopy resolved EGFP-Gαs localization on Flag-β2AR-containing endosomes in isoproterenol-treated cells, and we also observed EGFP-Gαs localization on additional compartments not containing internalized receptors (Fig. 1a, arrows indicate examples of EGFP-Gαs/Flag-β2AR colocalization). We quantified colocalization using Pearson correlation analysis, assessing pixel-based correlations between EGFP-Gαs and either the Flag-β2AR or mApple-EEA1 fluorescence signal, respectively, after agonist application. The Pearson correlation between EGFP-Gαs and Flag-β2AR decreased after agonist application (Fig. 1b), reflecting a loss of Flag-β2AR and EGFP-Gαs colocalization at the plasma membrane, while the correlation between EGFP-Gαs and mApple-EEA1 increased (Fig. 1c), reflecting Gαs redistribution to early endosomes. However, the correlation between EGFP-Gαs and Flag-β2ΑR plateaued at a level that remained significantly higher than that between a cytosolic EGFP control and Flag-β2ΑR (Fig. 1b), consistent with our observations of partial colocalization with receptors on endosomes. Together, these results indicate that activated β2ARs trigger EGFP-Gαs to rapidly redistribute from the plasma membrane to endomembranes, including to endosomes that also contain internalized β2ARs.

If Gαs redistributes to endomembranes by partitioning, we anticipated that inhibiting Gαs dissociation from membranes would reduce its accumulation on endosomes. We tested this prediction using a previously described Gαs/Gαi chimera that retains the ability to functionally couple to β2AR but is more stably attached to membranes by the addition of an irreversible myristoylation20,22 (membrane-pinned Gαs/i). As expected, the membrane-pinned Gαs/i construct did not detectably redistribute or associate with endomembranes in response to Iso (Fig. 1c, Supplementary Fig. 1a).

Efficient GPCR-G protein coupling requires Gαs to be associated with Gβγ2; thus we asked if Gβγ is present on the same endosomes using an mApple-labeled γ-subunit (mApple-Gγ2) coexpressed with Gβ1. Labeled Gβγ localized to the plasma membrane and multiple internal membranes, consistent with previous results28–30. This included membranes associated with EGFP-Gαs and containing internalized β2ARs (Fig. 1d, Supplementary Fig. 1c). Whereas endosomal localization of both Gαs and β2AR were clearly increased after β2AR activation by Iso, the localization of labeled Gβγ was not noticeably changed (Fig. 1d). These results support a model in which Gαs and β2AR colocalize on endosomes in an activation-induced manner, while these endosomes appear to be constitutively associated with Gβγ28. Accordingly, all of the protein components necessary for G protein coupling converge at endosomes after agonist-induced activation of the receptor.

Gαs returns to the plasma membrane after receptor inactivation

The ability of cells to respond to a subsequent agonist exposure would presumably require replenishment of Gαs at the plasma membrane, and previous studies indicate that the intracellular redistribution of Gαs triggered by β2AR activation is reversible after receptor inactivation20,21. We verified this in our hands by activating Flag-β2ΑR with Iso in cells coexpressing EGFP-Gαs and then applying the β2AR antagonist Alprenolol (Alp) in excess. We observed a pronounced reaccumulation of EGFP-Gαs on the plasma membrane after β2AR inactivation (Fig. 2a, Supplementary Fig. 2a), confirming reversibility of the Gαs redistribution process. We used nanoluciferase protein complementation (NanoBit) to quantify the reversible redistribution of Gαs by inserting LgBit into Gαs at the same position as EGFP (LgBit-Gαs) and measuring complementation with a plasma membrane-targeted SmBit construct (SmBit-mApple-CAAX) verified to appropriately localize to the plasma membrane (Supplementary Fig. 2b). In this assay, intracellular redistribution of LgBit-Gαs is indicated by a decrease in the luminescence signal (Fig. 2b). Iso-induced activation of Flag-β2AR produced such a decrease with a similar time course as the redistribution observed by microscopy (Fig. 2c) and this recovered to baseline after adding Alp (Fig. 2c). These results indicate that GPCR-triggered intracellular redistribution of Gαs is indeed reversible, replenishing Gαs at the plasma membrane after activation is terminated.

Intracellular redistribution of Gαs does not depend on GPCR endocytosis

Previous reports differ in whether the intracellular redistribution of Gαs triggered by β2ARs requires receptor endocytosis21,23,24, so we further investigated this question using improved experimental tools. As a first approach, we acutely blocked β2AR endocytosis with the dynamin inhibitor Dyngo4a31. Iso-induced internalization of Flag-β2AR was strongly suppressed by Dyngo4a treatment, but the intracellular redistribution of EGFP-Gαs was not detectably affected (Supplementary Fig. 3a). As a second approach, we suppressed receptor internalization by mutating essential serine phosphosites in the cytoplasmic tail of β2AR to alanine10,32,33 (β2AR-3S). HEK293 cells endogenously express β-adrenergic receptors34, but at a sufficiently low level not to produce detectable Iso-induced Gαs redistribution signal in our NanoBit assay (Supplementary Fig. 3b). This enabled us to compare the effects of β2AR-3S to WT β2AR by simple overexpression after confirming comparable levels of expression (Supplementary Fig. 3c). β2AR-3S triggered a rapid and pronounced redistribution of Gαs from the plasma membrane indistinguishable from that triggered by WT β2AR (Fig. 2c), and this redistribution was accompanied by a corresponding increase in EGFP-Gαs accumulation on endomembranes (Supplementary Fig. 3d). As a third approach, we blocked β2AR internalization using previously described β-arrestin 1/2 double knockout (βarr1/2 DKO) HEK293 cells16. After verifying inhibition of WT β2AR internalization (Supplementary Fig. 3e), we found that Iso application triggered robust and reversible intracellular redistribution of Gαs in these cells (Fig. 2d, Supplementary Fig. 3e). Moreover, re-expression of βARR2-mApple in this genetic background fully rescued internalization of Flag-β2ΑR without detectably changing the redistribution of Gαs (Fig. 2d, Supplementary Fig. 3e). These results provide several lines of evidence indicating that GPCR-triggered intracellular redistribution of Gαs does not require internalization of the triggering GPCR.

Intracellular redistribution of Gαs is triggered by multiple Gs-coupled GPCRs at native levels

We next asked if the ability to trigger intracellular redistribution of Gαs is shared by other Gs-coupled GPCRs. We focused on the adenosine-2B receptor (A2BR or ADORA2B) and vasoactive intestinal peptide-1 receptor (VIPR1 or VPAC1) as representatives of GPCR family A and B, respectively, that are natively expressed in HEK293 cells and differ in their endocytic trafficking properties34. Specifically, VIPR1 robustly internalizes after activation through an arrestin-independent mechanism16, β2AR undergoes arrestin-dependent internalization35,36 and the human A2BR is relatively resistant to agonist-induced internalization34 (Supplementary Fig. 4). We found both VIPR1 and A2BR to trigger a pronounced intracellular redistribution of EGFP-Gαs when overexpressed and activated by cognate agonist (VIP or NECA), similar to the redistribution triggered by overexpressed β2AR after activation by Iso (Supplementary Fig. 4). These results indicate that multiple GPCRs can trigger redistribution of EGFP-Gαs and provide further support for the conclusion that this process is not dependent on the trafficking properties of the triggering GPCR.

Because achieving GPCR-triggered redistribution of EGFP-Gαs in these assays required the receptor to be overexpressed, we wondered if Gαs redistribution requires supra-physiological expression of receptors or can be triggered also by native GPCRs. We reasoned that overexpressing EGFP-Gαs in cells that already express endogenous, unlabeled Gαs might limit the sensitivity of our assay for detecting redistribution triggered by endogenous receptors, particularly as Gαs is endogenously expressed already at a much higher level than the GPCRs tested29. To address this, we expressed EGFP-Gαs at a near-native level in cells lacking endogenous Gαs. We first knocked out endogenous Gαs by CRISPR-Cas9 editing of the GNAS gene (Gαs KO cells) and verified full knockout genetically and by immunoblot, as well as functionally by elimination of the Iso-induced cAMP elevation (Supplementary Fig. 5). We then isolated cell populations stably expressing EGFP-Gαs at a near-native level, as defined by labeled protein expression within 2-fold of endogenous Gαs detected in wild type cells, and we further verified that this level of expression functionally rescues the characteristic Iso-induced cytoplasmic cAMP response (Supplementary Fig. 6).

This near-endogenous level of EGFP-Gαs expression was detectable by confocal fluorescence microscopy but quite dim (Supplementary Fig. 6c), so we used total internal reflection fluorescence (TIRF) microscopy as a more sensitive and quantitative method to assess EGFP-Gαs redistribution. We detected intracellular redistribution of EGFP-Gαs by loss of fluorescence intensity from the evanescent field that selectively illuminates the basal plasma membrane relative to the cell interior. Using this approach, we observed a significant reduction of surface-localized EGFP-Gαs upon application of Iso but not vehicle. The magnitude of this effect was much lower than observed in β2AR-overexpressing cells (Fig. 3 and Supplementary Fig. 7), consistent with a considerably lower level of endogenous β2AR expression. VIP and NECA also triggered a comparable amount of intracellular redistribution of EGFP-Gαs through their endogenous receptors (Fig. 3 and Supplementary Fig. 7). Together, these results indicate that a variety of Gs-coupled GPCRs share the ability to trigger intracellular redistribution of Gαs under conditions of native or near-native expression.

VIPR1 produces active-state Gαs and Gαq/11 on the plasma membrane and Gαs on endosomes

The current understanding holds that Gs-mediated signaling requires Gαs to be in its active (GTP-bound) conformation2,37. The above experiments provided useful information about the overall distribution of Gαs but no information about its activation state. To address this, we utilized KB1691, a peptide shown previously to bind selectively to GTP-bound, active-state Gαs and to be a useful biosensor for detecting active-state Gαs in cells that overexpress both receptors and Gs37,38. We wondered if this peptide can be adapted to detect active-state Gαs at endogenous G protein expression levels. We focused on VIPR1 because this GPCR robustly triggers Gs-mediated signaling from both the plasma membrane and endosomes, resulting in sequential signaling phases that are temporally well-resolved16. We began by assessing VIP-stimulated recruitment of KB1691 to the plasma membrane, fusing KB1691 to SmBit-mApple (SmBit-mApple-KB1691) and measuring complementation with a plasma membrane-targeted LgBit (LgBit-CAAX, Fig. 4a). A clear VIP-induced recruitment signal was detected in cells expressing only endogenous G proteins (Fig. 4b). These results indicate that KB1691 is sufficiently sensitive to detect active-state Gαs accumulation at native expression levels, provided that the activating GPCR is overexpressed.

To test the specificity of sensor recruitment, we used knockout cells lacking Gαs and additionally deleted Gαolf, a close paralog of Gαs (Gαs/olf DKO cells) that we presumed would also engage the sensor (Supplementary Fig. 5). The VIP-induced biosensor response was markedly reduced in Gαs/olf DKO cells, verifying the ability of the sensor to detect active-state Gαs at native levels (Fig. 4b). We were surprised to observe a residual recruitment signal remaining in Gαs/olf DKO cells, with faster onset and shorter duration (Fig. 4b), despite the VIP-induced cAMP response being fully abolished in the same cells and functionally rescued by EGFP-Gαs re-expression as expected (Supplementary Fig. 5 and 6). Together, these results suggest that KB1691 detects not only active-state Gαs in the present experimental system, but also a kinetically distinct, non-Gs component that is activated by VIPR1.

As VIPR1 has been observed previously to initiate signaling through both Gs and Gq39, we wondered if the non-Gs component might represent active-state Gαq and/or Gα11. Consistent with this idea, the Gq/11 inhibitor YM-254890 (YM)40 eliminated the residual VIP-induced recruitment signal in Gαs/olf DKO cells (Fig. 4c). Furthermore, the residual, faster component detected by KB1691 was absent in Gαq/11 DKO cells (Supplementary Fig. 8a)41. Moreover, in contrast to parental cells, YM had no effect on KB1691 recruitment in Gαq/11 DKO cells (Supplementary Fig. 8a). Together, these data suggest that the non-Gs recruitment signal reflects active-state Gαq/11. To further test this interpretation, we adapted the Gαq/11 binding domain of p63RhoGEF, a previously described biosensor of active-state Gαq/1142, into our NanoBit assay platform (Supplementary Fig 8b). As predicted, VIPR1 caused detectable YM-sensitive recruitment of p63RhoGEF to the plasma membrane (Supplementary Fig 8c). In addition, the rapid peak of p63RhoGEF recruitment mirrored the kinetics of the YM-sensitive component detected by KB1691 (Fig. 4c,d, Supplementary Fig. 8c). Taken together, these data strongly suggest that the non-Gs signal produced by VIPR1 activation indeed represents active-state Gαq/11. We therefore defined the biosensor signal measured in the presence of YM as active-state Gαs and the YM-sensitive component as active-state Gαq/11, and we conclude that VIPR1 stimulates the accumulation of both active-state Gαs and active-state Gαq/11 on the plasma membrane in this system (Fig. 4d, Supplementary Fig. 8a).

We next asked if active-state G proteins can be detected also at endogenous levels on the endosome membrane. To address this, we modified the assay to measure complementation of SmBit-mApple-KB1691 with an endosome-targeted LgBit construct (endofin-LgBit, Fig. 4e). VIP indeed produced a robust endosomal recruitment signal (Fig. 4f). The kinetics of SmBit-mApple-KB1691 recruitment to endosomes were clearly slower than to the plasma membrane, as the endosome signal reached its maximum after 15 to 20 minutes of agonist application (Fig. 4f) while the plasma membrane signal peaked within several minutes (Fig. 4b). Interestingly, endosomal recruitment of the KB1691-derived sensor was abolished in Gαs/olf DKO cells (Fig. 4f) and was unaffected by either YM treatment (Fig. 4g, h) or Gαq/11 knockout (Supplementary Fig. 8d). These observations indicate that VIPR1-triggered accumulation of active-state Gαs indeed occurs at native levels on endosomes, as well as the plasma membrane. However, there appears to be a type of location bias in active-state Gαs accumulation between these membranes, as defined by VIPR1 increasing both active-state Gαs and Gαq/11 on the plasma membrane but selectively active-state Gαs on endosomes.

Active-state Gαs production on endosomes is endocytosis-dependent

We next asked if the production of active-state Gαs on endosomes depends on the presence of activated GPCRs in the endosome membrane. This is expected based on the present model that Gs activation at endosomes specifically requires a second GPCR-G protein coupling reaction occurring locally at the endosome limiting membrane4–6. However, as our results (Fig. 2, Supplementary Fig. 3) and the results of others21,23 indicate that intracellular redistribution of Gαs can be efficiently triggered by GPCRs in the absence of receptor internalization, we sought to explicitly determine if endosomal production of active-state Gαs indeed depends on the presence of active-conformation receptors in endosomes.

To address this question, we inhibited VIPR1 internalization by viral expression of dominant negative (K44E) mutant dynamin, as described previously16, and verified strong endocytic inhibition by fluorescence flow cytometry (Supplementary Fig. 9a, b). Mutant dynamin reduced the amount of active-conformation VIPR1 at endosomes after VIP application, as detected using a previously described active-conformation GPCR sensor (miniGs)16, and it increased the amount of activated VIPR1 remaining at the plasma membrane (Fig. 5a–c). This confirms the ability of endocytic inhibition to reduce the accumulation of activated VIPR1 in endosomes. Mutant dynamin did not fully block the endosomal activation signal (Fig. 5c), however, suggesting that internalization of only a small fraction of the total receptor pool is sufficient to produce a robust activation signal on endosomes. Nevertheless, mutant dynamin decreased the accumulation of active-state Gαs subunits on endosomes to a comparable degree as it decreased the measured accumulation of active-conformation receptors in the endosome membrane (Fig. 5d–f, Supplementary Fig. 9c, d). Together, these observations suggest that the production of active-state Gαs on endosomes, while apparently a highly sensitive process, is dependent on GPCR internalization and the presence of activated receptors in the endosome membrane.

Detection of VIPR1-mediated coupling to Gαs on endosomes

We next sought to localize the coupling reaction that produces active-state Gαs. To do so, we adapted the NanoBit assay to detect recruitment of Nb37 rather than KB1691. Nb37 is an nanobody that binds to the α-helical domain of Gαs when Gαs is stabilized by the activated receptor in a nucleotide-free state, corresponding to the catalytic intermediate thought to underlie physiological GPCR-G coupling43. Thus, we interpret Nb37 binding as a biosensor of GPCR-G protein coupling10, in contrast to KB1691, which we interpret as a biosensor of the GTP-bound, active-state G protein that is produced by the coupling reaction37. Nb37 was shown previously to detect coupling on endosomes in cells overexpressing both VIPR1 and G16 and we found it possible to detect coupling in cells expressing native G proteins using the same experimental design, provided that VIPR1 was overexpressed (Fig. 6a–c). VIP produced a robust Nb37 recruitment signal on both the plasma membrane and endosomes, and with similar sequential kinetics as the KB1691 recruitment signal (Fig. 6c). We conclude that local GPCR-G coupling indeed occurs on endosomes, and that it likely occurs continuously during prolonged agonist exposure to sustain active-state Gαs production.

We were surprised to also observe a residual Nb37 recruitment signal remaining in Gαs/olf DKO cells. Similar to results obtained with KB1691 (Supplementary Fig. 10a), this non-Gs signal was eliminated in the presence of YM and absent in Gαq/11 DKO cells (Supplementary Fig. 10b–d). Accordingly, we defined separate Gs and Gq/11 components of Nb37 recruitment according to YM-sensitivity, as with KB1691. Using this approach, we resolved with Nb37 both Gs and Gq/11 components of VIPR1-mediated coupling on the plasma membrane, but primarily a Gs component on endosomes (Fig. 6c, Supplementary Fig. 10c–f). These results further support the existence of location bias in G protein activation, based on VIPR1 coupling to both Gs and Gq/11 on the plasma membrane but primarily to Gs on endosomes.

The location bias of GPCR-G protein coupling on endosomes is GPCR-specific

As an additional approach to test the idea that endosomal Gs activation requires local coupling, we took advantage of the fact that the human A2BR naturally internalizes very weakly after agonist-induced activation when compared to VIPR134. If local coupling to activated receptors is required for endosomal G protein activation to occur, we predicted that A2BR would produce a relatively weak Gs coupling signal on endosomes. This was indeed the case. A2BR produced detectable coupling to Gs and Gq/11 at the plasma membrane, as indicated by both YM-insensitive and -sensitive components of Nb37 recruitment (Fig. 6d and Supplementary Fig. 10g, h), but little or no coupling to Gs at endosomes (Fig. 6d).

A2BR did produce a YM-sensitive component of Nb37 recruitment to endosomes, however, suggesting local activation selectively of Gq/11 (Fig. 6d). This was unexpected, in light of A2BR internalizing poorly after activation by NECA, but it supports and extends the concept of location bias in endosomal relative to plasma membrane G protein activation. Specifically, our results suggest that VIPR1 and A2BR are similar in their ability to activate both Gs and Gq/11 at the plasma membrane but, at endosomes, VIPR1 preferentially activates Gs while A2BR activates Gq/11. Accordingly, distinct GPCR family members appear to differentially bias local G protein activation on endosomes in a receptor-specific manner (Fig. 7).

Methods
Cell culture and transfections
HEK293 cells were purchased from ATCC (CRL-1573) and cultured in DMEM (Gibco 11965-092) and 10% FBS (R&D Systems, S12495) at 37°C and 5% CO2 in a humid environment. All cell lines used in this study were generated from HEK293 cells, with the exception of the HEK293A parental cells used in Supplementary Figs. 8 and 1041, the HEK293-derived GNAS/GNAL double knockout63 cells used in Supplementary Fig. 5, and the HEK293-derived GNAQ/GNA11 double knockout cells41 used in Supplementary Figs. 8 and 10. Polyclonal cells stably expressing Flag-β2AR34 were cultured in 500μg/mL geneticin (Gibco 10131027), and VIPR1 knockout cells stably expressing tet-inducible (TRE3G) Halo-VIPR141 were cultured in 2μg/mL puromycin. All cell lines were routinely screened for mycoplasma contamination (MycoAlert, Lonza LT07-318). Cells were transfected using Lipofectamine 2000 (ThermoFisher 11668019) according to the manufacturer’s protocol. For experiments in Fig. 5 and Supplementary Fig. 9c, d, cells were transfected with appropriate receptor and nanobit pairs and transduced after four hours with either mCherry or mCherry-Dynamin-1 K44E BacMam diluted in culture media.

DNA constructs and molecular cloning
All DNA constructs used in this study are listed in Supplementary Table 1. Novel constructs were constructed by standard InFusion (Takara Bio) or KLD (NEB) cloning techniques, following the manufacturers’ protocols, and sequences were confirmed by Sanger sequencing. mCherry and mCherry-Dynamin-1 K44E BacMam produced from pCMV-Dest (Thermo Fisher A24223) used in Fig. 5 and Supplementary Fig. 9 were produced according to the manufacturer’s protocol.

Generation of CRISPR KO cell lines
Single guide RNAs (sgRNAs) were designed with the Synthego CRISPR design tool (Supplementary Table 2). To generate ribonucleoproteins (RNPs), 3μL of 53.3μM sgRNA (Synthego) were mixed with 2μL of 40μM Cas9 (UC Berkeley Macrolab) and incubated at room temperature for 10 min. Cells (2.0 × 105) were prepared for electroporation with RNPs with the SF Cell Line 4D Nucleofector kit (Lonza) following the manufacturer’s protocol and electroporated in a 4D Nucleofector (Lonza) using program CM-130. After electroporation, monoclonal cell lines were established using standard techniques and genetic modifications were verified using either Sanger sequencing or next-generation sequencing (Amplicon-EZ, Azenta Life Sciences, see Supplementary Table 3 for NGS primers). For novel Gαs/olf DKO cells, modifications were done sequentially.

Generation of EGFP-Gαs KO cell lines
Gαs KO1 and KO2 cells were transfected with EGFP-Gαs and selected with 500μg/mL geneticin. After selection, cells expressing EGFP-Gαs were sorted into a polyclonal population using a FACSAria Fusion Flow Cytometer (BD Biosciences, KO1) or FACSAria III flow cytometer (BD Biosciences, KO2). Cells were cultured under continued selection.

Live cell confocal microscopy
Confocal imaging was performed using a fully automated Nikon Ti inverted microscope equipped with a CSU-22 spinning disk (Yokogawa), piezostage (MadCityLabs), 4-line Coherent OBIS laser launch (100mW at 405, 488, 561, and 640nM), a quad dichroic 405/491/561/640 (Yokogawa), and corresponding emission filters ET460/50m, ET525/50m, ET610/60m, ET700/75m in a filter wheel controlled by a Lambda 10-3B (Sutter) for channels DAPI/GFP/RFP/Cy5, respectively. Images were captured using an Apo TIRF 100x/1.49 oil objective lens (Nikon) and a Photometrics Evolve Delta EMCCD Camera (154nm/pixel) controlled with Nikon NIS Elements HC v. 5.21.03 software.

For live imaging, cells grown in either 6-well plates or 6 cm dishes were transfected 48 hours before imaging and plated into 35mm glass bottom microscopy dishes (Cellvis D35-20-1.5-N) coated with 0.001% (w/v) poly-L-lysine (Millipore Sigma P8920) 24 hours after transfection. Receptors were surface labeled with either monoclonal anti-Flag M1 antibody (Millipore Sigma F3040) labeled with Alexa Fluor 647 (Thermo Fisher A20186) or 200nM JF635-HTL64 for 10 min at 37°C and 5% CO2. After labeling, cells were washed three times and imaged in imaging media (DMEM (no phenol red, Gibco 31053-028) supplemented with 30mM HEPES pH 7.4) in a temperature- and humidity-controlled chamber at 37°C (OkoLab). Time-lapse images in Fig. 1, Supplementary Fig. 1, Supplementary Fig. 3, and Supplementary Fig. 4 were acquired by imaging cells at 20s intervals for 30 min, and agonist (specified in figure legends) was added after 5 min. In Supplementary Fig. 3a, cells were treated with either Dyngo4a (30μM, Abcam ab120689) or DMSO (0.1%) for 25 min prior to imaging, Alexa Fluor 647 coupled anti-Flag M1 antibody was added for the last 10 minutes of pretreatment, and cells were washed and imaged in imaging media containing either Dyngo4a or DMSO. In Supplementary Fig. 4b, cells were co-treated with 10μM H89 (Cell Signaling Technology 9844) and 200nM JF635-HTL64 (to surface label the receptors) for 10 min at 37°C and 5% CO2, and then washed and imaged in imaging media supplemented with 10μM H89. For time lapse images in Fig. 2 and Supplementary Fig. 2, cells were imaged at 20s intervals for 60min with 100nM Iso added at 5min and 10μΜ Alp added at 15min.

Images were processed for presentation in Fiji v2.1465. Pearson correlation analysis was performed using CellProfiler v4.2.666; briefly, cells were segmented based on the green channel for each frame and Pearson correlation was calculated between channels at each time point. Line scan analysis was performed in Fiji v2.1465; briefly, the fluorescence intensity along the indicated regions was measured and normalized to the average fluorescence intensity along the line and then plotted in GraphPad Prism.

Fixed imaging
Cells were transfected in 6-well plates 48h before fixation. After 24hours, cells were split onto coverslips coated with 0.001% poly-L-lysine (Millipore Sigma P8920) in 12-well plates and grown for an additional 24h. Cells were then surface labeled with monoclonal anti-Flag M1 antibody (Millipore Sigma F3040) labeled with Alexa Fluor 647 (Thermo Fisher A20186) for 10 min at 37°C and 5% CO2. After labeling, cells were washed two times with full media (DMEM + 10% FBS) and treated with either vehicle or Iso (1μM) for an additional 15min at 37°C and 5% CO2. Cells were then placed on ice, washed 1x with DPBS, and fixed at room temperature for 10 min in 3.7% formaldehyde in modified BRB80 (80mM PIPES pH 6.8, 1mM MgCl2, 1mM CaCl2). After fixation, cells were washed three times with DPBS, incubated in TBS for 20min, and washed an additional three times with DPBS. Dapi (1:5000) was included in the final wash. Cells were then mounted in ProLong Gold Antifade mounting medium and left to dry overnight in the dark. Slides were imaged using the confocal microscope described above using a Plan Apo VC 60x/1.4 oil objective lens. Images were processed for presentation using Fiji v2.1465. Line scan analysis was performed in Fiji v2.1465; briefly, the fluorescence intensity along the indicated regions was measured and normalized to the average fluorescence intensity along the line and then plotted in GraphPad Prism.

TIRF microscopy
For live cell TIRF microscopy, cells were imaged using the same methods as for live cell confocal microscopy. Images were acquired on a fully automated inverted Nikon Ti-E microscope controlled by Nikon NIS-Elements software (5.20.00 build 1423), a Nikon motorized stage equipped with a TIRF module with STORM lens (Nikon), Agilent MLC400 (405, 488, 561, 647nm) light source with NIDAQ interface (v18.00), and corresponding emission filters ET455/50m, ET525/50m, ET600/60m, ET705/72m in a filter wheel controlled by a Lambda 10-3B (Sutter) for channels DAPI/GFP/RFP/Cy5, respectively. Images were captured using an Apo TIRF 100x/1.49 objective (Nikon) with an Andor DU897 EMCCD camera and an OkoLab temperature controlled live stage insert. Time lapse images were acquired at 10s intervals for 25min at 37°C with agonist added at 5min.

Images were analyzed and processed for presentation using Fiji v2.1465. To quantify relative changes in surface fluorescence (F/F0), cells were manually segmented by drawing a region of interest (ROI) around the cell surface. The mean, background subtracted, fluorescence intensity (F) was measured at each time point and normalized to the average mean fluorescence intensity before agonist treatment (F0). F/F0 values for each cell within an individual movie (n = 2–6 cells per movie) were then averaged to calculate average F/F0 values for each individual movie. To calculate vehicle subtracted F/F0 values, the F/F0 values of vehicle control movies were averaged at each time point and subtracted from the F/F0 values at each time point for individual movies. For presentation in Fig. 3 and Supplementary Fig. 7, images were scaled to the pre-agonist timepoint and pseudo-colored using the viridis colormap in Fiji to visualize relative changes in fluorescence.

NanoBit luciferase complementation assays
Cells were grown in 6-well plates or 6 cm dishes and transfected with both receptor constructs (Flag-β2AR, Halo-VIPR1, or Halo-A2BR) and the appropriate LgBit- and SmBit-tagged constructs (in Nb37 assays shown in Fig. 6 and Supplementary Fig. 10, the SmBit (101) tag was used, while the SmBit (114) tag was used in all other assays67). After 24h, cells were washed, lifted, spun at 500xg for 3min and resuspended in assay buffer (20mM HEPES pH 7.4, 135mM NaCl, 5mM KCL, 0.4mM MgCl2, 1.8mM CaCl2) with 5μM coelenterazine-H (Research Product International C61500). Cells were plated (100μL) into untreated white 96-well plates (Corning 3912) and incubated at 37°C and 5 % CO2 for either 10 or 30min (for cells pretreated with DMSO (0.1%) or YM-254890 (1μM, Cayman Chemical 29735 or Tocris 7352), as indicated in figure legends) before reading. Luminescence was measured on either a Synergy H4 (BioTek, for data in Fig. 2 and Supplementary Fig. 3) or Spark (Tecan, for all other data) plate reader. For assays in Fig. 2, luminescence was read every 1 minute for a 5-minute baseline, after which either vehicle or Iso (100nM) was added and luminescence read for 10 minutes, followed by vehicle or Alp (10μM) addition and additional luminescence reading for 30 minutes. For assays in Supplementary Fig. 3b, luminescence was read every 1 min for a 5 min baseline, after which either vehicle or Iso (1μM) was added and luminescence read for an additional 30min. For all other assays, luminescence was read every 30s for a 5min baseline, after which vehicle or agonist (noted in figure legends) was added and luminescence measured for an additional 30min. For cells pretreated with DMSO or YM-254890, cells were kept in continual treatment for the duration of the assay.

To analyze data, the change in normalized luminescence was calculated by normalizing each well to its average baseline luminescence. Then, the average change of luminescence of vehicle-treated wells was subtracted from the average change in luminescence of agonist-treated cells. Data are represented as the vehicle subtracted change in luminescence of agonist-treated cells (ΔLum = Lumagonist - Lumvehicle). For endosome NanoBit assays, the first three time points (60s) after agonist addition were not shown, as a small amount (<10%) bleedthrough from the plasma membrane was observed intermittently, which we speculate represents a small amount of sensor mislocalization. This is not consistent with the time course of receptor internalization and does not affect the interpretation of results.

cADDis cAMP assays
Intracellular cAMP levels were measured using either Green Up cADDis cAMP biosensor (Montana Molecular U02006, Supplementary Fig. 5) or Red Up cADDis cAMP biosensor (Montana Molecular U0200R, Supplementary Fig. 6) following the manufacturer’s protocol. Briefly, 50,000 cells per well were plated into TC-treated black 96-well plates (Corning 3340) coated with 0.001% (w/v) poly-L-lysine (Millipore Sigma P8920) and transduced with cADDis BacMam. After 24h, cells were washed with assay buffer (20mM HEPES pH 7.4, 135mM NaCl, 5mM KCL, 0.4mM MgCl2, 1.8mM CaCl2) twice and incubated at 37°C in a temperature controlled plate reader (Tecan Spark for Green cADDis assays or BioTek Synergy H4 for Red cADDis assays). Baseline fluorescence was read with an excitation wavelength at 500nm (Green cADDis) or 558nm (Red cADDis) and emission wavelength at 530nm (Green cADDis) or 603nm (Red cADDis) for 5 min every 30s, after which agonist (noted in figure legends) was added and fluorescence read for an additional 30min. To calculate the change in intracellular cAMP (ΔF/F0), the average baseline fluorescence for each well was calculated (F0) and the change in fluorescence for each well (ΔF = F - F0) was normalized to F0.

Internalization by flow cytometry
VIPR1 knockout cells expressing tet-inducible (TRE3G) Halo-VIPR116 were plated in 6-well plates and then both transduced with either mCherry control or mCherry-Dynamin1 K44E Bacmam and induced with 1μg/mL doxycycline overnight. After 24h, cells were treated with 1μM VIP for 0 or 30min, washed two times with cold PBS-EDTA (UCSF Cell Culture Facility), and then labeled with 200nM JF635-HTL64 for 30min at 4°C. Cells were then washed three times with cold PBS-EDTA, lifted with TrypLE Express (Gibco 12604021), and then surface fluorescence was measured on a Cytoflex (Beckman Coulter) flow cytometer controlled by CytExpert v. 1.3.1.22 (Beckman Coulter). Data were analyzed using FlowJo v. 10.10.0 (B.D. Life Sciences). Populations were gated for cells expressing mCherry, and percent internalization was calculated as ((1-Ft)/Ft0)*100, where Ft represents the median background-subtracted fluorescence intensity at time t. Each biological replicate was calculated as the average of 3 technical replicates.

Immunoblotting
Cells were lysed in RIPA buffer (50mM Tris pH 7.4, 150mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with Roche cOmplete EDTA-free protease inhibitor tablets (Roche 04693159001) and lysate was boiled at 95°C for 5 min in NuPage LDS Sample Buffer (Thermo Fisher, NP0007) and 20mM DTT. For Flag-β2AR blots in Supplementary Fig. 3, lysate was incubated with LDS Sample Buffer and 20mM DTT for 1h at room temperature instead of boiled at 95°C. SDS-PAGE and western blots were performed using standard techniques with polyclonal rabbit anti-Gαs/olf antibody (LS-Bio LS-B4790, 1:1000, blocked in Tris-buffered saline, 5% milk, 0.1% Tween-20), a monoclonal rabbit anti-GAPDH antibody (D16H11, Cell Signaling Technology 5174S, 1:1000, blocked in LI-COR Intercept (TBS) blocking buffer (LI-COR 927-60001)), a monoclonal rabbit βARR1/2 antibody (D24H9, Cell Signaling Technology 4674, 1:1000, blocked in LI-COR Intercept (TBS) blocking buffer), or a monoclonal mouse anti-Flag M1 antibody (Millipore Sigma F3040, 1:1000, blocked in LI-COR Intercept (TBS) blocking buffer), followed by IRDye 800- or 680-linked anti-mouse or anti-rabbit IgG secondary antibodies (LI-COR Biosciences). Blots were imaged using an Odyssey Imager (v. 2.0.3, LI-COR Biosciences) and quantified using Fiji (v2.14).

Statistical analysis and reproducibility
Microscopy quantification data are presented as mean ± S.E.M. of individual dishes from at least three independent experiments, while cAMP and NanoBit data are presented as mean ± S.D. from at least three independent experiments. Each biological replicate in cAMP and NanoBit assays represents the average of at least two technical replicates. All images are representative of at least three biologically independent experiments, except for images in Supplementary Fig. 6c, which are representative of two independent experiments. Statistical tests and area under the curve calculations were performed using GraphPad Prism (v. 9 and v. 10).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript the authors study the movement of Gαs subunits and their activation on endosomes using conformational\nbiosensors. The manuscript is divided into two parts. The first part (Figures 1-3) looks at Gαs translocation to intracellular\nmembranes with an exclusive emphasis on trafficking to endosomes. The authors show that receptor activation at the\nplasma membrane leads to translocation of Gαs, no translocation of a Gγ subunit, and increased colocalization of Gαs with\nEEA1, a marker of early endosomes. They show that this does not depend on receptor endocytosis, is reversible and is\nmore difficult to observe after fixation. One of the main results is that translocation can be observed after activation of\nendogenous receptors, although this translocation is modest and apparently doesn’t result in a detectable increase in Gαs\non endosomes. Some of the methods used here have not been used previously to study this phenomenon, but none of the\nfindings or concepts are new. Early studies by Insel and colleagues in S49 cells relied on endogenous adrenergic receptors\nand demonstrated robust redistribution of Gαs (ref. 25). As the authors point out, the other key results verify findings of\nprevious studies. Just as important, these experiments shed no new light on signaling from endosomes as the present\nmanuscript says nothing about how the translocation phenomenon impacts signaling. Since the authors suggest that\nheterotrimers are required for coupling and apparently only Gαs translocates it is difficult to see how translocation supports\nsignaling.\nThe second part of the manuscript addresses an entirely different question and looks for active Gαs on endosomes using a\npeptide that binds to Gαs-GTP and a nanobody that binds to nucleotide-free Gαs. There was virtually no doubt that this\nhappens given prior functional studies and more direct experiments using Nb37 by the same group; the main novelty here is\nthe use of the KB1691 peptide in an endosome assay and the detection of endogenous Gαs-GTP after activation of\noverexpressed receptors. Differences between VIPR1 and A2BR are nice to see but are not particularly surprising given the\nknown properties of these receptors, and Fig. 6 suggests a quantitative rather than qualitative (as implied in Fig. 7b)\ndifference in how they activate G proteins at different locations. One of the most interesting new findings is robust signals\nfrom both Gαs sensors that persist in Gs/olf DKO cells and are blocked by YM. The authors hesitate to call these signals\nactive Gαq/11, presumably because of prior studies claiming specificity of these probes, but “non-Gs” is rather unsatisfying in\nlight of the documented specificity of the YM compound.\nAll in all, the manuscript contains some interesting results, but the large majority of these confirm previous studies, and the\nconceptual advance is rather limited.\nSpecific comments:\nThe authors claim that endosomal and plasma membrane signals show expected “sequential” kinetics but there are some\nanomalies that are difficult to explain. The time courses of plasma membrane and endosome active Gαs signals in Fig. 5e\nand f, and Fig. 6c are not that different; plasma membrane signals rise for several minutes, and endosome signals are more\nthan 50% of maximum at the first time point after VIP addition, even when endocytosis is inhibited. The time course of active\nreceptor appearance in endosomes is much slower. “Non-Gs” signals seem to be much faster and sometimes larger for\nthese Gs-coupled receptors. At the very least a more careful discussion of these issues is warranted.\nDynamin K44E is only partially effective in Fig. 5. Since the receptors here are overexpressed, these experiments should be\nsupplemented with receptors that are completely defective when it comes to endocytosis, and/or additional more effective\nblockers of receptor endocytosis. In any case the degree to which receptor endocytosis is blocked should be directly\nquantified.\nA related question, why was the β adrenergic receptor ignored for the second half of the manuscript, especially since the 3S\nmutant is in hand?\nThe authors conclude that VIPR1 activates Gq/11 (non-Gs) on the plasma membrane but not on endosomes, which seems\nto be the case in Fig. 4f (KB1691) but less convincingly in Fig. 6c (Nb37). If Gq/11 (non-Gs) is present on endosomes (Fig.\n6d), how does it avoid activation by VIPR1 at this site?\nThe authors conclude in the abstract that activation of endosomal Gαs depends on receptor endocytosis, but in fact they only\nshow that this activation is dependent on dynamin. Could dynamin K44E be inhibiting delivery of the peptide ligand to\nendosome-resident receptors? Experiments with endocytosis-deficient receptors could address this.\nTIRF images show DMSO controls but there are no DMSO traces in the grouped data. Given the small magnitude of\nreceptor-mediated translocation such controls are important in this instance.\nThe endosome time course traces in Figs. 4 and 5 are missing the first two time points after the addition of agonist, whereas\nthe plasma membrane traces show data for these time points. Why?\nPanels 2c and 2d are partially redundant.\nCan the authors provide some explanation for why KB1691 was replaced with Nb37 for the later experiments?\nThe authors should make a consistent distinction between Gs heterotrimers and Gαs subunits. For example, they claim in\nthe abstract that they show that plasma membrane events control endosomal Gs abundance. This should be Gαs.\n“Regulate” is a better description than “control” for how plasma membrane activation affects endosome Gαs abundance, as\nthe authors do not quantify the extent to which total endosome Gαs abundance changes after activation at the plasma\nmembrane.", "reviewer_2": "Wysomerski et al. Comments\nIn this manuscript, Wysolmerski and colleagues present data to propose mechanism by which endosomal G(s) G proteins\nare regulated. As acknowledged by the authors, there is significant accumulated evidence that supports the presence of G\nproteins in endosomes and G protein activation within these intracellular compartments. Here, the authors use very elegant\napproaches to show that i) these G proteins “arrive” at the endosomes without independently of the activating receptor but ii)\nthey require an active internalised receptor to be activated. These results are novel and expand our understanding of\nspatiotemporal GPCR signalling. The therapeutic implications of these findings remain future endeavours, but clearly such\nknowledge will help in that direction. Of note, during this review, a paper from the same author’s lab has been published and\nconceptually both papers seem related. It is suggested the authors emphasise novelty of the present work and relationship\nbetween both reports.\nI have a few questions about the results presented that require consideration prior to publication.\nProposed model of Gs accumulation in endosomes – how are the authors convinced that the Gs accumulated in the\nendosomes is of PM origin? Is it possible that PM signalling from GPCR/Gs triggers a signal that results in redistribution of\nGs from other (non-PM) compartments to accumulate in endosomes? Could the authors perform some tracking of Gs-GFP to\nassess this?\nThe authors suggest that KB1691 recruitment to endosomes, since it is not affected by YM, reflects Gs activation. As they\ndetected a non-Gs component at the PM, one would expect then that this endosomal recruitment of KB1691 is unaffected by\nGs/olf knockout?\nKB1691 and Nb37 revealing a non-Gs components – as far as this reviewer understands, both KB1691 and Nb37 were\noriginally described and demonstrated as selective for Gs G proteins. The results here seem to argue against that selectivity\nand propose that both these sensors can detect a “non-Gs component”. While the data seem to point in that direction, the\narguments for “location bias” seem premature without the understanding of what such “non-Gs” mechanism is. With the\ncurrent justification, this stands out as a major limitation of this work.\nOther comments:\n• While the interpretation of Fig1c is supported by the data, this would be further supported if the authors applied a mask to\nremove the PM region from their Pearson Correlation Coefficient calculation.\n• Supp Fig2 and 3 legends have scale bars in microM rather than microm.\n• As in ref 33, it would be recommended that they authors refer to the current patent on the KB1691 peptides.\n• Fig4 has one panel labelled PM and another Plasma membrane – use one for consistency", "reviewer_3": "Review of “Conformational biosensors delineate endosomal G protein regulation by GPCRs” by Brian Wysolmerski, Emily\nE. Blythe, Mark von Zastrow\nIn their manuscript, Wysolmerski and collaborators shed light on Gs redistribution and activation at the endosome following\nGPCR activation in HEK293 cells. They find that stimulation of endogenous β2AR, VIPR1 or A2BR causes a loss of Gs from\nthe plasma membrane. In the case of β2AR, they investigated this finding further and found that this loss is independent of\nreceptor internalization and that it corresponds to Gs redistribution to endosomes. In the case of VIP1R and A2BR, they\nshow that endosome-localized Gs is active and, only for VIP1R, they show that this activation is dependent on receptor\ninternalization. Finally, they propose a location bias mechanism in G protein activation by both VIPR1 and A2BR, where the\nproportion of Gs and non-Gs activation is both receptor- and compartment-dependent.\nThe results are potentially very interesting for the GPCR and cell biology community, however the manuscript suffers from the\nfollowing limitations:\n- The conclusions are presented as general mechanisms of GPCR functioning, but these are not supported by the data,\nwhich are receptor specific. Moreover, conclusions are based on experiments lacking sufficient number of repeats and from\nqualitative assessments lacking proper quantification.\n- The approaches used are not so novel. Nb37, NanoBit assay, KB1691 have been already described and used in several\noccasions (mentioned in the text by the authors). However, Nb37 and KB1691 here are used in a clever way to distinguish\nbetween GPCR-bound G protein versus active (=GTP bound) G protein. Similarly, the NanoBit assay was adapted to these\nspecific questions.\n- I don’t see the relevance of the finding that G proteins are redistributed to endosomes independently of receptor\nendocytosis. What does it mean physiologically speaking? What is the function of Gs redistribution to the endosome in\nabsence of receptor?\n- The finding that G proteins are active at the endosome only when the receptor is also there is somehow reassuring, but\nexpected I would say.\n- No downstream effect of location bias in G protein activation by VIPR1 and A2BR has been assessed.\n- No information on how G proteins and receptors couple at the endosome is provided. Do they traffic together? Is the\nreceptor internalized first and then the G protein recruited from the cytoplasm? Is through fusion of 2 endosomal\ncompartments (one containing the receptor and one showing G protein presence)?\n- Only G protein redistribution has been shown to happen at native levels of receptor, not G protein activation. The authors\nshould reconciliate these findings.\nApart from these concerns, please see below for other comments that should be addressed before the manuscript is suitable\nfor publication:\n1. p.3 “However, the presence of active-state Gαs on endosomes has only been hypothesized.” I guess the authors mean\nGTP-bound G protein. I this needs to be clearly stated as it can be perceived that all the work done by von Zastrow’s lab and\nothers using MiniG proteins and Nb37 does not point in that direction.\n2. p.3 “significantly differ in their ability to internalize after activation.” Is this a finding from this study or something already\nknown? If known, please add refs, if new… I don’t see a characterization of the 3 receptors internalization in this paper,\nunless I missed it.\n3. In Fig. 1, colocalization between G  and EEA1 is missing. Fig.1d is only representative/qualitative but no quantification is\nshown for Gαs G  colocalization at the EE.\n4. Also in Fig. 1 I’m surprised not to see G  at the PM in unstimulated cells. I was expecting complete colocalization with Gs.\nCould the authors comment on this?\n5. Could the experiment performed in Fig. 2 be done in a complementary way using LgBit-Gs and SmBit-endofin, to look at\nredistribution of Gs to and from the endosome upon Iso or Alp addition?\n6. In Fig. 2e, redistribution of Gs from the endosome to the membrane upon Alp addition is observed, even in absence of\nreceptor internalization. It would be great to hear what the authors think about this finding, if they could speculate on how Gs\ncan sense from the endosome the presence of an antagonist acting on receptors blocked at the plasma membrane.\n7. Why did the authors choose to block endocytosis of VIPR1 by overexpressing the dominant negative mutant of dynamin\nDyn1-K44E instead of using Dyngo-4a as it was done for β2AR?\n8. Please add quantification for confocal images shown in Supp. Fig. 1, 2, 3 and 4. The analysis should be conducted on a\nbigger sample, not just 4 cells, from more than 2 independent experiments.\n9. More repeats should be included for experiments that were repeated only twice. 4 cells are not sufficient. I suggest\nanalyzing 5 cells per repeat, for a total of 15 cells.\n10. There is a clear imbalance between the first part of the manuscript where all experiments are conducted using β2AR as\nmodel GPCR and mainly live-imaging techniques, and the second part of the paper where experiments are focused on\nVIP1R and A2BR using the NanoBit assay.\nSpecifically, the authors show that Gs redistribution to the endosomes is independent of receptor endocytosis only for β2AR.\nIt would be informative to know whether this is also true for other Gs coupled GPCRs such as VIPR1 and A2BR.\nSimilarly, was the NanoBit assay run for β2AR with either KB1691 or Nb37? The manuscript would benefit from adding\nthese data.\nFinally, since KB1691 is conveniently tagged with mApple, it would be good to include confocal images of KB1691\nlocalization before and after stimulation with either VIP or NECA (+ localization markers for PM and endosomes).\n11. In Fig. 7a, the authors talk about a general mechanism, but they demonstrate that redistribution of Gs to endomembranes\nis independent of receptor endocytosis only for β2AR. Furthermore, the representative GPCR is in purple, which in 7b\nrepresents VIPR1, for which receptor independent Gs redistribution to endosomes has not been shown. The scheme is\nconfusing from different aspects and I recommend changing it to reflect the findings described in the manuscript.\nMinor comments:\n12. Since in some experiments proteins are overexpressed and in some other experiments not, it would be cleared to add on\ncertain figure panel (e.g. Fig 1a) which tag was used, so it’s clear when proteins where overexpressed (e.g. Flag-b2AR, Gs-\nEGFP, etc).\n13. It is sometimes difficult to understand when the authors mean G protein activation or G protein-GPCR coupling. From\nwhat I read, G protein activation is monitored with KB1691, while G protein-GPCR coupling with Nb37. If that’s correct, I\nsuggest changing Supp. Figure 9 graph headers to (G protein-GPCR coupling, Nb37).\n14. Supp fig. 2a please add single channel images in black and white.\n15. The quality of some images is quite poor. Would it be possible to replace images in Fig. 1d +iso 20’ and Supp Fig. 4b\nwith better ones?\n16. p.9 “Signaling from endosomes requires Gαs associated with the endosome membrane to be in an active state.” Please\nadd a reference to this sentence.\n17. Personally, I do not like the word “abundance” used throughout the text, as to me, it implicates some quantitative\nmeaning, while here is it means purely presence of Gs. If the authors agree with my logic, could this word be changed to\n“presence” or “redistribution”?", "reviewer_4": "In this study by Wysolmerski et al., the authors reexamine previous findings related to Gs trafficking and endosomal activity\nand provide the first results exploring endosomal signaling with the new Gαs-GTP sensor from the Garcia-Marcos lab. These\nresults are timely because researchers studying location bias and intracellular signaling have surely thought about adapting\nthis biosensor to fit their research questions since its publication last year. It is certainly exciting to see that this biosensor,\nwhen adapted to measure endosomal signaling responses, can detect robust signals at endogenous G protein levels.\nHowever, the authors present some curious findings related to the specificity of two biosensors at the center of their study\n(KB1691 and Nb37). It feels as though they are concluding that the detection modules are not selective for Gαs while not\nruling out the possibility that other types of G protein activation may affect the concentration of components on endosomal\nmembranes. This distinction is important because the authors conclude that there is a non-Gs component being activated at\nendosomes for receptors like A2BR when it may be that Gs is indeed activated at endosomes, but Gq/11 activity at the\nplasma membrane modulates the possibility for Gs to be activated subsequently at endosomes. My suggestions can be\nfound below:\n1. YM acts as a molecular glue keeping Gαq and Gβγ together as an inactive heterotrimer (Mühle et al., PNAS, 2025). Can\nthe authors rule out that YM treatment doesn’t sequester Gβγ from Gαs? Have the authors performed the same experiments\nin Gαq/11KO cells? In a similar study from Vilardaga and colleagues (White et al., PNAS, 2020), Gαq/11 knockout cells were\nused and the authors observed a decrease in endosomal cAMP generation which they proposed was dependent on the\nliberation of Gβγ and PIP3 production to promote GPCR-arrestin binding (in this instance PTHR) and internalization. In the\nCell paper from Garcia-Marcos and colleagues where KB1691 was described for the first time (Janicot et al., Cell, 2024), I do\nnot think any Gq/11 inhibitors or knockout lines were used in the context of this sensor; their experiments to confirm G protein\nselectivity were based on representative tagged G proteins from the four major families and only tested PTX and YM on\nGi/o and Gq/11 respectively. I think that the authors from the current study need to provide more convincing evidence that this\nsensor is detecting something it is not, rather than the consequence of events that preceded downstream signaling at\nendosomes. For example, it could still be that KB1691 and Nb37 are Gαs-selective, but that endosomal Gαs activation is\naffected by Gq/11 activity at the plasma membrane – something that may be receptor-specific.\n2. While the results with VIPR and DynK44E suggest that endosomal production of active state Gαs is endocytosis-\ndependent, there could be other factors that regulate the presence of Gαs-GTP at endosomes like phosphodiesterases, RGS\nproteins and intrinsic GTPase activity. In other words, if VIPR internalization could be blocked completely, would we still\nobserve residual endosomal signaling and what could that be due to? Consider other receptors that have a greater\ndependence on arrestin/CME like V2R. Does this sensor have the dynamic range to measure the shaping of endosomal\nresponses by other proteins like PDEs and intracellular RGS proteins?\n3. Fig. 1 Gγ2 is geranylgeranylated and does not leave the membrane as readily as other gamma subunits (Masuho et al.,\nCell Systems, 2021). While there is a clear intracellular localization of Gγ2, the conclusion that this population of Gβγ\nsupports endosomal signaling is probably isoform-specific. In fact, it has also been proposed that Gβγ dimers internalized\nwith β-arrestin-bound receptors can enable a second round of Gαs activation for select receptors (Sokrat et al.,\nCommunications Biology, 2024). Given these previous findings, I suggest that the authors either broaden their conclusion\nabout how agonist-induced activation happens at endosomes or perform additional G gamma experiments with other\nsubtypes or receptors.\n4. Fig. 1C label EGFP as control or cytosolic EGFP. Same for Fig.5 and mCherry.\n5. It is unclear how subpanel 2C differs from the wildtype response in panel 2D. I would suggest reorganizing the subpanels\nto avoid duplication of results within the same figure because it is essentially the same experiment for WT in subpanels 2C\nand 2D."}, "author_response": "We thank the reviewer for the thoughtful comments. We agree that the first part of the manuscript is not conceptually new, and we don’t\nmean to imply otherwise. Our intention in this part is to place the work in context with present models in the field. We focus here on the\nβ2 adrenergic receptor because this is the GPCR that has been traditionally used for such studies. We then extend the present\nframework in 2 ways. First, we show a similar process of Gαs redistribution stimulated by two other Gs-coupled GPCRs. Second, we\ndemonstrate that these GPCRs, as well as with the β2 adrenergic receptor, are able to stimulate a rapid (within minutes) redistribution\nof Gαs from the plasma membrane at endogenous levels of receptor expression. We agree that the Insel group (Ransnas et al, cited)\npreviously provided biochemical evidence for endogenous Gαs redistribution stimulated by endogenous β2AR, but note that this\nrequired >1 hour of continuous agonist treatment while we show redistribution within several minutes. Thus it remains unclear whether\nor how those results would pertain to signaling (or perhaps they were detecting something else) but we simply cite without further\ndiscussion because we want to remain focused on redistribution occurring over a signaling-relevant time scale. We believe the present\nresults are the first to show this at endogenous protein levels.\nThe reviewer raised a concern about the degree of conceptual advance also in the second part of the study, which focuses on G protein\nactivation. We believe that there is considerable advance here and, in fact, that most of what we show in the second part is new. We do\nagree that the present model holds that Gs activation on endosomes requires a second coupling reaction on the limiting membrane,\nand that Nb37 recruitment to endosomes supports this model. We claim two key elements of novelty: First, to our knowledge, the\naccumulation of active-state Gαs on endosomes has not previously been demonstrated. We show this in the present study, and at\nendogenous levels of G protein expression. We think this is significant because G protein coupling and the accumulation of active-state\nGα subunits on a membrane are mechanistically distinct processes. Second, we demonstrate location bias in G protein coupling on\nendosomes relative to the plasma membrane. We agree this is perhaps the most novel and interesting aspect of the work, as the\nreviewer notes, and we have significantly strengthened this part in the revised manuscript.\nWe detail our responses and changes made in the revision below, in line with specific critiques raised by the reviewer.\nSpecific comments:\nThe authors claim that endosomal and plasma membrane signals show expected “sequential” kinetics but there are some\nanomalies that are difficult to explain. The time courses of plasma membrane and endosome active Gαs signals in Fig. 5e\nand f, and Fig. 6c are not that different; plasma membrane signals rise for several minutes, and endosome signals are more\nthan 50% of maximum at the first time point after VIP addition, even when endocytosis is inhibited. The time course of active\nreceptor appearance in endosomes is much slower. “Non-Gs” signals seem to be much faster and sometimes larger for\nthese Gs-coupled receptors. At the very least a more careful discussion of these issues is warranted.\nThis is a good point and important to clarify. Yes, we do think that the kinetics are different. The active-state Gαs (KB1691)\nincrease at the plasma membrane (Fig 5e) peaks before 10 minutes, but the endosome elevation (Fig 5f) reaches its maximum at about 15 min\nafter agonist application. We agree that active-state Gαs accumulation on the endosomes appears to be faster than the endosomal\naccumulation of active-state VIPR1 (mGs), but there is clearly some active-state receptor in the endosome at the time that we see\nactive-state G protein accumulation. Thus we speculate that this reflects only a fraction of the receptor pool being sufficient to activate\nthe endosomal complement of Gs. We are detecting active-state Gαs at endogenous levels, but need to over-express VIPR1 in order to\nsee this, so we think that this is plausible. Nevertheless, there is still a kinetic shift consistent with sequential activation at each\nmembrane location as proposed. We note and briefly discuss this point in the revised manuscript (p. 12 bottom).\nWe fully agree that the non-Gs component detected at the plasma membrane is indeed faster, and its magnitude is considerable. We\nfind this observation very interesting and note that it would be impossible to detect without the new methodology described in our study.\nAs the reviewer notes, we used the term ‘non-Gs’ rather than ‘Gq/11’ in the submitted manuscript. The reason is that we focused\nprimarily on detecting active Gαs and, at that time of submission, were not satisfied that we had fully defined the non-Gs component.\nThe simplest interpretation, as the reviewer notes, is that it represents Gq/11 based on its inhibition by YM. We have now verified this\nconclusion in two ways. First, we have added new data demonstrating a specific loss of the non-Gs component in Gq/11 DKO cells\n(Supplementary Figs. 8 and 10). Second, we have added new data using an established active-state Gαq/11 biosensor (p63RhoGEF,\nSupplementary Fig. 8), showing that this sensor detects the rapid component of activation consistent with what we previously defined\nas non-Gs, and this is lost on Gq/11 KO cells. In the revised manuscript we have included these new data as indicated, and have\nchanged to calling this component Gq/11 accordingly.\nDynamin K44E is only partially effective in Fig. 5. Since the receptors here are overexpressed, these experiments should be\nsupplemented with receptors that are completely defective when it comes to endocytosis, and/or additional more effective\nblockers of receptor endocytosis. In any case the degree to which receptor endocytosis is blocked should be directly\nquantified.\nA related question, why was the β adrenergic receptor ignored for the second half of the manuscript, especially since the 3S\nmutant is in hand?\nWe were also surprised that mutant dynamin, while it significantly inhibits both endosomal recruitment of mGs and KB1691, does so\nonly partially for both sensors. The virally expressed K44 mutant dynamin indeed inhibits VIPR1 internalization strongly. We published\nthis previously (Blythe 2024, as cited) and used the same strategy in the present study, but the reviewer is correct that we did not\ninclude data verifying this in the initial submission. In the revised manuscript, we have included flow cytometry data to verify this point\n(Supplementary Fig. 9a). Still, we find only a partial inhibition of mGs and KB1691 recruitment to endosomes as shown. We speculate\nthat this is because of receptor overexpression and that endocytosis concentrates receptors relative to the concentration in the plasma\nmembrane, such that only a small fraction of the overexpressed receptor complement is needed to produce detectable recruitment\nsignal measured by the biosensors. We believe this is the most parsimonious interpretation and are confident that there is significant\nendocytosis-dependence as claimed. In the revised text (Results section, p.13 bottom of page) we note and discuss this point.\nWe did not mean to ignore the β adrenergic receptor and, as noted above, we focused on this GPCR in the first part of the paper\nbecause it is the GPCR traditionally used for studies of Gαs redistribution. We focused on VIPR1 in the second part of the paper for two\nreasons. First, VIPR1 produces a stronger cAMP response after endocytosis (Blythe et al 2025 as cited). Second, we find VIPR1 more\ninteresting in the context of the G protein activation process because it can stimulate production of both active-state Gαq and\nactive-state Gαs. We have no evidence for such dual activation by β2AR and no evidence that the β2AR exhibits location bias at\nendosomes.\nRegarding the phospho-mutant receptors, in our hands we find that K44 mutant dynamin inhibits β2AR internalization more strongly\nthan the 3S mutation. We have so far been unable to identify a functional VIPR1 mutant that is endocytosis-deficient using a similar\nSer/Thr mutation strategy, and we also cannot use β-arr KO cells because VIPR1 internalization is arrestin-independent. For these\nreasons, we focused in the present study on the use of K44 mutant dynamin to inhibit receptor internalization.\nThe authors conclude that VIPR1 activates Gq/11 (non-Gs) on the plasma membrane but not on endosomes, which seems to be the\ncase in Fig. 4f (KB1691) but less convincingly in Fig. 6c (Nb37). If Gq/11 (non-Gs) is present on endosomes (Fig. 6d), how does it avoid\nactivation by VIPR1 at this site?\nThis is an excellent question, and we presently don’t know why VIPR1 fails to detectably couple to Gq/11 on endosomes. We are\nconfident in the biosensor data demonstrating the existence of this additional activation component on the plasma membrane. We\nspeculate that this may have to do with different lipid or protein compositions between the membranes, and this would be potentially in\nline with differences in G protein membrane binding properties shown years ago by the Wedegaertner lab. However, we have tested\nthis hypothesis in the present and believe that doing so would be beyond the realistic scope of the present study. In the present study,\nwe focus specifically on establishing the existence of this second component of G protein activation and its location-specificity.\nThe authors conclude in the abstract that activation of endosomal Gαs depends on receptor endocytosis, but in fact they only show that\nthis activation is dependent on dynamin. Could dynamin K44E be inhibiting delivery of the peptide ligand to\nendosome-resident receptors? Experiments with endocytosis-deficient receptors could address this.\nWe think this is unlikely because mutant dynamin expression does not block pinocytic delivery of fluid-phase markers to endosomes,\nand there is evidence that it may actually increase flux through this non-clathrin route (PMID 7559787). However, if it were the case that\nK44 inhibited peptide delivery, it would still be evidence for a role of receptor activation in endosomes. We agree that one could\ninvestigate this in more detail with endocytosis-mutant receptors but, as noted above, we have so far not successfully identified such a\nmutation for VIPR1.\nTIRF images show DMSO controls but there are no DMSO traces in the grouped data. Given the small magnitude of\nreceptor-mediated translocation such controls are important in this instance.\nThis is a good point. We are confident that the observed magnitudes of agonist-induced effects are significant but, in the initial\nsubmission, we showed only vehicle-subtracted curves because we thought it made the figure less ‘busy’. However, we agree that this\nis a plausible alternate interpretation in retrospect and, In the revised manuscript, we plot non-subtracted curves and include the vehicle\ncontrol as the reviewer suggests (Fig. 3 and Supplementary Fig. 7).\nThe endosome time course traces in Figs. 4 and 5 are missing the first two time points after the addition of agonist, whereas the plasma\nmembrane traces show data for these time points. Why?\nThis is correct. We systematically excluded the first three time points in the endosome nanobit data, corresponding to the first minute\nafter agonist application. The reason is that we occasionally see a small increase in the KB signal at these very early time points. We\nbelieve this cannot be from true accumulation in endosomes because it is seen in the first minute, and that it reflects either a small\namount of the FYVE domain (endofin) LgBit construct being mis-targeted to the PM or present on a very early endocytic intermediate\ncompartment. We do not see this consistently, only in some experiments, and thus favor a small amount of mis-targeting. We show\nrepresentative examples below, with all of the data points included in each case. In the left panel one sees no evidence of a small early\nelevation, but in the right panel there is a small blip. This observation is 1) sporadic, 2) very low in amplitude and 3) evident when very\nlittle receptor is present in endosomes. Therefore we do not think this is biologically significant and, even if it were, our interpretation of\nthe slower (consistent with endocytic dynamics) recruitment to endosomes would not change. In the revised manuscript, we have\nadded a brief explanation of this point in the Methods section (p. 25 middle of page).\nPanels 2c and 2d are partially redundant.\nThe data graphed in each panel were indeed different (corresponding to the same control condition across panels), but we agree they\nshow essentially the same thing. We have removed panel 2C in the revised manuscript for clarity.\nCan the authors provide some explanation for why KB1691 was replaced with Nb37 for the later experiments?\nWe believe that both sensors are important but that each senses something different. KB1691 binds selectively to active-state\n(GTP-bound) α-subunits (as demonstrated in Janicot et al. 2024, as cited) but Nb37 binds selectively to a nucleotide-free α-subunit\nconformation corresponding to a catalytic intermediate in the process of GPCR-mediated G protein activation (Westfield et al. 2011 and\nIrannejad et al. 2013, as cited). Thus we interpret KB1691 recruitment as a measure of active-state α-subunit accumulation on the\ntarget membrane and Nb37 recruitment as a measure of the coupling step that mediates G protein activation. The results obtained with\neach are consistent, and comport with our present understanding of how G protein activation occurs, but we believe that each\nbiosensor provides useful and distinct information. In the revised text we attempt to clarify this point with more precise language\nthroughout the Results section, and we have added text to explicitly note the difference in our interpretations of KB1691 and Nb37\nrecruitment results (p. 14 middle of page)\nThe authors should make a consistent distinction between Gs heterotrimers and Gαs subunits. For example, they claim in the abstract\nthat they show that plasma membrane events control endosomal Gs abundance. This should be Gαs.\nWe thank the reviewer for noting these inconsistencies and agree that this is compromised precision. In the revised manuscript, we\nhave corrected this and used consistent nomenclature throughout the revised manuscript.\n“Regulate” is a better description than “control” for how plasma membrane activation affects endosome Gαs abundance, as the authors\ndo not quantify the extent to which total endosome Gαs abundance changes after activation at the plasma membrane.\nThis is a good point. We have switched to this terminology throughout the revised manuscript.\nReviewer #2 (Remarks to the Author):\nWysomerski et al. Comments\nIn this manuscript, Wysolmerski and colleagues present data to propose mechanism by which endosomal G(s) G proteins are\nregulated. As acknowledged by the authors, there is significant accumulated evidence that supports the presence of G proteins in\nendosomes and G protein activation within these intracellular compartments. Here, the authors use very elegant approaches to show\nthat i) these G proteins “arrive” at the endosomes without independently of the activating receptor but ii) they require an active\ninternalised receptor to be activated. These results are novel and expand our understanding of spatiotemporal GPCR signalling. The\ntherapeutic implications of these findings remain future endeavours, but clearly such knowledge will help in that direction. Of note,\nduring this review, a paper from the same author’s lab has been published and conceptually both papers seem related. It is suggested\nthe authors emphasise novelty of the present work and relationship between both reports.\nWe are pleased that the reviewer appreciated our experimental approach and found our results generally interesting. The reviewer is\ncorrect that we reported data on Gαi/o subunit redistribution recently, and we cited this in the initial submission. We think that the\nproperties of Gαs are quite different. A key difference is that, in our hands, endosomal accumulation of active-state Gαi/o does not\ndepend on endocytosis of the activating GPCR whatsoever. We have emphasized this significant distinction in the revised Discussion\nsection (p.19 middle of page).\nI have a few questions about the results presented that require consideration prior to publication. Proposed model of Gs accumulation\nin endosomes – how are the authors convinced that the Gs accumulated in the endosomes is of PM origin? Is it possible that PM\nsignalling from GPCR/Gs triggers a signal that results in redistribution of Gs from other (non-PM) compartments to accumulate in\nendosomes? Could the authors perform some tracking of Gs-GFP to assess this?\nThis is an excellent question and we agree that it is possible, in principle, that the Gαs is coming from somewhere else. An ideal\napproach, as the reviewer suggests, would be to label Gαs at the plasma membrane and dynamically track it by imaging. We do this to\nfollow receptors in cells, but we have not successfully achieved this with Gα subunits. As an alternative approach to test this\nhypothesis, we have carried out additional experiments using a mutant Gαs that has been shown previously to be N-terminally\nmyristoylated and thereby stably tethered to the plasma membrane (Fig. 1 and Supplementary Fig. 1). Wedegaertner’s group (cited)\nshowed that this modification prevents agonist-induced redistribution of the α subunit to the cytoplasm in fixed preparations. Using live\nimaging, we verify this and also show that the accumulation of α subunits on endomembranes (including endosomes) is greatly\nreduced. These data support the hypothesis that endosomal Gαs originates from the plasma membrane, but clearly much remains to\nbe learned. These new data are included in Fig. 1c and Supp. Fig. 1a of the revised manuscript, and we have further commented on\nthis important point (adding a new paragraph) in the revised text (p. 6).\nThe authors suggest that KB1691 recruitment to endosomes, since it is not affected by YM, reflects Gs activation. As they\ndetected a non-Gs component at the PM, one would expect then that this endosomal recruitment of KB1691 is unaffected by\nGs/olf knockout?\nThe reviewer is correct that we detected a non-Gs component of KB1691 recruitment at the plasma membrane, and that we defined\nthis as such because it is unaffected by Gαs/olf knockout. The reviewer is also correct that KB1691 recruitment to endosomes is\ncompletely lost in Gαs/olf knockout cells. This is shown in Fig 4f and we have modified the text in the revised Results section to be\nmore clear on this important point.\nKB1691 and Nb37 revealing a non-Gs components – as far as this reviewer understands, both KB1691 and Nb37 were\noriginally described and demonstrated as selective for Gs G proteins. The results here seem to argue against that selectivity\nand propose that both these sensors can detect a “non-Gs component”. While the data seem to point in that direction, the\narguments for “location bias” seem premature without the understanding of what such “non-Gs” mechanism is. With the\ncurrent justification, this stands out as a major limitation of this work.\nWe agree that this was a significant limitation in the study as initially submitted. We were also surprised that a non-Gs component was\ndetected (and by both sensors), but were careful to use the term ‘non-Gs’ rather than ‘Gq/11’ in the submitted manuscript because, at\nthe time, we only had pharmacological support (YM) for this conclusion. To address this concern we have carried out additional\nexperiments to more fully define the non-Gs component. First, we now demonstrate a specific loss of the non-Gs component in Gq/11\nDKO cells, providing genetic evidence for supporting its definition as Gq/11. Second, we have adapted an established active-state\nGαq/11 biosensor (derived from p63RhoGEF) and show that this sensor indeed detects the non-Gs component with similar kinetics.\nTherefore we are now confident the non-Gs component represents Gαq/11, and we have revised the manuscript accordingly.\nOther comments:\n• While the interpretation of Fig1c is supported by the data, this would be further supported if the authors applied a mask to remove the\nPM region from their Pearson Correlation Coefficient calculation.\nWe agree that this would demonstrate a positive change in the Pearson coefficient of accumulation on receptor-containing endosomes\nas is evident in the individual images. We prefer not to do this, however, because the endosomal colocalization is visually evident in\nagonist-treated cells and we are concerned that removing the plasma membrane from the region analyzed might introduce unwanted\nbias to the analysis. We also find it interesting that the Pearson coefficient goes down to an intermediate level after agonist application,\nand we think that this is consistent with the visual evidence that the redistributed Gαs, while it clearly localizes to receptor-containing\nendosomes, does not localize there exclusively. Nevertheless, we agree that it would be helpful to provide explicit evidence for an\nincrease in localization at endosomes To address this, in the revised manuscript we include additional data showing colocalization of\nβ2AR, Gαs and labeled Gβγ on endosomes quantified with line scans (Supplementary Fig. 1c).\n• Supp Fig2 and 3 legends have scale bars in microM rather than microm.\n• As in ref 33, it would be recommended that they authors refer to the current patent on the KB1691 peptides.\n• Fig4 has one panel labelled PM and another Plasma membrane – use one for consistency\nWe thank the reviewer for noting these errors and have corrected them in the revised manuscript.\nReviewer #3 (Remarks to the Author):\nReview of “Conformational biosensors delineate endosomal G protein regulation by GPCRs” by Brian Wysolmerski, Emily E. Blythe,\nMark von Zastrow\nIn their manuscript, Wysolmerski and collaborators shed light on Gs redistribution and activation at the endosome following\nGPCR activation in HEK293 cells. They find that stimulation of endogenous β2AR, VIPR1 or A2BR causes a loss of Gs from the plasma\nmembrane. In the case of β2AR, they investigated this finding further and found that this loss is independent of receptor internalization\nand that it corresponds to Gs redistribution to endosomes. In the case of VIP1R and A2BR, they show that endosome-localized Gs is\nactive and, only for VIP1R, they show that this activation is dependent on receptor internalization. Finally, they propose a location bias\nmechanism in G protein activation by both VIPR1 and A2BR, where the proportion of Gs and non-Gs activation is both receptor- and\ncompartment-dependent.\nThe results are potentially very interesting for the GPCR and cell biology community, however the manuscript suffers from the following\nlimitations:\nWe are pleased that the reviewer found our results potentially very interesting, and we have done our best to address the concerns\nnoted.\n- The conclusions are presented as general mechanisms of GPCR functioning, but these are not supported by the data, which are\nreceptor specific. Moreover, conclusions are based on experiments lacking sufficient number of repeats and from qualitative\nassessments lacking proper quantification.\nWe agree that we cannot claim a general mechanism for all GPCRs, and that this is not supported by our data. In fact, our data show\nthat Gαs redistribution appears rather general but that there are differences among receptors in their coupling at different membranes.\nWe also acknowledge that some of the supplemental data were only n = 2. We have corrected this in the revised manuscript and added\nquantification in all, as suggested.\nThe approaches used are not so novel. Nb37, NanoBit assay, KB1691 have been already described and used in several occasions\n(mentioned in the text by the authors). However, Nb37 and KB1691 here are used in a clever way to distinguish between GPCR-bound\nG protein versus active (=GTP bound) G protein. Similarly, the NanoBit assay was adapted to these specific questions.\nWe are pleased that the reviewer found our adaptation of the biosensor approach interesting, and agree that the basic elements were\npreviously established (we specifically state this and include relevant citations). The important new aspects are 1) location-specific\ndetection, 2) detection of G protein activation and active-state Gα accumulation at endogenous expression levels, and 3) detection of\nlocation-biased coupling. To our knowledge none of this has been established previously.\n- I don’t see the relevance of the finding that G proteins are redistributed to endosomes independently of receptor endocytosis. What\ndoes it mean physiologically speaking? What is the function of Gs redistribution to the endosome in absence of receptor?\nWe agree that the ability of Gαs to redistribute without endocytosis of the activating receptor is interesting, but also note that this has\nbeen described previously. Our results verify and support this conclusion using new methods, relative to earlier reports. We think it\nimportant to establish this in our system because the production of active-state Gαs on endosomes differs in being\nendocytosis-dependent (Fig. 5). The functional significance of Gαs redistributing separately from activation is an excellent question that\nour study does not address. Our study focuses on establishing the difference that the reviewer notes, and revealing the additional and\nunanticipated selectivity in active-state Gα production. One can speculate that Gαs redistribution without receptors might have indirect\neffects by changing availability to other GPCRs, but we presently have no data to support or refute this. In the revised manuscript,\nwe have tried to clarify the focus of the present work and note that the functional significance of the distinction that the reviewer notes\nremains to be determined (p.18).\n- The finding that G proteins are active at the endosome only when the receptor is also there is somehow reassuring, but\nexpected I would say.\nWe agree, although note that there is evidence for an additional mechanism of activation that does not appear to work as expected,\nfor\nGi/o-class G proteins (Fisher et al. 2025, as cited), so we think it is worthwhile establishing that Gs indeed appears to work as\nexpected.\n- No downstream effect of location bias in G protein activation by VIPR1 and A2BR has been assessed.\nThis an excellent question that we have not yet addressed. Our goal in the present work is to establish the location of G protein\nactivation by these GPCRs and we believe the new data included in the revised manuscript help establish the existence of this location\nbias. However, we do not yet know the effect(s) of this bias on downstream signaling. We have explicitly noted this point in the revised\nDiscussion section (p.19 top of page).\n- No information on how G proteins and receptors couple at the endosome is provided. Do they traffic together? Is the\nreceptor internalized first and then the G protein recruited from the cytoplasm? Is through fusion of 2 endosomal\ncompartments (one containing the receptor and one showing G protein presence)?\nWe do not think that Gαs traffics together with the activated receptor. We show evidence supporting this conclusion (Fig. 2 and\nSupplementary Fig. 3) and also cite previous work from others consistent with it. We believe that the membrane trafficking model\nsuggested by the reviewer is plausible, but another plausible model is that Gαs partitions in and out of membranes to dynamically\nsample intracellular compartments after activation (this is a model supported also by Martin and Lambert 2016 as cited). Our results do\nnot distinguish these possibilities. Rather, our study focuses on 1) demonstrating that multiple Gs-coupled GPCRs are capable of\nstimulating dynamic redistribution of Gαs, 2) that they can do so at endogenous levels of receptor and G protein expression, and then\n3) on probing the subcellular locations of Gs activation and membrane accumulation of active-state Gαs. In the revised manuscript we\nhave attempted to better clarify this focus, and note in the discussion that there remain significant unresolved aspects of Gαs trafficking\n(p. 18).\n- Only G protein redistribution has been shown to happen at native levels of receptor, not G protein activation. The authors should\nreconciliate these findings.\nThis is correct. Our biosensor strategy is capable of detecting G protein activation and active-state α-subunit accumulation at native\nlevels of G protein expression, but we have so far been unable to detect this after activation of an endogenously expressed GPCR.\nThis is the case even for activation at the plasma membrane, so we believe that it reflects a limitation in sensitivity of our probes. A main\nnew advance in the present study is detecting G protein activation in a spatially resolved manner (at endogenous G proteins but\nadmittedly overexpressed GPCR), although we agree that it is important to continue working toward achieving such detection in a fully\nendogenous system. In the revised manuscript, we attempt to reconcile and better clarify this issue by emphasizing that the present\nneed for receptor overexpression , despite clear evidence (in previous work from others and us) that receptor signaling from multiple\nmembrane locations indeed does occur (starting on p. 11 middle of page).\nApart from these concerns, please see below for other comments that should be addressed before the manuscript is suitable for\npublication:\n1. p.3 “However, the presence of active-state Gαs on endosomes has only been hypothesized.” I guess the authors mean\nGTP-bound G protein. I this needs to be clearly stated as it can be perceived that all the work done by von Zastrow’s lab and\nothers using MiniG proteins and Nb37 does not point in that direction.\nWe agree that the evidence strongly points in this direction, and that our wording in retrospect was a bit strong. Indeed we and others\nhave detected membrane recruitment of Nb37 previously. We presently understand this probe to detect an intermediate in the process\nof Gαs activation (binds selectivity to nucleotide-free state), so we consider Nb37 to represent a sensor of coupling rather than\nactive-state Gαs (Nb37 binds poorly to the GTP-bound Gαs). We think KB1691 is useful because this probe has been shown to\nspecifically detect the true active-state (GTP-bound) Gαs. In the revised manuscript, we have clarified this distinction between the\nprobes (p.14 middle of page).\n2. p.3 “significantly differ in their ability to internalize after activation.” Is this a finding from this study or something already\nknown? If known, please add refs, if new… I don’t see a characterization of the 3 receptors internalization in this paper,\nunless I missed it.\nWe thank the reviewer for noting this. β2AR is well known to internalize via clathrin-coated pits and in an arrestin-dependent manner.\nWe recently reported that VIPR1 internalizes even more rapidly, also via clathrin-coated pits, but in an arrestin-independent manner\n(Blythe et al 2024 as cited). The lack of detectable internalization of A R is demonstrated in another manuscript that is presently in\n2B\nrevision, and we cited it as the Blythe et al BioRxiv posting. We do not claim an absolute lack of receptor endocytosis and, in the\nrevised manuscript, we include an additional image (Supplementary Fig. 4b) showing that human A R remains largely in the plasma\n2B\nmembrane after agonist application, and we have more explicitly indicated the basis of our descriptions in the revised text (p. 9 middle\nof page).\n.\n3. In Fig. 1, colocalization between G and EEA1 is missing. Fig.1d is only representative/qualitative but no quantification is\nshown for Gαs G colocalization at the EE.\nThe reviewer is correct, we label no more than three proteins in any image. We would like to label all four proteins noted\nsimultaneously, but are presently unable to image more than three channels without having to correct for artifacts of fluorophore\nspectral overlap (which we assiduously avoid for examination of colocalization). In considering the reviewer’s comment, we recognize\nthat we failed to explicitly show Gβγ colocalized with internalized receptors and Gαs. We can assure the reviewer that this is the case\nand apologize for missing this point. In the revised manuscript have included (Supplemental Figure 1d) an image of the internalized\nreceptor, Gαs, Gβγ. We also show a representative line scan of each channel. One can clearly see that Gαs and βγ overlap the\ninternalized receptor channel (and these are truly internalized receptors based on the labeling method used); however, Gαs and Gβγ\nare not restricted to receptor-containing endosomes and, in fact, are quite broadly distributed. This is consistent with previous studies\nfrom others describing endogenously labeled G protein localization (from the Lambert group) and its subcellular distribution by cell\nfractionation (from the Leonetti group; including specifically with EEA1). In the revised manuscript we note this broader distribution and\ncite these additional studies that support our conclusion (bottom of p. 6 and top of p. 7).\n4. Also in Fig. 1 I’m surprised not to see G at the PM in unstimulated cells. I was expecting complete colocalization with Gs.\nCould the authors comment on this?\nThis is an excellent point. Actually we do see evidence for Gβγ localization to the plasma membrane, as the reviewer predicts.\nHowever, it is not only at that location and it is generally easier to visualize punctate internal structures using confocal microscopy. PM\nlocalization is evident by line scan analysis, however (an example is shown in the new data in Supplementary Figure 1d). We also note\nthat Gβγ has previously been shown to localize intracellularly in the absence of receptor activation (Cho et al. 2022, Jang et al. 2024, as\ncited), and that differences in the relative localization of distinct Gβγ combinations have been observed (Masuho et al. 2021, as cited).\nWhile our results do not go into this level of detail, we believe our results are fully consistent with previous studies and have\nemphasized this in the revised text (bottom of p. 6 and top of p. 7).\n5. Could the experiment performed in Fig. 2 be done in a complementary way using LgBit-Gs and SmBit-endofin, to look at\nredistribution of Gs to and from the endosome upon Iso or Alp addition?\nYes we think so. We focused on assessing loss from the plasma membrane in Fig 2 because this is the result that we sought to verify,\nin context of the prevailing current view that Gαs dissociates from the plasma membrane and its increased concentration in the\ncytoplasm drives ‘sampling’ of intracellular membranes. However, the microscopy data indicate that this sampling process is not\nendosome-specific, as other internal membranes also visibly recruit Gαs. A concern with the complementary approach is how one\nwould interpret an increase in nanobit signal. An increase of the nanobit signal on endosomes could be explained simply as a\nconsequence of this increased cytoplasmic concentration. This is not an issue in the endosome nanobit assays using G protein\nactivation sensors because their concentration in the cytoplasm does not increase with receptor activation. If anything it decreases, due\nto membrane recruitment, and thus an increase in the endosome signal is not subject to the same caveat.\n6. In Fig. 2e, redistribution of Gs from the endosome to the membrane upon Alp addition is observed, even in absence of\nreceptor internalization. It would be great to hear what the authors think about this finding, if they could speculate on how Gs can\nsense from the endosome the presence of an antagonist acting on receptors blocked at the plasma membrane.\nThis is an interesting idea. The reviewer is correct that Alp is membrane-permeant and we have not ruled out communication back from\nthe endosome, as the reviewer proposes. However, we don’t presently think that retrograde communication would be needed to\nexplain the observed return of Gαs to the plasma membrane after Alp addition. The reason is that we believe Gαs dissociates from the\nplasma membrane after receptor activation and reparations through a cytoplasmic intermediate form (this goes back to the early\nobservations of Rasnas et al, and Rodbell even before that, both cited). If recovery of Gαs to the PM required retrograde\ncommunication, we would not expect the result shown. Instead, we think that antagonist application simply reverses the dissociation\nprocess from the PM, and this does not require any communication from the endosome.\n7. Why did the authors choose to block endocytosis of VIPR1 by overexpressing the dominant negative mutant of dynamin\nDyn1-K44E instead of using Dyngo-4a as it was done for β2AR?\nBoth Dyngo-4a and Dyn-K44E are useful inhibitors of endocytosis, and we generally observe similar results with both. We used\nDyn-K44E in the studies noted because we found Dyngo-4a to variably suppress the luminescence signal in our NanoBit assays. We\nbelieve this to be a technical off-target effect of Dyngo-4a, somehow it seems to variably inhibit some aspect of the luciferase reaction.\nWe have found Dyn-K44E to be preferable for NanoBit experiments because its expression does not affect the luminescence signal.\n8. Please add quantification for confocal images shown in Supp. Fig. 1, 2, 3 and 4. The analysis should be conducted on a\nbigger sample, not just 4 cells, from more than 2 independent experiments.\nWe thank the reviewer for noting this lack of quantification and have added line scans to quantify the images in Supplementary Figures\n1 through 4. We have also added a third replicate of the experiments in Supplementary Figure 2b and Supplementary Figure 4. For\nexperiments in Supplementary Figure 4b (A2bR images), we repeated the imaging in the presence of a PKA inhibitor (H89) to prevent\ncontractility induced by activation of overexpressed A R and improve the quality of images. The adenosine receptor images have been\n2B\nreplaced to reflect this experimental change, and we note this in the methods and figure legends (Methods, p. 22 and Supplementary\nFig. 4).\n9. More repeats should be included for experiments that were repeated only twice. 4 cells are not sufficient. I suggest\nanalyzing 5 cells per repeat, for a total of 15 cells.\nWe agree with the reviewer and thank them for pointing this out. The TIRF experiments in Figure 4 and Supplementary Figure 7\nrepresent data from at least 4 individual movies, with 2-6 cells per movie. F/F values for each cell within an individual movie were\n0\naveraged to calculate average F/F values for each individual replicate, so for all conditions, we analyzed at between 10 and 20 cells.\nmiddle of page, and Fig. 4 and Supplementary Fig. 7 legends).\n10. There is a clear imbalance between the first part of the manuscript where all experiments are conducted using β2AR as\nmodel GPCR and mainly live-imaging techniques, and the second part of the paper where experiments are focused on\nVIP1R and A2BR using the NanoBit assay.\nSpecifically, the authors show that Gs redistribution to the endosomes is independent of receptor endocytosis only for β2AR. It would be\ninformative to know whether this is also true for other Gs coupled GPCRs such as VIPR1 and A2BR.\nSimilarly, was the NanoBit assay run for β2AR with either KB1691 or Nb37? The manuscript would benefit from adding\nthese data.\nThese are all fair points and, in considering them, we recognize a need to further clarify our experimental focus and rationale.\nOur claim in the first part of the paper is that Gαs redistribution from the plasma membrane does not require internalization of the\nactivating GPCR. Our rationale for emphasizing the β2ΑR was 2-fold: First, this is the GPCR traditionally used for studies of Gαs\nredistribution, and our intention was to place the present results in context with the current understanding developed largely through the\nstudy of β2AR-induced effects. Second, the β2AR is experimentally advantageous because we have three independent methods for\nblocking its internalization (βarr DKO, β2AR-3S mutant and dynamin inhibition). We believe that A R provides further support for our\n2B\nconclusion because the human A R naturally does not internalize after agonist-induced activation; thus it enables us to assess Gαs\n2B\nredistribution in the absence of any experimental perturbation of trafficking. We would prefer not to make a strong claim for VIPR1\nbecause we have only one manipulation (dynamin inhibition) to block its internalization (it is not blocked by βarr DKO (Blythe et al.\n2024) or phospho-site mutations). In addition we think that, even if the properties of Gαs redistribution triggered by VIPR1 were\nsomehow different, such a difference would not change our conclusion that internalization of the activating GPCR is not required.\nOur claim in the second part of the paper is that Gs activation on the endosome membrane (assayed by the Nb37 biosensor) is\ndependent on the presence of activated receptors in the endosome, and that it produces an accumulation of active-state Gαs on the\nendosome membrane (assayed by the KB1691 biosensor). We previously reported that β2AR activates Gs on endosomes using a\nmicroscopy-based Nb37 recruitment assay (Irannejad et al. 2013). We focused here on VIPR1 because this GPCR more robustly\nstimulates Gs signaling from both the plasma membrane and endosomes (Blythe et al. 2024). The β2AR can also activate Gs at both\nlocations, but it does so less strongly than VIPR1 (Blythe et al. 2025). Therefore we have focused on VIPR1 in the present study, and\nbelieve that our results support the claimed conclusions.\nIn the revised manuscript, we have clarified our focus on the cell biology of Gs, rather than differences among the various Gs-coupled\nGPCRs we used to interrogate it. We explicitly acknowledge that there may be additional GPCR-specific biology that is not uncovered\nby our present work. We further clarify that our only strong claim about GPCR-specific differences exclusively concerns the last part of\nthe manuscript, where unexpected differences in G protein coupling selectivity at the endosome membrane relative to plasma\nmembrane are described.\nFinally, since KB1691 is conveniently tagged with mApple, it would be good to include confocal images of KB1691\nlocalization before and after stimulation with either VIP or NECA (+ localization markers for PM and endosomes).\nThis is a good point. We were unable to detect KB1691 recruitment visually by endogenous G proteins using confocal microscopy,\nat either the plasma membrane or endosomes. We think this is a detection sensitivity issue because we know that G protein activation\noccurs at both locations (previous studies with Nb37). This was the motivation for developing the NanoBit assay, which we find is more\nsensitive.\n11. In Fig. 7a, the authors talk about a general mechanism, but they demonstrate that redistribution of Gs to endomembranes\nis independent of receptor endocytosis only for β2AR. Furthermore, the representative GPCR is in purple, which in 7b\nrepresents VIPR1, for which receptor independent Gs redistribution to endosomes has not been shown. The scheme is\nconfusing from different aspects and I recommend changing it to reflect the findings described in the manuscript.\nWe thank the reviewer for this feedback. Our goal in Fig 7a was to provide a general framework, but we agree that we only support this\nexplicitly with 2 of the three GPCRs used in the present study (β2AR based on three independent endocytic inhibitors, and A2BR based\non its natural resistance to agonist-induced internalization). We agree that our initial scheme was confusing and may have overstated\nthe generality of the framework depicted. We do not wish to imply that all GPCRs behave the same way (and the G protein coupling\nselectivity data indicate that they do not). In the revised manuscript, we have changed the figure to more closely reflect our findings and\nchanged the color of the GPCR in Figure 7a to avoid the potential confusion noted by the reviewer. We have also clarified in the text (p.\n18) that there may exist still more GPCR-specific differences that are not uncovered by our present data .\nMinor comments:\n12. Since in some experiments proteins are overexpressed and in some other experiments not, it would be cleared to add on\ncertain figure panel (e.g. Fig 1a) which tag was used, so it’s clear when proteins where overexpressed (e.g. Flag-b2AR, Gs-\nEGFP, etc).\nWe agree with the reviewer that this would improve clarity and have revised the figures to include the tag name to clarify when a protein\nwas overexpressed.\n13. It is sometimes difficult to understand when the authors mean G protein activation or G protein-GPCR coupling. From\nwhat I read, G protein activation is monitored with KB1691, while G protein-GPCR coupling with Nb37. If that’s correct, I\nsuggest changing Supp. Figure 9 graph headers to (G protein-GPCR coupling, Nb37).\nThis is correct: we interpret KB1691 as a probe for the active-state (GTP-bound) Gα subunit, as established previously by Janicot et al.\nas cited. We interpret Nb37 as a probe for the G protein coupling reaction because Nb37 binds selectively to a nucleotide-free α-subunit\nconformation corresponding to the catalytic intermediate stabilized in GPCR-G protein coupling complex, as established previously by\nWestfield et al.as cited. We have clarified this distinction in the revised manuscript and figures, and we have attempted to correct any\nadditional inconsistencies in our language when describing the biosensor results (see Supplementary Fig. 10 and p. 14 middle of page).\n14. Supp fig. 2a please add single channel images in black and white.\nWe have added these images to the revised manuscript (Supplementary Figure 2a).\n15. The quality of some images is quite poor. Would it be possible to replace images in Fig. 1d +iso 20’ and Supp Fig. 4b\nwith better ones?\nWe have added a second example of Gαs + Gβγ images (Supplementary Fig. 1d) and replaced the images in Supplementary 4b. As\nnoted above (point 8), we found during the revision process that PKA inhibition (by pretreatment with H89) vastly improved the quality\nof imaging with overexpressed A R as, in the absence of PKA inhibition, activation of overexpressed A R leads to a large contractile\n2B 2B\nresponse. We have updated the revised text to reflect this change (see Methods p. 22 and supplementary Fig. 4 figure legend).\n16. p.9 “Signaling from endosomes requires Gαs associated with the endosome membrane to be in an active state.” Please\nadd a reference to this sentence.\nWe thank the reviewer for noting this omission. We have clarified this statement as “The current understanding holds that Gs-mediated\nsignaling requires Gαs to be in its active (GTP-bound) conformation“ and have added two references supporting this (p. 10).\n17. Personally, I do not like the word “abundance” used throughout the text, as to me, it implicates some quantitative\nmeaning, while here is it means purely presence of Gs. If the authors agree with my logic, could this word be changed to\n“presence” or “redistribution”?\nThis is a great point and we have changed the terminology throughout the revised manuscript and no longer use the word “abundance”.\nReviewer #4 (Remarks to the Author):\nIn this study by Wysolmerski et al., the authors reexamine previous findings related to Gs trafficking and endosomal activity and provide\nthe first results exploring endosomal signaling with the new Gαs-GTP sensor from the Garcia-Marcos lab. These results are timely\nbecause researchers studying location bias and intracellular signaling have surely thought about adapting this biosensor to fit their\nresearch questions since its publication last year. It is certainly exciting to see that this biosensor, when adapted to measure endosomal\nsignaling responses, can detect robust signals at endogenous G protein levels. However, the authors present some curious findings\nrelated to the specificity of two biosensors at the center of their study (KB1691 and Nb37). It feels as though they are concluding that\nthe detection modules are not selective for Gαs while not ruling out the possibility that other types of G protein activation may affect the\nconcentration of components on endosomal membranes. This distinction is important because the authors conclude that there is a\nnon-Gs component being activated at endosomes for receptors like A2BR when it may be that Gs is indeed activated at endosomes,\nbut Gq/11 activity at the plasma membrane modulates the possibility for Gs to be activated subsequently at endosomes.\nWe thank the reviewer for these thoughtful comments, and we are pleased that this reviewer found our results interesting and timely.\nWe were also surprised to detect a non-Gs component with both KB1691 and Nb37. We agree that the interpretation of this component\nis important and have made efforts to improve this, as detailed below.\nMy suggestions can be found below:\n1. YM acts as a molecular glue keeping Gαq and Gβγ together as an inactive heterotrimer (Mühle et al., PNAS, 2025). Can the authors\nrule out that YM treatment doesn’t sequester Gβγ from Gαs? Have the authors performed the same experiments in Gαq/11KO cells? In\na similar study from Vilardaga and colleagues (White et al., PNAS, 2020), Gαq/11 knockout cells were used and the authors observed a\ndecrease in endosomal cAMP generation which they proposed was dependent on the liberation of Gβγ and PIP3 production to promote\nGPCR-arrestin binding (in this instance PTHR) and internalization. In the Cell paper from Garcia-Marcos and colleagues where KB1691\nwas described for the first time (Janicot et al., Cell, 2024), I do not think any Gq/11 inhibitors or knockout lines were used in the context\nof this sensor; their experiments to confirm G protein selectivity were based on representative tagged G proteins from the four major\nfamilies and only tested PTX and YM on Gi/o and Gq/11 respectively. I think that the authors from the current study need to provide\nmore convincing evidence that this sensor is detecting something it is not, rather than the consequence of events that preceded\ndownstream signaling at endosomes. For example, it could still be that KB1691 and Nb37 are Gαs-selective, but that endosomal Gαs\nactivation is affected by Gq/11 activity at the plasma membrane – something that may be receptor-specific.\nWe agree with the reviewer that there are a number of possibilities for the origin of the non-Gs component. We do not think that it\nrepresents indirect activation of Gαs (such as by Gαq or βγ) because the non-Gs component is unaffected by Gαs/olf knockout (Fig. 4\n(KB1691) and Supplementary Fig. 10 (Nb37)). As the reviewer notes, we provided pharmacological (YM) but not genetic evidence for\ndefining this component as Gαq/11, and thus stuck with the more conservative term. In the revised manuscript we include the following\nnew data to address this: 1) We show that the non-Gs component is lost in Gαq/11 knockout cells (Supplementary Figs. 8 (KB1691)\nand 10 (Nb37)); 2) We further support the pharmacological definition by showing that YM has no effect on sensor recruitment in the\nGαq/11 knockout cells (Supplementary Figs. 8 and 10); and 3) We adapt an established active-state Gαq/11 binder to provide further\nsupport for the conclusion that the non-Gs component of biosensor recruitment indeed represents the production of active-state Gαq/11\n(Supplementary Fig. 8). By including these elements of new data, we now feel comfortable defining the non-Gs component as Gq/11.\nWe have modified the Results section to incorporate these new data and now explicitly conclude Gq/11 as the reviewer suggests.\n2. While the results with VIPR and DynK44E suggest that endosomal production of active state Gαs is endocytosis-dependent,\nthere could be other factors that regulate the presence of Gαs-GTP at endosomes like phosphodiesterases, RGS proteins and intrinsic\nGTPase activity. In other words, if VIPR internalization could be blocked completely, would we still observe residual endosomal\nsignaling and what could that be due to? Consider other receptors that have a greater dependence on arrestin/CME like V2R. Does this\nsensor have the dynamic range to measure the shaping of endosomal responses by other proteins like PDEs and intracellular RGS\nproteins?\nWe agree that there are likely other factors influencing G protein activity on endosomes. We also found it remarkable, as the reviewer\npoints out, that DynK44E does not fully block the detection of active-state VIPR1 or of active-state Gαs at endosomes. We do believe\nthat fully preventing active-state VIPR1 production in endosomes would block the accumulation of active-state Gαs on the endosome\nmembrane. However, we have not been able to explicitly test this hypothesis because, while DynK44E strongly inhibits agonist-induced\ninternalization of VIPR1 (as shown previously in Blythe 2024 as cited and now verified in the present study by flow cytometry in\nSupplementary Fig. 9), there is clearly still some receptor activity in endosomes under this condition. We also explicitly note this in the\nrevised Results section (p. 13 bottom of page).\n3. Fig. 1 Gγ2 is geranylgeranylated and does not leave the membrane as readily as other gamma subunits (Masuho et al., Cell\nSystems, 2021). While there is a clear intracellular localization of Gγ2, the conclusion that this population of Gβγ supports endosomal\nsignaling is probably isoform-specific. In fact, it has also been proposed that Gβγ dimers internalized with β-arrestin-bound receptors\ncan enable a second round of Gαs activation for select receptors (Sokrat et al., Communications Biology, 2024). Given these previous\nfindings, I suggest that the authors either broaden their conclusion about how agonist-induced activation happens at endosomes or\nperform additional G gamma experiments with other subtypes or receptors.\nThis is an excellent point and we recognize, in retrospect, that we overstepped in our conclusion and have revised it accordingly, stating\nthat only that Gβγ is associated with endosomes, and is evident both in untreated and agonist-treated cells, but make no conclusion\nabout whether this association is agonist-regulated (bottom of p.6 to top of p.7). We also thank the reviewer for noting Sokrat et al and\nhave modified the Discussion section to include this citation (p.18, middle of page) and to note that much remains to be learned about\nG protein trafficking.\n4. Fig. 1C label EGFP as control or cytosolic EGFP. Same for Fig.5 and mCherry.\nWe have corrected this.\n5. It is unclear how subpanel 2C differs from the wildtype response in panel 2D. I would suggest reorganizing the subpanels to avoid\nduplication of results within the same figure because it is essentially the same experiment for WT in subpanels 2C and 2D.\nThese are not the same data but we agree they show the same basic result. In the revised manuscript, we have removed panel 2c for\nclarity.\nResponse to Reviewers\nWe are pleased that reviewer 1 recognizes that we can now assign the YM-sensitive component definitively. This required including\nadditional data using Gq/11 KO cells and also developing an orthogonal biosensor strategy for detecting active Gq/11 at endogenous\nlevels. Reviewer 1 is correct that a number of results shown are generally consistent with previous work (including our own). We\nindicate and cite this explicitly, and include it only to place our new results in context with previous work. This reviewer appears not to\nrecognize other new contributions of our study: 1) that the previously described Gαs redistribution process precedes coupling on\nendosomes and is triggered by a discrete GPCR - G protein reaction at the plasma membrane; 2) explicit evidence that coupling on\nendosomes requires endocytosis of the receptor (the reviewer appears to think this is obvious but it is explicitly not the case for Gi as\nshown in PMID 40261932); 3) identifying a type of location-bias switching in the selectivity of G protein coupling on endosomes relative\nto the plasma membrane; and 4) demonstrating that this location-bias of endosomal G protein activation is itself GPCR-specific."}]

Decision letter