Characterization of the GTPγS release function of a G protein-coupled receptor

Domain: neuroscience
Published: 2025-12-17
Editorial outcome: minor_revision

Abstract
G protein-coupled receptor (GPCR) signaling is one of the most ubiquitous and sensitive forms of cell surface reception. GPCRs stabilize the nucleotide-free state of heterotrimeric guanine-nucleotide binding proteins (G proteins); however, this state is produced at the cost of relieving the G protein of its stable inhibitor, GDP. Upon agonist binding to receptor, the G protein binds GTP and signal transduction ensues. Herein we demonstrate that the agonist can also stimulate the release of GTP. This receptor-mediated mechanism permits dissociation and reassociation of the G protein as the receptor acts as a catalyst for two different reactions. We demonstrate that this mechanism requires a unique, selective active state in addition to the active state that promotes GDP release. The release reaction is competitive with antagonists and we demonstrate operational efficacy. Further, we show that agonists have the potential to preferentially stimulate GTP binding or GTP release. This release selectivity may serve as a form of receptor signaling and reshape our understanding of G protein-coupled receptor signaling. Finally, we demonstrate that these reactions can be recapitulated in human spinal cord dorsal horn, providing an avenue for investigating state selectivity in physiologically significant samples.

Introduction
Canonically, G protein-coupled receptors (GPCR) signal through the activation of G proteins1. G proteins are heterotrimeric by nature with monomeric Gα and dimeric Gβγ subunits. The heterotrimer binds guanosine diphosphate (GDP) and, when the receptor is stimulated by an agonist, an interaction between the receptor and the complex prompts the Gα subunit to release GDP. The nucleotide-free G protein (Gα apo) has higher affinity for guanosine triphosphate (GTP) and the active Gα forms; in this regard the GPCR serves as a guanine-nucleotide exchange factor (GEF). The G protein subsequently returns to the inactive Gα GDP state, following GTP hydrolysis, completing the signaling cycle. For the most part, the cycle is believed to be unidirectional where the role of the receptor is limited to stimulating GDP release, however, it is interesting to consider the receptor as a more dynamic partner in the signaling cascade.

Rhodopsin has been extensively studied as a prototypical GPCR and, like other GPCRs, rhodopsin activates a heterotrimeric G protein, transducin (Gt); the release of GDP is considered the rate-limiting step in G protein signaling where the rate of GTP binding is thought to be insaturable2–4. In early studies, nonhydrolyzable GTP-analogs such as guanosine-5’-O-[gamma-thio]triphosphate (GTPγS) were useful to study the activated Gα subunit5–7. Coincidently, the rate of nonhydrolyzable GTP binding occurs at the same rate that rhodopsin catalyzes the release of GDP8. While the fate of bound GTP was thought to rely on hydrolysis, these early studies showed that the release of a hydrolysis-resistant GTP analog could be catalyzed by photolyzed rhodopsin. It has been hypothesized that active-state GPCRs, including rhodopsin, catalyze the release of both GDP and GTP via a single active state9,10.

More generally GPCRs catalyze the dissociation of GDP from Gα, allowing Gα to readily bind GTP; however, some studies have suggested GTP release as a possible explanation for experimental data where the Gα apo species was observed to form, subsequent to the Gα GTP species11–13. As with rhodopsin, these studies involved measuring both the increase and release of nonhydrolyzable GTP binding over time in the presence of agonist and this has been reproduced in tissue14–21. Notably, these early release studies were performed in both the presence11–13 and absence14 of saturating exogenous nucleotide which would suggest that the reaction is permissive under a range of free nucleotide concentrations. Structural biology approaches have captured active state complexes revealing agonist-bound receptor engagement with Gα. In most cases, the G protein is stabilized in the nucleotide-free Gα apo state22–27. By contrast, NMR studies of receptor dynamics have revealed a more diversified energy landscape for active-state receptors to wander and receptor-G protein complexes to traverse28–32.

We present here, and in the companion manuscript (Stahl et al.33 in submission), the results of nearly a decade of studies on the role of the mu opioid receptor (MOR) in stimulating 35S-GTPγS release. We have discovered that the mu receptor exhibits active state-selective GTP release and that it is possible for agonist activity to be intrinsically GTP release-selective. Specifically, we present the observation that GEF activity, and therefore active-state affinity, can select for or against a G protein as a function of the nucleotide state (GTP binding or GTP release). This form of selectivity suggests that a requisite second active-state receptor is responsible for, and the selectivity-filter of, the GTP release mechanism. Further, it is possible for an agonist to exhibit a marked preference, or release selectivity, for inducing the dissociation of one or the other nucleotide from the G protein.

Results
Receptor-mediated regulation of GTPγS binding and release
The design of these initial experiments was chosen to mimic a pulse-chase approach34. Cell membranes were prepared from MOR expressing CHO cells and, in the pulse phase, stimulated with 1μM DAMGO in the presence of 0.1nM 35S-GTPγS (Fig. 1). At the 1-h time point (t0), this results in an agonist-mediated increase in the population of 35S-GTPγS labeled Giα captured upon rapid filtration. In the subsequent chase phase, the labeling reaction is quenched by a ten-fold dilution into buffer containing 1μM unlabeled GTPγS. For the next 60min, the residual 35S-GTPγS binding is captured on filters at the time points shown.

In the case where samples were diluted ten-fold a residual 100nM DAMGO remains from the pulse reaction. At the indicated times, this residual DAMGO produces significant dissociation of radiolabeled GTPγS binding from Gαi (Fig. 1). Importantly, the rate of dissociation as well as the magnitude of dissociation is increased by the addition of a saturating 10μM DAMGO (Fig. 1, 100nM DAMGO (10x dilution) rate: 0.016 (0.008–0.025) min−1; 10μM DAMGO rate: 0.048 (0.023–0.086), mean with 95% CI; p<0.0001 by extra sum-of-squares F test; t-test of 100nM DAMGO vs. 10μM DAMGO at 60min, p<0.05); this is in agreement with prior studies35. Inclusion of 10μM naloxone blocks the release induced by the residual DAMGO in the chase, highlighting the stability of the GTPγS-G protein complex when agonist cannot catalyze the reaction. Further, the naloxone stabilizes the complex as binding does not significantly differ between the t0 and all subsequent time points in the presence of naloxone (Fig. 1, one-way ANOVA of 10μM Naloxone vs. t0, ns, p>0.05) arguing against the occurrence of spontaneous release of GTPγS from the complex. The stability of the GTPγS-bound state has previously been reported for MOR stimulated GTPγS binding35. The DAMGO-mediated release prompts the question whether the agonist-mediated dissociation of GTP from Gαi could be agonist concentration-responsive.

To assess the concentration sensitivity of the response, the 60-min time point was selected as the logical end to the experiment as this reflected the same amount of time available to load 35S-GTPγS onto Gαi during the pulse phase. We note that while DAMGO produces a substantial decrease in 35S-GTPγS binding, the response plateaus by one hour (Fig. 1). The residual 35S-GTPγS binding is not likely due to rebinding of the 35S-GTPγS as the chase reaction includes a final concentration of 0.01nM 35S-GTPγS and 1μM unlabeled GTPγS (which is a 100,000-fold excess of unlabeled GTPγS to 35S-GTPγS). The residual binding is therefore interpreted to be a product of the dynamic nature of the receptor system and, in fact, this would be expected under these experimental conditions wherein the unlabeled Gαi GTPγS becomes a competitor for the receptor to mediate 35S-GTPγS release from Gαi-35S-GTPγS. This result is a distinct caveat of the single-turnover paradigm used to measure the release following G protein radionucleotide labeling.

As a starting point to assess the concentration-responsive nature of the release function, it was also imperative to be able to load the 35S-GTPγS on Gαi without including the agonist to avoid the presence of residual agonist in the chase. Therefore, we pursued an approach that elevates constitutive G protein activity, by removing sodium from the system, and avoids the introduction of agonist until the chase reaction. Sodium ions act as negative allosteric modulators at many GPCRs, including MOR, and the removal of sodium from the binding buffer (–Na+) leads to increased agonist affinity and elevated basal 35S-GTPγS binding36–39. This effect is demonstrated in Fig. 2A (see Binding Assay schematic) where removal of sodium leads to an increase in 35S-GTPγS binding in untransfected CHO cells (Parental) relative to the baseline in the presence of 100mM NaCl (Basal); notably, DAMGO has no effect in the presence of sodium in cells lacking MOR. The elevation of 35S-GTPγS binding in the sodium-free conditions likely reflects the activation of all endogenous sodium-regulated GPCRs in the CHO cells38. In the CHO-mMOR cells, both the inclusion of DAMGO and the removal of sodium (without DAMGO) leads to significant 35S-GTPγS binding. Both DAMGO and sodium-free 35S-GTPγS binding are blocked by pertussis toxin pretreatment, implicating the involvement of primarily Gα inhibitory G proteins in both conditions (Fig. 2B). Together, studies in the CHO parental line and using pertussis toxin support the role of both the receptor and Gαi in the 35S-GTPγS binding and release reactions. Beyond these controls, we do not rule in the involvement of additional membrane components as these reactions are not prepared or catalyzed in isolation. However, our data suggest that the agonist, receptor, and Gαi form at least the minimum ternary complex necessary for these reactions to occur.

We then asked if DAMGO could reverse the increase in 35S-GTPγS binding obtained in the sodium-free condition. For this study membranes (1mg protein) are preincubated/pulsed for 1h (see Release schematic in Fig. 2), in the absence of sodium with 0.1nM 35S-GTPγS and 10μM GDP to allow for constitutive loading of the G protein with 35S-GTPγS. The reaction is then diluted ten-fold, into buffer containing an excess of unlabeled GTPγS (1μM) and 100mM NaCl (to quench the constitutive Gαi loading) and allowed to proceed for an additional hour incubation/chase in the absence (Basal) or presence of 1μM DAMGO. In the MOR expressing CHO cells, DAMGO produces a significant decrease in 35S-GTPγS binding. This effect was observed to be receptor dependent as CHO-Parental membranes did not demonstrate DAMGO sensitivity (Fig. 2C). As was the case with the initial binding/pulse reaction, pretreatment of pertussis toxin blocks any observable release implicating the involvement of Gαi/o proteins (Fig. 2D).

While 35S-GTPγS is generally accepted to be nonhydrolyzable, or highly resistant to hydrolysis, it was a reasonable concern that the decrease in 35S-GTPγS could be due to phosphate hydrolysis. We, therefore, used a thin layer chromatography (TLC) approach to determine if there was cleavage of the radiolabeled thiophosphate group40. Due to the loading limits of the TLC plate (1–3μL), it was necessary to run these experiments in a much smaller volume at much higher reagent concentrations. For the pulse reaction, the assays were performed as described in the Binding schematic of Fig. 1 but with 300μg protein and 4.6nM 35S-GTPγS in the absence of sodium for 1h in 300μL assay buffer. Using 30μL of this reaction, Fig. 3A shows that under these conditions, GTPγS binding is still increased in the absence of sodium. For the chase reaction, the remaining membranes were then rapidly centrifuged to pellet the bound 35S-GTPγS and resuspended two times to wash away residual 35S-GTPγS. This pulsed protein was resuspended into 27μl of buffer containing 100mM NaCl and dilute 1:2 into chase reactions containing 1μM unlabeled GTPγS with and without 1μM DAMGO for an additional hour. A 2μL aliquot of the chase reaction was set aside for TLC analysis and the residual bound 35S-GTPγS was measured by rapid filtration. The inclusion of 1μM DAMGO produces a 50% reduction in bound 35S-GTPγS compared to vehicle (Fig. 3B). This reduction in binding was taken to reflect the same agonist-mediated loss of 35S-GTPγS binding observed in Figs. 1, 2.

Aliquots of the chase reactions were loaded onto a TLC plate (Fig. 3C). Since DAMGO produced a substantial reduction in residual 35S-GTPγS binding in these samples, an increase in the radioactivity migrating away from the point of application would occur if the decrease in 35S-GTPγS binding was due to thiophosphate hydrolysis. In a separate volume containing no protein but only buffers, CuSO4 was included as a positive control to degrade the thiophosphate, independent of enzymatic hydrolysis41. This is compared to the same sample without CuSO4. In all cases, the densitometry indicates only significant hydrolysis occurring in the CuSO4 (+Cu2+) lane (one-way ANOVA, Fig. 3D). These observations are in agreement with the findings of others that no significant hydrolysis of the tertiary thiophosphate is occurring during the reaction and, therefore, 35S-GTPγS must be dissociating from the G protein as an intact nucleotide triphosphate.

Together these findings demonstrate that the receptor acts as a control point for binding different states of the G protein. It is well known that the receptor plays a significant role in inducing the GTP binding reaction (receptor-mediated GDP dissociation and subsequent GTP binding). However, the role of the receptor in destabilizing the subsequent G protein-nucleotide interaction (receptor-mediated GTPγS release) is an unappreciated component of the GPCR signaling mechanism. In Fig. 1, we noted that the dilution of DAMGO (100nM residual) leads to a submaximal decrease in remaining 35S-GTPγS binding relative to the 10μM DAMGO treatment. Having optimized the system for agonist-independent nucleotide loading it was reasonable to investigate agonist concentration-dependent 35S-GTPγS release.

Ligand-mediated regulation of GTPγS binding and release
To further explore this apparent receptor-mediated mechanism in the GTPγS release paradigm, we compared the concentration-response measures of both the 35S-GTPγS binding and release functions. For these experiments, DAMGO stimulation of 35S-GTPγS binding and release was measured in MOR-expressing CHO cell membranes (Figs. 4A, B). DAMGO induces a concentration dependent increase in 35S-GTPγS binding as well as the release of GTP as revealed by the decrease in 35S-GTPγS bound (Fig. 4A). The saturable nature of the reactions is apparent as sigmoidal concentration-response curves with clear plateaus are presented for both responses. In the release experiments 35S-GTPγS loading was accomplished, as previously described, using the sodium-free approach described in Fig. 2. Non-linear regression analysis indicates no significant difference in DAMGO potency for binding or release suggesting that, in this cell line, DAMGO does not show selectivity for either of the two responses (Fig. 4A).

The competitive antagonist naloxone was applied at increasing concentrations in both experiments to produce a concentration-dependent rightward shift in the DAMGO response curves (Fig. 4B, C). The dextral displacement of DAMGO potency is a hallmark of competitive antagonists42. Using the classical approach, we estimate that naloxone has the same affinity (log Ki = −8.7 ± 0.1, the x-intercept in the Schild plot) for MOR in binding and release (Fig. 4B, C) as would be expected for a simple competitive interaction43. Together, the concentration dependency of each of these ligands furthers support the receptor-mediated nature of 35S-GTPγS binding and release. It is additionally apparent from the activity of DAMGO and naloxone that their intrinsic efficacy is largely similar between the two responses, with DAMGO demonstrating significant agonist activity and, conversely, naloxone lacking efficacy.

According to a two-state model of receptor activation, this data specifically excludes the possibility that the active state in one (binding or release) response is the inactive state in the other (release or binding) response44. This is because the drugs would need to acquire opposite activities in the two responses, which they clearly do not. That is, DAMGO would become an antagonist in one of the responses, and naloxone may become an agonist in one of the responses. Additionally, the observation that DAMGO potency and naloxone affinity are conserved for both reactions is strong evidence that these drugs act through a shared and accessible receptor population. Otherwise, we may observe a decrease in DAMGO potency or naloxone affinity as a result of the less accessible receptor population, which was also not the case.

Receptor-reserve and the Occupancy-Effector Relationship of GTPγS binding and release
Early on, the possibility that this was a system, or preparation, artifact was a central concern. Essentially, if the reverse reaction was the product of some population of Gαi-35S-GTPγS, that remained trapped at the receptor, then the G protein would not be available to perform its physiological responsibility and transduce signal downstream. However, if the release function was the result of the G protein being trapped on the receptor, then it would occur linearly as a product of agonist occupancy on the receptor. (A nonlinear occupancy-response relationship would be impossible.) It is possible to directly determine this relationship using the alkylating agent beta-funaltrexamine (β-FNA)45. As a MOR irreversible antagonist, β-FNA occludes the orthosteric binding site on the receptor and effectively removes the receptor’s ability to function in the system46,47. Radioligand binding studies show that β-FNA treatment produces a significant loss in 3H-naloxone binding in a concentration-dependent manner (Fig. 5A). To ensure that β-FNA did not lead to a nonselective degradation of receptors, MOR protein density was measured by western blot of the HA-tag on the N-terminal following β-FNA treatment. No difference in protein level was observed indicating that receptor levels remain constant in these preparations (Supplementary Fig. 1). Having demonstrated the selective loss of binding sites, it was reasonable to explore 35S-GTPγS binding and release in these preparations.

As a function of receptor alkylation, the DAMGO potency in both reactions shifts rightward (Figs. 5B, C). Raw data for binding and release following alkylation are included for comparison (Supplementary Fig. 2A, B). Also presented is the Occupancy-Response relationship where the change in 3H-naloxone binding sites is presented on the x-axis and the change in the maximum response for the binding and release function is presented on the y-axis (Supplementary Fig. 2C). In this representation both 35S-GTPγS binding and release have a nonlinear (clearly hyperbolic) occupancy-response relationship indicating that both responses are subject to substantial receptor-reserve. This approach has previously been established as a representation of the receptor-reserve of a system48–50.

It would, however, also be possible to demonstrate a nonlinear occupancy-response relationship if receptor alkylation was specific for some receptors that were occluded from the G protein population. (Essentially, if the total receptor population was not the same as the available receptor population.) In this case, 3H-naloxone binding sites would be depleted but 35S-GTPγS binding and release would still be the product of an artifactual MOR-Gαi-35S-GTPγS ternary complex. We then hypothesized that the change in potency from the alkylation reaction could be integrated into the occupancy-response analysis to demonstrate that the G protein was also not responding through an occupancy function. Using the analysis in (Fig. 5D), the (10^logEC50|control) / (10^logEC50|alkylation) was integrated into the abscissa (of Supplementary Fig. 2C) to produce the Δbinding*Δpotency relationship. From this representation, a linear relationship would be produced if loss of binding did not affect response potency (as the (10^logEC50|control) / (10^logEC50|alkylation) ratio would equal 1). This would indicate that alkylation was specific for an unavailable fraction of the total population with respect to the G protein.

A nonlinear Δbinding*Δpotency relationship thus implies that either alkylation affects the available fraction of the total population or that the available fraction is the total receptor population. In either case, the G protein must have access to the same available receptor population targeted for alkylation. This, then, demonstrates a mechanism where G proteins are not limited to a linear receptor-occupancy-effector relationship (i.e., pre-coupled) but are able to freely dissociate-reassociate with receptors for both the binding and release reaction. Further, this mechanism is shared for the binding and response function, indicating that receptors are able to interchange between G proteins, and potentially interact with multiple G proteins, not being limited to a static stoichiometry.

Ligand-selectivity for GTPγS binding and release
The clear result of the alkylation experiments is that the G protein is capable of moving in a bidirectional manner onto and off the receptor when bound to either GDP or GTP. Beyond a simple reversible binding reaction, by tracking the binding of the tertiary thiophosphate, we see that an active-state of the receptor is capable of coupling with either the GDP- or GTP-bound form of the G protein to induce nucleotide release. This complex relationship between the G protein and the receptor affords the exciting opportunity to investigate the state-selectivity of agonists. The μ opioid receptor has a large and structurally diverse collection of experimental and clinically relevant compounds, therefore, we compared the relative activity for the binding and release function for select agonists. Specifically, we hypothesized that some opioid compounds may demonstrate active state-selectivity for one or the other GTP binding reactions.

In this system, DAMGO did not exhibit selective preference for the binding or release reaction and thereby served as the control for full agonism in each system. The response of all other agonists could then be normalized to this maximum response, allowing for the comparison of changes in potency and efficacy. All agonists tested here exhibit clearly saturable response curves and Hill coefficients are all approximately 1 (Fig. 6). We applied three different models to analyze potency and efficacy in these experiments based on their status as the prevailing means of understanding ligand efficacy in pharmacology. First, the measure of Emax is an empirical estimate of agonist efficacy in a system and is referred to as Stephenson’s Efficacy (ε)51. Secondly, the determination of two-state Efficacy (α) is a mechanistic approach to measuring efficacy that is generally appropriate for partial agonists but incapable of dealing with full agonists52,53. Finally, the Operational Efficacy (τ) is likely the most relevant approach to estimating efficacy in this case as it is also non-mechanistic but incorporates a non-linear occupancy-response relationship which we have good confidence adequately describes both systems (Fig. 5D)54. Although none of these models is capable of correcting for the receptor reserve of a system (absent alkylation), simple estimates of changes in efficacy are still possible and informative of state selectivity. This is especially true for partial agonists or cases where full agonists in one system become partial in another system.

When comparing 35S-GTPγS binding and release, several agonists exhibit remarkable differences in both potency and efficacy (Fig. 6). The potency of morphine and fentanyl does not differ between the two responses. Interestingly, both agonists lose efficacy in the release reaction in every model fit. Conversely, nalbuphine exhibits a minimal increase in potency but gains efficacy in the release reaction. The observation that agonists can gain and lose efficacy in a given pathway is inconsistent with a two-state model of activation. Further, all three different models indicate that efficacy decreases for morphine and fentanyl and increases for nalbuphine. Beyond the changes in efficacy, most of the agonists display minimal to no change in potency. Buprenorphine exhibits a dramatic >10-fold increase in potency for the release reaction. This is strong evidence that rank order potency may also be a means of observing selection between the two active states and is consistent with a multi-state model of receptor activation55,56. According to two-state theory, rank order potency and/or efficacy are always conserved between systems if those systems are stimulated by a singular active-state receptor44. Potency and efficacy values are presented in Table 1.

35S-GTPγS binding and release in human spinal cord dorsal horn
Herein we have provided evidence that ligand-induced exchange occurs in transfected CHO cells that overexpress MOR. However, this biochemical approach can also be used to assess agonist activity in endogenous tissue. To demonstrate this, we dissected dorsal horn from the fourth lumbar region of human spinal cord, as this is known to have high MOR expression57 and is a site for epidural delivery of opioid analgesics58. Membranes were prepared and binding was performed in a conventional manner; the release reaction was loaded using 100nM DAMGO in the “pulse” reaction similar to that shown in Fig. 1 (Fig. 7A). Following the loading of 35S-GTPγS in the pulse reaction, a 15% increase in 35S-GTPγS loading can be achieved (Fig. 7B). Two potent MOR agonists, DAMGO and loperamide, produce a ~35% stimulation of 35S-GTPγS binding over baseline; both agonists produce release in the spinal cord preparations, but neither are capable of fully reversing the 15% loading (Fig. 7C, maximum reached is ~6%). (Raw data for DAMGO-mediated binding and release are presented in Supplementary Fig. 3.) Neither compound produced a significant shift in potency between the binding and release, but this may be due to the low sample number (n=3); regardless, given that these samples were procured as previously frozen tissue, we are encouraged that this approach could be used to compare agonist actions in physiologically relevant samples.

Methods
Materials
Compounds were from the following vendors: Sigma-Aldrich (St. Louis, MO, USA): morphine sulfate pentahydrate (M8777), naloxone hydrochloride (N7758), nalbuphine hydrochloride (N4396), and GDP (G7127). Cayman Chemical (Ann Arbor, MI, USA): fentanyl citrate (22659) and GTPγS (35098). Tocris Bioscience (Bristol, UK): DAMGO (Cat. 11711) and β-FNA (0926). Cell Signaling Technology (Danvers, MA): rabbit anti-HA (3724S, Lot 8), mouse anti-β-actin (3700S, Lot 21). Li-Cor Biotechnology (Lincoln, NE): goat anti-rabbbit 800CW (926-32211, Lot D30627-05), goat anti-mouse 680LT (926-68020, Lot D30418-15). 35S-GTPγS (NEG030H001MC) was from Revvity (Waltham, MA, USA). 3H-(-)naloxone was generously provided by the National Institute on Drug Abuse (NIDA) Drug Supply Program (NIDA; Bethesda, MD, USA). Human spinal cord sections (L4) were obtained from Anabios (San Diego, CA). Sections from three male donors (age 18, 22, and 35 years), snap frozen in LN2 upon collection, were used for coupling and release measures. Sections were stored in LN2, shipped on dry-ice, and stored at −80°C prior to use.

Cell lines
Most biochemistry experiments, except human spinal cord, were performed using membranes from Chinese Hamster Ovary cells (CHO-K1, ATCC CCL-61) expressing a N-terminal haemagluttinin-tagged (HA-tagged) form of the mouse μ opioid receptor (CHO-mMOR)53,73. (The CHO-K1 parental cell line was also used in one series of studies and were handled identically to CHO-mMOR cells.) Cells were cultured for harvesting in DMEM/F-12 supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 1μg/mL puromycin at 37°C in 5% CO2. Prior to collection, cells were incubated for two h in serum-free media. Following serum starve, cells were rinsed with phosphate-buffered saline (PBS) and removed from the plate by cell scraping in 5mM EDTA in PBS. Cell were pelleted at 2000 × g for two min and rinsed once in PBS. Cell pellets were stored at −80°C until use in biochemical assays. Pertussis toxin (cat# P2980-50ug, Millipore Sigma, St. Louis, MO) treated cells were incubated in standard media 100ng/mL pertussis toxin overnight. The following day, cells were rinsed with PBS and incubated in serum free buffer for 30min prior to scarping and pelleting. Cells were intentionally serum starved for a shorter time to ensure that pertussis toxin treatment did not reverse during the serum starvation.

Kinetic studies
In the initial studies, cell membranes were prepared by Teflon-on-glass homogenization in 10mM Tris HCl pH 7.4, 100mM NaCl, 1mM EDTA. Homogenate was centrifuged twice at 20K × g for 30min at 4°C. The resulting pellet was resuspended in assay buffer containing 50mM Tris HCl pH 7.4, 100mM NaCl, 5mM MgCl2, 1mM EDTA. Protein was quantified using the DC protein assay (BioRad, Hercules, CA). For the Pulse-Chase experiments presented in the pilot, 50μg/mL cell membranes were incubated with 10μM GDP, 0.1nM 35S-GTPγS, and 1μM DAMGO at 25°C temperature. The Pulse (loading) reaction was measured at the zero (t0) and one h (t0) time points. In the Chase reaction, aliquots of the loading reaction were dilute, as described, and incubated for the indicated timeframe at 25°C temperature. All samples were handled in this manner with dilution into a 2mL volume containing 10μM GDP, 1μM unlabeled GTPγS, and drug for the time indicated.

All reactions were quenched by rapid filtration through GF/B fiberglass filters on a 12-well manifold vacuum filtration system (Millipore Sigma, St Louis, MO). Filters were rinsed twice with ice cold diH2O and allowed to dry briefly. Filters were loaded into 7mL scintillation vials and 2mL of Safety-Solv scintillation cocktail. Vials were permitted to equilibrate, overnight, and scintillation was counted on an LS6500 liquid scintillation counter (Beckman Coulter, Brea, CA). All data was acquired and analyzed in disintegrations per minute (dpm).

Thin layer chromatography
35S-GTPγS loading and release studies were prepared specifically for the samples to be suitable for chromatographic processing and analysis. Specifically, it was necessary to process these samples in a manner that retained radioactivity density at levels high enough for detection on a phosphoimager. Initially, protein was processed as previously described. The initial loading reactions were Basal in the standard assay buffer (50mM Tris HCl pH 7.4, 100mM NaCl, 5mM MgCl2, 1mM EDTA) or sodium-free stimulation in 0mM NaCl buffer (50mM Tris HCl pH 7.4, 5mM MgCl2, 1mM EDTA). The loading reaction was run in a 300μL final volume (1mg/mL) with 10μM GDP and 4.3nM 35S-GTPγS for one h at 25°C temperature. Three aliquots of each loading reaction (3 × 10μL) were filtered and processed as described in the Kinetic Studies. The remainder of the sodium-free loading reaction was rapidly centrifuged (20K × g, 5min, 4°C) and rinsed in 100mM NaCl assay buffer. The final pellet was resuspended in 27μL 100mM NaCl assay buffer. Vehicle and 1μM DAMGO release reactions were measured by 1:2 dilution into 100mM NaCl assay buffer containing 10μM GDP and 1μM unlabeled GTPγS. Release reactions were allowed to proceed for an additional hour and filtered, again, as described. Filters were loaded into vials and counted by liquid scintillation on a scintillation counter.

The thin layer chromatography experiments were adapted from GTPase assays as the product of interest was the cleaved thiophosphate40. For TLC analysis, 2μL of the release reaction were dilute 1:2 into TLC running buffer (1mM KH2PO4, 4mM Na2HPO4 pH 7.4). Half of each dilution was counted by liquid scintillation on an LS6500 to insure equal plate loading. The other half of each dilution was spotted onto a PEI Cellulose TLC sheet (cat# M1055790001, Millipore Sigma, St. Louis, MO) and allowed to dry for one h. As controls for nucleotide phosphate hydrolysis, 35S-GTPγS (absent protein) was incubated +/- 1mM CuSO4 for one h in assay buffer. These conditions provided minimal and maximal limits for spontaneous (-Cu2+) and maximum catalytic hydrolysis of the phosphate bonds (+Cu2+)41. Dried plates were then placed in approx. one cm of running buffer and the front was allowed to migrate about 2/3 the distance of the plate. Following each run, the plate was wrapped in plastic wrap, exposed on BAS-MS Phosphor Screen, and frozen overnight in a dark box. The following day, the screen was developed on a GE Typhoon FLA 9500 phosphoimager (Cytiva, Marlborough, MA). Densitometry was measured using Fiji quantification software74.

35S-GTPγS binding and release studies
In general, 35S-GTPγS binding studies were carried out as previously published and as described in the Kinetic Studies previously described herein39. Cells were homogenized in 10mM Tris HCl pH 7.4, 100mM NaCl, and 1mM EDTA. Membranes were pelleted twice by centrifugation (20K × g, 4°C, 30min) and resuspension. After pelleting, membranes were resuspended in assay buffer (50mM Tris HCl pH 7.4, 100mM NaCl, 5mM MgCl2, 1mM EDTA). 35S-GTPγS binding was measured by incubation of membranes (10μg/well) with 10μg GDP, 0.1nM 35S-GTPγS, and drug as indicated. Reactions were performed for one h at 25°C in 200μL volumes in 96-well polypropylene plates.

35S-GTPγS release was measured using marginally different conditions. Specifically, cells were homogenized using the same procedure in sodium-free buffer (10mM Tris HCl pH 7.4, 1mM EDTA) and resuspended in sodium-free buffer (50mM Tris HCl pH 7.4, 5mM MgCl2, 1mM EDTA). 35S-GTPγS loading was accomplished in a large volume (20mL per plate) with 1mg protein, 10μM GDP and 0.1nM 35S-GTPγS for one h at 25°C. Following the sodium-free pulse conditions, preloaded membranes were diluted ten-fold into sodium containing buffer (50mM Tris HCl pH 7.4, 100mM NaCl, 5mM MgCl2, 1mM EDTA). The release reaction included 10μM GDP, 1μM unlabeled GTPγS, and drug (as indicated) and was run for one h at 25°C in a large volume 96-well plate.

Human spinal cord sections were prepared by gross removal of white matter and the dorsal half was homogenized using a tissue tearor (BioSpec Products, Model 985370, Bartlesville, OK, USA). Homogenate was drawn through a glass-on-glass homogenizer several times and pulled through an insulin needle (28-G). All tissue manipulation and homogenization were performed in homogenization buffer (10mM Tris (pH 7.4), 1mM EDTA) on ice. Membranes were pelleted by centrifugation at 20,000 × g for 30min at 4°C, resuspended and pelleted twice, followed by resuspension in assay buffer (50mM Tris (pH 7.4), 5mM MgCl2, 1mM EDTA). 35S-GTPγS binding was measured by incubation of membranes (10μg/well) with 10μg GDP, 0.1nM 35S-GTPγS, and drug as indicated. Reactions were performed for one h at 25°C in 200μL volumes in 96-well polypropylene plates.

35S-GTPγS release was measured in human spinal cord by loading pulse in a 2mL volume with 100nM DAMGO, 1mg protein, 10μM GDP and 1nM 35S-GTPγS for one h at 25°C. Loading controls, without DAMGO, were run for all samples. Following the pulse conditions, preloaded membranes were diluted 100-fold into assay buffer (50mM Tris HCl pH 7.4, 100mM NaCl, 5mM MgCl2, 1mM EDTA). The release reaction included 10μM GDP, 1μM unlabeled GTPγS, and drug (as indicated) and was run in a large volume 96-well plate. The release reaction was allowed to proceed for an addition hour at 25°C before termination. All reactions were terminated by rapid filtration through GF/B fiberglass filters on a 96-well Brandel MWXRI-96TI cell harvester (Brandel, Inc, Gaithersburg, MA). Filters were soaked in Microscint-20 (Revvity, Waltham, MA) on white OptiPlate-96 plates and counted on either a PerkinElmer TopCount scintillation counter (PerkinElmer, Downers Grove, IL) or a MicroBeta scintillation counter (Revvity, Waltham, MA). All counts were acquired and analyzed in dpm values. Graphical methods for binding and release are presented in the figures and in the companion manuscript.

Alkylation studies
Alkylation studies required special handling of the protein to ensure complete removal of the alkylating agent. For these experiments, pelleted cells CHO-mMOR cells were Teflon-on-glass homogenized in buffer (10mM Tris HCl pH 7.4, 100mM NaCl, 1mM EDTA) followed by rapid centrifugation (20,000 × g) for 30min at 4°C. Following centrifugation, harvested membranes were separated into aliquots and incubated in homogenization buffer or buffer including increasing concentrations of the alkylating agent, β-FNA46,47. Incubation, to permit β-FNA alkylation, proceeded for 30min at 25°C. After thirty minutes, all aliquots were centrifuged and resuspended (5min, 20K × g, 4°C) two times in homogenization buffer. After the third centrifugation, membranes were resuspended in assay buffer (50mM Tris HCl pH 7.4, 5mM MgCl2, 1mM EDTA). Following final resuspension, membranes were used for 35S-GTPγS binding, 3H-naloxone binding, and western blot protein quantification.

Radioligand binding studies
μ opioid binding was measured as previously reported39. Briefly, 10μg of membranes were combined with 6nM 3H-(-)naloxone in Tris pH 7.4, NaCl, 1mM EDTA. Incubations were run at 25°C for 1h. Non-specific binding was determined by the addition of 1μM cold naloxone in DMSO. Final DMSO concentrations were held constant at or below 0.1%. Bound receptor was harvested by rapid filtration through GF/B fiberglass filters on either a 12-well vacuum manifold or 96-well Brandel MWXRI-96TI cell harvester (Brandel, Inc, Gaithersburg, MA). and rinsed twice with ice cold 10mM Tris HCl pH 7.4. Filters were dried and loaded into scintillation vials with Safety-Solve or OptiPlate-96 plates with Microscint-20 (Revvity, Waltham, MA). Bound radioactivity was measured using a MicroBeta scintillation counter (Revvity, Waltham, MA). All counts were acquired, analyzed, and presented in dpm.

Western blot protein quantification
Receptor levels were determined by western blot using an anti-HA antibody since the mMOR was expressed with an HA-tag on the N-terminus. Aliquots from each alkylation treatment group were dilute into solubilization buffer (20mM Tris pH 7.4, 150mM NaCl, 2mM EDTA, 0.1% SDS). The subsequent soluble protein was quantitated using the DC protein assay and aliquoted for combination with XT buffer (BioRad, Hercules, CA) with 2-mercaptoethanol. The Precision Plus protein standard was run on all gels. Samples were run on 15 well 10% Bis-Tris pre-cast gels (cat# NP0316BOX, Thermo Scientific, Waltham, MA) for three h at 100V. Gels were mounted and transferred onto nitrocellulose membranes for two h at 20V on ice.

After transfer, nitrocellulose membranes were rinsed in TBST (10mM Tris pH 8.0, 150mM NaCl, 0.1% Tween-20) and blocked with 5% non-fat milk in TBST. Primary antibodies, anti-HA (1:1000 rabbit) and anti-β-actin (1:1000 mouse) in 5% non-fat milk in TBST, were added to the blocked membrane and incubated overnight at 4°C. Membranes were rinsed three times with TBST, blocked with 5% milk, and incubated for one h with secondary antibody, anti-rabbit 800 (1:10,000 goat) and anti-mouse 680 (1:10,000 goat). Membrane was rinsed three times with TBST and imaged using a Li-cor Odyssey scanner (LI-COR Biotechnology, Lincoln, NE). Densitometry was measured using Fiji quantification software. HA staining was normalized to β-actin as the protein loading control.

Data analysis
Nonlinear regression for stimulatory GTPγS binding, and normalized release data, was performed using the three-parameter equation:
Response = Basal + (Emax - Basal) / (1 + EC50 / X)
where, Basal is the system baseline, Emax is the maximum response of the system, EC50 is the midpoint of the agonist response curve in molar units, and X is the molar concentration of the agonist. For full agonists, the Emax was considered the same as agonist efficacy. Partial agonist efficacy is represented as the product of intrinsic efficacy (ε) and Emax (Partial agonist efficacy = ε · Emax). This intrinsic efficacy was fit for comparing changes in efficacy between GTPγS binding and release. For partial agonists where efficacy was compared, intrinsic efficacy is referred to as “Stephenson’s efficacy” as it is an observed efficacy that approximates the stimulus function51.

Alternatively, nonlinear regression for the inhibitory GTPγS release was performed using the three-parameter inhibition equation:
Response = Basal + (Emax - Basal) / (1 + X / IC50)
where, Basal is the system baseline, Emax is the maximum response of the system, IC50 is the midpoint of the agonist response curve in molar units, and X is the molar concentration of the agonist.

For the linearization analysis used for competitive Schild analysis43, EC50 and IC50 values were used and the standard (Dose Ratio – 1) was calculated as:
Dose Ratio – 1 = (EC50+antagonist / EC50control) – 1 or (IC50+antagonist / IC50control) – 1
where each Dose Ratio is the ratio of the molar EC50 or IC50 (for the curve of each antagonist concentration) to the EC50 or IC50 curves without antagonist. Each Dose Ratio was then converted to the logarithmic value and plotted Log[Antagonist] on the X-axis and Log(Dose Ratio-1) on the Y-axis. The equilibrium dissociation constant (log Ki) was estimated from this analysis as the X-intercept.

For the analysis of receptor alkylation, the loss of receptors was calculated as the ratio of 3H-naloxone binding between the control and alkylated membranes. This ratio was then applied as b in the adaptation of the operational model:
Response = Basal + (Emax - Basal) / (1 + (K / (τ · X))^n)
with X the molar agonist concentration, K the molar equilibrium affinity constant, n is the transducer slope, and τ the transduction ratio (or simply agonist efficacy). Of note, generally the Hill slope did not largely differ from one and was held constant as a shared value for each dataset fit. As the transduction constant is defined as τ = R T / K E, it is appropriate to modify τ by the alkylation ratio (i.e., q · τ or q · (R T / K E)). Using this modification, the change in system sensitivity due to alkylation, seen as a rightward shift in agonist potency and an absolute loss of maximal response, is incorporated into the equation as a decrease in τ as q is, by definition, less than one. This method permit the estimate of the agonist’s affinity75.

The occupancy-response relationship was initially plotted using the fractional B binding data from the alkylation groups on the x-axis (i.e., Bmax|alkylation / Bmax|control). The fractional E from the three-parameter fit of the normalized data for each alkylation group on the Y axis (i.e., Emax|alkylation / Emax|control). In this case, the control X values of one and zero would result when the total receptor population is either 100% available or 100% alkylated for binding. Likewise, the control Y values of zero and one would result from the population being available or alkylated for the measured response. The data from this representation can be fit to the equation:
E = X / (X + r - X · r)
where X is the size (fraction) of the receptor population available for binding after alkylation, E is the fractional response that remains after alkylation, and r is the EC50 / K. In this representation, when response follows a definite occupancy relationship, r reduces to one, and the equation reduces to E = X. Conversely, as EC50 shifts leftward from K, r becomes smaller than one, and the system exhibits a receptor reserve as the difference between EC50 and K.

This approach was further adapted to demonstrate the effector-occupancy relationship. In this new analysis, X is the product of fractional binding data from the alkylation groups (i.e., Bmax|alkylation / Bmax|control) and the potency ratio from the alkylation groups (i.e., EC50|control / EC50|alkylation). These newly defined X representation was plotted against the same Y values described for the occupancy-response relationship. Equation X was used to fit this representation, and linearity was tested using an F-test.

In the comparison of agonist efficacy, the operational model was used to estimate partial agonist efficacy54:
Response = Basal + (Emax - Basal) / (1 + (K / (τ · X))^n)
and all parameter definitions are identical between Eqs. 4 and 5. The only difference is that equation X lacks the alkylation parameter (b). This form of the equation is suitable for fitting and comparing partial agonist efficacy between systems but is not suitable for analyzing full agonists due to the lack of information regarding the occupancy-response relationship.

The two-state model of receptor activation was used to estimate partial agonist efficacy (α), or active-state selectivity, as previously described44,52,53. Data was fit, to determine α, with the equation:
Response = Basal + (Emax - Basal) / (1 + (K / X) · (1 + L) / (1 + L · (1 + X / K*)))
with X the molar agonist concentration, Basal the baseline response, Emax the system maximum response, K the molar inactive-state affinity constant, and L the isomerization constant. In all fitting of the two-state model, L was held constant at 0.01. This value was previously used and considered a reasonable assumption. An L value up to 0.1 (effectively a 10% baseline) produced no change in the relative activity of agonists in the two responses. The active-state selectivity, as presented, was greater than one for agonists, as it represented the active-state affinity as a function of the inactive-state affinity. The active-state affinity is the equilibrium affinity constant divided by alpha (K* = K / α). Further, α is the ratio of inactive- to active-state affinity (α = K / K*) or the agonist intrinsic efficacy53. This analysis was only appropriate for partial agonists as both inactive-state affinity and active-state selectivity cannot be estimated in a system that responds differently from occupancy (e.g., a receptor reserve).

For linear regression, data were analyzed for linearity with a slope constrained to unity. The X-intercept for the linearization was compared by an extra sum-of-squares F-test in Prism. For nonlinear regression, all parameters were shared between data sets when they represented system-dependent variables: Three-parameter equation (Basal, Emax), Two-state model (Basal, Emax, L), Operational model (Basal, Emax, n). However, parameters were permitted to float when they represented drug specific properties (e.g., Emax for a partial agonist). Curves were evaluated and best-fit parameter estimates with s.e.m. were compared for significance by an extra sum-of-squares F-test in Prism.

All data were plotted and analyzed using the Prism software (GraphPad Software, La Jolla, CA). All statistical comparisons are presented in Supplementary Table 1. All data presented are the result of multiple experiments (n≥3). The definition of all asterisks is included in the figure legends.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Thank you for the opportunity to review the Nature and Nature Comms papers from the Stahl/Bonn labs describing the novel phenomenon of GTP binding and GTP release at Gi/o proteins promoted by the mu-opioid receptor (MOR).\n\nThe Nature paper uses DAMGO as the reference agonist to describe the GTP-binding/release phenomenon and then two novel agonists that promote GTP release. Importantly using these two new agonists they demonstrate that co-administration of submaximal doses of the two GTP-release promoting agonists with morphine and fentanyl prolong the anti-nociceptive effects of morphine/fentanyl whilst minimising the cardiac and respiratory adverse effects (at least of fentanyl).\n\nThe Nature Comms article provides mechanistic details of the DAMGO mediate GTP-binding/release promoted by the MOR with particular examination of the impact of receptor reserve and the relationship between occupancy by, among other approaches, alkylation of the MOR.\n\nIt is my opinion that the Nature Comms paper lacks the potential physiological relevance of the GTP-binding and release which is covered by the Nature paper. The Nature paper lacks convincing evidence that the in vivo properties of the two novel agonists, muzepan1 and muzepan2, is in fact due to their GTP-release promoting activity. There may be a correlation between the GTP release properties of these agonists and in vivo enhancement of specific analgesic effects of fentanyl but this does not mean that this is the mechanisms by which muzepan1 and muzepan2 prolong fentanyl analgesia whilst minimising advise effects. The authors do for example overlook the potential importance of GAPs that increase the GTPase activity of G-proteins. Hence the kinetics of GTP hydrolysis in physiological settings (i.e. not in membranes under zero sodium) may be the over-riding factor that regulates G protein activity and not GTP-binding and release.\n\nMy suggestion is that the two papers are combined in a new submission to Nature Comms. That some of the pharmacological detail can be included as supplementary data allowing for the focus on the key data describing the GTP-binding and release and the fact that this may be a phenomenon promoted by more than the MOR but also other Gi/o coupled GPCRs. That there are a range of ligands (full and partial) that show differences in GTP-release/binding properties and that this MAY contribute to in vivo activity of these compounds – although this claim needs to be caveated. Finally the authors need to consider how important GTP-release/binding needs to be when considered in the context of GTP hydrolysis that in vivo will be subjected to enhanced kinetics by GAPs. If may be that GTPase activity (enhanced by GAPs) may, in a physiological context, be the important rate determining factor of the G protein cycle and not the binding and release of GTP.", "reviewer_2": "Bohn and Stahl present a carefully designed set of biochemical experiments to investigate the role of the mu-opioid receptor (MOR) in promoting GTPγS release from heterotrimeric G proteins. Using pulse-phase assays, the authors demonstrate that MOR activation can both facilitate GTPγS uptake and promote its subsequent release. The data are generally solid and well-presented, and the study addresses an intriguing and potentially novel aspect of GPCR signaling.\n\nHowever, a critical mechanistic issue requires further clarification. In this and prior studies reporting receptor-mediated GTPγS release from G proteins, the experimental systems include exogenous guanine nucleotides (e.g., GDP or non-hydrolyzable GTP analogs). This contrasts with the canonical function of activated GPCRs, where GDP release occur without the need for supplemental nucleotides. It is well-established that even in the absence of GPCR activation, bound GTPγS can be displaced by guanine nucleotides in solution, due to intrinsic conformational fluctuations of the α-helical domain (AHD) of Gα, which alternates between closed and open states. This raises the possibility that active GPCRs assist in AHD opening, but that this facilitation is significantly more effective in the GDP-bound state than in the GTPγS-bound conformation.\n\nThus, in the context of GTPγS binding, the ability of active GPCRs to induce nucleotide release may be limited unless aided by the presence of excess guanine nucleotides. This scenario could explain why receptor-mediated GTPγS release is observed in vitro but may not reflect a physiologically relevant process.\n\nMoreover, considering the cellular context, the cytosolic concentration of GTP is approximately ten-fold higher than that of GDP. Therefore, even if active GPCRs catalyze the release of GTP, re-binding of GTP to the Gα subunit would be rapid, resulting in a persistent active state. Additionally, activated receptors engage GDP-bound Gα as discussed above, allowing time for GTP hydrolysis in GTP-bound subunits before subsequent receptor interaction. These considerations suggest that receptor-mediated GTP release from G proteins, while detectable under in vitro conditions, may be negligible under physiological settings. Therefore, it is critical to experimentally demonstrate that the proposed GTP-release function of GPCRs has physiological relevance and leads to measurable effects on downstream signaling pathways.\n\nAdditional Minor Comments:\n1. In the Introduction (line 38), please include supporting references for the claims made.\n2. For consistency, please use \"MOR\" throughout the manuscript when referring to the mu-opioid receptor. Terms such as “mu receptor” appear in a few instances and should be standardized.\n3. The citations of references 8, 10, 11, 12, 17, and 19 should be reviewed for accuracy and relevance. For example, reference 10 concerns β2AR, while the manuscript text discusses rhodopsin. Additionally, these references do not appear to provide direct experimental evidence for receptor-mediated GTP release from G proteins, as suggested in the manuscript.", "reviewer_3": "This is an interesting study on some thoughts that had been discussed already in the early G-protein years in the labs of Al Gilman, Henry Bourne and Günter Schultz, and later studied - among others - by Moritz Bunemann et al: the idea is that the G protein cycle can operate not only in forward mode, but also in reverse mode, so that GTP is released from the active state of the G-protein rather then leading to G protein signaling by the dissociated G protein, wich would be terminated by GTP hydrolysis. To achieve this, a receptor has to induce GTP release, which is best studied with non-hydrolyzable GTP analogs, such as GTPgammaS.\n\nThe new aspects in this study - apart from detailed concentration-response relationships - is the evidence that the authors provide for a distinct active state of the receptor that can preferentially induce this reverse mode of the G protein cycle. This hypothesis is further elaborated in the partner manuscript that the authors have submitted to Nature.\n\nI think that this is a careful and very interesting study, and I only have two minor remarks - in addition to the question whether it is a good idea to publish the present data in another journal as the partner paper:\n1) I think a more extensive review of the old literature on G proteins acting in reverse mode would be appropriate\n2) As in the parallel manuscript, the authors discuss \"dose-response\" questions when they actually deal with \"concentration-response\" issues - please correct."}, "author_response": "Nature Communications manuscript NCOMMS-25-25118, “Characterization of the GTPγS release function of a G protein coupled receptor”\n\nReviewer #1 (Remarks to the Author):\nThank you for the opportunity to review the Nature and Nature Comms papers from the Stahl/Bonn labs describing the novel phenomenon of GTP binding and GTP release at Gi/o proteins promoted by the mu-opioid receptor (MOR).\n\nThe Nature paper uses DAMGO as the reference agonist to describe the GTP-binding/release phenomenon and then two novel agonists that promote GTP release. Importantly using these two new agonists they demonstrate that co-administration of submaximal doses of the two GTP-release promoting agonists with morphine and fentanyl prolong the anti-nociceptive eVects of morphine/fentanyl whilst minimising the cardiac and respiratory adverse eVects (at least of fentanyl).\n\nThe Nature Comms article provides mechanistic details of the DAMGO mediate GTP-binding/release promoted by the MOR with particular examination of the impact of receptor reserve and the relationship between occupancy by, among other approaches, alkylation of the MOR.\n\nIt is my opinion that the Nature Comms paper lacks the potential physiological relevance of the GTP-binding and release which is covered by the Nature paper.\n\nWe agree with the reviewer that the original Nature Comms submission could be strengthened by including the demonstration of potential physiological relevance. Therefore, we have added data from human spinal cord dorsal horn membrane preparations to demonstrate the utility of the assay.\n\nHerein we have provided evidence that ligand-induced exchange occurs in transfected CHO cells that overexpress MOR. However, this biochemical approach can also be used to assess agonist activity in endogenous tissue. To demonstrate this, we dissected dorsal horn from the fourth lumbar region of human spinal cord, as this is known to have high MOR expression55 and is a site for epidural delivery of opioid analgesics56. Membranes were prepared and binding was performed in a conventional manner; the release reaction was loaded using 100 nM DAMGO in the “pulse” reaction similar to that shown in Figure 1 (Figure 7a). Following the loading of 35S-GTPgS in the pulse reaction, a 15% increase in 35S-GTPgS loading can be achieved (Figure 7b). Two potent MOR agonists, DAMGO and loperamide, produce a ~35% stimulation of 35S-GTPgS binding over baseline; both agonists produce release in the spinal cord preparations, but neither are capable of fully reversing the 15% loading (maximum reached is ~6%). (Raw data for DAMGO-mediated binding and release are presented in Supp Figure3.) Neither compound produced a significant shift in potency between the binding and release, but this may be due to the low sample number (n=3); regardless, given that these samples were procured as frozen samples, we are encouraged that this approach could be used to compare agonist actions in physiologically relevant samples.\n\nThe Nature paper lacks convincing evidence that the in vivo properties of the two novel agonists, muzepan1 and muzepan2, is in fact due to their GTP-release promoting activity. There may be a correlation between the GTP release properties of these agonists and in vivo enhancement of specific analgesic eVects of fentanyl but this does not mean that this is the mechanisms by which muzepan1 and muzepan2 prolong fentanyl analgesia whilst minimising advise eVects. The authors do for example overlook the potential importance of GAPs that increase the GTPase activity of G-proteins. Hence the kinetics of GTP hydrolysis in physiological settings (i.e. not in membranes under zero sodium) may be the over-riding factor that regulates G protein activity and not GTP-binding and release.\n\nThis has been addressed in the response to the Nature manuscript.\n\nMy suggestion is that the two papers are combined in a new submission to Nature Comms. That some of the pharmacological detail can be included as supplementary data allowing for the focus on the key data describing the GTP-binding and release and the fact that this may be a phenomenon promoted by more than the MOR but also other Gi/o coupled GPCRs. That there are a range of ligands (full and partial) that show differences in GTP-release/binding properties and that this MAY contribute to in vivo activity of these compounds – although this claim needs to be caveated.\n\nWe strongly feel that the extent of information included in the Nature Comms paper warrants publication as an independent study because we have 1. Introduced a new mechanism of receptor action and we have shown that these measures are indeed mediated by the receptor by applying multiple receptor pharmacology approaches. 2. Proposed a new model (figure 8), and 3. We have now demonstrated the ability to measure the reaction in human tissue.\n\nFrom this mechanism we demonstrate a novel analysis for estimating eVector-occupancy and an extension of the two-state model (to a three-state model) of receptor activation. This is specifically necessary because of the diVerences in eVicacy we observe between the two responses. Together, these studies provide a suitable and reasonable amount of information to convey to the reader as a complete manuscript.\n\nBy presenting our process of studying this novel reaction, we aVord the reader a chance to understand the thought process that led to this discovery. This process included critical consideration of both historical and experimental results as well as the substantial use of classical pharmacology models and methods to provide structure and support for our conclusions. We believe that this makes the Nature Comms manuscript of fundamental importance to the field and that combining the Nature Comms manuscript with the Nature manuscript would detract from each manuscript and strengthening neither.\n\nFinally the authors need to consider how important GTP-release/binding needs to be when considered in the context of GTP hydrolysis that in vivo will be subjected to enhanced kinetics by GAPs. If may be that GTPase activity (enhanced by GAPs) may, in a physiological context, be the important rate determining factor of the G protein cycle and not the binding and release of GTP.\n\nThis has been addressed in the response to the Nature manuscript.\n\nReviewer #2 (Remarks to the Author):\nBohn and Stahl present a carefully designed set of biochemical experiments to investigate the role of the mu-opioid receptor (MOR) in promoting GTPγS release from heterotrimeric G proteins. Using pulse-phase assays, the authors demonstrate that MOR activation can both facilitate GTPγS uptake and promote its subsequent release. The data are generally solid and well-presented, and the study addresses an intriguing and potentially novel aspect of GPCR signaling.\n\nWe thank the reviewer for their recognition of our rigorous approach and for the appreciation of the novelty.\n\nHowever, a critical mechanistic issue requires further clarification. In this and prior studies reporting receptor-mediated GTPγS release from G proteins, the experimental systems include exogenous guanine nucleotides (e.g., GDP or non-hydrolyzable GTP analogs). This contrasts with the canonical function of activated GPCRs, where GDP release occur without the need for supplemental nucleotides. It is well-established that even in the absence of GPCR activation, bound GTPγS can be displaced by guanine nucleotides in solution, due to intrinsic conformational fluctuations of the α-helical domain (AHD) of Gα, which alternates between closed and open states.\n\nThe reviewer is entirely correct regarding the experimental design and the inclusion of exogenous nucleotide (as well as further adaptations such as buVer conditions and the inclusion of 5mM free MgCl ) in these studies. We and others have demonstrated the general correlation between this response and the physiological activity of opioid agonists. Although appropriately controlled, we concede the limitations of these experiments as they are separate measures of receptor activity not viewed in the native system.\n\nIn particular, the reviewer raises a key point that we were very concerned about, early on, in these experiments; mainly, the free nucleotide exchange that may occur as the system equilibrates during the chase reaction. However, a number of authors have demonstrated that the GTPγS-bound state is quite stable in the experimental timeframe presented. Our initial studies (Figure 1) demonstrate that, once labeled, the DAMGO-pulsed GTPγS-G protein complex does not spontaneously disintegrate upon a 10-fold dilution chase reaction in the presence of saturating Naloxone. Further, we observe the same stable complex when performing the pulse with the no sodium conditions which account for the bulk of the manuscript. As the nonhydrolyzable nucleotide is radiolabeled on the terminal phosphate, we believe this data is suVicient to support the conclusion that, for this system, the GTPγS-G protein complex is not in free nucleotide exchange (absent receptor stimulation). We happy acknowledge these considerations in the results section:\n\nThe inhibition of 35S-GTPgS dissociation by naloxone highlights the stability of the GTPgS-G protein complex. Cases of spontaneous free-nucleotide exchange have been observed in experimental systems, but we do not believe this confounds the results of these studies. The stability of the GTPgS-bound state has previously been reported for MOR stimulated GTPgS binding.33\n\nThis raises the possibility that active GPCRs assist in AHD opening, but that this facilitation is significantly more eVective in the GDP-bound state than in the GTPγS-bound conformation.\n\nWe believe that the receptor does exert influence over the AHD, either directly or indirectly, through the destabilizing role of the receptor for the nucleotide binding site. The reviewer correctly surmises that the “facilitation is significantly more eVective” when the G protein is bound to GDP. This is not a trivial supposition. In fact, it underscores the role of the receptor to induce GDP release. Fundamentally, this can be described as an energy barrier to the release of GDP that the agonist-bound receptor must overcome in order to induce a signaling event.\n\nWe respectfully suggest to the reviewer that “more eVective in the GDP-bound state” is only true for some agonists. In particular, we do not observe any diVerence in agonist activity for DAMGO but, for example, a loss of eVicacy for morphine and gain in eVicacy for nalbuphine. We highlight this divergence by employing the Stephenson’s, two-state, and operational models to estimate agonist eVicacy. Specifically, for these agonists to demonstrate changes in eVicacy, in both directions, for GTPγS release then, at a minimum, the energy barrier for the release of GTPγS is diVerent than that of GDP release. As such, agonists exhibit diVerent rank eVicacy for surmounting these separate energy barriers and a diVerent active-state of the receptor is responsible for each reaction (GDP release v GTPγS release). This active-state selectivity extends from the fact that, if a singular active-state were responsible for the two reactions, then all agonists would stabilize the same active-state in both reactions and all agonists would either gain or loose eVicacy “equally”.\n\nTo relate this back to the reviewer’s comments, if the “active GPCRs assist in AHD opening” then this would be permissible via a multiple active-state mechanism. We hypothesize that through this mechanism, the receptor may act both as a catalyst for nucleotide release (AHD opening) and as well as being sensitive to the instantaneous nucleotide-bound state of the G protein (GDP-bound or GTPγS-bound). This second form of sensitivity, and an associated state-dependence, would then avail the receptor to a more dynamic role in signal transduction where it can modulate the activity of the GTP-bound state.\n\nWe have added to the discussion to highlight the complexity of AHD opening and introduced this nucleotide sensor hypothesis:\n\nThe activation of the G protein proceeds through changes in the Gα subunit including a requisite movement of the alpha helical domain (AHD) away from the Ras domain and a receptor-interaction with the α5 helix to further destabilize the nucleotide binding site.59 However, Ras domain binding of nucleotide has been reported in the absence of the AHD60 and molecular dynamic simulation have estimated that alpha helical domain movement can occur, spontaneously, and independent of nucleotide release.4 It is very likely that movement of the AHD is also required for GTP release but, based on the observed changes in rank order agonist eLicacy, AHD migration alone is not likely to select for binding or release. Essentially, it is not clear how movement of the AHD would behoove certain agonists, while being disadvantageous to other agonists, in the binding reaction and for this relationship to be inverted in the release reaction. As such, we hypothesize that beyond simply reacting to the diLerence in nucleotide-bound state of the G protein, the receptor acts as a sensor of the specific nucleotide-bound state of the G protein. This reverse flow of information from the G protein would situate the receptor as a “first and next step” in the G protein signaling cascade permitting the receptor to exert continued control over the fidelity and texture of the signal.\n\nThus, in the context of GTPγS binding, the ability of active GPCRs to induce nucleotide release may be limited unless aided by the presence of excess guanine nucleotides. This scenario could explain why receptor-mediated GTPγS release is observed in vitro but may not reflect a physiologically relevant process.\n\nWe agree with the reviewer on the qualification that active GPCRs induce nucleotide release in the presence of excess guanine nucleotide. This was specifically reported for rhodopsin where [α-32P]GDP release was measured in the absence and presence of GTP (Fung and Stryer, 1980). In these experiments, GDP release was not observed upon photolysis, in the absence of exogenous GTP addition, but was observed in the presence of exogenous GTP. As GEFs, both receptors and rhodopsin are subject to the system availability of nucleotide to accomplish their exchange function. This is probably at least minimally comparable to a physiological condition as there is very likely never a point in time when the G protein exists absent free nucleotide.\n\nIn this manuscript, we have sought to introduce the segregation of mechanism for the GTP binding and GTP release functions. Using radiolabeled GTPγS we are able to estimate the role of the receptor for both activities and highlight the receptor-mediated nature of each reaction. By demonstrating diVerences in agonist eVicacy we are able to extend the current model of receptor activation to include the action of a second active state in the system and connect this second active state with a specific change to the system.\n\nBased on the concerns of the reviewers regarding the strength of the Nature Comms manuscript and the physiological relevance of these studies, we have extended the current study with the observation of this reaction in human spinal cord. This data highlights both the physiological implications of the current study and the ubiquity of the approach we have employed to study this reaction.\n\nMoreover, considering the cellular context, the cytosolic concentration of GTP is approximately ten-fold higher than that of GDP. Therefore, even if active GPCRs catalyze the release of GTP, re-binding of GTP to the Gα subunit would be rapid, resulting in a persistent active state. Additionally, activated receptors engage GDP-bound Gα as discussed above, allowing time for GTP hydrolysis in GTP-bound subunits before subsequent receptor interaction.\n\nWe agree with the reviewer that the free GTP concentration in cytosol is suVicient to saturate the nucleotide binding site on the G protein. However, the release of the GTP from Gα would likely cost the system energy. (We would remark that based on structural studies, the switch regions of the Gα subunit more closely resemble the inactive state (Gα ) than the active state (Gα ).) This cost in system energy (and to the switch region) provide a window where the G protein is not free but constrained both spatially and thermodynamically. We hesitate to extend this hypothesis too far, but we suggest in the discussion that there is the possibility for conservation of high energy nucleotides (the feastR hypothesis). We have added context to this section of the discussion to highlight the free nucleotide dependency of this hypothesis and the broader comment that saturating nucleotide levels may detract from this line of thinking:\n\nHowever, this type of mechanism would be subject to the free nucleotide concentration as saturation of the apo state would be expected to be rapid at the estimate physiological nucleotide concentration. An additional consideration is the residence time of the Gα subunit on the receptor as the extension of the apo-state lifetime would come at the cost of further delaying both signal propagation and the hydrolytic recovery of the Gα ground state.\n\nThese considerations suggest that receptor-mediated GTP release from G proteins, while detectable under in vitro conditions, may be negligible under physiological settings. Therefore, it is critical to experimentally demonstrate that the proposed GTP-release function of GPCRs has physiological relevance and leads to measurable eVects on downstream signaling pathways.\n\nWe agree with the reviewer on the value of these experiments to connect the GTP release reaction with downstream signaling pathways. However, these agonists have been extensively studied in multiple signaling pathways for decades by multiple investigators. By introducing this new concept, we invite the formation of testable hypotheses regarding the connection of release preference to signaling properties observed.\n\nAdditional Minor Comments:\n1. In the Introduction (line 38), please include supporting references for the claims made.\n\nWe have adjusted the language to better convey our view of the evolving role of the receptor in G protein signal transduction.\n\nChange: For the most part, the cycle is believed to be unidirectional where the role of the receptor is limited to stimulating GDP release, however, it is interesting to consider the receptor as a more dynamic partner in the signaling cascade.\n\n2. For consistency, please use \"MOR\" throughout the manuscript when referring to the mu-opioid receptor. Terms such as “mu receptor” appear in a few instances and should be standardized.\n\nWe are happy to make this correction.\n\n3. The citations of references 8, 10, 11, 12, 17, and 19 should be reviewed for accuracy and relevance. For example, reference 10 concerns β2AR, while the manuscript text discusses rhodopsin. Additionally, these references do not appear to provide direct experimental evidence for receptor-mediated GTP release from G proteins, as suggested in the manuscript.\n\nThe reviewer is correct, we have adjusted the language for these references to express their relationship to the current study:\n\nChange: Coincidently, the rate of nonhydrolyzable GTP binding occurs at the same rate that rhodopsin catalyzes the release of GDP.8\n\nChange: It has been hypothesized that active-state GPCRs. including rhodopsin, catalyze the release of both GDP and GTP via a single active state.9,10\n\nChange: More generally GPCRs catalyze the dissociation of GDP from Ga, allowing Gαapo to readily bind GTP; however, some studies have suggested GTP release as a possible explanation for experimental data where the Ga species was observed to form, subsequent to the Ga species.11,12,13 As with rhodopsin, these studies involved measuring both the increase and loss of nonhydrolyzable GTP binding over time in the presence of agonist and this has been reproduced in tissue.14-17, 18-20\n\nChange: The original Reference 17 has been removed.\n\nReviewer #3 (Remarks to the Author):\nThis is an interesting study on some thoughts that had been discussed already in the early G-protein years in the labs of Al Gilman, Henry Bourne and Günter Schultz, and later studied - among others - by Moritz Bunemann et al: the idea is that the G protein cycle can operate not only in forward mode, but also in reverse mode, so that GTP is released from the active state of the G-protein rather then leading to G protein signaling by the dissociated G protein, wich would be terminated by GTP hydrolysis. To achieve this, a receptor has to induce GTP release, which is best studied with non-hydrolyzable GTP analogs, such as GTPgammaS.\n\nThe new aspects in this study - apart from detailed concentration-response relationships - is the evidence that the authors provide for a distinct active state of the receptor that can preferentially induce this reverse mode of the G protein cycle. This hypothesis is further elaborated in the partner manuscript that the authors have submitted to Nature.\n\nI think that this is a careful and very interesting study, and I only have two minor remarks - in addition to the question whether it is a good idea to publish the present data in another journal as the partner paper:\n1) I think a more extensive review of the old literature on G proteins acting in reverse mode would be appropriate\n\nWe thank the reviewer for the opportunity to expand on this dimension of the paper. We completely agree that this is both useful to the reader and appropriate to give credit where credit is due. We have added substantially to the introduction and discussion to highlight previous work related to the “reverse mode” of signaling. We have added to the discussion to specifically highlight this work and its relation to both the ternary complex model and our new representation of the three-state model.\n\nIn particular, the reverse reaction is superimposed into the two-state coupling model to highlight the necessary adaptation required by the present findings. This model has previously been presented, in particular, as a reverse mechanism involving Ga GTP reassociation with the receptor.65 In fact, reverse mode was observed to be productive both for nucleotide release and for modifying G protein signaling activity as a coupling mechanism.65,66 Importantly this was found to be agonist concentration-dependent as well as nucleotide-specific where the concentration and form of the applied exogenous guanine nucleotide were central to the productivity of the receptor-G protein interaction. Interestingly, in an early study in the β2-receptor, epinephrine/Ga -pulsed stimulation of adenylyl cyclase was reversed by a subsequent epinephrine-chase reaction.67 Further, in the reconstitution of the receptor:adenylate cyclase system, it was observed that epinephrine and GTP, together, could reverse epinephrine-GppNHp stimulated cyclase activity.68 These studies coalesce on a mechanism where the receptor continues to convey information to the G protein and, in di`erent ways, may modulate the e`ectors activity.\n\nTaken together, Ga -receptor reassociation do not contradict the two-state coupling model in any way. Rather, it substantially underscores the validity of the model and adds to it in a way that more closely approximates reality. With the inclusion of this new reversible and state-selective mechanism, it was possible to integrate the two reactions as the Three-State Coupling Model (Figure 8). This model highlights the di`erent roles of the two active states (R* induces Ga -> Ga ; R** induces Ga -> Ga ). Formally, we propose the separation of alpha (a) into two e`icacy parameters where these e`icacy parameters are related to the two di`erent active states with their functions: GDP release (a1) and GTP release (a2).\n\n2) As in the parallel manuscript, the authors discuss \"dose-response\" questions when they actually deal with \"concentration-response\" issues - please correct.\n\nThe reviewer is completely correct. We are happy to make this change to the manuscript as requested. We call the reviewers attention to the “Dose Ratio” we present in the Schild analysis. We agree with the reviewer that this is an inappropriate use of the term “Dose” but this terminology is standard in the application of Schild’s analysis (Arunlakshana and Schild, 1959; A Pharmacology Primer, Kenakin, 2025). We believe that the term “Dose Ratio” should be kept in the manuscript for clarity. In all other places, the use of dose has been changed to concentration."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The article has been substantially revised in line with the reviewers suggestions. It is well written and presents a compelling argument for re-thinking of the two state model of G protein activation to include novel features of GTP binding and release. The authors also present how GTP-binding and release can be exploited pharmacologically with two new ligands that preferentially affect this process and show analgesic properties with reduced side effects. The authors in my view have taken into account the reviewers comments and it is my opinion that there is nothing substantive to be gained by further rounds of revision. The only comment I have is that this is designed to be a companion article for a Nature paper. It is still my opinion that the articles should be combined into a single Nat Comms paper.", "reviewer_2": "The reviewer remains unconvinced whether GTP release by the receptor is an active process, analogous to receptor-mediated GDP release, and whether this GTP release is physiologically relevant.\n\nIn the case of GDP release, it has been widely demonstrated that incubation of an agonist-activated receptor with a GDP-bound Gi heterotrimer (in the absence of free GDP or GTP) induces GDP dissociation.\n\nTo convincingly demonstrate receptor-mediated GTP release, an in vitro system with purified receptors and GTP-bound Gα subunits should be employed, under conditions devoid of free GDP or GTP. The membrane-based system used by the authors contains other proteins and membrane components that may confound interpretation.\n\nAs previously noted by the reviewer, any GTP-released, nucleotide-free Gα subunits would be rapidly reoccupied by GTP under physiological conditions. Although the authors added an experiment using human spinal cord membranes, this system again involves membranes and does not effectively address the physiological implications of receptor-mediated GTP release in a more defined or cellular context.\n\nIn Figure 1, naloxone did not induce GTP release, while the “dilution control” exhibited spontaneous GTP release. This suggests that naloxone may inhibit spontaneous GTP release, a mechanism that warrants further explanation. Additionally, since the “dilution control” itself shows GTP release, the authors’ statement that “The inhibition of 35S-GTPγS dissociation by naloxone highlights the stability of the GTPγS-G protein complex” may not be entirely accurate.\n\nIn Figure 2D, the authors aim to investigate whether the MOR–Gi interaction that mediates GTP release involves the same interface as for GDP release. Would it be informative to treat with pertussis toxin after GTP loading to test this more directly?\n\nMinor comments:\n1. Some statements require reference citations:\n   o Line 64: “For nearly … release.”\n   o Line 482: “Biochemical studies … of rhodopsin.”\n   o Line 508: “however, we demonstrate … (Stahl et al., Nature).”\n2. Line 66: “mu receptor” should be revised to “mu opioid receptor (MOR)”.\n3. Line 91: “1uM” should be formatted as “1 μM”.\n4. Line 91: “0.1nM” should be formatted as “0.1 nM”.\n5. Line 53: “Gαapo” — ensure consistent formatting and terminology for “apo” throughout the manuscript.", "reviewer_3": "Comments to the revised manuscripts by Stahl&Bohn et al.\nNature manuscript 2025-04-08323, \"GTP release-selective agonists prolong opioid analgesic efficacy\"\nNature Communications manuscript NCOMMS-25-25118, “Characterization of the GTPγS release function of a G protein coupled receptor”\n\nI am taking the liberty on commenting on both manuscripts together, since I believe (as noted in my comments to the original submissions) that they belong closely together.\n\nIn their rebuttal letters the authors have made a series of convincing arguments that a) the original data plus the results of new experiments make a convincing case for a previously unknown state of the MOR that promotes GTP release from Gi, b) the same agonists that specifically promote this GTP release have distinct pharmacological properties in vivo. I think that there is a very good likelihood that the two effects are closely and most likely causally related. I am still not sure whether I understood why promoting G-protein release requires a new initiation of an ARG complex, or whether the specific state of the MOR might just make the G protein in reverse mode – but at any rate this is a clear demonstration of a specific state of a receptor that ultimately results in GTP release by a G protein.\n\nI believe, therefore, that the authors do indeed show something fundamentally new. This is because the data clearly indicate a specific “novel” state of a GPCR that produces distinct downstream events, both in terms of G-protein behavior and in terms of pharmacological effects in vivo. Given that we deal with findings quite different from the usual bias discussion (i.e. either b-arrestin vs. G protein or between different G-proteins – but always with the same types of effects), I believe that these observations are of fundamental importance, both from a biochemical and a pharmacological point of view.. Therefore, while I agree with referee 1 in noting that the correlation does not formally prove a mechanistic link, I do believe that the data and the model are interesting enough to recommend publication in Nature.\n\nI am also happy with the responses that the authors have given to the other referees and with the changes that they have made in response to these comments. Notably, I find it obvious that there are many, many more experiments that one might wish for, which were proposed by the other referees – ranging from structural analyses (that would demonstrate the binding site of mucepans as well as the character of the specific state of the MOR) to experiments studying chronic opiate animal models (which would indeed be clinically most interesting). However, such studies would in my opinion go well beyond what a single lab can achieve and also beyond what one would hope for in one or two publications.\n\nSo I am essentially happy with the data as they stand. My only remaining concern is one that I would leave to the editors – how to best present these data and whether and how it might be better to combine the stories."}, "author_response": "Nature Communications manuscript NCOMMS-25-25118A, “Characterization of the GTPγS release function of a G protein coupled receptor”\n\nReviewer #1 (Remarks to the Author)\nThe article has been substantially revised in line with the reviewers suggestions. It is well written and presents a compelling argument for re-thinking of the two state model of G protein activation to include novel features of GTP binding and release. The authors also present how GTP-binding and release can be exploited pharmacologically with two new ligands that preferentially affect this process and show analgesic properties with reduced side effects. The authors in my view have taken into account the reviewers comments and it is my opinion that there is nothing substantive to be gained by further rounds of revision. The only comment I have is that this is designed to be a companion article for a Nature paper. It is still my opinion that the articles should be combined into a single Nat Comms paper.\n\nWe thank the referee for their careful consideration of our manuscript, and we agree with them that this does present an opportunity to reconsider the implications of the two state model of G protein activation. We feel that the reader will be best served by presenting the data in the current form as these submissions both complement each other and distinguish different aspects of our findings.\n\nReviewer #2 (Remarks to the Author):\nThe reviewer remains unconvinced whether GTP release by the receptor is an active process, analogous to receptor-mediated GDP release, and whether this GTP release is physiologically relevant.\n\nIn the case of GDP release, it has been widely demonstrated that incubation of an agonist-activated receptor with a GDP-bound Gi heterotrimer (in the absence of free GDP or GTP) induces GDP dissociation.\n\nTo convincingly demonstrate receptor-mediated GTP release, an in vitro system with purified receptors and GTP-bound Gα subunits should be employed, under conditions devoid of free GDP or GTP. The membrane-based system used by the authors contains other proteins and membrane components that may confound interpretation.\n\nWe thank the referee for their critical and constructive input on our manuscript. The referee has clearly spent time considering our hypothesis and the implications of this work. We agree that GDP release has been demonstrated, extensively, in both the absence and presence of free nucleotide. Similar experiments have previously been performed to measure agonist-mediated GTP dissociation. We have cited a number of these studies in the introduction but many of these were performed in the presence of exogenous nucleotide (GDP or GTP). However, Cassel and Selinger, 1977 (JCycNuclRes 3:11-22) observed the agonist-mediated release of 3H-GppNHp by isoproterenol in the absence of exogenous nucleotide. Further, they demonstrate in the same experiments that isoproterenol produced a decrease in persistent GppNHp activation of adenylyl cyclase in turkey erythrocytes. We have added to the introduction to note that the experimental conditions of Cassel and Selinger were performed in the absence of exogenously applied nucleotide:\n\nNotably, these early release studies were performed in both the presence11—13 and absence14 of saturating exogenous nucleotide which would suggest that the reaction is permissive under a range of free nucleotide concentrations.\n\nAdditionally, Citri and Schramm (JBC 1982 257(22):13257-13262) and Hekman et al. (EMBO 1984 3:3339-3345) employed purified and reconstituted receptor, G protein, and adenylyl cyclase to demonstrate that both GppNHp- and GTPγS-activation of G protein could be reversed by the additions of GTP (via subsequent hydrolysis of GTP->GDP). This was ligand dependent as the effect was produced by isoproterenol but not by propranolol. We have added these details to the discussion:\n\nFurther, in the reconstitution of the receptor|G protein|adenylate cyclase system, it was observed that epinephrine and GTP, together, could reverse GppNHp-bound G protein-stimulated cyclase activity.16,15 As a reconstituted system, this suggests that the receptor and activated G protein are the minimum system requirements necessary to recover the release reaction.\n\nRegarding other proteins and membrane components in the membrane-based system we have employed, we present data from CHO parental cells and pertussis toxin controls to support the receptor-mediated and Gα selective nature of these studies. These experiments, as well as the alkylation studies, were intended to support this minimum system requirement for the two reactions. We concede that we do not rule out additional actors in these experiments and we have remarked on this in the results section:\n\nTogether, studies in the CHO parental line and using pertussis toxin support the role of both the receptor and Gα in the 35S-GTPγS binding and release reactions. Beyond these controls, we do not rule out the involvement of additional membrane components as these reaction are not prepared or catalyzed in isolation. However, our data suggests that the agonist, receptor, and Gα form at least the minimum ternary complex necessary for these reactions to occur.\n\nAs previously noted by the reviewer, any GTP-released, nucleotide-free Gα subunits would be rapidly reoccupied by GTP under physiological conditions. Although the authors added an experiment using human spinal cord membranes, this system again involves membranes and does not effectively address the physiological implications of receptor-mediated GTP release in a more defined or cellular context.\n\nWe agree with the referee that the cytosolic concentration of GTP is very high and would rapidly saturate any free nucleotide binding site. However, if the receptor-mediated dissociation reaction of GTP is anything other than zero it is possible for the receptor to play a continued role in the G protein cycle as the Gα would remain a substrate for receptor signaling. At a minimum, this would reframe receptor activity in the two state model as noted. We acknowledge this possibility in the discussion and we have expanded this section to address this caveat. We speculate that this mechanism would have the possibility to add texture to the shape of the signal while still being subject to the same thermodynamic constraints.\n\nWe speculate that rhodopsin signaling could offer an even more exciting possibility. Biochemical studies suggest that depletion of transducin may limit the signaling capacity of rhodopsin.3 This is because transducin exists in limited pools which rhodopsin must both consume to signal and conserve to be able to signal. In fact, this depletion effect becomes even more substantial when the geometries of the lipid bilayer are considered in the approach of the substrates to the catalyst.3 In addition, the concentration of rhodopsin in rod outer segments is ~4-8mM;60 this would surpass the free GTP concentration by approximately five-fold.61,62 If the GTP release function were to act as a buffer to the system, it could quantitatively increase the density of transducin (as both Gα and Gα would then be catalytic substrates). This could permit the free nucleotide pool to be maintained, at least fractionally, as not every coupling event would lead to guanosine triphosphate consumption. We note that, in most physiological cases, the free nucleotide concentration would rapidly saturate the displaced nucleotide but this may still permit the receptor to extend its influence over the shape of the G protein signal. Further, by maintaining the availability of both substrates (GTP and transducin) for rhodopsin the release reaction may provide a control point for rhodopsin to manage energy expenditure of, and impart additional control over, the cellular processing of visual stimulus.\n\nIn Figure 1, naloxone did not induce GTP release, while the “dilution control” exhibited spontaneous GTP release. This suggests that naloxone may inhibit spontaneous GTP release, a mechanism that warrants further explanation. Additionally, since the “dilution control” itself shows GTP release, the authors’ statement that “The inhibition of 35S-GTPγS dissociation by naloxone highlights the stability of the GTPγS-G protein complex” may not be entirely accurate.\n\nThe use of “dilution control” was intended to be descriptive of the experiment but is technically incorrect. The dilution control is accurately described as 100nM DAMGO and, based on the data in Figure 4, is an approximately EC concentration of DAMGO in the 90 release assay. As such, the dilution control is not spontaneous GTPγS dissociation but rather 100nM DAMGO stimulated release. Naloxone, therefore, blocks this unavoidable residual DAMGO concentration that contaminates the release reaction. In fact, this “contamination” was the inspiration for the sodium-free loading conditions that were outlined in Figure 2 and employed subsequently throughout the manuscript. We have adjusted the text and the Figure 1 Legend to make this clearer to the reader:\n\nIn the case where samples were diluted ten-fold a residual 100 nM DAMGO remains from the pulse reaction. At the indicated times, this residual DAMGO produces significant dissociation of radiolabeled GTPS binding from G (Figure 1). Importantly, the rate of dissociation as well as the magnitude of dissociation is increased by the addition of a saturating 10 µM DAMGO (Figure 1, 100 nM DAMGO rate: 0.016 (0.008 – 0.025) min-1; 10 µM DAMGO rate: 0.048 (0.023 – 0.086), mean with 95% CI; p<0.0001 by extra sum-of-squares F test; t-test of 100 nM DAMGO vs. 10 µM DAMGO at 60 min, p<0.05); this is in agreement with prior studies.34 Inclusion of 10µM naloxone blocks the release induced by the residual DAMGO in the chase, highlighting the stability of the GTPS-G protein complex when agonist cannot catalyze the reaction. Further, the naloxone stabilizes the complex as binding does not significantly differ between the t and all subsequent time points in the presence of naloxone (Figure 1, one-way ANOVA of 10µM Naloxone vs. t , ns, p>0.05) arguing against occurrence of spontaneous release of GTPS from the complex. The stability of the GTPS-bound state has previously been reported for MOR stimulated GTPS binding.34\n\nThe DAMGO-mediated release prompts the question whether the agonist-mediated dissociation of GTP from G could be agonist concentration-responsive.\n\nFigure 1 Legend:\nFigure 1. Time-course for decay of 35S-GTPγS binding following agonist stimulation of the mouse µ-opioid receptor. Baseline (t ) and 1µM DAMGO -60 (1 hour, t ) show the agonist-mediated nucleotide loading in the pulse phase 0 (###p<0.001, unpaired two-tailed t-test). In the subsequent chase phase, a loss of radiolabeled nucleotide binding relative to t , is apparent for 100nM 0 DAMGO (ns, p>0.05, *p<0.05, **p<0.01 one-way ANOVA with Dunnett’s post-hoc test) and in the presence of 10µM DAMGO (**p<0.01, ***p<0.001; ****p<0.0001). Notably, 10µM naloxone completely prevents decay of 35S-GTPγS binding out to 60 minutes (ns, p>0.05, all points vs. t , 0 one-way ANOVA). All data points are presented as the average of three experiments with the bars presenting mean and s.e.m.\n\nIn Figure 2D, the authors aim to investigate whether the MOR–Gi interaction that mediates GTP release involves the same interface as for GDP release. Would it be informative to treat with pertussis toxin after GTP loading to test this more directly?\n\nWe agree with the reviewer that this will be an exciting and important experiment, however, our initial experiments were performed by treating intact cells with pertussis toxin prior to homogenization and protein preparation. Treating membranes with pertussis toxin is possible and would be informative, as the referee notes, but this would require substantial changes to our current experimental design. Moreover, these experiments were initially intended to demonstrate the Gα selectivity of the binding and release reaction and we will need to run a number of different controls to fully address our interest in the receptor-G protein interface. We see this as an exciting next step in our research into the binding and release reactions.\n\nMinor comments:\n1. Some statements require reference citations:\n   o Line 64: “For nearly … release.”\n   This has been rewritten for clarity:\n   We present here, and in the companion manuscript (Stahl et al, in submission), the results of nearly a decade of studies on the role of the mu opioid receptor (MOR) in stimulating 35S-GTPγS release. We have discovered that the mu receptor exhibits active state-selective GTP release and that it is possible for agonist activity to be intrinsically GTP release-selectivity.\n\n   o Line 482: “Biochemical studies … of rhodopsin.”\n   This has been adapted and references added for clarity:\n   We speculate that rhodopsin signaling could offer an even more exciting possibility. Biochemical studies suggest that depletion of transducin may limit the signaling capacity of rhodopsin.3 This is because transducin exists in limited pools which rhodopsin must both consume to signal and conserve to be able to signal. In fact, this depletion effect becomes even more substantial when the geometries of the lipid bilayer are considered in the approach of the substrates to the catalyst.3 In addition, the concentration of rhodopsin in rod outer segments is ~4-8mM;60 this would surpass the free GTP concentration by approximately five-fold.61,62 If the GTP release function were to act as a buffer to the system, it could quantitatively increase the density of transducin (as both Gα and Gα would then be catalytic substrates). It could permit the free nucleotide pool to be, at least fractionally, maintained as not every coupling event would lead to guanosine triphosphate consumption. We note that, in most physiological cases, the free nucleotide concentration would rapidly saturate the displaced nucleotide but this may still provide the receptor to impart texture to the shape of the G protein signal. Further, by maintaining the availability of both substrates for rhodopsin activity may provide a control point for rhodopsin to manage energy expenditure of, and impart control over, the cellular processing of a visual stimulus.\n\n   o Line 508: “however, we demonstrate … (Stahl et al., Nature).”\n   This has been corrected to be consistent throughout the manuscript.\n\n2. Line 66: “mu receptor” should be revised to “mu opioid receptor (MOR)”.\n   This has been corrected.\n\n3. Line 91: “1uM” should be formatted as “1 μM”.\n   This has been corrected.\n\n4. Line 91: “0.1nM” should be formatted as “0.1 nM”.\n   This has been corrected.\n\n5. Line 53: “Gαapo” — ensure consistent formatting and terminology for “apo” throughout the manuscript.\n   This has been corrected to be consistent throughout the manuscript.\n\nIn the corrections of the previous submission, reference #19 was incorrectly modified in the response to the referee. The correct reference has been inserted.\n\nReviewer #3 (Remarks to the Author):\nComments to the revised manuscripts by Stahl&Bohn et al.\nNature manuscript 2025-04-08323, \"GTP release-selective agonists prolong opioid analgesic efficacy\"\nNature Communications manuscript NCOMMS-25-25118, “Characterization of the GTPγS release function of a G protein coupled receptor”\n\nI am taking the liberty on commenting on both manuscripts together, since I believe (as noted in my comments to the original submissions) that they belong closely together.\n\nIn their rebuttal letters the authors have made a series of convincing arguments that a) the original data plus the results of new experiments make a convincing case for a previously unknown state of the MOR that promotes GTP release from Gi, b) the same agonists that specifically promote this GTP release have distinct pharmacological properties in vivo. I think that there is a very good likelihood that the two effects are closely and most likely causally related. I am still not sure whether I understood why promoting G-protein release requires a new initiation of an ARG complex, or whether the specific state of the MOR might just make the G protein in reverse mode – but at any rate this is a clear demonstration of a specific state of a receptor that ultimately results in GTP release by a G protein.\n\nI believe, therefore, that the authors do indeed show something fundamentally new. This is because the data clearly indicate a specific “novel” state of a GPCR that produces distinct downstream events, both in terms of G-protein behavior and in terms of pharmacological effects in vivo. Given that we deal with findings quite different from the usual bias discussion (i.e. either b-arrestin vs. G protein or between different G-proteins – but always with the same types of effects), I believe that these observations are of fundamental importance, both from a biochemical and a pharmacological point of view.. Therefore, while I agree with referee 1 in noting that the correlation does not formally prove a mechanistic link, I do believe that the data and the model are interesting enough to recommend publication in Nature.\n\nI am also happy with the responses that the authors have given to the other referees and with the changes that they have made in response to these comments. Notably, I find it obvious that there are many, many more experiments that one might wish for, which were proposed by the other referees – ranging from structural analyses (that would demonstrate the binding site of mucepans as well as the character of the specific state of the MOR) to experiments studying chronic opiate animal models (which would indeed be clinically most interesting). However, such studies would in my opinion go well beyond what a single lab can achieve and also beyond what one would hope for in one or two publications.\n\nSo I am essentially happy with the data as they stand. My only remaining concern is one that I would leave to the editors – how to best present these data and whether and how it might be better to combine the stories.\n\nWe thank the referee for their input and we feel that all three referees have made the manuscript stronger and easier for the reader to approach. We appreciate the referee’s recommendation for publication."}]

Decision letter