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

Article https://doi.org/10.1038/s41467-025-66516-y Characterization of the GTPγS release function of a G protein-coupled receptor Received: 2 April 2025 Laura M. Bohn & Edward L. Stahl Accepted: 6 November 2025 1234567890():,; 1234567890():,; Check for updates 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. 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 GαGDP subunit to release GDP. The nucleotide-free G protein (Gαapo) has higher affinity for guanosine triphosphate (GTP) and the active GαGTP forms; in this regard the GPCR serves as a guanine-nucleotide exchange factor (GEF). The G protein subsequently returns to the inactive GαGDP 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 GtαGTP 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 Gαapo to readily bind GTP; however, some studies have suggested GTP release as a possible explanation for experimental data where the Gαapo species was observed to form, subsequent to the GαGTP species11–13. As with rhodopsin, these studies involved measuring both the increase and release of nonhydrolyzable GTP binding over Department of Molecular Pharmacology and Physiology, University of South Florida Morsani College of Medicine, Tampa, Florida, USA. e-mail: ; Nature Communications | (2025)16:11193 1 Article https://doi.org/10.1038/s41467-025-66516-y 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 Gαapo state22–27. By contrast, NMR studies of receptor dynamics have revealed a more diversified energy landscape for activestate 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 pulsechase approach34. Cell membranes were prepared from MOR expressing CHO cells and, in the pulse phase, stimulated with 1 μM DAMGO in the presence of 0.1 nM 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 1 µM unlabeled GTPγS. For the next 60 min, the residual 35S-GTPγS binding is captured on filters at the time points shown. GTPγS Pulse-Chase Experiment 100nM DAMGO(10x dilution) + DAMGO 10µM + Naloxone 10µM 35S-GTPγS Bound, dpm 14000 ### 12000 ns ns 10000 ns ns 8000 ** *** * **** 6000 4000 ** **** 2000 0 t-60 t0 DAMGO 1µM Pulse 10 20 30 60 Dissociation Time, min (initiated @ t0) Chase Fig. 1 | Time-course for decay of 35S-GTPγS binding following agonist stimulation of the mouse µ-opioid receptor. Baseline (t-60) and 1 µM DAMGO (1 h, t0) show the agonist-mediated nucleotide loading in the pulse phase (###p < 0.001, unpaired two-tailed t-test). In the subsequent chase phase, a loss of radiolabeled nucleotide binding relative to t0, is appar (...truncated)