Two-step structural changes in M3 muscarinic receptor activation rely on the coupled Gq protein cycle

Kim, Yong-Seok; Yeon, Jun-Hee; Ko, Woori; Suh, Byung-Chang

doi:10.1038/s41467-023-36911-4

Download PDF

Article
Open access
Published: 08 March 2023

Two-step structural changes in M3 muscarinic receptor activation rely on the coupled G_q protein cycle

Nature Communications volume 14, Article number: 1276 (2023) Cite this article

3500 Accesses
2 Citations
38 Altmetric
Metrics details

Subjects

Abstract

G protein-coupled receptors (GPCRs) regulate diverse intracellular signaling pathways through the activation of heterotrimeric G proteins. However, the effects of the sequential activation–deactivation cycle of G protein on the conformational changes of GPCRs remains unknown. By developing a Förster resonance energy transfer (FRET) tool for human M3 muscarinic receptor (hM3R), we find that a single-receptor FRET probe can display the consecutive structural conversion of a receptor by G protein cycle. Our results reveal that the G protein activation evokes a two-step change in the hM3R structure, including the fast step mediated by G_q protein binding and the subsequent slower step mediated by the physical separation of the Gα_q and Gβγ subunits. We also find that the separated Gα_q-GTP forms a stable complex with the ligand-activated hM3R and phospholipase Cβ. In sum, the present study uncovers the real-time conformational dynamics of innate hM3R during the downstream G_q protein cycle.

Structural and dynamic insights into supra-physiological activation and allosteric modulation of a muscarinic acetylcholine receptor

Article Open access 23 January 2023

Molecular mechanism of muscarinic acetylcholine receptor M3 interaction with Gq

Article Open access 23 March 2024

Structures in G proteins important for subtype selective receptor binding and subsequent activation

Article Open access 27 May 2021

Introduction

G protein-coupled receptors (GPCRs) are one of the largest families of membrane receptors, with more than 800 members encoded by about 3% of human genes^1,2,3. They are widely expressed in cells organizing tissues and organs, and regulate cellular and physiological processes by transmitting various extracellular signals, including light, temperature, neurotransmitters, hormones, and odor substances, into cells^4,5. The GPCR signals are transmitted to membrane-associated effectors like ion channels and enzymes through several heterotrimeric (Gαβγ) G proteins. As mediators, G proteins play an important role in enabling GPCR signaling to have high flexibility, sensitivity, and specificity⁵. For this reason, the way in which GPCRs and G proteins interact with each other to induce this signaling efficiency in living cells has been studied extensively^6,7,8.

When GPCRs are activated by an agonist, the receptors combine with inactive heterotrimeric G proteins, followed by GDP release from the Gα subunit and GTP binding to the site, leading to the transient stabilization of the GPCR–G protein complexes⁹. Through biochemical and biophysiological methods, the interaction sites of these active-state GPCR–G protein complexes have been clarified in terms of the relationships between GPCR and Gα^10,11,12, GPCR and Gβγ^13,14,15,16, and Gα and Gβγ^10,17,18. In more recent studies, high resolution structures of those complexes have been defined by cryogenic electron microscopy^{19,20,21,22,23,24,25}.

Although they have been less studied compared with the active-state GPCR–G protein complexes, some resting-state complexes called preassembled inactive-state complexes have also been explored recently using ligand-binding and biochemical studies^24,26,27,28 as well as biophysical studies in live cells^{29,30,31,32,33}. These studies suggested that the preassembled complex formation may accelerate the onset of signaling by GPCRs and thus increase the GPCR sensitivity³³. However, it has not yet been determined which subunit of heterotrimeric G proteins interacts with GPCRs in their preassembly or whether the preassembly is maintained during the receptor activation without further structural changes. Moreover, some studies have looked into the different receptor conformations during the different stages of the G protein activation cycle^34,35, though the real-time effects of the G protein activation cycle on the innate conformation of GPCR remain unknown.

In this study, we develop a Förster resonance energy transfer (FRET) construct of human muscarinic acetylcholine receptor M3, hM3R-YFP-CFP, which enables the analysis of the conformational transition of hM3R that occurs during the coupled G_q protein cycling processes as well as the cascade from hM3R to phospholipase Cβ (PLCβ) within cells. Using this FRET construct, we find that the activation–deactivation cycle of G_q protein evokes multistep conformational changes of hM3R, which depend on the time-dependent association, separation, and dissociation of G_q protein subunits. We also provide evidence that the separated Gα_q and Gβγ subunits continuously stay on the agonist-activated hM3R, steadily activate PLCβ, and release from the receptor with different time constants. Our results demonstrate that the downstream G_q protein cycle inversely and dynamically regulates the upstream hM3R structure.

Results

A single hM3R-YFP-CFP FRET construct revealed a two-step hM3R activation signal

To investigate the signaling properties of M3R, a hM3R FRET construct, hM3R-YFP-CFP was constructed by replacing the third intracellular loop (ICL3) of wild-type hM3R with YFP and tagging CFP to the C-terminus (Fig. 1a) without damaging the selective recognition sites of G_q/11-proteins (Supplementary Fig. 1a–c). The receptor was activated by applying the muscarinic receptor agonist oxotremorine-M (Oxo-M). In contrast to the previously reported mouse M1 muscarinic FRET probe (mM1R-YFP-CFP)³⁶, binding of Oxo-M to this hM3R-YFP-CFP decreased the FRET ratio (FRETr) signal (Fig. 1b). In addition, hM3R-YFP-CFP showed ~3-fold larger FRETr responses and a ~5-fold higher signal-to-noise ratio (SNR) compared to mM1R-YFP-CFP (Fig. 1b–d). Photostability was also improved, and thus, the FRETr signal was maintained longer without a significant decrease compared to mM1R-YFP-CFP (Supplementary Fig. 1d, e). When the FRETr of the hM3R-YFP-CFP construct was measured in intact HEK293T cells under the faster perfusion system with higher frequency (f = 10 Hz), we found a two-step reduction of the FRETr signal with faster (step 1) and then slower (step 2) responses by Oxo-M treatment (Fig. 1e, top). There was a short delay (0.08 ± 0.05 s, n = 5) before the step 1. The time constant τ values of step 1 and step 2 were 0.22 ± 0.02 s and 1.92 ± 0.30 s (n = 5), respectively (Fig. 1f, top). However, the relative responsiveness of the two steps was almost equal at around 50% (Fig. 1g, top). Elevation of the sampling frequency to 100 Hz decreased the photostability but did not divide steps 1 and 2 further (Supplementary Fig. 1f). The FRETr change of the receptor was also measured after the application of the natural ligand acetylcholine (Ach). Consistent with the data of Oxo-M, 1 μM Ach induced two-step activation of hM3R-YFP-CFP with faster step 1 and slower step 2 responses (Fig. 1e–g, bottom). Both Oxo-M and Ach changed the FRETr signal in a concentration-dependent manner, but with significantly different EC₅₀ values of 0.007 μM for Ach and 0.112 μM for Oxo-M (Supplementary Fig. 2). The results suggest that Ach had a higher affinity for hM3R-YFP-CFP receptor compared to Oxo-M, which was similar to the responses of other M3R FRET sensors³⁷.

**Fig. 1: hM3R-YFP-CFP FRET construct shows a two-step activation.**

To characterize the two steps of the hM3R-YFP-CFP FRETr response, we manipulated intracellular GTP concentrations in the whole-cell configuration of the cells (Fig. 1h). In cells intracellularly perfused with the physiological level of GTP (0.1 mM), Oxo-M application induced almost the same two-step reduction in the FRETr signal of hM3R-YFP-CFP as those of intact cells (Fig. 1i, left). The cells patched with a pipette solution containing 1 mM of the GDP analog guanosine 5-O-(2-thiodiphosphate) (GDPβS) as a competitive antagonist of GTP binding to G proteins also displayed a two-step FRETr reduction with a similar proportion of step 1 and step 2 in the overall response (Fig. 1i, right, j). The time constant of the step 1 response was not changed, but the response of step 2 was nearly 5 times slower than that of cells with 0.1 mM of GTP (Fig. 1k). These results suggest that step 1 is likely related to the conformational change following the activation of hM3R-YFP-CFP by agonist binding, while step 2 represents the additional conformational changes of the receptor following the G protein activation. To check whether the internalization of the receptor affects the FRETr change, hM3R-YFP-CFP expression on the HEK293T cell membrane was visualized with a confocal microscope. We confirmed that there were no detectable changes in the fluorescence distribution of hM3R-YFP-CFP at the plasma membrane before and after the receptor activation (Fig. 1l).

To examine whether Oxo-M binding to hM3R-YFP-CFP normally evokes downstream intracellular signaling through the activation of coupled G_q protein, PLC-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) was measured in cells co-transfected with the PIP₂ probe RFP-labeled pleckstrin homology domain of PLCδ₁ (RFP-PH). Cells transfected with the wild-type mM1R or hM3R plasmid showed a strong translocation of RFP-PH from the plasma membrane to cytosol by Oxo-M treatment (Supplementary Fig. 3a–c). There was no significant movement of RFP-PH in cells expressing mM1R-YFP-CFP, confirming that mM1R-YFP-CFP cannot trigger downstream signaling pathways, as reported in a previous study³⁶ (Supplementary Fig. 3d). However, Oxo-M induced strong translocation of RFP-PH in cells expressing hM3R-YFP-CFP, suggesting that the activation of hM3R-YFP-CFP by Oxo-M can stimulate downstream signaling pathways through G_q protein activation (Supplementary Fig. 3e). There was a slight difference in the relative RFP-PH changes at the plasma membrane and cytosol, but the half time of activation (T₅₀) showed very similar kinetics to those of wild-type hM3R (Supplementary Fig. 3f, g). Those results were further confirmed in the experiments with Ach (Supplementary Fig. 4a, b). Though there was a considerable decrease in the relative fluorescence changes at the PM and cytosol (Supplementary Fig. 4c), the kinetics for the PIP₂ hydrolysis were not changed in the FRET receptor (Supplementary Fig. 4d). Therefore, the results suggest that hM3R-YFP-CFP can transmit external signals to downstream pathways through the activation of the G_q protein with similar kinetics to that of wild-type hM3R.

Two-step activation of hM3R-YFP-CFP can be explained by G_q protein activation

Based on the similarity between hM3R-YFP-CFP and wild-type hM3R in terms of kinetics of PIP₂ depletion, we further examined the molecular basis of the two steps. First, we hypothesized that the pathways corresponding to the fast step 1 of hM3R-YFP-CFP were related to a conformational change following Oxo-M binding to the receptor and subsequent interaction of the activated receptor with the heterotrimeric G_q protein. Thus, we performed FRET experiments in cells transfected with hM3R-CFP, Gβ1-YFP, and cognate G protein subunits (Fig. 2a). The results showed a single-step FRETr response composed of a fast signal upon Oxo-M treatment as reported in previous G_q-coupled receptors^36,38 (Fig. 2b, left, c, gray). The τ of the step (0.23 ± 0.04 s) (Fig. 2d, gray) was very similar to the step 1 τ of hM3R-YFP-CFP (Fig. 1k, left, gray), suggesting that the step 1 reaction includes fast coupling between hM3R and G_q protein after Oxo-M binding to the receptor. We conducted the same experiment yet 0.1 mM of GTP in the pipette solution was replaced with 1 mM of GDPβS to cross-check the similarity between the responses (Fig. 2b, right). The results showed that there was also a single FRETr signal response with almost the same time constant as that with 0.1 mM GTP (Fig. 2c, d, green) or step 1 reaction of hM3R-YFP-CFP (Fig. 1k, left), confirming that intracellular GTP concentration does not affect the step 1 reaction. We also measured the difference in the SNR of step 1 between hM3R-YFP-CFP and hM3R-CFP plus Gβ1-YFP (Fig. 2e). The SNR value (169 ± 23) of the step 1 response of hM3R-YFP-CFP was nearly twice those of hM3R-CFP plus Gβ1-YFP with 0.1 mM of GTP (90 ± 14) or with 1 mM of GDPβS (69 ± 8) (Fig. 2e; Supplementary Fig. 5a, b). There was no significant difference in the SNR between cells intracellularly perfused with GTP or GDPβS. Finally, Oxo-M induced the coupling of hM3R-CFP and Gβ1-YFP in a concentration-dependent manner with the EC₅₀ value of ~2 μM (Supplementary Fig. 6a, c). When we measured the coupling between Gβ1-YFP and hM3R-BFP-CFP where the YFP was replaced by blue fluorescence protein (BFP), there was no significant change in the EC₅₀ value (Supplementary Fig. 6b, c). The τ_ON value was not changed, but the SNR level decreased because of the partial reduction in the FRET signal amplitude (Supplementary Fig. 6d). These results suggest that the insertion of YFP to the ICL3 could attenuate the potency for the G_q protein coupling to our M3R FRET reporter.

**Fig. 2: Two-step activation kinetics of hM3R-YFP-CFP FRET correspond to hM3R-G_q protein interaction and Gα_q and Gβγ dissociation, respectively.**

We also performed FRET experiments between Gα_q and Gβγ subunits in cells expressing Gα_q-CFP and Gβ1-YFP with wild-type hM3R, Gγ2, and GRK2 (Fig. 2f). GRK2 has been known to interact with G_q protein subunits^39,40 to regulate the downstream signaling pathways. The results showed a single-step reduction of FRETr with 0.25 ± 0.08-s delay (n = 6) upon Oxo-M treatment (Fig. 2g, left, h, gray). The average τ value of this step was 1.45 ± 0.19 s (Fig. 2i gray), which was similar to that of the step 2 response of hM3R-YFP-CFP (Fig. 1k, right, gray). We conducted the same experiment in the presence of 1 mM of GDPβS instead of GTP (Fig. 2g, right). The results showed that with GDPβS, the response was much slower (10.38 ± 1.46 s) (Fig. 2i, green) compared to that of cells with GTP (Fig. 2i, gray) but similar to that of the step 2 response of hM3R-YFP-CFP with GDPβS (Fig. 1k, right, green). We also found that there was no significant difference in the SNR between hM3R-YFP-CFP (step 2) and Gα_q-CFP plus Gβ1-YFP under the GTP or GDPβS condition (Fig. 2j; Supplementary Fig. 5c, d), suggesting that the step 2 FRET response of hM3R-YFP-CFP corresponds to the slow dissociation of Gα_q and Gβγ subunits after activation on the receptor. Using confocal microscopy we confirmed that the cells expressing hM3R-CFP plus Gβ1-YFP or Gα_q-CFP plus Gβ1-YFP showed normal PIP₂ hydrolysis through G_q protein activation (Supplementary Fig. 7a, c and 7b, d, bottom). Moreover, hM3R activation did not release any Gα_q and Gβγ subunits from the plasma membrane to the cytosol (Supplementary Fig. 7a, c and 7b, d, top), suggesting that both subunits were independently tethered to the plasma membrane after separation.

Oxo-M dissociation evoked two-step deactivation kinetics in hM3R-YFP-CFP

We also measured the deactivation kinetics of hM3R-YFP-CFP after Oxo-M washout. As shown in Supplementary Fig. 8a, hM3R-YFP-CFP deactivation showed a two-step FRETr recovery with faster step 1 and slower step 2 reactions. To determine the nature of these two steps, FRETr was also measured during the receptor deactivation in cells expressing hM3R-CFP plus Gβ1-YFP or Gα_q-CFP plus Gβ1-YFP (Supplementary Fig. 8b, c). As a result, the deactivations were observed as a single step in both experiments. The deactivation τ of hM3R-CFP plus Gβ1-YFP (0.53 ± 0.04 s) showed a value very similar to that of step 1 (0.47 ± 0.08 s) of hM3R-YFP-CFP deactivation (Supplementary Fig. 8d), whereas that of Gα_q-CFP plus Gβ1-YFP (21.37 ± 1.34 s) was similar to that of step 2 (20.02 ± 2.29 s) of hM3R-YFP-CFP deactivation (Supplementary Fig. 8e). In terms of percent distribution of each step in the total recovery, both hM3R-CFP plus Gβ1-YFP and Gα_q-CFP plus Gβ1-YFP showed a similar distribution value of around 88% (Supplementary Fig. 8f, g). We further confirmed that, consistent with previous findings³⁶, the coexpression of GRK2 significantly increased the signal amplitude and SNR of the FRETr response between Gα_q-CFP and Gβ1-YFP but did not change the kinetics of receptor-induced activation or deactivation (Supplementary Fig. 9). GRK2 expression neither affect the kinetics nor the peak responses of FRETr in hM3R-YFP-CFP, but decreased the SNR by slightly enhancing the noise level (Supplementary Fig. 10).

hM3R-YFP-CFP can make a complex with the separated Gβγ subunits

To further elucidate how hM3R communicates with G_q proteins to transmit external signals to the downstream pathways, we constructed two constitutively active Gα_q mutant forms: Gα_q(Q209L) and Gα_q(R183C) (Fig. 3a). Previous studies have reported that diseases such as uveal melanoma result from continuous GPCR activity through Gα_q mutation (Q209L or R183C) and loss of GTPase activity of the subunit^41,42,43. First, we examined if the additional expression of wild-type G_q proteins affects the Oxo-M-induced FRETr signal of hM3R-YFP-CFP (Supplementary Fig. 11). As shown in Fig. 3b and Supplementary Fig. 12, the overexpression of wild-type G_q proteins did not change the two-step kinetics for activation and deactivation, the percentage recovered, or the SNR of hM3R-YFP-CFP. However, in cells co-transfected with mutant Gα_q(Q209L or R183C) and cognate G protein subunits, only a single step of activation and recovery was identified in the FRETr experiment (Fig. 3c), showing a comparable time constant to step 1 (ON or OFF) of wild-type G_q protein (Fig. 3d). Because of the absence of step 2, the FRETr was almost completely recovered within a single step (Fig. 3e). Consistently, after the intracellular perfusion of the nonhydrolyzable GTP analog guanosine 5-O-(3-thiotriphosphate) (GTPγS), Oxo-M evoked a single-step activation of FRETr probably because GTPγS activated the G proteins almost irreversibly⁴⁴ (Supplementary Fig. 13).

**Fig. 3: hM3R-YFP-CFP shows only step 1_ON and 1_OFF in presence of constitutively active Gα_q proteins.**

Next, we performed the FRETr experiment between hM3R-CFP and Gβ1-YFP in cells co-expressing mutant Gα_q(Q209L or R183C) and Gγ2. The results showed that Oxo-M induced a single-step FRETr change with similar time constants and percentage recovered, but significantly lower SNR compared to those of wild-type Gα_q (Fig. 3f–h; Supplementary Fig. 14). These results suggest that, upon Oxo-M binding to the receptor, hM3R can independently interact with the separated Gβγ subunits though its binding affinity is weaker than that for heterotrimeric G protein. Interestingly, the results also showed that the interaction between the hM3R and Gβγ subunit was sustained during the application of the agonist and recovered immediately after Oxo-M washout. Finally, a FRETr experiment was performed in cells expressing hM3R, mutant Gα_q(Q209L or R183C)-CFP, Gβ1-YFP, Gγ2, and GRK2. The results showed no changes in FRETr by Oxo-M application, except in wild-type Gα_q (Fig. 3i–k), indicating that the mutant Gα_q(Q209L or R183C) and Gβγ subunits were dissociated in the resting condition and thus responded to receptor activation separately.

Inhibition of GDP release from Gα_q selectively blocked the step 2 response of hM3R-YFP-CFP

YM-254890 (YM) has been studied as a candidate drug for diverse diseases, such as thrombosis, asthma, and melanoma⁴³. Preclinical studies have shown that YM’s main mechanism of action is the inhibition of GDP release from the Gα_q protein⁴³. We examined the effects of YM on the FRETr response of the hM3R and G_q proteins. As seen in Fig. 4a, left, in the presence of YM, only the step 1 (ON and OFF) FRETr response was detected upon Oxo-M application in cells expressing hM3R-YFP-CFP and wild-type G_q proteins. When the YM was removed in the middle of hM3R-YFP-CFP activation with Oxo-M, the cells showed a slow step 2_ON FRETr response up to about 31% of the total FRETr activation (step 1_ON + step 2_ON) and a step 2_OFF response up to about 30% of the total deactivation (step 1_OFF + step 2_OFF) (Fig. 4a, b, red). However, the results of experiments with mutant Gα_q(Q209L) revealed that there was a normal step 1 FRETr response by Oxo-M but that the step 2 response was not detected in hM3R-YFP-CFP even after washing out YM (Fig. 4c, d). This could have been due to the pre-separation of constitutively active mutant Gα_q and βγ subunits before Oxo-M treatment. There was no significant difference in the τ values of step 1 (ON and OFF) between wild-type G_q and G_q(Q209L) (Fig. 4e). These results suggest that YM did not affect the interaction of G_q protein with Oxo-M-activated hM3R but selectively inhibited the step 2 response by blocking the reaction of GDP release from the Gα_q protein and the following GTP binding and separation of the heterotrimeric G_q protein to Gα_q and βγ subunits (Fig. 4f).

**Fig. 4: YM-254890 (YM) blocks the step 2 stage of hM3R-YFP-CFP activation.**

The molecular mechanism of the single-step FRET response under YM treatment was further studied in cells expressing hM3R-CFP plus Gβ1-YFP. Consistently, as shown in Fig. 4g, h, YM did not affect the interaction between hM3R-CFP and Gβ1-YFP by Oxo-M. Moreover, the interaction between hM3R-CFP and Gβ1-YFP in cells expressing mutant G_q(Q209L) was normal. We also examined the effects of YM in cells expressing Gα_q-CFP plus Gβ1-YFP. In those cells, YM inhibited the activation of the G_q protein and thus blocked the separation of Gα_q-CFP and Gβ1-YFP (Fig. 4i, left). Then, after the removal of the YM, there was a slow FRETr decrease by the separation of Gα_q and βγ subunits. Interestingly, a minor but significant decrease in FRETr was detected after Oxo-M application even in the presence of YM, suggesting that the association of heterotrimeric G_q protein with ligand-activated hM3R may result in the structural rearrangement between Gα_q and Gβγ subunits although they are still closely associated with each other. In cells expressing mutant Gα_q(Q209L)-CFP plus Gβ1-YFP, there were no changes in FRETr after YM washout, supporting the idea that Gα_q(Q209L)-CFP and Gβ1-YFP were already separated and not affected by either YM or receptor activation (Fig. 4i, j). We additionally examined whether this Gα_q(Q209L)-CFP, which has no interaction with Gβγ, is normally anchored to the plasma membrane. The confocal microscopy imaging showed no difference in the fluorescent expression level of Gα_q-CFP and Gβ1-YFP in the plasma membrane or cytosol depending on whether Gα_q was mutated in the resting state (Supplementary Fig. 15a–f). Moreover, in both conditions, receptor activation did not change those signal levels (Supplementary Fig. 15a–d). However, there was a difference in the regulation of PIP₂ levels. Oxo-M hydrolyzed the PIP₂ and increased the intracellular level of RFP-PH in cells expressing wild-type Gα_q-CFP and Gβ1-YFP (Supplementary Fig. 15a). In contrast, in cells expressing mutant Gα_q(Q209L)-CFP and Gβ1-YFP, the cytosolic level of RFP-PH was already elevated regardless of receptor activation, confirming that Gα_q(Q209L)-CFP can activate PLC even without receptor stimulation (Supplementary Fig. 15b).

Two-step structural change in hM3R-YFP-CFP depends on G protein coupling

Existing fluorescence-based M3 sensors commonly show a single-step FRET change, which represents the conformational activation of a receptor by agonist binding^{37,38,45,46,47}. To examine whether the two-step FRET signal observed in hM3R-YFP-CFP, especially in step 1, involves receptor activation in addition to G_q-protein coupling, we applied a class A GPCR mutation strategy that blocks Gα binding to our hM3R-YFP-CFP construct without impairing receptor activation by the ligand⁴⁸. The three amino acid residues at positions 3x46 in TM3, 6x37 in TM6, and 7x53 in TM7 (notation represents the GPCRdb numbering scheme⁴⁹ from GPCRdb [http://www.gpcrdb.org]⁵⁰) are conserved in all class A GPCRs and important for the cytosolic release of 6x37, which comes in contact with a universally conserved leucine residue (position G.H5.25; notation taken from “The Common G Protein Numbering Scheme”⁵¹) of the Gα subunit⁴⁸ upon activation. This leucine residue plays a pivotal role in the coupling of Gα to GPCRs⁵². Through modeling of the 3D structure of M3R(3A) using both I-TASSER and SWISS-Model, we confirmed that the allosteric effect of alanine substitution on the structure or operation of the receptor’s agonist-binding sites⁵³ was insignificant⁴⁸ (Fig. 5a, left; Supplementary Fig. 16, left). To block the G_q protein coupling of hM3R-YFP-CFP, we substituted the corresponding I162 (3x46), L493 (6x37), and Y544 (7x53) residues with alanine in the construct. We found that the mutant hM3R(3A)-YFP-CFP was normally expressed in the cell membrane, but PIP₂ was not hydrolyzed even after the receptor was activated by Oxo-M or Ach (Fig. 5b, c). Next, the FRET signal of hM3R(3A)-YFP-CFP was measured after the treatment with these two agonists. Unlike the wild-type hM3R-YFP-CFP, hM3R(3A)-YFP-CFP displayed no considerable FRETr changes (Fig. 5d, e). These results suggested that hM3R(3A)-YFP-CFP could not interact with the heterotrimeric G_q protein; this result was consistent with previous findings on class A receptors⁴⁸. We also conducted FRET experiments between hM3R(3A)-CFP and Gβ1-YFP. As expected, the 3A mutant receptor did not interact with G_q proteins (Fig. 5f, g). Therefore, the FRETr signal of hM3R-YFP-CFP represents the conformational changes induced by G_q protein coupling but not the structural activation of the receptor by ligand binding. To convincingly show that the step 1 of hM3R-YFP-CFP is caused by G_q coupling and not by ligand binding-induced conformational changes, we also examined the activity of single-leucine mutant (3x50) M3R which has been shown to block G_q signaling but still undergo ligand-induced conformational rearrangement and arrestin-coupling⁵⁴. As a result, the mutant hM3R(R166L^3x50)-YFP-CFP displayed no considerable PIP₂ hydrolysis and FRETr changes (Supplementary Fig. 17).

**Fig. 5: hM3R(3A) is not able to bind G_q protein.**

Independent binding of Gβγ to hM3R may require the coupling of Gα_q-GTP to the receptor

To further understand the coupling mechanism between hM3R and separated Gβγ subunits, we examined the independent Gβγ interaction with the mutant hM3R(3A). First, consistent with the results shown in Fig. 5f, g, the findings indicated that coupling was normal between the wild-type hM3R-YFP and Gα_q-CFP but completely abolished between hM3R(3A)-YFP and Gα_q-CFP (Fig. 6a, b). Similarly, Gα_q(Q209L)-CFP coupled with the wild-type hM3R-YFP but not with hM3R(3A)-YFP (Fig. 6c, d). Interestingly, the maximum change in the relative FRETr signal was strongly reduced in cells expressing the wild-type hM3R-YFP plus Gα_q(Q209L)-CFP compared with that in cells expressing the wild-type hM3R-YFP plus wild-type Gα_q-CFP (WT Gα_q-CFP: 0.030 ± 0.002; Gα_q(Q209L)-CFP: 0.014 ± 0.003, ***p = 0.0006; Fig. 6b, d). Next, we measured the FRETr signal in cells expressing hM3R(3A)-YFP-CFP, Gα_q(Q209L), and the rest of the wild-type cognate G protein subunits. Unlike the responses of the wild-type hM3R-YFP-CFP (Fig. 6e, f, left), the FRETr signal did not decrease in hM3R(3A)-YFP-CFP (Fig. 6e, f, right). Finally, we examined the direct binding of the separated Gβγ to hM3R in cells co-transfected with Gα_q(Q209L). As shown in Fig. 6g, h, FRETr did not change in cells expressing the mutant hM3R(3A)-CFP. These results suggest that the separated Gβγ subunits could bind only to receptors coupled with Gα_q-GTP.

**Fig. 6: hM3R(3A) does not interact with Gβγ subunits independently.**

Sustained assembly of hM3R, Gα_q, and PLCβ₁ during receptor activation

To determine the operating role of Gα_q in the regulation of hM3R conformation, we performed FRETr experiments in cells transfected with hM3R-YFP, Gα_q-CFP, and cognate G protein subunits. The receptor activation upon Oxo-M application showed a single-step FRETr increase, suggesting the interaction of hM3R-YFP with Gα_q-CFP (Fig. 7a, d). Interestingly, the increased FRETr signal was maintained during the Oxo-M treatment. After Oxo-M removal, the FRETr signal returned to baseline through two-step kinetics with almost equal proportions (Fig. 7a, f). To investigate this two-step recovery, we first conducted the same FRETr experiments in the presence of YM (Supplementary Fig. 18a). There was a single-step increase in FRETr like without YM, with a τ of around 0.2 s (Fig. 7e; Supplementary Fig. 18f, left). This was very similar to the step 1 τ (~0.21 s) of hM3R-YFP-CFP, confirming that the fast step 1_ON response of hM3R-YFP-CFP is mediated by the coupling of hM3R with heterotrimeric inactive G_q protein. Importantly, in the presence of YM, there was only a single-step FRETr recovery (Supplementary Fig. 18a), with the time constant similar to the step 1_OFF of hM3R-YFP plus Gα_q-CFP (Fig. 7g) and step 1_OFF of hM3R-YFP-CFP (Supplementary Fig. 8a, d). Based on these results, we can speculate that the two-step recovery of FRETr in cells with hM3R-YFP and Gα_q-CFP can be defined as the step 1_OFF and step 2_OFF responses in hM3R-YFP-CFP, where step 1_OFF may indicate the release of Gβγ subunit from hM3R after the uncoupling of Oxo-M from hM3R and step 2_OFF could be a result of the dissociation of Gα_q-CFP from hM3R-YFP after the hydrolysis of GTP to GDP by the GTPase activity of Gα_q (Fig. 7a). This speculation was further tested using the mutant Gα_q(Q209L). As shown in Fig. 7b, f, no significant step 2_OFF response was observed after the Oxo-M washout. Consistently, the step 1_ON response decreased in cells expressing Gα_q(Q209L) because of the independent binding of Gβγ to the receptor (Fig. 7c). However, the kinetics of step 1_ON and 1_OFF remained unchanged (Fig. 7e, g). These results further supported that the slow step 2_OFF in the FRETr response of hM3R-YFP plus Gα_q-CFP was due to the slow dissociation of Gα_q-CFP from hM3R-YFP. Through confocal microscopy imaging, we further confirmed that the cells transfected with hM3R-YFP, Gα_q-CFP, and cognate G protein subunits also possessed a normal signaling cascade to PLCβ activation and PIP₂ hydrolysis by receptor activation (Supplementary Fig. 19a). In addition, the hM3R signaling to PLCβ activation was sustained until washout of the agonist (Supplementary Fig. 19b).

**Fig. 7: hM3R, Gα_q, and PLC form a heterotrimer during receptor activation.**

To investigate the interaction of activated Gα_q-GTP with its effector PLCβ, we performed FRETr experiments in cells transfected with hM3R, Gα_q-CFP, YFP-PLCβ₁, and cognate G protein subunits. The receptor activation upon Oxo-M application showed a single-step FRETr increase that was stronger but slower than that of hM3R-YFP and Gα_q-CFP, suggesting that the coupling between hM3R and Gα_q is completed faster than the complex formation of Gα_q and PLCβ₁ (Fig. 7h, i). Surprisingly, the increased FRETr signal between Gα_q-CFP and YFP-PLCβ₁ was also maintained during the activation of the receptor (Fig. 7h). When checking the deactivation process in Gα_q-CFP plus YFP-PLCβ₁, a single-step decrease was measured (Fig. 7h, j). The kinetics of this single-step deactivation was found to be slower than the step 1_OFF reaction of hM3R-YFP plus Gα_q-CFP (Fig. 7g left) or hM3R-YFP-CFP (Supplementary Fig. 8a, d) but faster than the step 2_OFF reaction of hM3R-YFP plus Gα_q-CFP (Fig. 7g, right) or hM3R-YFP-CFP (Supplementary Fig. 8a, e).

Considering the stable interaction between active hM3R and Gα_q-GTP (Fig. 7a), the results suggested that hM3R, Gα_q, and PLCβ₁ formed a stable heterotrimeric complex during receptor activation. To test this possibility, we examined the direct interaction between hM3R-CFP and YFP-PLCβ₁. As shown in Fig. 7k, Oxo-M strongly increased the FRETr signal in cells expressing the two probes, supporting the conclusion that a stable complex formed between M3R and PLCβ₁ upon receptor activation. The analysis of the FRETr signals revealed two-step responses during activation and deactivation (Fig. 7l, m, right). Interestingly, the kinetics of the fast steps 1_ON and 1_OFF was similar to that of receptor activation (Fig. 7l, m, left). The kinetics of step 2_OFF was close to that of the step 2_OFF of hM3R-YFP and Gα_q-CFP (Fig. 7g, right), suggesting that PLCβ₁ might dissociate from hM3R when Gα_q left the receptor. Together, the results demonstrate that PLCβ₁ is released from Gα_q-GTP more slowly than the step 1_OFF reaction and that this release ends before the completion of the step 2_OFF reaction (Fig. 8). Finally, to check the function of YM, we conducted the same FRETr experiments with Gα_q-CFP plus YFP-PLCβ₁ in the presence of YM (Supplementary Fig. 18b–f). There was no FRETr change by receptor activation, indicating that YM successfully inhibited G_q activation by blocking the GDP release from Gα_q. The results further confirmed that the inactive G_q protein cannot interact with PLCβ₁.

**Fig. 8: Schematic diagram of G_q protein cycle-mediated conformational changes of hM3R.**

Discussion

Various GPCR studies using FRET probes, bioluminescence resonance energy transfer (BRET) probes, or cpGFP (circular permutated green fluorescent protein) have been conducted to observe the intramolecular conformational change of receptors by ligand binding^{36,37,38,46,55,56,57,58,59,60,61}. However, to observe the GPCR signaling pathways after receptor activation, additional experiments^36,38,62,63 are required utilizing new photo probes corresponding to each pathway step. In those step-by-step experiments for the signaling cascades, the continuity of the entire structural deformation of GPCR is inevitably damaged. In this study, we overcame these limitations by constructing a M3R FRET sensor hM3R-YFP-CFP. Using the construct, we discovered that the structure of hM3R reversibly changed through a multistep process according to the activation cycle of the G_q protein. In addition, the FRET results among M3R, Gα_q, and PLCβ suggested that the ligand-activated M3R formed a sustained heterotrimeric complex with Gα_q and PLCβ.

Here, we reported the two-step activation and deactivation of M3R. All existing fluorescence-based M3 sensors commonly displayed receptor activation through a single-step change in fluorescent signals. In previous studies, the activation of a receptor generally corresponds to the conversion of the M3R structure from an inactive state to an active one after agonist binding and not to the structural changes induced by G protein coupling. However, hM3R-YFP-CFP exhibited completely different properties from the existing sensors in that it detected G protein coupling after the activation stage and thus released the activation cycle of the G protein as a two-step FRET signal. This finding was further confirmed by the experiments involving the receptors with mutated Gα-binding sites without the impaired activation of the receptor⁴⁸. The distinction in the target response of our receptor sensor might be attributed to the differences in receptor construction methods. For example, a fluoresceine arsenical hairpin binder (FlAsH)-based M3 FRET sensor^37,38 shows normal downstream signaling similar to our hM3R-YFP-CFP but only detects the receptor activation through the FRET response, and the direction of response is increase that is opposite to ours. These differences might be due to the location and size of the inserted amino-acid motif CCPGCC, where the amino-acid residues from 271 and 465 (193 amino acids) of ICL3 were replaced with CCPGCCE (7 amino acids); in our construct, the residues between 270 and 485 (214 amino acids) were replaced with SacII-EYFP-AgeI (243 amino acids) in ICL3. In addition, the sensor required an additional binding of FlAsH to the inserted CCPGCC. Thus, such differences possibly led to the differential positioning of acceptor fluorescence (FlAsH or EYFP) relative to CFP between these two FRET sensors; eventually, the stage of signaling pathways and the direction of the FRET response varied. Similarly, GACh2.0 (ACh2.0)⁴⁶ and GRAB_ACh3.0 (ACh3.0)⁴⁵ were constructed by inserting cpGFP into ICL3; when the receptor becomes activated, cpGFP transforms into an intact GFP structure and emits fluorescence. Unlike hM3R-YFP-CFP, this sensor is a dead form of downstream signaling and therefore can only detect receptor activation. This difference is also likely due to the locations of the inserted cpGFP instead of YFP. For ACh2.0, the residues between amino acids 253 and 493 (239 amino acids) of ICL3 were replaced with linker-cpGFP-linker (301 amino acids); for ACh3.0, the residues between amino acids 259 and 491 (231 amino acids) of ICL3 were replaced with linker-cpGFP-linker (246 amino acids). The ICL2 and ICL3 parts of GPCRs have conserved amino acids, which are essential for recognizing a certain type of G protein^24,64. However, these amino acids were lost during cpGFP insertion; this loss likely caused these cpGFP-based sensors to become the dead form of downstream signaling.

Using hM3R-YFP-CFP, we demonstrated that a single-receptor FRET probe can reveal the multistep intramolecular conformational changes of a receptor during the coupled G protein cycle. We confirmed that, in the activation process, the association of ligand-activated hM3R with the inactive G_q protein displayed the fast step 1 FRETr signal and the following dissociation of Gα_q and Gβγ subunits under receptor coupling was responsible for the slower step 2 FRETr signal (Fig. 8). We could not obtain any independent FRETr change by ligand binding itself in hM3R-YFP-CFP even at a high frequency resolution (100 Hz) and fast solution exchange system (~20 ms). Previous studies have shown that ligand–M3R coupling is completed within 100 ms^37,62, thus it is possible that the ligand binding to the external surface of our hM3R FRET construct generates relatively insignificant FRETr change.

In our disease model studies using the constitutively active Gα_q(Q209L or R183C) forms, there was a single fast step of FRETr change in hM3R-YFP-CFP alone and in hM3R-CFP plus Gβ1-YFP. Since the Gβγ subunits exist independently in resting cells after decoupling from the constitutively active Gα_q(Q209L or R183C), the results indicate that the ligand-activated hM3R can associate with the separated Gβγ subunits. In addition, the hM3R-Gβγ coupling remains unchanged during the YM application, confirming that the fast step 1 FRETr signal of hM3R-YFP-CFP in the presence of Gα_q(Q209L or R183C) is mostly mediated by the intermolecular interaction with the Gβγ subunit. We also found that agonist binding to hM3R itself changes the interaction between Gα_q and Gβγ subunits before dissociation. As shown in Fig. 4i, j, even in the presence of the GDP release inhibitor YM, there was a minor but significant FRETr decrease between Gα_q and Gβγ subunits after Oxo-M application. These initial structural changes between Gα_q and Gβγ subunits upon binding to ligand-activated hM3R may be important for GDP release from the inactive Gα_q and subsequent GTP binding.

We confirmed that the loss of G protein coupling ability in the triple-alanine mutant (3x46/6x37/7x53) vasopressin V2 receptor⁴⁸ can be reproduced in M3R, which is the same class A GPCR. By constructing hM3R(3A), we revealed that hM3R-YFP-CFP only detected the downstream signals related to the activation cycle of G_q protein but not the precedent receptor activation step by ligand binding. This was further confirmed by additional experiments using single-leucine mutant (3x50) M3R. Previous study reported that the mutant M3R(R165L^3x50) still had normal agonist-binding affinity and arrestin-dependent signaling pathways⁵⁴. Nevertheless, our hM3R(R166L^3x50)-YFP-CFP showed no significant PIP₂ hydrolysis and FRETr changes (Supplementary Fig. 17).

Another significant finding is the two-step deactivation of FRETr in cells expressing hM3R-YFP and Gα_q-CFP after washout of the agonist. The fast step 1 and the subsequent slower step 2 recoveries seem to be due to the sequential interaction changes by the decoupling of the Gβγ subunit from hM3R following agonist dissociation and the dissociation of Gα_q-CFP from hM3R after the slow dephosphorylation of GTP to GDP by GTPase activity, respectively. These results suggest that, in the recovery process from receptor activation, Gα_q-GTP alone can be coupled with the receptor during the step 2 period even in the absence of the Gβγ subunit on the receptor.

Several studies related to the inactive-state preassembly of GPCRs and G proteins have been reported recently^{24,26,27,28,29,30,31,32,33}. However, the results have been contradictory even when using the same M3R^33,65. The discrepancy might be due to the fact that the studies could not directly probe the stability of the protein–protein complex⁵. In the present study, we showed that there was a significant FRETr increase between hM3R-CFP and Gβ1-YFP, indicating that the activated hM3R interacts with heterotrimeric G_q protein to form a tight complex. However, the FRETr signal amplitudes and the SNR were much smaller compared to the step 1 responses of hM3R-YFP-CFP. Those weak FRETr signals compared to noises can be caused by the preassembly of inactive hM3R with the Gβγ subunit of G_q protein in the resting state, although the association is not as tight as the condition under receptor activation. Thus, we speculate that agonist binding to hM3R may reorganize the preassembled complex to a closer conformation between the two probes.

It has generally been accepted that the activated Gα-GTP subunit dissociates from the receptor to yield a Gα-GTP monomer–effector complex, while the separated Gβγ subunit stays complexed with the receptor, which is now free to modulate other intracellular signaling molecules independently. However, some heterotrimeric G_i and G_s proteins maintain assembly with receptors in the early stage of activation^30,32. Here, our study demonstrates that Gα_q-GTP does not leave hM3R after dissociation from βγ subunits, and it continuously activates the downstream PLCβ molecule after forming a complex with hM3R until washout of the agonist. Interestingly, in our study, the increased FRETr signal between Gα_q-CFP and YFP-PLCβ₁ or between hM3R-CFP and YFP-PLCβ₁ was also maintained steadily during the hM3R activation. These results are consistent with the sustained PIP₂ depletion in the plasma membrane during the hM3R activation by the agonist (Supplementary Fig. 19). Therefore, in line with the previous studies showing that G protein can form a constitutive complex with effectors^{66,67,68,69,70}, our results suggest that hM3R, Gα_q, and PLCβ form a stable heterotrimeric complex together induced by the agonist and cause the steady PIP₂ hydrolysis until washout of the agonist.

Methods

Constructs

In the plasmid (Addgene plasmid # 45547) encoding the human M3D receptor, two point mutations (C149Y and G239A) were created to generate wild-type human M3R (hM3R). This hM3R was appended to pcDNA 3, which includes cerulean, a variant of enhanced cyan fluorescent protein (ECFP), to generate hM3R-Cerulean. The restriction enzyme EcoRI recognition site mediated this appendage between Leu590 at the C terminus of hM3R and Met1 at the N terminus of cerulean. The cerulean of this hM3R-Cerulean was substituted with enhanced yellow fluorescent protein (EYFP) to generate hM3R-EYFP. To create the intramolecular fluorescent probe hM3R-EYFP-Cerulean, EYFP replaced a segment between Ala270 and Lys485 in the third intracellular loop of hM3R-Cerulean. The recognition sites of restriction enzymes SacII and AgeI mediated this replacement. Three amino-acid point mutations (I162A, L493A and Y544A) of these hM3R-Cerulean, hM3R-EYFP, and hM3R-EYFP-Cerulean were created to generate hM3R(3A)-Cerulean, hM3R(3A)-EYFP, and hM3R(3A)-EYFP-Cerulean. One amino-acid point mutation (R166L) of the hM3R-EYFP-Cerulean was created to generate hM3R(R166L^3x50)-EYFP-Cerulean. Point mutations of the existing human Gα_q³⁶ and mouse Gα_q-ECFP^36,71,72 were created to generate Gα_q(Q209L), Gα_q(R183C), Gα_q(Q209L)-ECFP, and Gα_q(R183C)-ECFP. All constructs were made using adaptations of the QuikChange (Agilent Technologies, Santa Clara, CA) mutagenesis protocol and restriction enzyme kits (Enzynomics, Daejeon, South Korea) and were verified by automated sequencing (Macrogen, Seoul, South Korea). The sequences of primers used for plasmid cloning are listed in Supplementary Table 1. Mouse M1R-YFP-CFP³⁶, mouse Gα_q-ECFP^36,71,72, bovine Gβ₁-EYFP³⁶, rat EYFP-PLCβ₁^36,72, and human pleckstrin homology (PH) domain probe RFP-PH(PLCδ₁)⁷³ were obtained from B. Hille (University of Washington School of Medicine, Seattle, WA). PH(PLCδ₁)-ECFP and PH(PLCδ₁)-EYFP^36,74 were obtained from K. Jalink (The Netherlands Cancer Institute, Amsterdam, Netherlands). Plasmids containing unlabeled mouse M1R, human Gα_q, Gβ₁, Gγ₂, and bovine GPCR kinase 2 (GRK2)³⁶ were obtained from B. Hille. Here, we refer to fluorophores simply as CFP or YFP regardless of whether regular or enhanced fluorescent proteins were used. We confirm that all used unique biological materials are readily available from the corresponding author.

Cell culture

Human embryonic kidney tsA-201 cells (large T-antigen transformed HEK293 cells (HEK293T cells); RRID: CVCL_2737) were a kind gift from Dr. Bertil Hille at University of Washington. The identity of this cell line has been authenticated by STR analysis and has recurrently tested negative for mycoplasma contamination using PCR (Cosmogenetech, Daejeon, South Korea). All experiments were performed on transiently transfected HEK293T cells. The 2-ml transfection medium contained 10 μl of Lipofectamine-2000 and 0.2–0.8 μg of each plasmid. In the G protein studies, for better membrane expression of any G protein subunit probe, cells were always transfected with three G protein subunits (α, β, and γ) together. The next day, the cells were plated onto poly-L-lysine-coated #0 glass coverslip chips, and fluorescent cells were studied 36–48 h after transfection.

Förster resonance energy transfer (FRET)

Our FRET system was described in our previous publications^75,76,77. FRET between CFP and YFP was measured by a photomultiplier tube (PMT)-based photometry system (Till Photonics GmbH). Regular pulses of indigo light (426–450 nm) from a homemade LED-based monochromator excited the fluorescent proteins. Emission, which passed through a 40×, NA 0.95 dry immersion objective lens (IX71; Olympus), was separated into short (460–500 nm) and long (520–550 nm) wavelengths by a dichroic mirror (505DCLP) and band-pass filters (D480-40 for short wavelengths and ET535-30 for long wavelengths; Chroma Technology) and detected by two PMTs connected with an FDU-2 fluorescence detection unit (Till Photonics GmbH). Donor and acceptor signals obtained by photometry system were transferred to a data acquisition board (PCI-6221; National Instruments, or EPC-10; HEKA Elektronik). Timing control, signal acquisition, and real-time calculation of FRET ratio (FRETr) including background compensation, were performed using a homemade program or PatchMaster (HEKA Elektronik) that also controlled the monochromator through the data acquisition interface. To correct bleed-through of emission of CFP into the YFP detector, cells expressing only CFP were used to obtain the ratio of the detected signal in short and long wavelength emission channels³⁶. The calculated ratio (cFactor = CFP/YFP = 0.45) was used to correct the raw YFP emission signal. The bleed-through of YFP light into the CFP detector was only 0.02 and was neglected. The FRETr was thus calculated as follows:

$${{{{{\rm{FRETr}}}}}}=({{{{{\rm{YFP}}}}}}{{{{{\rm{c}}}}}}-{{{{{\rm{cFactor}}}}}}\times {{{{{\rm{CFP}}}}}}{{{{{\rm{c}}}}}})/{{{{{\rm{CFP}}}}}}{{{{{\rm{c}}}}}},$$

where YFP_C is the signal from YFP excited as a result of FRET (YFP emission by CFP excitation), CFP_C is the CFP emission detected by the short wavelength PMT, and YFP_C is the YFP emission detected by the long wavelength PMT.

The whole-cell configuration of the patch-clamp method using an EPC-10 patch-clamp amplifier at room temperature was used to manipulate intracellular concentration of GTP, GDPβS or GTPγS. Pipettes were pulled from glass micropipette capillaries (World Precision Instruments) using a Flaming/Brown micropipette puller (P-97; Sutter Instrument Co.). Pipette resistance was 1.3–3.0 MΩ, and series resistance was between 3.4 and 6.0 MΩ with 60% compensation. The pipette solution composition was described in Solutions and materials section. The holding potential in all experiments was −80 mV. The raw data were processed with Excel 2016 (Microsoft).

Confocal imaging

HEK293T cells were imaged 2 d after transfection on poly-L-lysine-coated chips with a LSM 700 confocal microscope (Carl Zeiss) at room temperature. The images were scanned with a 40 × objective lens at 1024 × 1024 or 512 × 512 pixels using digital zoom on a cover glass-bottom dish filled with the external solution. Image processing and measurement of fluorescence intensity were carried out using Zeiss ZEN 2.3 SP1 software. To analyze relative fluorescence intensity, the fluorescence intensity of a region of interest (ROI) at each time point was divided by the average of points 30-s period before Oxo-M or Ach treatment. The half time of activation curve (T₅₀) was calculated from sigmoidal curve fitting. All images were transferred from LSM4 to JPG format. The raw data were processed with Excel 2016 (Microsoft).

Solutions and materials

Cells were subjected to a continuous slow bath flow of Ringer’s solution. The fresh external Ringer’s solution was supplied to the chamber by the valve-controlled gravity perfusion system VC3-4CLG from ALA Scientific Instruments (Farmingdale, New York). This supplied solution was simultaneously sucked out by the vacuum pump system DR 4-000-007 from Drummond Scientific Company (Broomall, Pennsylvania). The external Ringer’s solution exchange for drug treatment was accomplished by a theta tube moved laterally by a step-driven motor (SF-77C) from Warner Instruments (Holliston, Massachusetts) and was complete within 20 ms. The external Ringer’s solution used for FRET and confocal imaging contained the following (in mM): 160 NaCl, 2.5 KCl, 2 CaCl₂, 1 MgCl₂, 10 HEPES, and 8 glucose, adjusted to pH 7.4 with NaOH. The pipette solution for FRET under the normal GTP concentration (0.1 mM) condition contained the following (in mM): 175 KCl, 5 MgCl₂, 5 HEPES, 0.1 BAPTA, 3 Na₂ATP, and 0.1 Na₃GTP, adjusted to pH 7.4 with KOH. For the case of the GTP depletion condition, the 0.1 mM Na₃GTP was substituted with 1 mM GDPβS. For the case of the nonhydrolyzable GTP analog experiments, the 0.1 mM GTP was substituted with 0.1 mM GTPγS. When using Oxo-M as an agonist, a concentration of 10 μM was used. Oxo-M, Ach and poly-l-lysine were from Sigma-Aldrich (St. Louis, Missouri). DMEM, Lipofectamine-2000, and penicillin/streptomycin were from Invitrogen (Waltham, Massachusetts). Fetal bovine serum was from Gemini Bio-Products (West Sacramento, California). YM-254890 was from Cayman Chemical (Ann Arbor, Michigan).

In silico modeling using I-TASSER and SWISS-Model

For the predicted three-dimensional (3D) structure of M3R with mutating Gα-binding sites, molecular modeling was executed by using the cryo-EM structure of rat M3R (PDB ID: 4DAJ)⁵³ as a template from Protein Data Bank (PDB) because there is no cryo-EM structure of human M3R. In Swiss-Model, we used the structure with PDB ID: 4U14 determined by X-ray crystallography as template⁷⁸. We submitted the full sequence of rat M3R(3A) (derived from NM_012527.2) and obtained models predicted from I-TASSER online server V5.2 (http://zhanglab.ccmb.med.umich.edu/I-TASSER/)⁷⁹ and SWISS-Model web server released from July 2022 (https://swissmodel.expasy.org/). Excluding the flexible intracellular loops, transmembrane domains with Gα-binding sites of cryo-EM M3R and in silico M3R(3A) model were superimposed by matchmaker of University of California, San Francisco (UCSF) chimera. The amino-acid residues representing orthosteric agonist-binding sites^53,80 and Gα-binding sites^48,52 were indicated. Structure visualization and modifications were made using UCSF chimera.

Data analysis

All data were analyzed using Excel 2016 (Microsoft), Igor Pro-6.0 (WaveMetrics), or GraphPad Prism 8 (GraphPad Software). The measured FRETr traces were normalized as follows. The average FRETr signal of 5-s period before the agonist treatment was set to the initial value 1. The average FRETr signal of 5-s period before the washing out of drugs was set to the minimum 0 or maximum 2 value according to the direction of the reactions. To measure the relative FRETr in a time course, the data at each time point were divided by the average FRETr signal of 5-s period before the agonist treatment. To perform a fitting and determine a time constant (τ) and ΔFRETr amplitude of activation in the “hM3R-YFP-CFP” experiments, when the starting point of agonist treatment was regarded as the zero time point, the time duration from 0 to 1.2 s after agonist addition was designated as “Step 1_ON” and the 1.3–15 s time period during the agonist treatment was designated as “Step 2_ON”. To perform a fitting and determine a time constant (τ) of deactivation in the “hM3R-YFP + Gα_q-CFP” experiments, 0 − 3.6 s was designated as “Step 1_OFF” and 3.7–30 s was designated as “Step 2_OFF”. Except for the cases previously described, a fitting was performed with a least-squares criterion to determine time constants (τ) of activation and deactivation and the maximum ΔFRETr amplitude. The signal-to-noise ratio (SNR) was calculated as the maximum ΔFRETr amplitude divided by the standard error of the baseline ΔFRETr. Percent (%) ON of FRETr of hM3R-YFP-CFP was calculated as 100× (ΔFRETr amplitude of each activation step (step 1 or step 2)/maximum ΔFRETr amplitude). Percent (%) ON of FRETr of the other constructs with single-step activation was calculated as 100× (the average of the amplitudes (in contrast to the initial value 1) of points of 2.5-s period before washing out of agonist in each normalized FRETr/the average of the amplitudes (in contrast to the initial value 1) of points of 2.5-s period before agonist washing out in normalized average FRETr). Percent (%) OFF of FRETr of hM3R-YFP-CFP was calculated as 100× (ΔFRETr amplitude of each deactivation step (step 1 or step 2)/maximum ΔFRETr amplitude of activation). Percent (%) OFF of FRETr of the other constructs with single-step deactivation was calculated as 100× (ΔFRETr amplitude of deactivation/maximum ΔFRETr amplitude of activation). Statistics in the text and figures represent mean ± SEM. Statistical comparisons between the means of two groups were analyzed using Student’s t-test and Welch’s t-test according to their variances. Otherwise, Mann–Whitney U test was used if the data did not follow normal distribution. Statistical significances of ΔFRETr changes before and after receptor activations were analyzed using paired t-test. Statistical comparisons between the means of three or more independent groups under one independent variable were analyzed using one-way ANOVA or Welch’s ANOVA according to their variances. Statistical comparisons between the means of three or more independent groups with equal variance under two independent variables were analyzed using two-way ANOVA. Differences were considered significant at the *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 levels. Source data used in data analysis are provided with this paper.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The data that support this study are available from the corresponding authors upon request. The published cryo-EM structure of rat M3R can be accessed using accession code 4DAJ. The published X-ray crystal structure of rat M3R can be accessed using accession code 4U14. Source data are provided with this paper.

References

Klabunde, T. & Hessler, G. Drug design strategies for targeting G-protein-coupled receptors. ChemBioChem 3, 928–944 (2002).
Article CAS PubMed Google Scholar
Fredriksson, R., Lagerstrom, M. C., Lundin, L. G. & Schioth, H. B. The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol. Pharmacol. 63, 1256–1272 (2003).
Article CAS PubMed Google Scholar
Pin, J. P., Kniazeff, J., Prezeau, L., Liu, J. F. & Rondard, P. GPCR interaction as a possible way for allosteric control between receptors. Mol. Cell. Endocrinol. 486, 89–95 (2019).
Article CAS PubMed Google Scholar
Lagerstrom, M. C. & Schioth, H. B. Structural diversity of G protein-coupled receptors and significance for drug discovery. Nat. Rev. Drug Discov. 7, 339–357 (2008).
Article PubMed Google Scholar
Calebiro, D., Koszegi, Z., Lanoiselee, Y., Miljus, T. & O’Brien, S. G protein-coupled receptor-G protein interactions: a single-molecule perspective. Physiol. Rev. 101, 857–906 (2021).
Article CAS PubMed Google Scholar
Rybin, V. O., Xu, X., Lisanti, M. P. & Steinberg, S. F. Differential targeting of β-adrenergic receptor subtypes and adenylyl cyclase to cardiomyocyte caveolae. A mechanism to functionally regulate the cAMP signaling pathway. J. Biol. Chem. 275, 41447–41457 (2000).
Article CAS PubMed Google Scholar
Insel, P. A. et al. Compartmentation of G-protein-coupled receptors and their signalling components in lipid rafts and caveolae. Biochem. Soc. Trans. 33, 1131–1134 (2005).
Article CAS PubMed Google Scholar
Sungkaworn, T. et al. Single-molecule imaging reveals receptor-G protein interactions at cell surface hot spots. Nature 550, 543–547 (2017).
Article ADS CAS PubMed Google Scholar
De Lean, A., Stadel, J. M. & Lefkowitz, R. J. A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase-coupled beta-adrenergic receptor. J. Biol. Chem. 255, 7108–7117 (1980).
Article PubMed Google Scholar
Kjeldgaard, M., Nyborg, J. & Clark, B. F. The GTP binding motif: variations on a theme. FASEB J. 10, 1347–1368 (1996).
Article CAS PubMed Google Scholar
Rasmussen, S. G. et al. Crystal structure of the β₂ adrenergic receptor-Gs protein complex. Nature 477, 549–555 (2011).
Article ADS CAS PubMed PubMed Central Google Scholar
Glukhova, A. et al. Rules of engagement: GPCRs and G proteins. ACS Pharmacol. Transl. Sci. 1, 73–83 (2018).
Article CAS PubMed PubMed Central Google Scholar
Wu, G., Benovic, J. L., Hildebrandt, J. D. & Lanier, S. M. Receptor docking sites for G-protein betagamma subunits. Implications for signal regulation. J. Biol. Chem. 273, 7197–7200 (1998).
Article CAS PubMed Google Scholar
Herrmann, R. et al. Sequence of interactions in receptor-G protein coupling. J. Biol. Chem. 279, 24283–24290 (2004).
Article CAS PubMed Google Scholar
Herrmann, R., Heck, M., Henklein, P., Hofmann, K. P. & Ernst, O. P. Signal transfer from GPCRs to G proteins: role of the Gα N-terminal region in rhodopsin-transducin coupling. J. Biol. Chem. 281, 30234–30241 (2006).
Article CAS PubMed Google Scholar
Herrmann, R. et al. Rhodopsin-transducin coupling: role of the Gα C-terminus in nucleotide exchange catalysis. Vision Res. 46, 4582–4593 (2006).
Article ADS CAS PubMed Google Scholar
Oldham, W. M. et al. Mechanism of the receptor-catalyzed activation of heterotrimeric G proteins. Nat. Struct. Mol. Biol. 13, 772–777 (2006).
Article CAS PubMed Google Scholar
Dror, R. O. et al. SIGNAL TRANSDUCTION. Structural basis for nucleotide exchange in heterotrimeric G proteins. Science 348, 1361–1365 (2015).
Article ADS CAS PubMed PubMed Central Google Scholar
Zhang, Y. et al. Cryo-EM structure of the activated GLP-1 receptor in complex with a G protein. Nature 546, 248–253 (2017).
Article ADS CAS PubMed PubMed Central Google Scholar
Liang, Y. L. et al. Phase-plate cryo-EM structure of a class B GPCR-G-protein complex. Nature 546, 118–123 (2017).
Article ADS CAS PubMed PubMed Central Google Scholar
Liang, Y. L. et al. Phase-plate cryo-EM structure of a biased agonist-bound human GLP-1 receptor-Gs complex. Nature 555, 121–125 (2018).
Article ADS CAS PubMed Google Scholar
Garcia-Nafria, J., Lee, Y., Bai, X., Carpenter, B. & Tate, C. G. Cryo-EM structure of the adenosine A2A receptor coupled to an engineered heterotrimeric G protein. Elife 7, e35946 (2018).
Article PubMed PubMed Central Google Scholar
Tsai, C. J. et al. Cryo-EM structure of the rhodopsin-Gα_i-βγ complex reveals binding of the rhodopsin C-terminal tail to the Gβ subunit. Elife 8, e46041 (2019).
Article PubMed PubMed Central Google Scholar
Maeda, S., Qu, Q., Robertson, M. J., Skiniotis, G. & Kobilka, B. K. Structures of the M₁ and M₂ muscarinic acetylcholine receptor/G-protein complexes. Science 364, 552–557 (2019).
Article ADS CAS PubMed PubMed Central Google Scholar
Garcia-Nafria, J. & Tate, C. G. Cryo-EM structures of GPCRs coupled to G_s, G_i and G_o. Mol. Cell. Endocrinol. 488, 1–13 (2019).
Article CAS PubMed Google Scholar
Neubig, R. R. Membrane organization in G-protein mechanisms. FASEB J. 8, 939–946 (1994).
Article CAS PubMed Google Scholar
Rebois, R. V. & Hebert, T. E. Protein complexes involved in heptahelical receptor-mediated signal transduction. Recept. Channels 9, 169–194 (2003).
Article CAS PubMed Google Scholar
Hein, P. & Bunemann, M. Coupling mode of receptors and G proteins. Naunyn Schmiedebergs Arch. Pharmacol. 379, 435–443 (2009).
Article CAS PubMed Google Scholar
Bunemann, M., Frank, M. & Lohse, M. J. G_i protein activation in intact cells involves subunit rearrangement rather than dissociation. Proc. Natl Acad. Sci. USA 100, 16077–16082 (2003).
Article ADS PubMed PubMed Central Google Scholar
Gales, C. et al. Real-time monitoring of receptor and G-protein interactions in living cells. Nat. Methods 2, 177–184 (2005).
Article CAS PubMed Google Scholar
Nobles, M., Benians, A. & Tinker, A. Heterotrimeric G proteins precouple with G protein-coupled receptors in living cells. Proc. Natl Acad. Sci. USA 102, 18706–18711 (2005).
Article ADS CAS PubMed PubMed Central Google Scholar
Gales, C. et al. Probing the activation-promoted structural rearrangements in preassembled receptor-G protein complexes. Nat. Struct. Mol. Biol. 13, 778–786 (2006).
Article CAS PubMed Google Scholar
Qin, K., Dong, C., Wu, G. & Lambert, N. A. Inactive-state preassembly of G_q-coupled receptors and G_q heterotrimers. Nat. Chem. Biol. 7, 740–747 (2011).
Article CAS PubMed PubMed Central Google Scholar
Huang, S. K. et al. Delineating the conformational landscape of the adenosine A_2A receptor during G protein coupling. Cell 184, 1884–1894.e1814 (2021).
Article CAS PubMed Google Scholar
Gregorio, G. G. et al. Single-molecule analysis of ligand efficacy in β₂ AR-G-protein activation. Nature 547, 68–73 (2017).
Article ADS CAS PubMed PubMed Central Google Scholar
Jensen, J. B., Lyssand, J. S., Hague, C. & Hille, B. Fluorescence changes reveal kinetic steps of muscarinic receptor-mediated modulation of phosphoinositides and Kv7.2/7.3 K⁺ channels. J. Gen. Physiol. 133, 347–359 (2009).
Article CAS PubMed PubMed Central Google Scholar
Ziegler, N., Batz, J., Zabel, U., Lohse, M. J. & Hoffmann, C. FRET-based sensors for the human M₁-, M₃-, and M₅-acetylcholine receptors. Bioorg. Med. Chem. 19, 1048–1054 (2011).
Article CAS PubMed Google Scholar
Hoffmann, C. et al. Comparison of the activation kinetics of the M₃ acetylcholine receptor and a constitutively active mutant receptor in living cells. Mol. Pharmacol. 82, 236–245 (2012).
Article CAS PubMed Google Scholar
Wolters, V., Krasel, C., Brockmann, J. & Bunemann, M. Influence of Gα_q on the dynamics of M₃-acetylcholine receptor-G-protein-coupled receptor kinase 2 interaction. Mol. Pharmacol. 87, 9–17 (2015).
Article PubMed Google Scholar
Floser, A. et al. Disentangling bias between G_q, GRK2, and arrestin3 recruitment to the M₃ muscarinic acetylcholine receptor. Elife 10, e58442 (2021).
Article PubMed PubMed Central Google Scholar
Van Raamsdonk, C. D. et al. Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi. Nature 457, 599–602 (2009).
Article ADS PubMed Google Scholar
Van Raamsdonk, C. D. et al. Mutations in GNA11 in uveal melanoma. N. Engl. J. Med. 363, 2191–2199 (2010).
Article PubMed PubMed Central Google Scholar
Campbell, A. P. & Smrcka, A. V. Targeting G protein-coupled receptor signalling by blocking G proteins. Nat. Rev. Drug Discov. 17, 789–803 (2018).
Article CAS PubMed PubMed Central Google Scholar
Harrison, C. & Traynor, J. R. The [³⁵S]GTPγS binding assay: approaches and applications in pharmacology. Life Sci. 74, 489–508 (2003).
Article CAS PubMed Google Scholar
Jing, M. et al. An optimized acetylcholine sensor for monitoring in vivo cholinergic activity. Nat. Methods 17, 1139–1146 (2020).
Article CAS PubMed PubMed Central Google Scholar
Jing, M. et al. A genetically encoded fluorescent acetylcholine indicator for in vitro and in vivo studies. Nat. Biotechnol. 36, 726–737 (2018).
Article CAS PubMed PubMed Central Google Scholar
Tateyama, M. & Kubo, Y. Analyses of the effects of Gq protein on the activated states of the muscarinic M₃ receptor and the purinergic P2Y₁ receptor. Physiol. Rep. 1, e00134 (2013).
Article PubMed PubMed Central Google Scholar
Venkatakrishnan, A. J. et al. Diverse activation pathways in class A GPCRs converge near the G-protein-coupling region. Nature 536, 484–487 (2016).
Article ADS CAS PubMed PubMed Central Google Scholar
Isberg, V. et al. Generic GPCR residue numbers - aligning topology maps while minding the gaps. Trends Pharmacol. Sci. 36, 22–31 (2015).
Article CAS PubMed Google Scholar
Isberg, V. et al. GPCRdb: an information system for G protein-coupled receptors. Nucleic Acids Res. 44, D356–D364 (2016).
Article CAS PubMed Google Scholar
Flock, T. et al. Universal allosteric mechanism for Gα activation by GPCRs. Nature 524, 173–179 (2015).
Article ADS CAS PubMed PubMed Central Google Scholar
Liu, Q. et al. Ligand recognition and G-protein coupling selectivity of cholecystokinin A receptor. Nat. Chem. Biol. 17, 1238–1244 (2021).
Article CAS PubMed PubMed Central Google Scholar
Kruse, A. C. et al. Structure and dynamics of the M3 muscarinic acetylcholine receptor. Nature 482, 552–556 (2012).
Article ADS CAS PubMed PubMed Central Google Scholar
Nakajima, K. & Wess, J. Design and functional characterization of a novel, arrestin-biased designer G protein-coupled receptor. Mol. Pharmacol. 82, 575–582 (2012).
Article CAS PubMed PubMed Central Google Scholar
Hein, P. et al. G_s activation is time-limiting in initiating receptor-mediated signaling. J. Biol. Chem. 281, 33345–33351 (2006).
Article CAS PubMed Google Scholar
Rochais, F. et al. Real-time optical recording of β₁-adrenergic receptor activation reveals supersensitivity of the Arg389 variant to carvedilol. J. Clin. Investig. 117, 229–235 (2007).
Article CAS PubMed PubMed Central Google Scholar
Ferrandon, S. et al. Sustained cyclic AMP production by parathyroid hormone receptor endocytosis. Nat. Chem. Biol. 5, 734–742 (2009).
Article CAS PubMed PubMed Central Google Scholar
Reiner, S., Ambrosio, M., Hoffmann, C. & Lohse, M. J. Differential signaling of the endogenous agonists at the β₂-adrenergic receptor. J. Biol. Chem. 285, 36188–36198 (2010).
Article CAS PubMed PubMed Central Google Scholar
Sleno, R. et al. Designing BRET-based conformational biosensors for G protein-coupled receptors. Methods 92, 11–18 (2016).
Article CAS PubMed Google Scholar
Bourque, K. et al. Distinct conformational dynamics of three G protein-coupled receptors measured using FlAsH-BRET biosensors. Front. Endocrinol. 8, 61 (2017).
Article Google Scholar
Che, T. et al. Nanobody-enabled monitoring of kappa opioid receptor states. Nat. Commun. 11, 1145 (2020).
Article ADS CAS PubMed PubMed Central Google Scholar
Lohse, M. J., Nuber, S. & Hoffmann, C. Fluorescence/bioluminescence resonance energy transfer techniques to study G-protein-coupled receptor activation and signaling. Pharmacol. Rev. 64, 299–336 (2012).
Article CAS PubMed Google Scholar
Zhou, Y., Meng, J., Xu, C. & Liu, J. Multiple GPCR functional assays based on resonance energy transfer sensors. Front. Cell Dev. Biol. 9, 611443 (2021).
Article PubMed PubMed Central Google Scholar
Wess, J. Molecular basis of receptor/G-protein-coupling selectivity. Pharmacol. Ther. 80, 231–264 (1998).
Article CAS PubMed Google Scholar
Azpiazu, I. & Gautam, N. A fluorescence resonance energy transfer-based sensor indicates that receptor access to a G protein is unrestricted in a living mammalian cell. J. Biol. Chem. 279, 27709–27718 (2004).
Article CAS PubMed Google Scholar
Davare, M. A. et al. A β₂ adrenergic receptor signaling complex assembled with the Ca²⁺ channel Ca_V1.2. Science 293, 98–101 (2001).
Article CAS PubMed Google Scholar
Rebois, R. V. et al. Heterotrimeric G proteins form stable complexes with adenylyl cyclase and Kir3.1 channels in living cells. J. Cell Sci. 119, 2807–2818 (2006).
Article CAS PubMed Google Scholar
Halls, M. L. & Cooper, D. M. Sub-picomolar relaxin signalling by a pre-assembled RXFP1, AKAP79, AC2, β-arrestin 2, PDE4D3 complex. EMBO J. 29, 2772–2787 (2010).
Article CAS PubMed PubMed Central Google Scholar
Nagi, K. & Pineyro, G. Kir3 channel signaling complexes: focus on opioid receptor signaling. Front. Cell. Neurosci. 8, 186 (2014).
Article PubMed PubMed Central Google Scholar
Kankanamge, D. et al. Dissociation of the G protein betagamma from the G_q-PLCβ complex partially attenuates PIP₂ hydrolysis. J. Biol. Chem. 296, 100702 (2021).
Article CAS PubMed PubMed Central Google Scholar
Hughes, T. E., Zhang, H., Logothetis, D. E. & Berlot, C. H. Visualization of a functional Gα_q-green fluorescent protein fusion in living cells. Association with the plasma membrane is disrupted by mutational activation and by elimination of palmitoylation sites, but not be activation mediated by receptors or AlF4. J. Biol. Chem. 276, 4227–4235 (2001).
Article CAS PubMed Google Scholar
Scarlata, S. & Dowal, L. The use of green fluorescent proteins to view association between phospholipase C beta and G protein subunits in cells. Methods Mol. Biol. 237, 223–232 (2004).
CAS PubMed Google Scholar
Suh, B. C., Inoue, T., Meyer, T. & Hille, B. Rapid chemically induced changes of PtdIns(4,5)P₂ gate KCNQ ion channels. Science 314, 1454–1457 (2006).
Article ADS CAS PubMed PubMed Central Google Scholar
van der Wal, J., Habets, R., Varnai, P., Balla, T. & Jalink, K. Monitoring agonist-induced phospholipase C activation in live cells by fluorescence resonance energy transfer. J. Biol. Chem. 276, 15337–15344 (2001).
Article PubMed Google Scholar
Keum, D., Baek, C., Kim, D. I., Kweon, H. J. & Suh, B. C. Voltage-dependent regulation of Ca_V2.2 channels by G_q-coupled receptor is facilitated by membrane-localized β subunit. J. Gen. Physiol 144, 297–309 (2014).
Article CAS PubMed PubMed Central Google Scholar
Keum, D., Kruse, M., Kim, D. I., Hille, B. & Suh, B. C. Phosphoinositide 5- and 3-phosphatase activities of a voltage-sensing phosphatase in living cells show identical voltage dependence. Proc. Natl Acad. Sci. USA 113, E3686–E3695 (2016).
Article CAS PubMed PubMed Central Google Scholar
Park, C. G., Yu, W. & Suh, B. C. Molecular basis of the PIP₂-dependent regulation of Ca_V2.2 channel and its modulation by Ca_V β subunits. Elife 11, e69500 (2022).
Article PubMed PubMed Central Google Scholar
Thorsen, T. S., Matt, R., Weis, W. I. & Kobilka, B. K. Modified T4 lysozyme fusion proteins facilitate G protein-coupled receptor crystallogenesis. Structure 22, 1657–1664 (2014).
Article CAS PubMed PubMed Central Google Scholar
Yang, J. & Zhang, Y. I-TASSER server: new development for protein structure and function predictions. Nucleic Acids Res. 43, W174–W181 (2015).
Article ADS CAS PubMed PubMed Central Google Scholar
Levay, M. et al. NSC23766, a widely used inhibitor of Rac1 activation, additionally acts as a competitive antagonist at muscarinic acetylcholine receptors. J. Pharmacol. Exp. Ther. 347, 69–79 (2013).
Article CAS PubMed Google Scholar

Download references

Acknowledgements

We thank the many laboratories that kindly provided the plasmids. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Science, Information and Communications Technology, and Future Planning) (2022R1A2C1006560 [B.-C.S.]) and the Basic Science Research Program (2020R1A4A1019436 [B.-C.S.]).

Author information

Authors and Affiliations

Department of Brain Sciences, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Daegu, 42988, Republic of Korea
Yong-Seok Kim, Jun-Hee Yeon, Woori Ko & Byung-Chang Suh

Authors

Yong-Seok Kim
View author publications
You can also search for this author in PubMed Google Scholar
Jun-Hee Yeon
View author publications
You can also search for this author in PubMed Google Scholar
Woori Ko
View author publications
You can also search for this author in PubMed Google Scholar
Byung-Chang Suh
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

B.-C.S. and Y.-S.K. conceptualized the project and designed the experiments; Y.-S.K. performed most of the experiments; J.-H.Y. performed amino-acid point mutations to construct the hM3R(3A) expression vectors and the hM3R(R166L^3x50)-YFP-CFP expression vector; W.K. performed In silico modelling using I-TASSER and SWISS-Model; Y.-S.K. analyzed the results; Y.-S.K. wrote the original draft. All authors edited the manuscript. B.-C.S. supervised the project and wrote the final paper.

Corresponding author

Correspondence to Byung-Chang Suh.

Ethics declarations

Competing interests

Y.-S.K. and B.-C.S. have a pending patent for hM3R-YFP-CFP and use there of: South Korean patent application number 10-2022-0147739. The other authors declare no competing interests.

Peer review

Peer review information

Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Reporting Summary

Source data

Source Data

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Kim, YS., Yeon, JH., Ko, W. et al. Two-step structural changes in M3 muscarinic receptor activation rely on the coupled G_q protein cycle. Nat Commun 14, 1276 (2023). https://doi.org/10.1038/s41467-023-36911-4

Download citation

Received: 30 July 2022
Accepted: 23 February 2023
Published: 08 March 2023
DOI: https://doi.org/10.1038/s41467-023-36911-4

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.