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. 2024 Oct 3;146(41):28527–28537. doi: 10.1021/jacs.4c11250

Coupling and Activation of the β1 Adrenergic Receptor - The Role of the Third Intracellular Loop

Xingyu Qiu †,‡, Kin Chao §, Siyuan Song †,‡, Yi-Quan Wang ∥, Yi-An Chen ∥, Sarah L Rouse §, Hsin-Yung Yen †,∥,*, Carol V Robinson †,‡,*
PMCID: PMC11487556  PMID: 39359104

Abstract

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G protein-coupled receptors (GPCRs) belong to the most diverse group of membrane receptors with a conserved structure of seven transmembrane (TM) α-helices connected by intracellular and extracellular loops. Intracellular loop 3 (ICL3) connects TM5 and TM6, the two helices shown to play significant roles in receptor activation. Herein, we investigate the activation and signaling of the β1 adrenergic receptor (β1AR) using mass spectrometry (MS) with a particular focus on the ICL3 loop. First, using native MS, we measure the extent of receptor coupling to an engineered Gαs subunit (mini Gs) and show preferential coupling to β1AR with an intact ICL3 (β1AR_ICL3) compared to the truncated β1AR. Next, using hydrogen–deuterium exchange (HDX)-MS, we show how helix 5 of mini Gs reports on the extent of receptor activation in the presence of a range of agonists. Then, exploring a range of solution conditions and using comparative HDX, we note additional HDX protection when ICL3 is present, implying that mini Gs helix 5 presents a different binding conformation to the surface of β1AR_ICL3, a conclusion supported by MD simulation. Considering when this conformatonal change occurs we used time-resolved HDX and employed two functional assays to measure GDP release and cAMP production, with and without ICL3. We found that ICL3 exerts its effect on Gs through enhanced cAMP production but does not affect GDP release. Together, our study uncovers potential roles of ICL3 in fine-tuning GPCR activation through subtle changes in the binding pose of helix 5, only after nucleotide release from Gs.

Introduction

G protein-coupled receptors (GPCRs) constitute the largest families of transmembrane proteins, with around 800 receptors classified into five major classes based on their structural features. The molecular basis of GPCR activation and modulation is intricate, owing to their highly diverse structural features for ligand recognition and the convergence of their G protein-coupling. GPCRs share a similar architecture of seven-transmembrane domains with loops connecting the transmembrane helices on both sides of the lipid bilayer. These loops are thought to play essential roles in ligand binding to the receptor.1,2 The intracellular loop 3 (ICL3) connects TM5 and TM6, the two helices known to participate in G protein coupling.

Upon agonist stimulation, TM5 and TM6 of a GPCR undergo significant conformational changes to create a binding cavity for the recruitment of G proteins.1,2 Several lines of evidence suggest that participation of ICL3 in this interaction influences the activation pathway of the receptor.3−11 For example, previous research has indicated that ICL3 of the β2 adrenergic receptor (β2AR) plays a role in conferring the coupling selectivity of the Gs protein.4,5 Substitution of the C-terminal parts of β2AR ICL3, by the sequence of the α2 Adrenergic receptor (α2 AR) ICL3, drastically weakened the coupling of β2AR to Gs.4−6 Using an assay based on accumulation of second messengers (cAMP and InsP1), a recent study also revealed that absence of ICL3 promotes GPCR recruitment of noncognate G proteins.11 Additionally, peptides from the ICL3 of both the α2AR and the Dopamine D2 receptor demonstrated a strong affinity to Go and Gi.11 These synthetic peptides were also shown to facilitate GTPase activity of Gi/o implying a possible role for ICL3 in Gi/o activation.3,7−10 Moreover, ICL3 was found to participate in the binding of GPCRs to β-arrestin.12−17 Together, these different observations indicate that ICL3 has unique functions in both the canonical and noncanonical signaling pathways of GPCRs. However, due to its flexibility, ICL3 is usually removed in structural studies or sometimes replaced by fusion proteins such as T4L lysozyme and BRIL for thermo-stabilization purposes. As intact ICL3 of β1AR has not been observed in previous high-resolution structural studies,18 the impact of this loop on coupling is not fully understood.

Considering where ICL3 might exert an effect in the established steps of GPCR signaling, which starts with engagement of helix 5 from the G protein with the GPCR,19 the receptor then induces a conformational change of the G protein to release GDP (Figure 1A). The G protein then binds to GTP and dissociates from the receptor for downstream signaling and cyclic AMP (cAMP) production. In the presence of ligands, previous structural studies of β2AR presented evidence for initial engagement of the receptor with a GDP-bound G protein, or an intermediate complex.19−22 To our knowledge, the impact of ICL3 on this intermediate complex has not been investigated.

Figure 1.

Figure 1

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Native MS analysis of β1AR (Truncated β1AR and β1AR_ICL3) in complex with mini Gs. (A) Schematic of the G protein signaling process. An agonist binds to the extracellular side of the GPCR to induce the initial engagement of the receptor to the G protein. This results in the formation of an intermediate complex and a conformational change of Gs then mediates GDP release. GTP binding to the nucleotide free complex forms the active G protein (Gs.GTP) which then dissociates from the complex for downstream signaling. (B) Peaks in the mass spectrum are assigned to β1AR constructs and their complexes coupled to mini Gs after incubation for 20 min in the presence of isoprenaline (5-fold excess). Apo truncated β1AR, apo β1AR_ICL3, colored pale blue and green respectively, are incubated with an equivalent conconcentration of mini Gs (all at 2.5 μM) to establish between the receptor constructs for mini Gs. Truncated β1AR-mini Gs and β1AR_ICL3 mini Gs complexes are colored darker blue and green, respectively. The complex:monomer ratio of β1AR_ICL3 is significantly higher than the ratio of truncated β1AR-mini Gs:truncated β1AR (bar chart (C) upper right). Error bars are generated according to the standard deviation calculated from three independent repeats. A student’s t-test was used to evaluate the statistically significant differences (p < 0.01).

We set out to define the effects of ICL3 on G protein coupling on the formation and stability of the intermediate complex and on cAMP production using a combination of native MS and HDX. Both MS approaches have been employed previously for GPCRs, quantifying the basal activity of β1AR and the conformational changes of various GPCRs.19,23,24 Here, we apply these approaches to reveal the extent of β1AR coupling to mini Gs (the engineered GTPase domain of the Gαs protein25). First in the presence of different agonists and then with and without ICL3, HDX-MS reveals the extent of G-protein engagement. Subsequently by varying the concentration of the receptors for HDX-MS, and performing molecular dynamics (MD) simulations, we show that upon coupling ICL3 can change the binding pose of helix 5 of mini Gs. By performing a cAMP accumulation assay, we then found the promotional effect of ICL3 on signal transduction of β1AR. We also applied a GDP binding assay, time-resolved HDX, and native MS, to demonstrate that ICL3 of β1AR changes the coupling mode of Gs helix 5, downstream of the intermediate complex, specifically at the nucleotide-free state of the complex.

Results and Discussion

ICL3 promotes β1AR coupling to mini Gs

To investigate the influence of ICL3 on GPCR-G protein coupling, we recorded native mass spectra of the two receptor constructs (described below) with and without ICL3, and mini Gs (Figure 1B). Mini Gs has been reported to form stable complexes with several GPCRs, including the adenosine A2A receptor (A2AR) and β1AR, and to increase the agonist affinity of receptors allosterically.25 The structure of the A2AR coupled to mini Gs revealed similar activation features observed in the β2AR–Gs complex such as outward movement of TM6 and interaction between helix 5 of Gs with the intracellular side of the receptor.26,27

We selected β1AR for our investigation expressing the construct for truncated β1AR (β1AR without an intact ICL3) from M. gallopavo β114-E130W (β1AR), based on the previously published thermostabilized β1AR44-m23 construct.28,29 Compared to the β1AR44-m23, our construct (β114-E130W) omits two point mutations at TM5 and TM6 (Y227A and A282L), which allow β114 to couple to G proteins in the presence of an agonist.25 The additional mutation, E130W of β1AR, promotes functional expression of β1AR as well as allows for the receptor to be purified without a bound ligand. For β1AR with an intact ICL3 (defined here as β1AR_ICL3) we used the M. gallopavo β114E130WIC3 where the ICL3 sequence was added back in to β114-E130W (Supplementary Figure 1). The mass spectrometry coupling experiments are based on a method described previously.30 Briefly, truncated β1AR is incubated with β1AR_ICL3 at a 1:1 molar ratio (2.5 μM) with mini Gs (2.5 μM) and a 5-fold excess of isoprenaline for 20 min. Native mass spectra are then recorded under conditions such that both apo β1AR and β1AR_ICL3 are detected (Figure 1B). Results show that the peaks assigned to the β1AR_ICL3 mini Gs complex are significantly more intense than those observed for the truncated β1AR-mini Gs complex. (Peak intensities of the monomers and complexes were quantified by using UniDec software.) We compared the ratio of the peak intensities assigned to the complex: apo receptor for the two receptors (Figure 1C). Since we are comparing ratios rather than absolute peak intensities, changes in ionization efficiency can be discounted. The significantly higher intensity ratio for β1AR_ICL3:β1AR_ICL3 mini Gs compared to truncated β1AR:truncated β1AR-mini Gs implies that ICL3 promotes the coupling of β1AR to mini Gs. The next step is to understand how this enhanced coupling is achieved.

Regions of mini Gs helix 5 involved in the activation of β1AR

We conducted HDX-MS of the receptor and mini Gs to locate regions of the G protein involved in coupling. First we identified multiple peptides of mini Gs following pepsin digestion and obtained a sequence coverage of 91% (Supplementary Figure 2). We then considered the concentration of mini Gs needed for full-complexation of the receptor in solution. Previous experiments revealed specific binding of mini Gs to β1AR with an apparent KD value of 200 nM.31 Recording native MS we observed full complex formation, in the absence of ICL3, at a receptor concentration of 1.5 μM, 1.2 equiv of mini Gs, consistent with the high affinity of this construct (Supplementary Figure 3). We therefore used this ratio of receptor to mini Gs (1.2:1) to conduct HDX-MS analysis.

Next we compared the HDX properties of mini Gs incubated with apo β1AR and activated with isoprenaline (coupling method as reported30 and reproduced in the Supporting Information). Following complex formantion (incubation time 20 min with 60 s of labeling) a significant reduction of deuterium uptake in the presence of isoprenaline was observed, compared to the apo form of β1AR without isoprenaline. On average a reduction of 20–30% and 10–20% was observed for the C- and N-terminal peptides respectively of helix 5 (Figure 2A–B). This observation suggests that after incubation both the N and C terminal ends of helix 5 of mini Gs are strongly protected, the effect being more pronounced for the C-terminus (residues 219–216 IQRMHLRQ) given its greater burial in the hydrophobic core of the receptor. We next explored three further deuterium labeling times (10, 250, and 1000 s) using the same incubation time. Under all conditions we observed protection of helix 5, the effect reducing with longer labeling times (Supplementary Figure 4). These results are together consistent with the insertion of helix 5 into the hydrophobic transmembrane region of the receptor upon complex formation.

Figure 2.

Figure 2

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HDX protection of helix 5 in mini Gs reports on the activity of truncated β1AR in the presence of agonists. (A–B) Deuterium uptake spectra of peptides (A) “IQRMHLRQ” and (B) “RRIFNDCRD” from mini Gs helix 5 in complex with β1AR (without ICL3) with and without isoprenaline, after 60 s labeling time and an incubation time of 20 min. Heat maps of the degree of HDX difference between apo mini Gs and mini Gs in complex with truncated β1AR (‘HDX percent β1AR-mini Gs - HDX percent of apo β1AR-mini Gs). Regions are highlighted based on the structure of mini Gs coupled to a class A receptor (A2AR), for illustrative purposes.27 (C–D) Deuterium uptake is plotted as a function of labeling time in the presence of four drugs and compared to the apo form of the receptor. (C) 209–217 assigned to the N-terminal residues of mini Gs helix 5. (D) 218–226 assigned to the C-terminal residues of mini Gs helix 5. Error bars are generated according to the standard deviation calculated from three independent repeats. A student’s t test was used to evaluate the statistically significant differences (p < 0.01). (E) Structures of the four drugs used here.

As a control experiment, we also conducted HDX-MS analysis on mini Gs/i since β1AR couples to Gs.32 (Mini Gs/i is a chimeric protein with helix 5 of Gi.) After incubation for 20 min of mini Gs/i with isoprenaline-bound β1AR, and after labeling for 60 s, no deuterium uptake change was observed for helix 5 of mini Gs/i (Supplementary Figure 5). Since the structure of β1AR coupled to Gi33 shows that helix 5 of Gi interacts differently with the receptor, our HDX-MS result, which does not capture the interaction of helix 5 of mini Gi with β1AR, does not rule out a weak interaction in a distinct conformation (Figure 2A-B). Nevertheless, our results suggest that HDX-MS could potentially reveal the extent and selectivity of β1AR coupling to mini Gs. If this is the case, then we hypothesize that a series of ligands with different agonist effects would lead to differences in HDX protection because of the extent of their coupling.

We tested three drugs, dobutamine, salbutamol, and carazolol, in addition to isoprenaline for their ability to induce HDX protection in helix 5 of mini Gs. The three additional drugs we selected are (i) Carazolol, a nonselective antagonist to both β1 and β2 adrenoreceptors;34 (ii) Dobutamine, a partial agonist of β1AR35 used for the treatment of cardiogenic shock; and (iii) Salbutamol, a partial agonist of β2AR,36 used to relieve symptoms of asthma by relaxing smooth muscle in the airway. We added the drugs individually to a solution of β1AR and mini Gs and recorded the HDX protection focusing on helix 5 of mini Gs (Figure 2C–D). Comparing the drugs to the solution of apo β1AR with mini Gs we found that for carazolol (antagonist), there is no difference in protection of helix 5. By contrast, isoprenaline-bound β1AR yielded the strongest protection, while β1AR bound to dobutamine or salbutamol conferred less protection. These HDX results suggest that the conformational state of helix 5 of mini Gs is not only a sensitive reporter of complex engagement but also serves as a proxy for the activity of β1AR (Supplementary Figure 5C).

ICL3 influences the coupling of mini Gs helix 5

To examine the effects of ICL3 on coupling, we used the HDX-MS approach described above. We compared (i) the active state of β1AR_ICL3 + isoprenaline, (ii) the active state of truncated β1AR + isoprenaline, and (iii) inactive β1AR (− isoprenaline). All three were incubated individually with mini Gs for 20 min. Comparing N- and C-terminal helix 5 peptides from the three experimental conditions we observe strikingly greater protection in the active β1AR_ICL3 compared to β1AR (absence of ICL3) and the inactive states (− isoprenaline) (Figure 3 and Supplementary Figure 5D-E). Comparing both activated complexes, mini Gs–β1AR_ICL3 and mini Gs-β1AR complex, shows that ICL3 facilitates further protection of helix 5 over that conferred to the mini Gs-β1AR complex. Both activated complexes, in turn, exhibit greater protection than the intactive one. This observation is in line with our proposal above that HDX protection of helix 5 is a reporter for the activation of β1AR (Figure 2C–D) and demonstrates that ICL3 promotes further the activity of β1AR.

Figure 3.

Figure 3

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ICL3 increases HDX protection of Gs helix 5 upon β1AR coupling with truncated and β1AR_ICL3. (A–B) Deuterium uptake spectra of mini Gs helix 5 peptides (A) “IQRMHLRQ” C-ter and (B) “RRIFNDCRD” N-ter in complex with β1AR _ICL3 or the truncated construct (both activated and in 1.2 equiv excess) and the inactive truncated β1AR. Deuterium uptake is plotted as a function of labeling time (250 s) and shown for (A) the C-terminus and (B) the N-terminus of Gs helix 5. (C–D) D uptake is plotted as a function of deuterium labeling time from 10 to 1000 s for these two peptides. Error bars are generated according to the standard deviation calculated from three independent repeats. A student’s t test was used to evaluate the statistically significant differences (p < 0.05*, p < 0.01**). Heat maps of the HDX fractional uptake difference between the truncated β1AR and β1AR _ICL3 complexes at a deuterium labeling time of 250 s (i.e., [% HDX mini Gs-β1AR_ICL3] – [% HDX mini Gs-β1AR_truncated]). Differences are displayed on the structure of mini Gs coupled to A2AR, for illustrative purposes.27

To explore further the origin of this additional HDX protection in helix 5, and how it might change as equilibrium is perturbed, we manipulated the concentration of the receptor (0 μM to 24 μM), fixed the concentration of mini Gs (20 μM) and isoprenaline (100 μM), and the labeling time (250 s). The deuterium uptake of both the N-terminal and C-terminal peptides of mini Gs helix 5 decreases as the receptor concentration increases. In other words, protection increases as the concentration of the receptors increases (Figure 4A-B). In both cases, for the truncated β1AR and β1AR_ICL3, 0.6 equiv of receptor was sufficient to reach saturation of protection for helix 5. However, the extent of protection to the C-terminus of helix 5 was greater for β1AR_ICL3 than for β1AR at all concentrations tested. Together, these HDX results support our observations from native MS wherein mini Gs preferentially couples to β1AR_ICL3. We suggest therefore that the enhanced protection observed in the presence of ICL3 must arise from a conformational change of mini Gs helix 5 during complex formation, which contributes to its increased binding affinity.

Figure 4.

Figure 4

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Impact of ICL3 on HDX protection of mini Gs helix 5 and the binding orientation of mini Gs. (A–B) Concentration-dependent HDX-MS measured only for mini Gs helix 5 in complex with β1AR_ICL3 or the truncated construct (deuterium labeling time: 250 s). The concentration of mini Gs is fixed at 20 μM and deuterium uptake is plotted as a function of receptor concentration (from 0 to 24 μM) for two selected mini Gs helix 5 peptides: (A) the C terminal residues and (B) the N terminal residues. The error bars are generated according to the standard deviation calculated from three independent repeats. A student’s t-test was used to evaluate the statistically significant differences (p < 0.01). (C–D) Coarse-grained molecular dynamics simulations: In the presence of ICL3, helix 5 of mini Gs tends to adopt a more closed binding orientation (C), compared to the orientation preferred by truncated β1AR (D). (E) The final frames of 4 × 2 μs atomistic molecular dynamics simulations. The left-hand panel shows the hydrogen bond between R221 of mini Gs helix 5 and Q237 of β1AR for β1AR_ICL3, where 3 out of 4 repeats maintained a hydrogen bond with the terminal charged NηH2+ in R221 and 1 repeat with the weaker NεH in R221. The right-hand panel shows the hydrogen bond for truncated β1AR, where 3 out of 4 repeats formed hydrogen bonds with the weaker NεH in R221, and in one repeat, the hydrogen bond is completely broken, allowing a more prounced change in orientation of mini Gs. The zoomed-in view (rhs) shows the time series of the hydrogen bond breakage in the truncated β1AR (red to blue: initial to final frames shown at 200 ns intervals).

MD reveals differences in mini Gs during coupling

To obtain structural insights into the effect of ICL3 on the orientation of mini Gs, we carried out coarse grained molecular dynamics (cgMD) simulations for the β1AR-mini Gs complex with and without ICL3. (MD modeling is based on the M. gallopavo β1AR cryo EM structure of dobutamine−β1-AR–Gs (8DCR)37Supplementary Figure 6A-B.) During cgMD simulations of the complex, in a model plasma membrane, two distinct orientations of mini Gs, relative to truncated β1AR, were observed: one in which the initial cryo-EM orientation was maintained (Supplementary Figure 6C), and another in which mini Gs is shifted such that it no longer packs against TM5 and TM6 of β1AR (Supplementary Figure 6D). We then compared these truncated structures with those in the presence of ICL3 (highlighted in Figure 4C). Over the course of the 5 μs simulation, the angle formed by the orientation of helix 5 of mini Gs, to the plane by three residues in β1AR_ICL3, is consistently lower than that for the same residues in β1AR (Supplementary Figure 6E). Representative structures show that β1AR_ICL3 couples in a more “closed” orientation than truncated β1AR in all 5 cgMD replicates (Figure 4D). Specifically, ICL3 was observed to pack against mini Gs as well as to interact with the model plasma membrane. In the absence of ICL3 mini Gs adopted a shifted, “open” orientation in which helix 5 of mini Gs is exposed to solvent. Furthermore, our atomistic MD simulation shows that ICL3 enhances a hydrogen bond interaction between the terminal charged NηH2+ in R221, equivalent to R385 of wild-type Gs, on helix 5 of mini Gs and Q237 of β1AR (Figure 4E, Supplemenary Figure 7B-E). In contrast, in the case of truncated β1AR, a weaker hydrogen bond interaction with NεH in R221 was observed, and in one repeat, the complete breakage of the hydrogen bond was seen, resulting in a more shifted mini Gs state. Together, our MD results are in line with our HDX-MS study, which indicates that β1AR_ICL3 yields a more protected patch on helix 5 of mini Gs, specifically at the sequence encompassing R221 from I219 to Q226 (IQRMHLRQ).

ICL3 of β1AR promotes coupling after GDP release

Both the native MS and HDX-MS experiments reported above captured the nucleotide-free state of mini Gs in complex with β1AR, after 20 min of incubating the receptor and mini Gs protein. To investigate the possible influence of ICL3 on proposed intermediate states of coupling, i.e., to the GDP-bound mini Gs, we need to explore earlier time points in the coupling reaction. We therefore performed time-resolved HDX-MS (or pulsed HDX). We fixed the deuterium labeling time at 10 s and varied the incubation time of activated receptor and mini Gs from 10 s to 1 h at 20 °C. A short deuterium labeling time (10 s) should minimize additional coupling taking place during the labeling period. Under these conditions, we found that ICL3 did not induce significant protection on helix 5 of mini Gs compared to the construct without ICL3 at the first two coupling incubation times (10 s and 1 min) (Supplementary Figure 8). After 1 h of coupling incubation, a small extent of protection induced by ICL3 was captured. We surmise that a labeling time of 10 s is too short and the HDX differences too subtle for us to draw conclusions.

We therefore repeated the experiment above but this time using a longer labeling time of 60 s. Under these conditions we found that helix 5 of mini Gs increased in protection gradually as the coupling incubation time increased from 10 s to 20 min at 20 °C, reaching a saturation level after 20 min (Figure 5A-B). This observation implies that we can capture discrete stages of β1AR complex formation on a time scale of several minutes. Surprisingly, we observed that the deuterium uptake of helix 5 when coupled to truncated β1AR or β1AR_ICL3 showed no difference at the initial stages of complex formation (10 and 60 s). Differences in helix 5, but no other regions of mini Gs, only become apparent after 5 min of coupling and become saturated at 20 min. We align these results with our native MS analysis, wherein coupling is fully realized with no GDP binding detected (incubation time 20 min at 20 °C) (Supplementary Figure 3) and note that the extent of coupling does not change but HDX protection increases gradually. Collectively therefore our data suggest that ICL3 induces a protected patch on helix 5 after the initial stages of complex formation (>1 min) in the nucleotide-free β1AR-mini Gs complex.

Figure 5.

Figure 5

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The effect of ICL3 on β1AR coupling to mini Gs occurs postnucleotide-release. (A–B) Time-resolved HDX-MS of helix 5 in complex with β1AR_ICL3 or the truncated construct without ICL3 (Deuterium labeling time: 60 s). Deuterium uptake is plotted as a function of coupling incubation time (time after agonist addition) from 10 s to 1 h for 2 selected peptides covering (A) the C terminus and (B) the N terminus of mini Gs helix 5. Error bars are generated according to the standard deviation calculated from three independent repeats. A student’s t-test was used to evaluate the statistically significant differences (p < 0.01). (C) Schematic to show how BODIPY FL GDP can be used to monitor the release of GDP from the GPCR-mini Gs complex. (D) GDP release assay for truncated β1AR (Blue) and β1AR_ICL3 (Orange). The Relative fluorescence intensity (FL intensity) % is plotted as a function of time upon isoprenaline activation. The value is calculated from (Recorded FL intensity – Basal FL intensity)/(Highest FL intensity – Basal FL intensity) × 100%. Recorded FL intensity represents the average FL intensity (A.U) recorded in a 15 ms period. Basal FL intensity represents the average fluorescence intensity at the final 300 scans. The highest FL intensity stands for the average fluorescence intensity during the initial 15 scans (0–15 ms). Error bars are generated according to the standard deviation calculated from three independent repeats. (E–F) cAMP accumulation assay for HEK293 with overexpressed β1AR_ICL3, Truncated β1AR, and empty HEK293 as a control (gray) shows that ICL3 promotes the canonical signaling of β1AR. (E) Glosensor Luminescence (RLU) of control (gray), Trun β1AR (blue) and β1AR_ICL3 (orange) is plotted against the time after addition of isoprenaline (full agonist) to HEK293T. (F) Glosensor Luminescence (RLU) of β1AR_ICL3, Trun β1AR, and control after 30 min incubation with isoprenaline is plotted as bar chart. Error bars are generated according to the standard deviation calculated from three independent biological repeats (control), four independent biological repeats (β1AR_ICL3), and five independent biological repeats (Truncated β1AR). A one-way ANOVA test was used to evaluate the statistically significant differences (p < 0.01). ICL3 was found to significantly promote Gs signaling of β1AR.

To explore further the influence of ICL3 on GDP release, we set up an orthogonal fluorescence assay to explore the time scale of GDP release during β1AR-mini Gs coupling. We compared the GDP release rate triggered by truncated β1AR with that of β1AR_ICL3 using BODIPY FL GDP, a modified GDP analogue with a Bodipy dye connected to the 2′ position of the ribose ring via an aminoethylcarbamoyl linker. The fluorescence of Bodipy dye is suppressed by purine (in GDP) via electron transfer quenching in solution. When the analogue binds to a GTPase, such as mini Gs, the interaction between the G Protein and the purine reduces the efficiency of electron transfer quenching, leading to the promotion of fluorescence. Upon the addition of an active GPCR, the release of GDP, triggered by the receptor coupling event, results in a decrease in the fluorescence (Figure 5C). Thus, GDP release can be captured by monitoring changes in fluorescence intensity.

To conduct the GDP release assay, an excess of β1AR (either truncated or with ICL3) was incubated with mini Gs and BODIPY FL GDP in a 96 well plate prior to the fluorescence assay (see Supporting Information for detailed conditions). Immediately after the addition of isoprenaline, fluorescence intensity was monitored over time at 20 °C. GDP release during coupling was successfully captured via a decay in fluorescence as a function of time, decreasing until around 60 s (Figure 5D). This observation implies that GDP release is complete within ∼60 s at 20 °C. A Kolmogorov–Smirnov test for the two distributions showed that the p value was larger than 0.95. Importantly, the GDP release curves for truncated β1AR and the β1AR_ICL3 follow the same distribution implying that the initial stages of GDP release are not impacted by the presence or absence of ICL3.

To achieve full coupling of mini Gs to β1AR, we used a mini Gs mutant (I208A) throughout our study. This mutation not only enhances the affinity of mini Gs for β1AR but also makes the complexes resistant to GTP-induced dissociation. Given that this mutation could potentially affect our GDP release assay, as a control, we also performed a GDP release assay using mini Gs without the alanine mutation at I208 (mini Gs I208). First we considered the affinity of the two β1AR constructs for mini Gs I208 via native MS (Supplementary Figure 9A-B). The coupling experiments were conducted separately for the two constructs due to the relatively unstable nature of the mini Gs I208 complex with the two receptors. Despite the relatively low coupling affinity of the I208A mutant, our native MS results demonstrate that the presence of ICL3 enhances the coupling of β1AR to mini Gs I208. Comparing the GDP release rates for β1AR, with and without ICL3, coupled to mini Gs I208 the results are closely similar allowing us to conclude that although coupling is less efficient the mutation does not impact GDP release rates (Supplementary Figure 9C).

Considering further this GDP release assay, wherein no difference between β1AR and β1AR_ICL3 was observed for wild-type or I208 mini Gs, and our time-resolved HDX-MS data in which the first two coupling incubation times showed no effect of ICL3, we surmise that rather than influencing coupling at the early intermediate complex stage, ICL3 exerts its major effect later on the nucleotide-free state of the complex. To explore the mechanism further we therefore conducted a cell-based cAMP production assay to investigate the impact of ICL3 on β1AR signaling through the Gs pathway. To do this we used the Glosensor assay to compare HEK293 cells with overexpression of β1AR_ICL3 and the truncated construct. β1AR_ICL3 showed significantly stronger luminescence intensity (p < 0.0001) compared with truncated β1AR. This assay allows the inference that ICL3 plays an essential role in the canonical signaling of β1AR downstream of the early coupling events.

To capture early coupling of a GDP-bound state via native MS we woud need to exlore complex formation in <60 s (Figure 5D). To do this, we first incubated β1AR and mini Gs and, after buffer exchange, added isoprenaline so that the solution could be analyzed immediately with a minimal incubation time. Under these conditions, the GDP-bound complex could be observed in native mass spectra (Figure 6A) allowing us to test whether β1AR_ICL3 has an effect on the affinity of this intermediate state. To compare the affinity of the two β1AR complexes to GDP, under the exact same MS and solution conditions, truncated β1AR, β1AR_ICL3 and mini Gs were incubated at a 1:1:2 ratio with excess isoprenaline. Native mass spectra were recorded, and the extent of mini Gs-GDP versus apo mini Gs binding were assessed. The spectra are complicated with multiple split peaks due to nucleotide and metal ion binding. However, we are able to measure the ratio of GDP-bound (β1AR-mini Gs or β1AR_ICL3 mini Gs) to GDP-free (β1AR-mini Gs or β1AR_ICL3 mini Gs) (Figure 6B). Comparable intensity ratios were observed (Figure 6B inset). Overall, this native MS result further supports our conclusions made from time-resolved HDX-MS and the GDP release assays that together show that ICL3 does not have a significant impact on the affinity of the complex during the initial stages of the coupling process, prior to GDP release. Rather the effect of ICL3 is realized on cAMP accumulation during the later stages of coupling.

Figure 6.

Figure 6

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(A–B) Native mass spectra of the β1AR-mini Gs-GDP complex. (A) The ratio of truncated β1AR:mini Gs was 1:1 (As we discussed above the spectrum was recorded immediately after addition of agonist < 10 s) (B) The ratio of truncated β1AR: β1AR_ICL3:mini Gs was 1:1:2. The peaks are assigned to truncated β1AR-mini Gs complex (pale green), the GDP-bound truncated β1AR- mini Gs complex (dark green), the β1AR_ICL3 mini Gs complex (light blue), and the GDP-bound β1AR_ICL3-mini Gs complex (dark blue). Peak intensity is quantified (inset bar graph) and shows that ICL3 does not change the binding affinity of the receptor to mini Gs GDP. (C) Illustrative figure showing that ICL3 does not influence the initial engagement of helix 5 of Gs to β1AR, at the intermediate state, prior to GDP release. Instead, ICL3 increases the affinity of β1AR to Gs by inducing different coupling poses of helix 5 of the G protein at a later stage, after GDP release.

Conclusions

Herein, we investigated the intramolecular regulation of β1AR activity by ICL3 using native-MS, HDX-MS, a cell-based cAMP accumulation assay, and a GDP release assay. Native MS analysis showed that mini Gs preferentially couples to β1AR with an intact ICL3 loop (β1AR_ICL3) compared to β1AR without ICL3. Previous structural studies report extensive interactions between helix 5 of a G protein and intracellular regions of TM3, TM5, and TM6 of a GPCR.38−42 With HDX-MS analysis, we found that helix 5 of Gs can be used to probe the activity of GPCRs by qualitatively revealing the efficacy of various drugs to activate β1AR. We consider helix 5 of Gs to be a reliable reporter of the extent of activation since it forms part of the binding interface with the receptor.

It is interesting to compare our data with a previous NMR study that showed how chemical shift changes of M2235.54 and M2966.41 on β1AR correlated with ligand efficacy. The movement of these two residues was assigned to opening of the cytoplasmic end of β1AR for G protein coupling with the degree of conformational change likely regulated by drugs with different efficacy.36 NMR analyses of other class-A receptors, including β2AR and A2AR, led to the proposal that a distinct intermediate may exist between the inactive and active conformations.21,40−42 Nevertheless, detailed mechanisms of partial agonism have not been confirmed.1 Our study further suggests that the binding pose of helix 5 of mini Gs, on the intracellular side of partial agonist bound β1AR, is different from the fully activated β1AR-mini Gs complex (Supplementary Figure 5C). Strikingly, under treatment of a full agonist, β1AR_ICL3 induces further protection of helix 5, upon complex formation, beyond that of agonist-bound β1AR. We propose therefore that ICL3 can induce a distinct binding orientation of helix 5 upon complex formation, that is not stabilized in the truncated construct. MD simulations give insights into the structural dynamics of complex formation between β1AR/β1AR_ICL3 and mini Gs and are consistent with our propsoal of two binding orientations of helix 5 of mini Gs. In one of the binding poses (with ICL3) helix 5 shows a more compact interaction with the receptor. We propose that the presence of ICL3 of β1AR shifts the proportion of helix 5 to a relatively closed conformation which is stabilized by a hydrogen bond located within a highly protected patch (219–226) for enhanced coupling and alignment of the unstructured loop along the membrane.

Previous structural studies reported the existence of a transient intermediate complex in which GDP remains bound to the complex.19−22 A very recent study using time-resolved cryo-EM shows in intricate detail conformational changes of a GPCR-G protein complex after the addition of GTP.46 By contrast, our time-resolved HDX, with distinct β1AR-mini Gs incubation times, explores the role of ICL3 in the initial stages of G protein engagement, before the addition of GTP. Interestingly, rather than the higher protection of the helix 5 motif, as observed for long incubation times, no significant difference in deuterium exchange of helix 5 in complex with β1AR or β114_ICL3 is observed in the first 60 s of incubation. This observation was further supported by our GDP release assay, revealing that the rate of GDP release is not influenced by truncation of ICL3. Combined with our cell based signaling assay, ICL3 was proven to promote the upregulation of cAMP by agonist bound β1AR. Together these results imply that the regulatory effect of ICL3 occurs postnucleotide release on downstream signaling events.

In conclusion, we have shown that activation of β1AR via agonists leads to a level of engagement with mini Gs that is enhanced by the presence of ICL3. We propose that this enhanced engagement is achieved by changing the binding pose of mini Gs helix 5 to produce a proected patch on a more compact structure following GDP release (Figure 6C). Overall, therefore these results provide new insights into the function of this unstructured loop, post GDP release, in stabilizing coupling then promoting the cAMP downstream signaling. Collectively they shed new light on the intramolecular regulation of GPCR activation, with important consequences for drug design.

Methods

Extended experimental and method details can be found in the Supporting Information (PDF). For protein expression and purification, β1AR, β1AR_ICL3 were overexpressed in Sf9 insect cells utilizing recombinant baculoviruses prepared using the dual expression vector pFastBac (Thermo Fisher). The engineered minimal G proteins, mini Gs construct R414 and mini Gs/i construct R43 were cloned into the pET15b plasmid for overexpression in E. coli. These proteins were purified by histogram affinity chromatography. For native MS analysis, β114, β114_ICL3, and mini Gs were buffer exchanged into MS Buffer (two times the CMC of Fos-Choline and 200 mM ammonium acetate) and analyzed on a Q-Exactive UHMR Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific). Raw native MS spectra were deconvoluted and quantified by using UniDec software. HDX-MS experiments were performed on equipment from Waters Corporation, Manchester, UK. Purified receptors and mini Gs were diluted to concentrations of interest (the HDX process being sensitive to protein concentration47) using the equilibration buffer with different compositions. The cAMP production assay and Time-resolved GDP assay was performed by FLUOstar Omega Microplate Reader (BMG). MD Simulations were done using the GROMACS package with version 2022.4.48 Trajectory analysis was carried out using the gromacs tool and VMD.49

Acknowledgments

Research in the C.V.R. laboratory is supported by a Wellcome Trust Grant 221795/Z/20/Z. Research in the S.L.R. laboratory is supported by Medical Research Council Grant MR/T017961/1.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.4c11250.

  • The Supporting Information contains the supplementary Figures S1–S9 and details of the experimental procedures. (PDF)

The authors declare the following competing financial interest(s): C.V.R. is a cofounder of and consultant at OMass Therapeutics. The remaining authors declare no competing interests.

Supplementary Material

ja4c11250_si_001.pdf (1.2MB, pdf)

References

  1. Weis W. I.; Kobilka B. K. The molecular basis of G protein–coupled receptor activation. Annu. Rev. Biochem. 2018, 87 (1), 897–919. 10.1146/annurev-biochem-060614-033910. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Flock T.; Hauser A. S.; Lund N.; Gloriam D. E.; Balaji S.; Babu M. M. Selectivity determinants of GPCR–G-protein binding. Nature 2017, 545 (7654), 317–322. 10.1038/nature22070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Wade S. M.; Scribner M. K.; Dalman H. M.; Taylor J. M.; Neubig R. R.; Wade S. M.; Scribner M. K.; Dalman H.; Taylor J. M.; Neubig R. R. Structural requirements for G(o) activation by receptor-derived peptides: activation and modulation domains of the alpha 2-adrenergic receptor i3c region. Mol. Pharmacol. 1996, 50 (2), 351–358. [PubMed] [Google Scholar]
  4. O’Dowd B. F.; Hnatowich M.; Regan J. W.; Leader W. M.; Caron M. G.; Lefkowitz R. J. Site. Site-directed mutagenesis of the cytoplasmic domains of the human beta 2-adrenergic receptor. localization of regions involved in G protein-receptor coupling. J. Biol. Chem. 1988, 263 (31), 15985–15992. 10.1016/S0021-9258(18)37546-X. [DOI] [PubMed] [Google Scholar]
  5. Liggett S. B.; Caron M. G.; Lefkowitz R. J.; Hnatowich M. Coupling of a mutated form of the human beta 2-adrenergic receptor to GI and GS. requirement for multiple cytoplasmic domains in the coupling process. J. Biol. Chem. 1991, 266 (8), 4816–4821. 10.1016/S0021-9258(19)67722-7. [DOI] [PubMed] [Google Scholar]
  6. Ozcan O.; Uyar A.; Doruker P.; Akten E. D. Effect of intracellular loop 3 on intrinsic dynamics of human β2-adrenergic receptor. BMC Struct. Biol. 2013, 13, 29. 10.1186/1472-6807-13-29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Taylor J. M.; Jacob-Mosier G. G.; Lawton R. G.; Remmers A. E.; Neubig R. R. Binding of an alpha 2 adrenergic receptor third intracellular loop peptide to G beta and the amino terminus of G Alpha. J. Biol. Chem. 1994, 269 (44), 27618–27624. 10.1016/S0021-9258(18)47029-9. [DOI] [PubMed] [Google Scholar]
  8. Dalman H. M.; Neubig R. R. Two peptides from the alpha 2A-adrenergic receptor alter receptor G protein coupling by distinct mechanisms. J. Biol. Chem. 1991, 266 (17), 11025–11029. 10.1016/S0021-9258(18)99122-2. [DOI] [PubMed] [Google Scholar]
  9. Wagner T.; Oppi C.; Tocchini Valentini G. P. Differential regulation of G protein α-subunit gtpase activity by peptides derived from the third cytoplasmic loop of the α2-adrenergic receptor. FEBS Lett. 1995, 365 (1), 13–17. 10.1016/0014-5793(95)00435-C. [DOI] [PubMed] [Google Scholar]
  10. Maiek D.; Münch G.; Palm D. Two sites in the third inner loop of the dopamine D2 receptor are involved in functional G protein-mediated coupling to adenylate cyclase. FEBS Lett. 1993, 325 (3), 215–219. 10.1016/0014-5793(93)81076-C. [DOI] [PubMed] [Google Scholar]
  11. Sadler F.; Ma N.; Ritt M.; Sharma Y.; Vaidehi N.; Sivaramakrishnan S. Autoregulation of GPCR signalling through the third intracellular loop. Nature 2023, 615 (7953), 734–741. 10.1038/s41586-023-05789-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cheng Z. J.; Zhao J.; Sun Y.; Hu W.; Wu Y. L.; Cen B.; Wu G. X.; Pei G. B-arrestin differentially regulates the chemokine receptor CXCR4-mediated signaling and receptor internalization, and this implicates multiple interaction sites between β-arrestin and CXCR4. J. Biol. Chem. 2000, 275 (4), 2479–2485. 10.1074/jbc.275.4.2479. [DOI] [PubMed] [Google Scholar]
  13. Mukherjee S.; Palczewski K.; Gurevich V. v.; Hunzicker-Dunn M. B-arrestin-dependent desensitization of luteinizing hormone/choriogonadotropin receptor is prevented by a synthetic peptide corresponding to the third intracellular loop of the receptor. J. Biol. Chem. 1999, 274 (19), 12984–12989. 10.1074/jbc.274.19.12984. [DOI] [PubMed] [Google Scholar]
  14. Krupnick J. G.; Gurevich V. v.; Schepers T.; Hamm H. E.; Benovic J. L. Arrestin-rhodopsin interaction. multi-site binding delineated by peptide inhibition. J. Biol. Chem. 1994, 269 (5), 3226–3232. 10.1016/S0021-9258(17)41852-7. [DOI] [PubMed] [Google Scholar]
  15. Wu G.; Krupnick J. G.; Benovic J. L.; Lanier S. M. Interaction of arrestins with intracellular domains of muscarinic and α2-adrenergic receptors. J. Biol. Chem. 1997, 272 (28), 17836–17842. 10.1074/jbc.272.28.17836. [DOI] [PubMed] [Google Scholar]
  16. Gelber E. I.; Kroeze W. K.; Roth B. L.; Gray J. A.; Sinar C. A.; Hyde E. G.; Gurevich V.; Benovic J.; Roth B. L. Structure and function of the third intracellular loop of the 5-hydroxytryptamine2A receptor: The third intracellular loop is α-helical and binds purified arrestins. J. Biol. Neurochem. 1999, 72 (5), 2206–2214. 10.1046/j.1471-4159.1999.0722206.x. [DOI] [PubMed] [Google Scholar]
  17. Degraff J. L.; Gurevich V. v.; Benovic J. L. The third intracellular loop of α2-adrenergic receptors determines subtype specificity of arrestin interaction. J. Biol. Chem. 2002, 277 (45), 43247–43252. 10.1074/jbc.M207495200. [DOI] [PubMed] [Google Scholar]
  18. Warne T.; Serrano-Vega M. J.; Baker J. G.; Moukhametzianov R.; Edwards P. C.; Henderson R.; Leslie A. G. W.; Tate C. G.; Schertler G. F. X. Structure of a β1-adrenergic G-protein-coupled receptor. Nature 2008, 454 (7203), 486–491. 10.1038/nature07101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Du Y.; Duc N. M.; Rasmussen S. G. F.; Hilger D.; Kubiak X.; Wang L.; Bohon J.; Kim H. R.; Wegrecki M.; Asuru A.; Jeong K. M.; Lee J.; Chance M. R.; Lodowski D. T.; Kobilka B. K.; Chung K. Y. Assembly of a GPCR-G protein complex. Cell 2019, 177 (5), 1232–1242. 10.1016/j.cell.2019.04.022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Liu X.; Xu X.; Hilger D.; Aschauer P.; Tiemann J. K. S.; Du Y.; Liu H.; Hirata K.; Sun X.; Guixà-González R.; Mathiesen J. M.; Hildebrand P. W.; Kobilka B. K. Structural insights into the process of GPCR-G Protein Complex Formation. Cell 2019, 177 (5), 1243–1251. 10.1016/j.cell.2019.04.021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gregorio G. G.; Masureel M.; Hilger D.; Terry D. S.; Juette M.; Zhao H.; Zhou Z.; Perez-Aguilar J. M.; Hauge M.; Mathiasen S.; Javitch J. A.; Weinstein H.; Kobilka B. K.; Blanchard S. C. Single-molecule analysis of ligand efficacy in β2ar–G-protein activation. Nature 2017, 547 (7661), 68–73. 10.1038/nature22354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Chung K. Y.; Rasmussen S. G. F.; Liu T.; Li S.; Devree B. T.; Chae P. S.; Calinski D.; Kobilka B. K.; Woods V. L.; Sunahara R. K. Conformational changes in the G protein GS induced by the B2 adrenergic receptor. Nature 2011, 477 (7366), 611–615. 10.1038/nature10488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Grison C. M.; Lambey P.; Jeannot S.; Del Nero E.; Fontanel S.; Peysson F.; Heuninck J.; Sounier R.; Durroux T.; Leyrat C.; Granier S.; Bechara C. Molecular Insights into Mechanisms of GPCR Hijacking by Staphylococcus Aureus. Proc. Natl. Acad. Sci. U. S. A. 2021, 118 (42), e2108856118 10.1073/pnas.2108856118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Yen H. Y.; Jazayeri A.; Robinson C. V. G Protein-Coupled Receptor Pharmacology—Insights from Mass Spectrometry. Pharmacol. Rev. 2023, 75 (3), 397–415. 10.1124/pharmrev.120.000237. [DOI] [PubMed] [Google Scholar]
  25. Carpenter B.; Tate C. G. Engineering a Minimal G Protein to Facilitate Crystallisation of G Protein-Coupled Receptors in Their Active Conformation. PEDS 2016, 29 (12), 583–594. 10.1093/protein/gzw049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rasmussen S. G. F.; Devree B. T.; Zou Y.; Kruse A. C.; Chung K. Y.; Kobilka T. S.; Thian F. S.; Chae P. S.; Pardon E.; Calinski D.; Mathiesen J. M.; Shah S. T. A.; Lyons J. A.; Caffrey M.; Gellman S. H.; Steyaert J.; Skiniotis G.; Weis W. I.; Sunahara R. K.; Kobilka B. K. Crystal structure of the β2 adrenergic receptor–GS protein complex. Nature 2011, 477 (7366), 549–555. 10.1038/nature10361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Carpenter B.; Nehmé R.; Warne T.; Leslie A. G. W.; Tate C. G. Structure of the adenosine A2A receptor bound to an engineered G protein. Nature 2016, 536 (7614), 104–107. 10.1038/nature18966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Warne T.; Moukhametzianov R.; Baker J. G.; Nehmé R.; Edwards P. C.; Leslie A. G. W.; Schertler G. F. X.; Tate C. G. The structural basis for agonist and partial agonist action on a β1-adrenergic receptor. Nature 2011, 469 (7329), 241–244. 10.1038/nature09746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Roth C. B.; Hanson M. A.; Stevens R. C. Stabilization of the human β2-adrenergic receptor TM4–TM3–TM5 helix interface by mutagenesis of GLU1223.41, a critical residue in GPCR structure. J. Mol. Biol. 2008, 376 (5), 1305–1319. 10.1016/j.jmb.2007.12.028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Yen H. Y.; Hoi K. K.; Liko I.; Hedger G.; Horrell M. R.; Song W.; Wu D.; Heine P.; Warne T.; Lee Y.; Carpenter B.; Plückthun A.; Tate C. G.; Sansom M. S. P.; Robinson C. V. PtdIns(4,5)P2 stabilizes active states of gpcrs and enhances selectivity of G-protein coupling. Nature 2018, 559 (7714), 423–427. 10.1038/s41586-018-0325-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nehme R.; Carpenter B.; Singhal A.; Strege A.; Edwards P. C.; White C. F.; Du H.; Grisshammer R.; Tate C. G. Mini-g proteins: Novel tools for studying gpcrs in their active conformation. PLoS One 2017, 12 (4), e0175642 10.1371/journal.pone.0175642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Madamanchi A. β-Adrenergic Receptor Signaling in Cardiac Function and Heart Failure. McGill Journal of Medicine. 2007, 10 (2), 99–104. [PMC free article] [PubMed] [Google Scholar]
  33. Alegre K. O.; Paknejad N.; Su M.; Lou J. S.; Huang J.; Jordan K. D.; Eng E. T.; Meyerson J. R.; Hite R. K.; Huang X. Y. Structural basis and mechanism of activation of two different families of G proteins by the same GPCR. Nat. Struct. Mol. Biol. 2021, 28 (11), 936–944. 10.1038/s41594-021-00679-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Innis R. B.; Corrêa F. M. A.; Snyder S. H. Carazolol, an Extremely Potent β-Adrenergic Blocker: Binding to β-Receptors in Brain Membranes. Life. Sci. 1979, 24 (24), 2255–2264. 10.1016/0024-3205(79)90102-4. [DOI] [PubMed] [Google Scholar]
  35. Tuttle R. R.; Mills J. Dobutamine. Development of a New Catecholamine to Selectively Increase Cardiac Contractility. Circ. Res. 1975, 36 (1), 185–196. 10.1161/01.RES.36.1.185. [DOI] [PubMed] [Google Scholar]
  36. Solt A. S.; Bostock M. J.; Shrestha B.; Kumar P.; Warne T.; Tate C. G.; Nietlispach D. Insight into Partial Agonism by Observing Multiple Equilibria for Ligand-Bound and Gs-Mimetic Nanobody-Bound B1-Adrenergic Receptor. Nat. Commun. 2017, 8 (1), 1795. 10.1038/s41467-017-02008-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Su M.; Paknejad N.; Zhu L.; Wang J.; Do H. N.; Miao Y.; Liu W.; Hite R. K.; Huang X. Y. Structures of B1-Adrenergic Receptor in Complex with Gs and Ligands of Different Efficacies. Nat. Commun. 2022, 13 (1), 4095. 10.1038/s41467-022-31823-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Kang Y.; Kuybeda O.; de Waal P. W.; Mukherjee S.; van Eps N.; Dutka P.; Zhou X. E.; Bartesaghi A.; Erramilli S.; Morizumi T.; Gu X.; Yin Y.; Liu P.; Jiang Y.; Meng X.; Zhao G.; Melcher K.; Ernst O. P.; Kossiakoff A. A.; Subramaniam S.; Xu H. E. Cryo-EM Structure of Human Rhodopsin Bound to an Inhibitory G Protein. Nature 2018, 558 (7711), 553–558. 10.1038/s41586-018-0215-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Koehl A.; Hu H.; Maeda S.; Zhang Y.; Qu Q.; Paggi J. M.; Latorraca N. R.; Hilger D.; Dawson R.; Matile H.; Schertler G. F. X.; Granier S.; Weis W. I.; Dror R. O.; Manglik A.; Skiniotis G.; Kobilka B. K. Structure of the μ-Opioid Receptor-Gi Protein Complex. Nature 2018, 558 (7711), 547–552. 10.1038/s41586-018-0219-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Draper-Joyce C. J.; Khoshouei M.; Thal D. M.; Liang Y. L.; Nguyen A. T. N.; Furness S. G. B.; Venugopal H.; Baltos J. A.; Plitzko J. M.; Danev R.; Baumeister W.; May L. T.; Wootten D.; Sexton P. M.; Glukhova A.; Christopoulos A. Structure of the Adenosine-Bound Human Adenosine A1 Receptor-Gi Complex. Nature 2018, 558 (7711), 559–563. 10.1038/s41586-018-0236-6. [DOI] [PubMed] [Google Scholar]
  41. García-Nafría J.; Nehmé R.; Edwards P. C.; Tate C. G. Cryo-EM Structure of the Serotonin 5-HT1B Receptor Coupled to Heterotrimeric Go. Nature 2018, 558 (7711), 620–623. 10.1038/s41586-018-0241-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Xia R.; Wang N.; Xu Z.; Lu Y.; Song J.; Zhang A.; Guo C.; He Y. Cryo-EM Structure of the Human Histamine H1 Receptor/Gq Complex. Nat. Commun. 2021, 12 (1), 2086. 10.1038/s41467-021-22427-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Ye L.; van Eps N.; Zimmer M.; Ernst O. P.; Scott Prosser R. Activation of the A 2A Adenosine G-Protein-Coupled Receptor by Conformational Selection. Nature 2016, 533 (7602), 265–268. 10.1038/nature17668. [DOI] [PubMed] [Google Scholar]
  44. Imai S.; Yokomizo T.; Kofuku Y.; Shiraishi Y.; Ueda T.; Shimada I. Structural Equilibrium Underlying Ligand-Dependent Activation of B2-Adrenoreceptor. Nat. Chem. Biol. 2020, 16 (4), 430–439. 10.1038/s41589-019-0457-5. [DOI] [PubMed] [Google Scholar]
  45. Staus D. P.; Strachan R. T.; Manglik A.; Pani B.; Kahsai A. W.; Kim T. H.; Wingler L. M.; Ahn S.; Chatterjee A.; Masoudi A.; Kruse A. C.; Pardon E.; Steyaert J.; Weis W. I.; Prosser R. S.; Kobilka B. K.; Costa T.; Lefkowitz R. J. Allosteric Nanobodies Reveal the Dynamic Range and Diverse Mechanisms of G-Protein-Coupled Receptor Activation. Nature 2016, 535 (7612), 448–452. 10.1038/nature18636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Papasergi-Scott M. M.; Pérez-Hernández G.; Batebi H.; Gao Y.; Eskici G.; Seven A. B.; Panova O.; Hilger D.; Casiraghi M.; He F.; Maul L.; Gmeiner P.; Kobilka B. K.; Hildebrand P. W.; Skiniotis G. Time-Resolved Cryo-EM of G-Protein Activation by a GPCR. Nature 2024, 629 (8008), 1182–1191. 10.1038/s41586-024-07153-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Masson G. R.; Burke J. E.; Ahn N. G.; Anand G. S.; Borchers C.; Brier S.; Bou-Assaf G. M.; Engen J. R.; Englander S. W.; Faber J.; Garlish R.; Griffin P. R.; Gross M. L.; Guttman M.; Hamuro Y.; Heck A. J. R.; Houde D.; Iacob R. E.; Jørgensen T. J. D.; Rand K. D. Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments. Nat. Methods. 2019, 16 (7), 595–602. 10.1038/s41592-019-0459-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Abraham M. J.; Murtola T.; Schulz R.; Páll S.; Smith J. C.; Hess B.; Lindahl E. Gromacs: High Performance Molecular Simulations through Multi-Level Parallelism from Laptops to Supercomputers. SoftwareX 2015, 1-2, 19–25. 10.1016/j.softx.2015.06.001. [DOI] [Google Scholar]
  49. Humphrey W.; Dalke A.; Schulten K. VMD: Visual Molecular Dynamics. J. Mol. Graph. 1996, 14 (1), 33–38. 10.1016/0263-7855(96)00018-5. [DOI] [PubMed] [Google Scholar]

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