Critical Residues in the Gβ Subunit Mediate Selective Recruitment of the Gαq Heterotrimer to Activated G Protein-Coupled Receptors

Abstract

G protein-coupled receptors (GPCRs), the largest family of membrane proteins, serve as primary targets for over 30% of FDA-approved drugs. While signaling bias among Gα subtypes is well characterized, emerging evidence highlights that Gβγ subtypes govern the coupling of the Gα heterotrimer to GPCRs. However, whether Gβγ subtype selectivity depends on the receptor or the Gα subunit and the underlying mechanism remain unresolved. Here, we focused on Gαq-coupled GPCRs and investigated Gβγ preferences using bioluminescence resonance energy transfer assays to assess Gβγ selectivity along with Gαq-GPCR coupling and calcium flux assays to measure downstream Ca²⁺ mobilization. By characterizing seven Gαq-coupled receptors, including the muscarinic acetylcholine receptor family (M1 mAChR, M3 mAChR, M5 mAChR) and four other Gαq-coupled GPCRs (AT1R, NTSR1, KISS1R, hM3Dq), the Gβ1/2γ2 combinations were identified as the preferred subtypes for Gαq coupling. Structural analysis and site-directed mutagenesis assays revealed that three key residues in Gβ2 (F111, V178, and G179), distinct from Gβ4, are crucial for enhancing Gαq coupling. Notably, Gβ4 exhibited preferential recruitment of the Gαq heterotrimer in response to pilocarpine relative to acetylcholine, suggesting that pilocarpine may function as a Gβ4-biased agonist. These findings advance our understanding of Gβγ-mediated subtype selectivity and open avenues for developing biased ligands in drug discovery.

GPCRs constitute a large class of eukaryotic seven-transmembrane receptors and represent the largest family of membrane proteins. GPCRs detect a diverse array of ligands and stimuli, enabling them to transduce extracellular signals into the cell through interactions with downstream effectors such as G proteins and β-arrestins. These signaling pathways regulate a broad range of physiological processes including neurotransmission, immune responses, and metabolic homeostasis. Due to their widespread expression and pivotal roles in human physiology, GPCRs make up the largest family of therapeutic targets. Over 30% of FDA-approved drugs, totaling over 700 agents, act on GPCRs. , As the primary effector proteins downstream of GPCRs, G proteins mediate signal transduction through dynamic coupling with activated receptors.

G proteins are heterotrimeric complexes comprising Gα subunits and tightly associated Gβγ dimers. Distinct effector proteins downstream of GPCRs exhibit marked differences in signaling efficacy and temporal dynamics, leading to preferential activation of specific pathwaysa phenomenon termed “signaling bias”. This bias arises from selective stabilization of receptor conformations that favor coupling to one effector over others, despite simultaneous availability of multiple effectors. The generation of diverse intracellular signaling profiles with varying effectmediated through spatiotemporal regulation of downstream effectorsintroduces remarkable complexity to GPCR signaling networks. Conventional ligand action theory posits that agonists activate all downstream pathways proportionally through equilibrium-unbiased interactions. In contrast, recent studies have revealed that ligands interact with receptors in diverse ways. Biased ligands can selectively activate G proteins or β-arrestin, stabilize receptor conformation with specific Gα subtypes, or modulate GPCR kinases, thereby potentiating or inhibiting particular signaling pathways within the GPCR-mediated network. Notably, accumulating evidence indicates that receptor-selective coupling to heterotrimeric G proteins serves as a critical determinant of pathway-specific signaling responses.

Human G proteins are organized into 16 Gα subunits, categorized into four evolutionarily conserved subfamilies: Gαs, Gαi/o, Gαq/11, and Gα12/13. The human genome encodes 5 Gβ subunits (Gβ1–5) and 12 Gγ subunits (Gγ1–12), generating diverse Gβγ subtype combinations and influencing Gα coupling efficiency. For instance, opioid receptors coupled to Gαi predominantly signal through Gβ1γ1/7 dimers, mediating pain, addiction, and hyperglycemia, whereas adrenergic receptors engaging Gαs show preferential utilization of Gβ1γ1/2 complexes. This molecular complexity presents both challenges and opportunities for biased ligand development.

Muscarinic acetylcholine receptors (mAChRs) were first cloned in 1986 and produced five distinct subtypes (M1 mAChR–M5 mAChR). Functionally, M1 mAChR, M3 mAChR, and M5 mAChR subtypes are excitatory receptors, and coupling primarily with Gαq/11 leads to the increase of calcium mobilization. They can regulate neural functions including neuronal excitability, synaptic plasticity, and acetylcholine (ACh) release modulation. There are a few other Gαq/11-coupled GPCRs, which are considered as pivotal drug targets. For example, the neurotensin receptor (NTSR1) is associated with Parkinson’s disease and schizophrenia, while the angiotensin II type 1 receptor (AT1R) is linked to hypertension. The Transcriptional Reporter Universal Platform for Analysis of Transduction Pathways in Heterologous systems (TRUPATH) was developed to study GPCR signaling dynamics. It has been developed for high-throughput screening in vitro, notably identifying β3γ9 as preferential subtype combination for Gαq during Gβγ dissociation. However, this phenomenon was exclusively detected in NTSR1. The selectivity of Gβγ subtypes accompanying Gαq coupling and the underlying structural mechanism remain understudied, representing a critical gap in GPCR pharmacology.

Gβ has 5 subtypes, in which Gβ1–4 share higher sequence similarity (between 80% and 90%) while Gβ5 has about 50% similarity to other subtypes. Moreover, the Gγ subunit unforms a stable complex with Gβ5 but specifically binds Gβ1–4, thus leading to less stable binding of Gβ5 to Gα as well. The selection of the Gγ2 and Gγ9 subunits was guided by empirical evidence from distinct experimental paradigms: The systematic screening via the TRUPATH platform revealed Gγ9 as the optimal subunit for the NTSR1-Gαq dissociation system. Also, the M1 mAChR-Gαq combination system demonstrated Gγ2 prominent coupling efficacy in our previous study. Therefore, in this study, we employ Gβ1–4 and Gγ2/9 to conduct a mechanistic investigation into the subtype preference of Gβγ in Gαq-coupled GPCRs.

This study interrogated the Gβγ subtype selectivity in Gαq-coupled GPCR signaling using BRET and calcium flux assays. Analysis of seven Gαq-coupled receptors (M1/3/5 mAChRs, AT1R, NTSR1, KISS1R, and hM3Dq) identified Gβ1/2γ2 as the predominant subtypes in Gαq-mediated signaling. Structural and mutational data pinpointed three Gβ2 residues (F111, V178, and G179), different from Gβ4, critically affected Gαq coupling efficiency. Intriguingly, pilocarpine preferentially recruits Gβ4-coupled Gαq heterotrimers compared to ACh, implicating its role as a Gβ4-biased agonist. We aim to elucidate the mechanistic principles underlying the Gβγ-driven receptor preference for heterotrimeric G protein coupling, providing pharmacological insights into the rational design of pathway-selective ligands.

1. Materials and Methods

2.1. cDNA Constructs

cDNAs, namely, M1-Rluc8, M3-Rluc8, M5-Rluc8, NTSR1-Rluc8, and AT1R-Rluc8, were designed to fuse Rluc8 into the C-terminus of the receptor while incorporating a flexible SGGGS linker. The plasmids for Gβ1–4 were all obtained from Addgene. mVenus-Gγ2 and mVenus-Gγ9 contained mVenus (fluorescent protein) fused to the N-terminus of Gγ2 and Gγ9 through a short flexible linker sequence (GSAGT). All constructs were cloned into pcDNA3.1 and synthesized by Sangon Biotech (Shanghai, China).

2.2. Cell Culture

This study used HEK-293T cells, a cell line derived from human embryonic kidney cells. HEK-293T cells were purchased from the ATCC. HEK-293T cells were cultured in DMEM (BasalMedia, Shanghai, China) containing 10% FBS (LONSERA, Canelones, Uruguay), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, Thermo Scientific, Waltham, MA, USA) and transfected with plasmids in a humidified atmosphere at 37 °C and 5% CO₂. 24 h after transfection, subsequent experiments can be conducted.

2.3. BRET Assays

HEK-293T cells were seeded in a 25 cm² culture flask containing 5 mL of medium at 37 °C and 5% CO₂. 24 h later, the cells were trypsinized and seeded into black flat-bottomed 96-well plates at a cell density of 2 × 10⁴/100 μL per well (Corning Inc., Corning, NY, USA). The expression constructs were transfected using Lipofectamine 3000 (0.15 μL/well) reagents (Thermo Scientific, Waltham, MA, USA), with a total of 0.1 μg/well, and the cells were incubated at 37 °C and 5% CO₂ for 24 h.

The culture medium was aspirated, and the cells were washed three times with 100 μL of Tyrode's solution (Tyrode's solution containing 150 mM NaCl, 4 mM KCl, 2 mM MgCl₂, 2 mM CaCl₂, 20 mM HEPES, and 10 mM glucose, with pH 7.4). Then, the Tyrode solution was aspirated again, and 50 μL of Tyrode solution was added. Subsequently, the solution was incubated at room temperature for 5 min. 10 μL of freshly prepared luciferase substrate (adding coelenterazine h, final concentration of 5 μmol/L, Hangzi Biotechnology, Shanghai, China) was added, and the solution was mixed. Then, 50 μL of different concentrations of agonists, such as acetylcholine or pilocarpine, BaoGongTeng A, angiotensin II, neurotensin, kisspeptin-10, and deschloroclozapine (MedChemExpress, Monmouth-Junctio, NJ, USA). To measure the BRET ratio after adding the agonist, the microplate reader (Pherastar FSX, BMG LABTECH, Offenburg, Germany) was turned on and the filter was set to BRET1, and the dual emissions were measured at 475 and 530 nm wavelengths (BRET1 Settings: Donor filter: 473–30 nm; Acceptor filter: 535–30 nm). All experiments were conducted under standard culture conditions (37 °C, 5% CO₂) in continuous acquisition mode with an integration time of 0.2 s per well. Baseline BRET signals were recorded for 10 min prior to ligand stimulation, followed by real-time monitoring for 10 min of poststimulation. The BRET signal exhibited a time-dependent enhancement, reaching a plateau phase at 420 s.

2.4. Calcium Mobilization Assay

We used Rhod-2 AM (Yeasen Biotechnology, Shanghai, China) to detect intracellular calcium influx. HEK-293T cells were evenly spread on the plate at a cell density of 2 × 10⁴/100 μL per well. When the cell density reached 70–80%, the plasmids used in the corresponding experiments were transfected into the cells and placed at 37 °C and 5% CO₂. After 24 h of culture, the culture medium was discarded, and buffer (Hanks and HEPES) was used to dilute Rhod-2 AM into a working solution of 5 μmol/L. 50 μL of Rhod-2 AM working solution was added to each well to cover the cells. The cells were incubated at 37 °C for 30 min without light. The dye was discarded, and the cells were washed three times with buffer without probes. Then, 50 μL of buffer was added to cover the cells and place them at 37 °C for 20 min before using the PHERAstar FSX to detect the cells. Cells were excited at 557 nm to measure the fluorescence level, and the emission was recorded at 581 nm.

2.5. Gβ2/4 Mutation Plasmid Constructs

To introduce single or multiple point mutations into Gβ2/4, a pair of primers must first be designed. These primers are used in PCR amplification to replace the base at the mutation site of the wild-type gene. The primers should be centered around the site of mutation and include complementary sequences on both sides, with a total length of about 35 bp. During the amplification process of the mutant plasmid, a high-fidelity enzyme (Vazyme Bio, China) was used for PCR amplification. After the PCR product was obtained, it should be immediately placed on ice. Then, 1 μL of DpnI (Takara Bio, Japan) was added to the PCR reaction mixture, mixed well, and incubated at 37 °C for 1–2 h. DpnI recognizes methylated sites and cleaves them. Since the original plasmid template extracted from DH5α Escherichia coli (Sangon Biotech, Shanghai, China) is generally methylated, DpnI can digest the original wild-type plasmid template while leaving the amplified mutant plasmid intact. Finally, after digestion, the product was transformed into DH5α E. coli. By plating and selecting single colonies, followed by shaking the culture and sequencing, the plasmid containing the mutant gene was obtained.

2.6. AlphaFold3 Modeling Analysis

First, the sequence files of Gαq, Gβ2, Gβ4, and Gγ2 proteins were retrieved from the National Center for Biotechnology Information (NCBI) database. These sequences were then imported into AlphaFold3 to model GαqGβ2γ2 and GαqGβ4γ2 complexes, and the resulting PDB files were analyzed using PyMOL to identify differential amino acids at the binding interfaces between Gαq and Gβ, as well as between Gβ and Gγ2. The sequence of the Gβ2 mutant was imported into AlphaFold3 to model its trimeric complex with Gαq and Gγ2. The resulting PDB file was analyzed using the MOE to calculate the RMSD values of the Gβ2 mutant against wild-type Gβ2 and Gβ4. The results were further analyzed by using GraphPad Prism 9.0.

2.7. Western Blot

Wild-type HEK-293 cells and cells stably transfected with the [M1-Rluc8]-Gαq-Gβ-[mVenus-Gγ] system were cultured in DMEM supplemented with 10% FBS for 24 h. The cells were scraped off, and total protein lysates were prepared using RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate) containing protease inhibitors. Proteins (20 μg/lane) were resolved on 10% SDS-PAGE gels and transferred to PVDF membranes. Membranes were probed with the following primary antibodies: anti-Gαq (1:1000, Cat No.13927–1-AP, Proteintech), anti-Gβ4 (1:2000, Cat No.13780–1-AP, Proteintech), anti-Gβ2 (1:1000, Cat No.11693–1-AP, Proteintech), and anti-β-actin (1:10000, Cat No.66009–1-Ig, Proteintech) as a loading control. Horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000, Cell Signaling Technology) were used for chemiluminescence detection (Amersham ImageQuant 800, GE Healthcare). Western blot band intensities were quantified using ImageJ software (NIH) and normalized to β-actin. Statistical significance was assessed using two-way ANOVA (GraphPad Prism 9.0), where P < 0.05 is considered significant.

2.8. Fluorescence Quantification of mVenus-Tagged Gγ2

HEK293 cells transfected with the [M1-Rluc8]-Gαq-Gβ-[mVenus-Gγ] system and wild-type control cells were cultured for 24 h. The fluorescence intensity of the mVenus-tagged Gγ2 subunit was measured using a Pherastar FSX microplate reader (BMG LABTECH, Offenburg, Germany) with excitation and emission wavelengths set to 515 and 528 nm, respectively. Statistical analysis was performed using GraphPad Prism 9.0.

2.9. Data Analysis

All concentration–response curves were analyzed by using a three-parameter logistic model in GraphPad Prism 9.0. BRET dose–response data were evaluated via two strategies: (1) normalization to a reference agonist or (2) baseline-corrected net BRET (calculated as the Emax-fit minus baseline-fit values). EC₅₀ and Emax parameters were determined by simultaneously fitting data across biological replicates. BRET ratios were derived from wavelength-corrected measurements using the formula (mVenus emission at 535 nm, 30 nm bandwidth)/(Rluc8 emission at 475 nm, 30 nm bandwidth) – (RLuc8 emission at 535 nm/RLuc8 emission at 475 nm). Experiments followed randomized designs. Results are presented as mean ± SEM and analyzed by one-way ANOVA (statistical significance threshold: p < 0.05).

Results

3.1. M1 mAChR Prefers Gβ1/2γ2 Subtypes When Recruiting the Gαq Heterotrimer

All muscarinic acetylcholine receptor subtypes can be activated by orthosteric agonists, including the endogenous ACh and exogenous pilocarpine (Pilo) and BaoGongTeng A (BGT-A) (Figure A). Upon M1 mAChR activation, it recruits a Gαq heterotrimer, reducing the distance between M1-Rluc8 and mVenus-Gγ and allowing resonance energy transfer and a detectable change in the signal (Figure B). By using combinations with Gβ1/2/3/4 and Gγ2/9, we established the [M1-Rluc8]-Gαq-Gβ-[mVenus-Gγ] system to evaluate the efficiency of Gβγ subtypes when Gαq heterotrimer coupling to M1 mAChR.

1.

Screening of Gαq binding to M1 mAChR systems. (A) 2D chemical structures of the three agonists; (B) BRET1 detection diagram; M1 mAChR demonstrates preferential coupling to specific Gβ and Gγ subunits upon stimulation with (C) ACh, (D) Pilo, and (E) BGT-A, as measured by the BRET1 assay (n = 3, mean ± SEM); quantitative assessment of pEC₅₀ values via the BRET assay for (H) Gγ and (I) Gβ subunits using three distinct agonists (n = 3, mean ± SEM); (J) schematic diagram of calcium flow detection downstream of Gαq; M1 mAChR exhibits preferential coupling to specific Gβ and Gγ subunits in response to stimulation by (K) ACh, (L) Pilo, and (M) BGT-A, as determined by Ca²⁺ mobilization (n = 3, mean ± SEM); comprehensive evaluation of ΔCa²⁺ mobilization by (N) Gγ subunits and (O) Gβ subunits through three agonists (n = 3, mean ± SEM); quantitative assessment of pEC₅₀ values via Ca²⁺ mobilization analysis for (P) Gγ and (Q) Gβ subunits using three distinct agonists (n = 3, mean ± SEM).

Moreover, we validated the expression levels of the key components in our system. Western blot analysis confirmed substantially lower endogenous Gαq and Gβ levels in wild-type HEK-293 cells compared to transfected groups expressing the [M1-Rluc8]-Gαq-Gβ-[mVenus-Gγ] complex. Despite 90% sequence homology between Gβ2/Gβ4 limiting antibody specificity, standardized immunoblot quantification demonstrated equivalent subtype expression (Supplementary Figure 1A, B). Transfected cells exhibited consistent Gαq/Gβ expression and comparable mVenus-tagged Gγ2 levels (Supplementary Figure 1C, D), validating our system’s balanced Gαβγ protein expression and elimination of subunit-specific expression artifacts.

We observed that Gγ9 showed less sensitivity compared to Gγ2 and Gβ1/2 were significantly higher response than Gβ3/4 (Figure C–E). Significantly reduced agonist potency was observed for Gγ9 compared to Gγ2 in agonist-induced activation (pEC₅₀: 3.5 vs 6.0 (ACh); 2.4 vs 5.5 (Pilo); Figure H). Concurrently, diminished ΔBRET ratios were recorded (ΔBRET ratio: 0.1 vs 0.007 (BGT-A); Figure F), indicating inherently weaker coupling efficiency. Among the Gβ subunits, Gβ1 and Gβ2 exhibited comparable response values, whereas Gβ3 demonstrated reduced magnitudes (50–54% of Gβ1/2 levels), with Gβ4 showing the lowest responses (36–39% relative to Gβ1/2) under ACh activation (Figure G). Regarding agonist potency, the Pilo group exhibited Gβ1/2 superiority over Gβ3/4 (pEC₅₀: 5.4 vs 4.0), whereas other ligand pairs showed comparable ratios (Figure I). Experimental results demonstrated that the Gβ1/2γ2 subunit exhibited higher sensitivity when Gαq was coupled to the M1 mAChR.

We measured changes in Ca²⁺ concentration using a calcium flow assay after activating Gαq with agonists (Figure J). The result corroborated BRET observations, revealing that M1 mAChR exhibited marked preference for Gγ2 over Gγ9 and Gβ1/2 over Gβ3/4 (Figure K–M). Statistical evaluation of ΔCa²⁺ mobilization and pEC₅0 values substantiated the Gβ1/2γ2 heterotrimer demonstrating maximal coupling efficiency among the tested combinations (Figure P, Q). The BRET kinetic analyses (Supplementary Figure 1E–H) revealed that Gβ1/2γ2 exhibited both rapid-onset and sustained BRET responses under stimulation with 1 μM ACh or 10 μM Pilo, achieving signal stabilization at 420 s. This time-dependent enhancement pattern further corroborates the preferential engagement of Gβ1/2γ2-containing heterotrimers during receptor activation.

In summary, we concluded that Gαq coupling to M1 mAChR exhibits preferences for Gβ1/2γ2 subtypes and that this preference is independent by agonists.

3.2. Gαq-Coupled mAChRs Exhibit Conserved Gβγ Subtype Selectivity

We extended our investigation to M3 mAChR and M5 mAChR, both coupled to Gαq. By examining receptor binding to Gαq and downstream calcium signaling under ACh and Pilo stimulation (Figure A–D; I–L), the results confirmed that the Gβγ preference for Gβ1/2γ2 was consistent with M1 mAChR findings.

2.

Screening of Gαq binding mAChR systems. (A) M3 mAChR and (C) M5 mAChR demonstrate preferential coupling to specific Gβ and Gγ subunits upon stimulation with ACh as measured by the BRET1 assay (n = 3, mean ± SEM); (B) M3 mAChR and (D) M5 mAChR exhibit preferential coupling to specific Gβ and Gγ subunits in response to stimulation by ACh as determined by Ca²⁺ mobilization (n = 3, mean ± SEM); quantitative assessment of the ΔBRET ratio and pEC₅₀ values via the BRET assay for (E) Gγ subunits and (F) Gβ subunits in mAChRs (ACh) (n = 3, mean ± SEM); quantitative assessment of ΔCa²⁺ mobilization and pEC₅₀ via Ca²⁺ mobilization analysis for (G) Gγ subunits and (H) Gβ subunits in mAChRs (ACh) (n = 3, mean ± SEM).

This confirms that Gαq-coupled muscarinic acetylcholine receptors share a specific subunit preference, a property unaffected by changes in the agonists. Notably, although the three Gαq-coupled receptor subtypes share identical Gβγ preference spectra, their signaling outputs diverge quantitatively. M1 mAChR and M3 mAChR showed similar responses under ACh or Pilo stimulation. Notably, M5 mAChR demonstrated significantly enhanced signaling outputs, exhibiting approximately 2-fold greater maximal response in Gβ1/2γ2 coupling efficiency compared to M1 mAChR and M3 mAChR subtypes under ACh activation (ΔBRET ratio: Gβ1γ2 = 0.12 vs 0.14 vs 0.28; Gβ2γ2 = 0.11 vs 0.13 vs 0.2; pEC₅₀: Gβ1/2γ2 = 6.0 vs 5.3 vs 6.9; Figures G, E, F). This signal magnification effect was transmitted through the Gαq signaling pathway and quantifiable via downstream calcium-fluid-derived signaling (ΔCa²⁺ mobilization: Gβ1γ2 = 0.9 vs 0.9 vs 1.5; Gβ2γ2 = 0.85 vs 0.75 vs 1.5; pEC₅₀: Gβ1/2γ2 = 6.8 vs 6.5 vs 6.8; Figures O, G, H). Similar results can be obtained with Pilo stimulation (Supplementary Figure 2A–H).

3.3. GPCRs Coupled with Gαq Have a Preference for Gβ1/2γ2

We selected four Gαq-coupled GPCRs to investigate whether the Gβ1/2γ2 selectivity is shared among other Gαq-coupled GPCRs: AT1R, NTSR1, KISS1R, and the engineered hM3Dq receptor (Figure A). The hM3Dq receptor, derived from human M3 mAChR, is specifically activated by deschloroclozapine (DCZ) and exclusively signals through the Gαq pathway. Then, we established a BRET system for AT1R and NTSR1 to directly analyze their coupling efficiency to Gαq proteins (Figure B, C). The results revealed that NTSR1 showed optimal receptor preference for heterotrimeric G protein coupling to Gβ1/2γ2 (ΔBRET ratio: Gβ1γ2 vs Gβ2γ2 vs Gβ4γ2 = 0.067 vs 0.064 vs 0.035; Figure E). Notably, the ΔBRET ratios for the Gγ9 subtype were barely zero (Figure D), and AT1R also exhibited a similar trend.

3.

Screening of Gαq binding other GPCR systems. (A) Schematic diagram of GPCRs and their ligands for four nonmuscarinic receptors; (B) AT1R and (C) NTSR1 demonstrate preferential coupling to specific Gγ and Gβ subunits upon stimulation with ligands as measured by the BRET1 assay (n = 3, mean ± SEM); quantitative assessment of the ΔBRET ratio by (D) Gγ subunits and (E) Gβ subunits in AT1R and NTSR1 (n = 3, mean ± SEM); quantitative assessment of ΔCa²⁺ mobilization by (F) Gγ subunits and (G) Gβ subunits in four GPCRs (n = 3, mean ± SEM).

The results of downstream calcium concentration changes also confirmed that Gβ1/2γ2 was the best Gβγ subtype in NTSR1, AT1R, KISS1R, and hM3Dq (Supplementary Figure 3A–D) and the ΔCa²⁺ mobilization of NTSR1 (Gβ2γ2 vs Gβ2γ9 vs Gβ4γ2 is 0.64 vs 0.23 vs 0.45; Figure F, G). A summary of the pEC₅₀ values from the above experiments revealed that Gβ1/2γ2 preference was consistently maintained at the best agonistic potency in every receptor (Supplementary Figure 3E–H). In summary, we found that in addition to the muscarinic receptor family, other Gαq-coupled GPCRs also exhibit the highest coupling efficiency with Gβ1/2γ2. This finding highlights the selective interaction between Gα subtypes and Gβγ subtypes, providing insights into the complexity and diversity of GPCR downstream signaling.

3.4. Group III Residues in Gβ Influenced the Coupling Efficiency of Gαq to M1 mAChR

The four Gβ subtype proteins share similar structures, conformations, and amino acid lengths, complicating investigations into the Gβ1/2γ2 subtype selectivity mechanisms. Our prior findings showing distinct coupling efficiencies between Gβ2 and Gβ4 in Gαq-coupled GPCRs prompted us to focus on these subtypes. We hypothesize that residue differences between Gβ4 and Gβ2 at the Gαq-Gβγ interface may affect the binding efficiency, leading to reduced coupling by Gβ4. Using AlphaFold3, we modeled the GαqGβ2γ2 and GαqGβ4γ2 complexes, identifying one distinct residue at the Gαq-Gβ interface and six divergent residues at the Gβ-Gγ2 interface (Figure A). Then, we mutated all divergent residues in Gβ4 to their Gβ2 counterparts and performed BRET assays to quantify M1 mAChR coupling. However, the Gβ4 mutant showed no improved coupling efficiency (ΔBRET ratio: Gβ4 vs mutant = 0.016 vs 0.017; Figure B), suggesting that these residues at the interface exhibit limited impact on Gβ specificity.

4.

Identification of difference residues modulating Gβ2/Gβ4 coupling efficiency with Gαq. (A) Modeling and analysis of divergent amino acids at Gαq-Gβ2/4-Gγ2 interfaces; (B) BRET1-based functional analysis of β4 chimeric mutants (interaction interface residues) (n = 3, mean ± SEM); (C) schematic grouping diagram based on amino acid sequence similarities and differences among three Gβ subtypes: group I: residues identical across all three Gβ subunits (Gβ1, Gβ2, Gβ4); group II: 12 residues conserved in Gβ1/Gβ2 but divergent in Gβ4; group III: 14 residues conserved in Gβ1/Gβ4 but divergent in Gβ2; group IV: 11 residues conserved in Gβ2/Gβ4 but divergent in Gβ1; group V: 8 residues unique to each Gβ subunit (no cross-subtype conservation); (D) mutation sites of β4 mutants; (E–H) BRET1-based functional analysis of β4 chimeric mutants (n = 3, mean ± SEM).

We hypothesized that noninterface residues induce conformational changes governing Gβγ selectivity, so we compared the full-length amino acid sequences of Gβ1, Gβ2, and Gβ4 (Supplementary Figure 4A). Residues across Gβ1/2/4 were classified into five groups according to identity: group I: residues identical across all three Gβ subunits (Gβ1, Gβ2, Gβ4); group II:12 residues conserved in Gβ1/Gβ2 but divergent in Gβ4; group III: 14 residues conserved in Gβ1/Gβ4 but divergent in Gβ2; group IV: 11 residues conserved in Gβ2/Gβ4 but divergent in Gβ1; group V: 8 residues unique to each Gβ subunit (no cross-subtype conservation) (Figure C). A three-step sequential mutation experiment was based on the above categorization, replacing Gβ4 amino acids with their Gβ2 counterparts. Details of the mutation sites and contents are shown in Figure D.

We examined whether the group II residues influenced the coupling efficiency. Site-directed substitution of these residues showed no significant changes in M1 mAChR binding (Figure E). Then, we mutated the residues of group V in Gβ4, which were based on the mutant from the first step. However, the results showed no significant changes (Figure F). While neither of the first two mutation experiments improved Gβ4 coupling efficiency, they helped narrow the scope of our investigation and focus on group III. Then, we mutated two clusters (T178, T179, and T181 and G216, T223, and A242) of closely positioned amino acids in group III on the second-step mutants. The results showed that each mutation group modestly improved Gβ4 coupling efficiency (Figure G), The β4 (II&V&III-A) variant exhibited a 2-fold enhancement compared to Gβ4 (ΔBRET ratio: Gβ4 vs mutant = 0.021 vs 0.042). Moreover, the β4 (II&V&III–B) mutant demonstrated only smaller improvements (ΔBRET ratio: Gβ4 vs mutant = 0.021 vs 0.031). However, combining the two mutations yielded no improvement (Figure H). These findings suggest that single-point mutations on each of the residues of group III may be necessary for further investigation.

3.5. Four Residues in Group III Influenced Gβ2 Coupling Efficiency

We performed targeted mutagenesis of the Gβ2 residues in group III, replacing these sites with their Gβ4 counterparts (Figure A). Under ACh stimulation of the M1 mAChR, BRET assays revealed altered response values at sites F111, S125, V178, and G179 compared to wild-type Gβ2 (Figure B, C; Supplementary Figure 5A, B). Compared to wild-type β2, the β2­(V178T), β2­(F111Y), and β2­(S125N) variants exhibited attenuated responses (25.4%, 22.4%, and 27.0%, respectively), with the β2­(G179T) mutant showing the most significant change (39.1% decrease relative to wild-type β2). Furthermore, Ca²⁺ mobilization assays downstream of M1 mAChR validated that site-specific mutations in Gβ2 impair Gαq-mediated signaling transduction (Figure J, K). These findings identified that the residues of F111, S125, V178, and G179 influenced Gβ2 coupling efficiency. Under Pilo activation, the β2­(G179T) mutant showed 29.0% reduction in coupling efficiency, consistent with ACh responses (Figure D, E). These results demonstrated that the core site group, key to determining Gβ2 subunit selectivity, is not influenced by agonists.

5.

Identification of critical amino acid residues modulating Gβ2 coupling efficiency through site-directed mutagenesis. (A) The detailed amino acid information on group III Gβ2 mutation sites; BRET1-based functional analysis of β2 mutants generated by site-directed mutagenesis under stimulation with (B) ACh or (D) Pilo (n = 3, mean ± SEM); comprehensive evaluation of relative ΔBRET ratios in β2 mutants generated by site-directed mutagenesis under stimulation with (C) ACh or (E) Pilo, ΔBRET ratios normalized to Gβ2 (baseline = 1.0); Gβ4 and Gβ2 mutants analyzed against this reference (n = 3, mean ± SEM); BRET1-based functional analysis of β2 mutants (G179 residue) under stimulation with (F) ACh or (H) Pilo (n = 3, mean ± SEM); comprehensive evaluation of relative ΔBRET ratio in β4 mutants (G179 residue) under stimulation with (G) ACh or (I) Pilo (n = 3, mean ± SEM); (J) detection of Ca²⁺ mobilization of M1 mAChR in β2 mutants generated by site-directed mutagenesis in response to ACh stimulation (n = 3, mean ± SEM); (K) quantitative assessment of ΔCa²⁺ mobilization via the Ca²⁺ mobilization assay for β2 mutants generated by site-directed mutagenesis in M1 mAChR (ACh) (n = 3, mean ± SEM).

We hypothesized that the β2­(G179T) mutation due to the larger threonine side chain increases steric hindrance, likely impairing Gαq binding and altering protein conformation. Therefore, we engineered site-directed mutations at position 179 of Gβ2, substituting glycine with lysine, tryptophan, and threonine, which have larger molecular weights. The results demonstrated that mutations at position 179 significantly reduced the response values in both ACh and Pilo assays. Specifically, the β2­(G179W) mutant (introducing tryptophan, the largest amino acid) exhibited reductions of 36.8% and 36.6% in coupling efficiency for ACh and Pilo groups, respectively (Figure F–I), providing conclusive evidence that residue G179 critically governs Gβ2 subunit selectivity.

We conducted another mutation experiment, performing single mutations of residues in group III, replacing Gβ4 with their Gβ2 counterparts. However, the results showed no improvement in the low coupling efficiency of Gβ4 (Supplementary Figure 5C–F). These findings confirmed that the effect of single mutations in group III is specific to Gβ2 and insufficient for Gβ4, because we found that targeted mutations of group III amino acids (T178 V, T179G) in β4­(II&V) improved a little coupling efficiency compared to Gβ4 (ΔBRET ratio: Gβ4 vs β4­(II&V) T178 V vs β4­(II&V) T179G = 0.028 vs 0.037 vs 0.042; Supplementary Figure 5G, H).

3.6. Residues F111, V178, and G179 Serve as Critical Determinants of Gβ2 Coupling Efficiency

We hypothesized that double mutations of the residues (F111, S125, V178, and G179) can decrease Gβ2 activity and further impaired coupling efficiency. Using Gβ2 as a template, we performed double mutations, and the results showed that F111Y-G179T and V178T-G179T mutants showed a greater reduction in response values compared to the G179 single mutation. Under the stimulation of ACh, the levels of β2­(F111Y&G179T) and β2­(V178T&G179T) mutants were further decreased (28.2–40.4% of β2­(G179T) levels;). With Pilo, the decreases were 0.4% and 20.3%, respectively (Figure A–D). These results suggest that F111 and V178 are also critical for Gβ2 selectivity and further confirm that G179 is a key determinant of Gβ2 selectivity.

6.

Identification of critical amino acid residues modulating the Gβ2 coupling efficiency through double site mutagenesis. BRET1-based functional analysis of β2 mutants generated by double-site mutagenesis under stimulation with (A) ACh or (C) Pilo (n = 3, mean ± SEM); comprehensive evaluation of relative ΔBRET ratio in β2 mutants generated by double-site mutagenesis under stimulation with (B) ACh or (D) Pilo (n = 3, mean ± SEM).

To validate the universal effect of residues F111, V178, and G179 in attenuating Gβ2 coupling efficiency across Gαq-coupled GPCRs, we conducted BRET assays in M3 mAChR, M5 mAChR, AT1R, and NTSR1 systems (Supplementary Figures 7 A–D). These experiments demonstrated that mutations at these positions significantly reduced receptor coupling efficiency (Supplementary Figure 7E). Subsequent calcium mobilization analyses in M1 mAChR and NTSR1 further confirmed that Gβ2 mutations impair Gαq-mediated signal transduction (Supplementary Figure 7F–H). Collectively, these findings establish F111, V178, and G179 as evolutionarily conserved residues critical for the Gβ2 functionality in diverse Gαq-coupled GPCR signaling pathways.

However, the double mutations of these sites on Gβ4 showed no increase in response values (Supplementary Figure 6A–D), demonstrating that group III mutations are insufficient to improve Gβ4 coupling efficiency.

4. Discussion

Our study adds significant evidence that Gβγ subtypes contribute to receptor preference for heterotrimeric G protein coupling in Gαq-coupled GPCRs, expanding the current understanding of signaling bias. , Through a comparative analysis of seven Gαq-coupled receptors, we observed preferential engagement of Gβ1/2γ2 subtypes across receptor families. However, the concrete coupling efficiency varied across receptors. The mutation data highlight conserved residues in Gβ2 (F111, V178, and G179) as determinants of Gβ2 coupling efficiency, but the molecular mechanisms that determine the preference of Gβ1/2γ2 are not completely elucidated. In particular, we found that pilocarpine is a class of Gβ4-preferring ligands compared to acetylcholine, enhancing our understanding and application of preferred ligands.

Our study uncovered conserved Gβ1/2γ2 subtype preference across Gαq coupled GPCRs. It is also consistent with the currently reported M1 mAChR-Gβγ subtype , and provides rationalization for the reason for the Gβ1/2γ2 subunit being chosen in most of the present GPCR structural analysis. However, quantitative analysis revealed that M5 mAChR displays higher Gβ1/2γ2 coupling efficiency compared to M1 mAChR and M3 mAChR subtypes under equivalent expression conditions and persisted through downstream Gαq-mediated signaling outputs. We suspect that evolutionary conservation of sequence homology within the mAChR family (79% transmembrane domain identity) underlies their shared Gβγ subtype preference, while receptor-specific conformational variations (particularly in intracellular loop 3) account for coupling efficiency differences. Specifically, we observed that NTSR1 and AT1R display negligible coupling efficiency with Gγ9-containing combinations across all tested Gβ subunits, where AT1R showed only half of the coupling efficiency of NTSR1. These distinct coupling profiles likely arise from activation-induced conformational changes unique to each receptor structure. ,

Research has revealed specific Gβγ selectivity among other Gα. The β1-adrenergic receptors (β1ARs) in cardiomyocytes predominantly couple with Gαsβ1γ1 heterotrimers to enhance myocardial contractility and heart rate. Conversely, dopamine D1 receptors (D1Rs) in the striatum specifically utilize the Gαolfβ2γ7 combination, which regulates motor control and reward-related behaviors. These findings demonstrate that the selective recruitment of specific Gβγ subunit combinations can modulate G protein activity, thereby impacting the efficiency of signal transduction downstream of GPCR activation.

The residues of F111, V178, and G179 play critical roles in determining Gβ2 coupling efficiency in this study. We employed AlphaFold3 to model the heterotrimer of Gβ2 mutants bound to Gαq and Gγ2 (Supplementary Figure 8A). However, the comparison of RMSD values between the Gβ2 mutant, Gβ2, and Gβ4 revealed values below 1 (Supplementary Figure 8B, C), indicating minor structural differences and high similarity among the three proteins. This suggests that these three residues have no large conformational changes.

Using the Genome Aggregation Database (gnomAD) and ClinVar, we identified pathogenic missense mutations (F111, V178, and G179) clustered within the WD40 domain of Gβ2a region critical for Gαq interaction. Although clinical annotations for these variants remain ambiguous and no studies have focused on these residues, functional studies demonstrate that single-residue substitutions reduce Gβ2 coupling efficiency by 22.4–39.1%, directly impairing GPCR signal transduction efficacy. Notably, these findings resemble the reported Gβ2-L77R mutation associated with the global developmental delay. Furthermore, the K78R mutation in Gβ1 has been shown to impair G protein coupling efficiency, leading to developmental delay and generalized seizures. These findings indicate that single-site amino acid substitutions in Gβ2 impair its structural and functional integrity, which consequently disrupt GPCR signaling pathways, potentially through compromised interactions with Gαq or receptor interfaces.

Through analysis of M1 mAChR with three distinct ligands (ACh, Pilo, and BGT- A), we observed that although Gβ1/2γ2 was the preferred subtype, Pilo showed substantially reduced coupling efficiency compared to ACh and BGT-A. This indicates that while Gβγ subtype preference remains consistent, the efficacy of subtype engagement varies between ligands. Like TRV130 (Oliceridine), an FDA-approved postoperative analgesic developed for the μ-opioid receptor (MOR), it potently biases the agonist Gαi/o pathway. This is also consistent with the theory that structural changes in ligands can modulate the downstream effectors of GPCRs. ,

Moreover, published studies on Gαq signaling consistently report high Gαq activation efficacy for ACh (EC₅0 ∼10–30 nM) and moderate efficacy for Pilo (EC₅0 ∼1–3 μM) in systems with physiological receptor densities. In our BRET-based Gq recruitment assays, the observed pEC₅0 values (ACh: 6.0 ± 0.3; Pilo: 5.5 ± 0.4) indeed appear to be reduced relative to these benchmarks. This discrepancy likely arises from fundamental differences between direct Gαq coupling detection (BRET) and downstream calcium flux readouts. BRET measures proximal receptor-Gαq interactions, requiring near-complete receptor occupancy to achieve maximal energy transfer. In contrast, calcium signaling is amplified through PLC-IP₃ cascades, where even partial Gαq activation can trigger sufficient IP₃ production to saturate endoplasmic reticulum calcium release. This hierarchical amplification explains why calcium assays report higher apparent ligand potency. Our BRET data thus reflect a more stringent measure of ligand-receptor-Gαq ternary complex formation, while calcium flux captures system-level amplification inherent to native signaling contexts.

Interestingly, we found that when we normalized the Emax value of Pilo to ACh, Gβ4 was nearly twice that of Gβ2, indicating a significant difference between the two subtypes (Supplementary Figure 9A). These findings suggest that although both agonists prefer Gβ2 over Gβ4, Pilo shows a stronger preference for Gβ4 compared to ACh. Furthermore, Gβ2 mutations increase pilocarpine preference for Gβ2 (Supplementary Figure 9B). This result enhances our understanding of the biased ligand and demonstrates that it not only exists between G proteins and β-arrestins but also depends on Gβ subtypes. Current research on biased ligands targeting specific Gβγ subtypes remains exploratory, yet several compounds modulating Gβγ pathways have been identified. Notably, M119 demonstrates the highest affinity for Gβ1γ2 and selectively disrupts Gαi1-Gβγ interactions. Concurrently, gallein suppresses inflammatory responses and tumor metastasis by inhibiting Gβγ-PLCβ binding.

Our findings show that Gβ1/2γ2 is the best subtype of Gαq-GPCR and the mutations in specific Gβ2 residues significantly affect its coupling efficiency but still have some limitations. While critical residues (F111, V178, and G179) in Gβ2 for Gαq coupling were identified, their structural dynamics during activation await atomic-resolution analysis (e.g., cryo-EM or simulations). This study failed to exclude potential interference from endogenous Gβ subunits, despite their low expression in HEK-293 cells. Future studies should generate all Gβ subtype knockout cell lines and employ Gβ subtype-specific knockout animal models to validate the systemic impact of Gβ1/2γ2 subspecies on physiology and pathology.

5. Conclusions

This study establishes Gβ1/2γ2 as the universal Gβγ subtype preference for Gαq-coupled GPCRs, and we identified a triresidue (F111-V178-G179) governing Gαq-Gβ2γ2 coupling efficiency, where combinatorial substitutions produce lower Gβ2 coupling efficiency. Moreover, that pilocarpine prefers Gβ4 compared to acetylcholine is discovered. These findings offer a strong experimental basis for further research on “signaling bias” and will significantly advance the development of targeted drugs.

Glossary

ABBREVIATIONS

GPCRs

G protein-coupled receptors

mAChRs

muscarinic acetylcholine receptors

M1 mAChR

M1 muscarinic acetylcholine receptor

PLC-β

phospholipase C-β

IP₃

inositol trisphosphate

DAG

diacylglycerol

NTSR1

neurotensin receptor

AT1R

angiotensin II type 1 receptor

ACh

acetylcholine

Pilo

pilocarpine

BGT-A

BaoGongTeng A

DCZ

deschloroclozapine

β1ARs

β1-adrenergic receptors

D1Rs

dopamine D1 receptors

MOR

μ-opioid receptor

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.5c00167.

• Relative expression levels and BRET kinetic analysis of Gαqβγ proteins in the M1 mAChR-Gαq coupling system; BRET analysis and calcium mobilization of M3 mAChR and M5 mAChR in response to pilo stimulation; calcium mobilization and pEC₅₀ statistics of AT1R, NTSR1, KISS1R, and hM3Dq; full-length amino acid sequences of Gβ1, Gβ2, and Gβ4; detection of critical amino acid residues modulating Gβ2/4 coupling efficiency through site-directed mutagenesis; BRET analysis and calcium mobilization of M1/3/5 mAChR, AT1R, and NTSR1 in Gβ2 F111/V178/G179 mutants; heterotrimer structure analysis of Gβ2 mutants binding Gαq and Gγ2; comprehensive evaluation of the normalized Emax ratio (Pilo/ACh) in Gβ2 mutants (PDF)

‡.

Y.Y. and D.W. contributed equally.

The authors declare no competing financial interest.