Dynamic monomer-dimer transition in ligand-induced apelin receptor activation

Domain: biology
Published: 2026-04-03
Editorial outcome: major_revision

Abstract
G-protein-coupled receptors (GPCRs) are significant signal transducers that exist as monomers and in multiple oligomeric forms. However, molecular mechanism driving their dynamic interconversion to regulate intricate signaling in class A GPCRs remains elusive, compounding our understanding of their related pathophysiological functions. Here, we present a set of 12 assemblies of the apelin receptor (APLNR), including dimeric apo state, agonistic small molecule- or nanobody-bound state of monomeric and dimeric APLNR with and without G-proteins, providing a detailed dynamic view of the monomer-dimer transition. High-resolution cryo-EM structures reveal that different ligands induce varying degrees of pre-dissociation of dimers in the absence of G-protein, with G-protein coupling facilitating the transition from dimeric to monomeric receptor. These insights enhance our understanding of the dynamic regulation of class A GPCRs between monomeric and dimeric forms and advance the rational drug design strategies aimed at selectively modulating of APLNR signaling.

Introduction
While Class C GPCRs are obligate dimers for their function, Class A GPCRs may exist as monomers or oligomers1-6, exhibiting distinct agonist affinity, efficacy, as well as the signaling profiles and pharmacological functions7-10. However, it remains unclear whether these forms mediate signaling transduction in an interconvertible manner or independently (Fig. 1a), compounding our understanding of complex mechanism of downstream signaling. Further studies on the molecular basis and functional implications of monomer–dimer interconversion in class A GPCRs are essential for achieving precise modulation of downstream signaling and understanding their pathophysiologic roles.

APLNR is a model system that may be suitable for investigating the dynamic transition between monomeric and dimeric forms of class A GPCR, supported by both transfected cell studies and cryo-EM structures4,11,12. However, these structures are inadequate for fully capturing the dynamics of the dimer-monomer transition, given their limited resolution and insufficient determination of multiple conformations. Moreover, the relationship between monomeric and dimeric forms and pathophysiologic functions has not been elucidated yet. APLNR is regard as a promising target due to its critical role in maintaining cardiovascular health and preventing muscle weakness13-18. Nonetheless, apelin-induced β-arrestin-dependent signaling contributes to detrimental cardiac hypertrophy, driving interests in the development of G-protein-biased APLNR agonists as potential heart failure therapies19-22. It is suggested that APLNR dimers exhibit distinct pathophysiological functions compared to monomers, indicating differences in the selective signaling pathways associated with various forms23-26. Therefore, it is crucial to elucidate how diverse ligands recognition induce the formation of specific monomeric or dimeric states, ultimately impacting signaling and therapeutic effects. AMG986, also named as BGE-105 and Azelaprag, is the most potent small molecule agonist in activating both G-protein and β-arrestin signaling of APLNR, advancing rapidly in clinical trials for anti-obesity treatment27-29. However, a Phase 2 clinical trial evaluating AMG986 was recently discontinued owing to potential off-target liver toxicity. In parallel, JN241-9 stands as the pioneering single-domain antibody agonist targeting class A GPCR, derived from JN24130. The greater size of nanobody agonist contributes to the alignment of particles in cryo-EM data processing, overcoming micelle interference, thereby facilitating the capture of monomeric APLNR in the absence of G-protein. We have selected these two agonists to conduct a comprehensive study on the activation mechanisms and functions of dimers and monomers.

In this study, we resolved 12 cryo-EM density maps of wild type full-length APLNR, encompassing monomeric and dimeric forms of the apo state, agonists-bound state in the presence or absence of G-proteins, 9 of which achieve high resolution between 2.4 and 3.0 Å and enable the accurate structure modelling (Supplementary Tables 1 and 2). These results reveal multiple conformational states of APLNR dimers and monomers, in both apo and active forms, which provide potential pathways for transitions between these states. Furthermore, our preliminary functional studies suggest a possible role of cholesterol in stabilizing the APLNR dimers, as well as how different ligands modulate the dimer-to-monomer transition, providing insights into drug development specifically targeting class A GPCRs between monomers and dimers.

Results
Signaling and structural determination of APLNR dimers and monomers during activation process

Previous structural studies of the APLNR have indicated that the π-π interactions network formed by F1013.24 (where the superscript refers to the Ballesteros-Weinstein transmembrane numbering system31) plays a central role in stabilizing the dimeric interface11. To investigate the differences in downstream signaling mediated by monomeric and dimeric forms, we first assessed the signaling profile of wild type APLNR and the dimer-reduced form (FC1013.24A) using AMG986 and JN241-9, by conducting a NanoBiT assay and a GloSensor cAMP assay to detect G-protein activity and the bioluminescence resonance energy transfer (BRET) assay to detect β-arrestin recruitment. AMG986 showed roughly 10-fold greater potency in activating various G-proteins, including Gi, Gq and G13, as well as β-arrestin, compared to JN241-9 (Supplementary Figs. 1a-g). Additionally, AMG986 induced a more rapid activation rate than JN241-9 (Supplementary Fig. 1d). Though negligible differences in EC50 or Emax from G-protein signaling, the dimer-reduced form (F1013.24A) showed a higher Emax than the dimeric form in the β-arrestin signaling (Supplementary Fig. 1f), consistent with previous results11, which may be indicative that the dimer exhibits G-protein bias compared to the monomer. No detectable change in either Hill-slope or pEC50 is evident for either AMG986 or JN241-9 between dimers (WT) and the dimer-reduced form (F1013.24A), suggesting that ligand binding allostery, if present, is not detectable. Moreover, cAMP accumulation results showed that the dimer-reduced form appear to exhibit ~25% higher constitutive activity than that of dimeric form (Supplementary Figs. 1d-e, p < 0.001), prompting us to investigate the regulatory mechanism of dimerization induced by different potent ligands.

Determination of cryo-EM structures for class A GPCRs without G-protein is impeded by their small dimensions and inherent instability. Moreover, the crystallization process is rendered challenging when the structures exhibit flexibility, particularly when intermediate-state GPCRs manifest a diversity of conformations. Typically, receptors are often fused with proteins like BRIL to increase stability, which may not accurately represent their native conditions32,33. Here, we successfully achieved high-resolution cryo-EM structures of agonists-bound class A GPCR in various conformations without G-protein, providing potential insights into the dynamic process of activation. Specifically, dimeric receptors with increased size contributes significantly to achieve high-resolution structures in the absence of G-protein (Fig. 1b and Supplementary Figs. 1h-k). Furthermore, to overcome the difficulty of obtaining monomeric GPCR structures hindered by micelle interference, we selected JN241-9, a single-domain antibody, as an agonist suitable for structural alignment. The JN241-9–APLNR monomer species was identified during 2DA and 3D classification, albeit at relatively low resolution. Data processing statistics revealed a 5:1 ratio of dimer to monomer, with three distinct conformations of the JN241-9–dimer identified, differing only in the ligand-binding pose (termed by ligand-bound states LBS1a, LBS1b and LBS1c, respectively) (Fig. 1b and Supplementary Fig. 2a). Additionally, the two AMG986-bound states of APLNR dimer without G-protein are distinguished by unique conformations of TM6 and helix 8 (H8) in one protomer (termed by LBS2a and LBS2b, respectively) (Supplementary Fig. 2b). These results indicate that the different ligands may induce different conformational states. Furthermore, we determined the structures of the APLNR–Gi1 complexes with the two agonists. Data processing statistics clearly demonstrate that monomeric complexes greatly exceed dimeric complexes, though dimeric formation is preferred in the absence of G-protein (Supplementary Figs. 2 and 3), implying that most dimers have dissociated during activation. Notably, the two protomers are designated as protA and protB, respectively (Fig. 1b). Data processing revealed that AMG986-bound dimeric APLNR–Gi1 complexes adopt two conformations in protB (termed by ACT2a and ACT2b, respectively), aligning with those structures without G-protein (Supplementary Figs. 2b and 3b).

Ligands recognition and conformational comparison in different states

We first compared two AMG986-bound states of APLNR dimer without G-protein (LBS2a and LBS2b) against the apo state to identify structural differences (Fig. 2a). In both apo and AMG986–dimerLBS2a structures, the receptor adopts an inactive conformation, characterized by TM6 remaining in the closed state. In contrast to LBS2a, protB in LBS2b adopts an active-like conformation, with TM6 undergoing an outward movement and H8 bending inward to engage the intracellular pocket, in line with the previously reported fully active conformation (Fig. 2a)11. Our observations further reveal that protB progressively moves away from protA across the transition states from apo to LBS2b, represented by the movement of TM3 and TM4 in the intracellular half of protB when aligned with protA (Fig. 2b). While hydrophobic interactions involving the “FGTFF” motif (APLNR residues 97-101) contribute to dimeric stabilization in the extracellular half (Supplementary Fig. 4a), the intracellular half of the dimer interface might be stabilized by more extensive interactions mediated by cholesterol clusters, particularly involving TM1, TM2, and TM4 (Supplementary Fig. 4b). Cα distance measurements between TM1–TM4 residues of protB and nearby protA residues at the intracellular interface revealed a gradual increase from the apo state to LBS2b, with the most notable change at F782.53 (Fig. 2c and Supplementary Figs. 4c-f). These results suggest that in the absence of G-protein, APLNR dimers bound to ligands such as AMG986 exhibit a tendency to dissociate into monomers.

Upon comparing the ligand-binding pockets of the AMG986–dimer (LBS2a and LBS2b) and the AMG986–monomer–Gi complex, we noted that AMG986 in the protB of LBS2b adopts a similar binding pose to that in the AMG986–monomer–Gi complex (Fig. 2d). AMG986 in protB of LBS2b displays a lateral shift toward Y2997.43 relative to its position in LBS2a, thereby triggering a downward shift of key residues associated with activation, including F782.53, W2616.48 and Y2997.43, ultimately resulting the outward movement of TM6 in protB of LBS2b (Fig. 2d). Additionally, we observed three distinct conformations of JN241-9 as aforementioned, while the receptor remained unchanged in its inactive conformation. The pivotal residue Y105, located in the CDR3 region of nanobody JN241-9 and inserting into the very bottom of the ligand pocket, undergoes a rotation from pointing towards the crevice between TM4/5 and ECL2 of APLNR in the LBS1a conformation to facing the intracellular direction in the LBS1b conformation (Figs. 1b and 2e). When comparing the ligand-binding pockets of JN241-9–dimer with and without G-protein, we noted that in LBS 1b, the Y105 of JN241-9 is positioned closer to Y2997.43, resembling the fully active conformation. However, no alterations in key motifs or TM6 conformational changes were observed in LBS1b (Fig. 2e). In the fully active-state structure of JN241-9, Y105 exhibits a deeper insertion toward Y2997.43, facilitating the shift of F782.53, W2616.48 and Y2997.43 into their activation conformations. Furthermore, comparison of the ligand-binding pockets in protB showed that AMG986 penetrates more deeply than JN241-9, thereby driving the allosteric transition of F782.53, W2616.48 and Y2997.43, which could explain its capacity to induce the opening of TM6 (Fig. 2f). These findings indicate that AMG986 can independently induce one protomer within the APLNR dimer to adopt an active conformation, in contrast to JN241-9, which requires G-protein engagement to promote receptor activation. We also observed that the ligand engagement induced conformational changes of key motifs in receptors across the entire activation process, including the rearrangement of the “Na+ pocket”, “PVF motif”, “NPxxY motif”, “DRY motif”, which align with the known mechanisms of GPCR activation (Supplementary Fig. 5).

G-protein coupling promotes a dimer-to-monomer transition of APLNR

According to statistics and analysis of cryo-EM data processing, while two agonists-bound APLNR in the absence of G-protein exhibits more dimers than monomers, the proportion of monomer–G-protein complexes in the fully active state far exceeds dimer–G-protein complexes, indicating that most of dimers dissociate into monomers when introducing G-protein (Fig. 3a, Supplementary Figs. 2 and 3). The protB in JN241-9–dimer–Gi complex adopts an inactive-state configuration and H8 in protB is disordered. By comparing the contact areas between the receptor and the G-protein in monomeric complex and dimeric complex, we found that the buried surface area of monomer (1213 Å2) is significantly greater than that of dimer (917.7 Å2), suggesting that G-protein binding to the monomer is more stable (Fig. 3b). In addition, we compared JN241-9- and AMG986-bound dimerLBSb to their monomeric complex aligning with protA, and observed that the binding of heterotrimeric G-protein would generate a clash with protB in the dimerLBSb, therefore prompting the dissociation of protB (Fig. 3c).

Meanwhile, there are still a small fraction of cryo-EM particles corresponding to dimer–G-protein complexes, accounting for 1/14 of monomeric complexes activated by JN241-9 and 1/19 of monomeric complexes activated by AMG986 (Fig. 3a). The high-resolution structure of the JN241-9–dimer–Gi complex shows a 5.7-degree downward rotation of G-protein measured by αN helix to allow for the stable binding of protB. Notably, the position of the αN helix in the JN241-9–dimer–Gi complex closely resembles that observed in the apelin–dimer–Gi complex12 (Fig. 3b and Supplementary Fig. 7e). Additionally, cholesterol clusters at the dimerization interface were observed to elastically connect the two protomers like an adhesive sponge (Fig. 3d). Compared to the JN241-9-dimerLBS1b, the JN241-9–dimer–Gi complex exhibits a counterclockwise rotation of protB based on TM4 from the bottom view, which moves away from protA (Fig. 3d). The cholesterol density near TM1 is disorder in dimeric complex, resulting in a decreased protB-cholesterol interacting area of 1750 Å2 in the dimeric complex compared to that of 2031 Å2 in the JN241-9–dimerLBS1b, which suggest that protB tends to dissociate from protA when bound to G-protein (Fig. 3e). Furthermore, the kinetic GloSensor cAMP assay revealed that signaling response associated with monomeric activation (F101A) was greater than that of the dimeric form (WT) during the initial 15 minutes. However, both responses converged thereafter, further suggesting that most dimers transition into monomers upon receptor activation (Supplementary Fig. 1d).

Careful cryo-EM data processing of the JN241-9-bound APLNR–Gi1 complex identifies a class of dimers that were not bound to Gi-protein, from which we further uncover three distinct fractions of particles, whose conformations are consistent with the G-free specimens. The particle number ratios of these conformations change from 47.5% (LBS1a), 12.9% (LBS1b) and 39.6% (LBS1c) to 27.6%, 17.1% and 55.3%, respectively. The increasement of the proportions of LBS1b and LBS1c states further indicates that these two conformations are closer to the active state conformation in comparison with LBS1a state (Fig. 3a, Supplementary Figs. 2a and 3a).

The extent of dimer dissociation upon APLNR activation is ligand-dependent

Different from JN241-9, the sole action of AMG986 without G-protein is capable of achieving the partial activation of one protomer in the APLNR dimer, thus prompting a tendency for the protomers to dissociate from each other (Fig. 2 and Supplementary Fig. 4). Inspiringly, the structures of AMG986–dimer–Gi complexes adopt either the active or the inactive conformations in protB, aligning with those dimeric structures in the absence of G-protein (Supplementary Fig. 6a). These findings suggest that ligand-specific modulation of dimeric conformations may contributes to varying degrees of dimer dissociation upon activation.

Comparison of the AMG986–dimerLBS2b with the monomeric complex reveal that the binding of G-protein would generate a more severe clash between Gβγ in the monomeric complex and H8 as well as ICL1 of protB in the AMG986–dimerLBS2b, compared to the JN241-9-bound APLNR structures (Fig. 3c). This implies that the AMG986-bound dimer has a higher propensity to dissociate during activation than the JN241-9-bound dimer. Additionally, comparing the two agonists-bound dimeric complexes highlights a counterclockwise rotation of about 27° in the entire G heterotrimer observed in the AMG986-bound structure (Supplementary Fig. 6b).

To date, multiple agonist-bound APLNR dimer structures in the presence or absence of Gi-protein have been resolved11,12. Accordingly, we compared the APLNR structures resolved in this study with previously reported structures. Pairwise alignment of the entire dimeric assemblies revealed relatively small Cα root mean square deviation (RMSD) values (<1.0 Å) among G-protein-free dimers (Supplementary Fig. 7a). In contrast, Cα RMSDs between structures with and without G-protein coupling exhibited larger deviations (1.0 to 2.5 Å), largely attributable to conformational shifts in TM6 and H8 (Supplementary Fig. 7a). Moreover, Cα RMSDs among G-protein-coupling dimers also showed relatively larger deviations (1.0 to 2.1 Å), suggesting that different ligands induce distinct dimer conformations in the active state (Supplementary Fig. 7a). To assess the extent of dimer dissociation, we compared the Cα RMSDs of protB aligned on protA (Supplementary Fig. 7b). Comparison between G-protein-bound and G-protein-free dimers revealed a remarkably wide range of Cα RMSDs (2.3 to 6.3 Å), particularly in comparisons involving the ELA–Gi complex (PDB: 7W0N)11, further indicating different ligands induce varying degrees of dimer dissociation during activation (Supplementary Figs. 7b-d).

Methods
Constructs

To prepare the cryo-EM sample of AMG986- and JN241-9-bound APLNR without G-protein, human wild type full-length APLNR (UniProt: P35414) was cloned into pFastBac1 vector (Invitrogen) with an N-terminal hemagglutinin (HA) signal sequence connected to a Flag tag. To further improve APLNR expression, a thermostabilized BRIL was fused to the N-terminus after the Flag-tag. A double MBP tag followed by a C-terminal His tag was fused into the C terminal of APLNR with a linker containing a TEV protease cleavage site. Unlike the construct for JN241-9-bound APLNR, LgBit was added at the end of TEV protease site in the construct of AMG986-bound APLNR. To prepare the cryo-EM sample of AMG986- and JN241-9-bound APLNR–Gi1 complexes, an N-terminal HA signal sequence connected to a Flag-tag was fused to the N-terminal APLNR. Human dominant-negative (DN) Gαi1 was generated by site-directed mutagenesis to decrease the nucleotide-binding affinity and increase the stability of G-protein60. Human Gβ1 was cloned into pFastBac dual vector with Gγ2. In addition, the scFv16, which encodes the single-chain variable fragment of mAb16, was subcloned into pFastBac plasmid.

To analyze G-protein signaling through NanoBit assay and cAMP accumulation assay, a Flag-tag was fused to the N-terminal of full-length and various mutants of APLNR. Additionally, Gαi1 was fused with LgBit, and Gγ was fused with SmBiT, and all three constructs were cloned into pcDNA3.1 plasmids. For β-arrestin signaling analysis, the C-terminus of the APLNR receptor was fused with the Rluc8 fragment, the Venus fragment was inserted into the N-terminus of β-arrestin2 and then both were cloned into pBiT1.1 plasmids (Promega).

Expression and purification of scFv16

The secreted scFv16 protein was expressed and purified using the bac-to-bac system. Briefly, the scFv16 with a C-terminal histidine-tag was overexpressed in Trichoplusia ni Hi5 insect cells for 48 h and purified by Ni-NTA resin. The eluted protein was cleaved by 3C protease and further purified by gel filtration chromatography using a Superdex 200 Increase 10/300 GL column (GE Healthcare). The purified scFv16 fractions were concentrated and stored at -80 °C until use.

Expression and purification of JN241-9

JN241-9 with a 6× His tag at the C-terminal was expressed in the periplasm of E. coli BL21 cells. Briefly, JN241-9 target gene was transformed in the bacterium and amplified in TB culture medium. When OD600 reached 0.7-1.2, 1 mM IPTG was added to induce expression followed by overnight incubation at 28 °C. JN241-9 was purified by Ni-NTA resin. The eluted protein was collected and then desalted using a HiPrep 26/10 Desalting column. Fractions of JN241-9 were concentrated to ~8 mg/ml and quickly frozen in the liquid nitrogen with 10% glycerol and stored at −80 °C.

Expression and purification of agonists-APLNR

APLNR constructs were expressed in sf9 insect cells using the bac-to-bac Baculovirus Expression System (Invitrogen). The cells were then incubated for 48 h at 27 °C and subsequently collected by centrifugation at 1,000 × g for 30 min. Cell precipitates were stored at -80 °C until further use. The cell pellets were suspended in buffer containing 20 mM HEPES pH 7.5, 2 mM MgCl2, 100 mM NaCl, protease inhibitor cocktail (Bimake) and agonist (100 nM AMG986 or 1 μM JN241-9). The membranes were solubilized with 0.5% (w/v) lauryl maltose neopentyl glycol (LMNG) and 0.1% (w/v) cholesterol hemisuccinate (CHS) for 2 h at 4 °C and separated by centrifugation at 30,000g for 30 min. Following incubation with amylose resin (NEB), and the protein was eluted with 20 mM maltose. TEV protease was then added for 1 h to remove the 2×MBP protein. After concentrated, the protein was loaded onto Superose 6 Increase 10/ 300 GL (GE Healthcare) column with running buffer containing 20 mM HEPES, pH 7.5, 100 mM NaCl, 0.00075% (w/v) LMNG, 0.00025% (w/v) glyco-diosgenin (GDN), 0.0002% (w/v) CHS and agonist (100 nM AMG986 or 1 μM JN241-9). The monomeric peak fractions were collected and concentrated to 10-15 mg/ml for electron microscopy experiments.

Expression and purification of agonists–APLNR–Gi1 Complexes

APLNR, Gαi1 and Gβγ constructs were co-expressed at the ratio of 1:1:1 in sf9 insect cells using the bac-to-bac Baculovirus Expression System (Invitrogen). The cells were then incubated for 48 h at 27 °C and subsequently collected by centrifugation at 1,000 × g for 30 min. For the purification of APLNR–Gi1 complexes, the cell pellets were suspended in buffer containing 20 mM HEPES pH 7.5, 2 mM MgCl2, 100 mM NaCl and protease inhibitor cocktail (Bimake), followed by addition of 1.2 mg scFv16 antibody, 12 mU/ml apyrase (Sigma), and agonist (100 nM AMG986 or 1 μM JN241-9). The mixture was incubated for 1 h at room temperature for complex formation. After that, the membranes were solubilized with 0.5% (w/v) LMNG and 0.1% (w/v) CHS for 2 h at 4 °C. The supernatant was then separated by centrifugation at 30,000g for 30 min. Following incubation with anti-FLAG affinity resin, the protein was eluted with 0.2 mg/ml Flag peptide. Add 10% (w/w) scFv16 antibody and incubated for 15 min at room temperature. The elution was concentrated with a 100 kDa cut-off concentrator (Millipore) and loaded onto a Superose 6 increase 10/300 GL column (GE Healthcare) with a running buffer containing 20 mM HEPES pH 7.5, 100 mM NaCl, 0.00075% LMNG, 0.0002% (w/v) CHS, 0.00025% (w/v) GDN and agonist (100 nM AMG986 or 1 μM JN241-9). The monomeric peak fractions were collected and concentrated to 10-15 mg/ml for electron microscopy experiments.

Cryo-EM grid preparation and data collection

To prepare cryo-EM grids, purified complexes (3 μL, ~10 mg/ml) were applied individually to the grids that were subsequently flash frozen in liquid ethane using Vitrobot Mark IV (Thermo Fisher Scientific) under 100% humidity at 4 C. For AMG986-, JN241-9-bound APLNR and AMG986-, JN241-9-bound APLNR–Gi1 complexes, cryo-EM data collection was performed on a Titan Krios at 300 kV accelerating voltage. Micrographs were recorded using a Falcon 4 direct electron detector in super-resolution mode with a pixel size of 0.93 Å using EPU software. Image stacks were obtained at a dose rate of about 13.24 electrons per Å2 per second with a defocus ranging from -1.0 to -2.5 μm. The total exposure time was 3.92 s, and 40 frames were recorded per micrograph.

Cryo-EM data processing

Dose-fractionated image stacks were aligned using MotionCor2.161, implemented in Relion 4.0. Contrast transfer function (CTF) parameters for each micrograph were estimated by Gctf v1.1862. The following data processing was performed using RELION 4.0 and CryoSPARC v4.0.363,64.

For the JN241-9-bound APLNR complex, a total of 13,193,854 particles were auto-picked in Relion 4.0. High-quality particle projections from multiple rounds of 2D classification and Heterogenous Refinement were utilized for ab-initio reconstruction to generate initial reference maps. A good subset of 2,817,890 particle projections was subjected to Heterogenous Refinement and 2D classification and two distinct particle populations were discerned, corresponding to the APLNR monomer and APLNR dimer. A subsequent round of 3D classification for APLNR dimer yielded three particle subsets: JN241-9-dimerLBS1a, JN241-9–dimerLBS1b and JN241-9–dimerLBS1c. These subsets of 97,020 particles, 101,362 particles and 138,605 particles were subjected to 3D refinement, CTF refinement, and Bayesian polishing in Relion, generating three maps with a global resolution of 2.7 Å.

For the AMG986-bound APLNR complex, a total of 14,104,471 particles were auto-picked. Particles from multiple rounds of 2D classification and Heterogenous Refinement were utilized for ab-initio reconstruction to generate initial reference maps. A good subset of 2,271,602 particle projections was subjected to Heterogenous Refinement and 2D classification, identifying APLNR monomer and APLNR dimer populations. Further 3D classification yielded two particle subsets: AMG-dimerLBS2a and AMG-dimerLBS2b. These subsets of 306,883 particles and 288,225 particles were subjected to 3D refinement, CTF refinement, and Bayesian polishing in Relion, generating two maps with a global resolution of 2.4 Å for AMG–dimerLBS2a and 2.5 Å for AMG–dimerLBS2b.

For the JN241-9-bound APLNR–Gi1 complex, a total of 16,153,222 particles were auto-picked. Particles from multiple rounds of 2D classification and Heterogenous Refinement were utilized for ab-initio reconstruction to generate initial reference maps. 4,170,567 particles were subjected to Heterogenous Refinement and 2D classification and four distinct particle populations were discerned: JN241-9–monomer, JN241-9–dimer JN241-9–monomer–Gi and JN241-9–dimer–Gi. A subsequent round of 3D classification for JN241-9–dimer also yielded three particle subsets: LBS1a, LBS1b and LBS1c. Final subsets of 187,827 particles for JN241-9–monomer–Gi and 198,168 particles for JN241-9–dimer–Gi were subjected to 3D refinement, CTF refinement, and Bayesian polishing in Relion, generating two maps with a global resolution of 2.5 Å and 3.0 Å, respectively.

For the AMG986-bound APLNR–Gi1 complex, a total of 6,477,441 particles were auto-picked. Particles from 2D classification and Heterogenous Refinement were utilized for ab-initio reconstruction to generate initial reference maps. A good subset of 1,257,292 particle projections was subjected to Heterogenous Refinement and 2D classification and four populations were discerned: AMG986–monomer, AMG986–dimer AMG986–monomer–Gi and AMG986–dimer–Gi. The final subset of 63,229 particles for AMG986–monomer–Gi were subjected to 3D refinement, CTF refinement, and Bayesian polishing in Relion, generating a map with a global resolution of 2.8 Å.

Comprehensive refinement metrics are detailed in Supplementary Tables 1 and 2.

Model building and refinement

The model of Apelin receptor in complex with single-domain antibody (PDB: 6KNM) was used to generate the initial template of receptor30. The atomic coordinates of Gi and scFv16 from the structure of MM07-bound APLNR–Gi1 complex (PDB: 8XZH) were used to generate the initial template of the G-protein complex19. Models were docked into the cryo-EM density map using UCSF Chimera65. Agonist coordinates and geometry restraints were generated using a phenix.elbow66. The docked model was subjected to flexible fitting using Rosetta 2019.35 and iterative manual adjustment in Coot and real-space-refined in Phenix67. The final refinement statistics were validated using the module ‘comprehensive validation (cryo-EM)’ in Phenix66. The goodness-of-fit of the model to the map was determined using a global model-versus-map Fourier shell correlation. The refinement statistics are provided in supplementary information. Structural figures were created using UCSF Chimera and the UCSF Chimera X package65,68.

NanoBiT-G-protein dissociation assay

G-protein activation was measured using a NanoBiT G-protein dissociation assay.2 HEK293T cells were seeded in a 6-well plate and plasmid transfection was performed with a mixture of 600 ng Gαi, 500 ng Sm-Gβ, 500 ng Gγ2, 200 ng APLNR plasmid (per well). After 6 h of transfection, cells were digested and resuspended in complete DMEM medium (5% FBS, 1% antibiotic) and plated in 96-well flat-bottomed white microplates at a volume of 100 μl per well. Next day, the cells were washed twice with D-PBS and incubated in 40 μl of 5 μM coelenterazine A solution diluted with HBSS (assay buffer) for 40 min at room temperature. After baseline luminescence was measured for 3 cycles (Tecan), test ligands (5×, diluted in the HBSS assay buffer) were added to the cells (10 μl per well) and incubated for 3-5 min at room temperature before the second measurement was taken. The ligand-induced signal ratio was normalized to the baseline luminescence, and fold-change signals over vehicle treatment were used to show the G-protein dissociation response. Concentration-response curves were fitted using the “log (agonist) vs. response (Three parameters)” model in GraphPad Prism 8 to obtain EC₅₀ values.

cAMP accumulation assay

Human embryonic kidney 293T (HEK293T) cells were seeded in a 6-well plate and transfected with a plasmid mixture consisting of 1,500 ng receptor and 500 ng cAMP biosensor GloSensor-22F (Promega). After 6 h, transfected cells were plated onto a 96-well plate. After another 12 h, cells were washed twice with D-PBS and then incubated in CO2-independent media (assay buffer) containing 2% GloSensor cAMP Reagent (Promega) and 1 μM Forskolin at a volume of 40 μl per well. MβCD with various concentrations was added to assay buffer when investigating the stabilized effect of cholesterols. The transfected cells were incubated for 30 min in 37 °C CO2 incubator and another 30 min at room temperature in the dark before measurements for baseline luminescence (Spark Multimode microplate reader, TECAN). Next, test ligands (5×, diluted in CO2-independent media containing 1 μM Forskolin) were added to the cells (10 μl per well) and incubated for 15-20 min at room temperature before the second measurement was taken. Concentration-response curves were fitted using the “log (agonist) vs. response (Three parameters)” model in GraphPad Prism 8 to obtain EC₅₀ values.

Bioluminescence resonance energy transfer (BRET) assay

BRET assay was used to detect β-arrestin recruitment. HEK293T cells were transiently co-transfected with APLNR-Rluc8 and Venus-β-arrestin2 (1:4). The transfected cells were seeded into 96-well flat-bottomed white microplates at a volume of 100 μl per well. After 24 h, cells were washed twice with D-PBS and then maintained in the BRET buffer (containing 10 mM HEPES and 0.1% BSA) at a volume of 45 ml per well for 30 min in 37 °C CO2 incubator. Luciferase substrate coelenterazine h (5 μM) was then added to the transfected cells. Coelenterazine and cells were incubated together in the dark for 10 min and the plate was placed into the microplate reader to read the base value. Transfected cells were then stimulated with ligands (5 μl) at different concentrations and read with the microplate reader again to get the BRET signal value. Both the BRET signal value and the base value were determined as the ratio of light emitted by Venus-tagged biosensors and light emitted by Rluc8-tagged biosensors. The BRET acceptor (520-560 nm) and BRET donor (460-485 nm) emission signals were measured using the Spark Multimode microplate reader (Tecan). Venus’s fluorescence was measured before reading luminescence and calculated as average fluorescence from each control well. The BRET signal from the same well was determined as the ratio of the light emitted by Venus (520-560 nm) over that emitted by RLuc8 (460-485 nm). Concentration-response curves were fitted using the “log (agonist) vs. response (Three parameters)” model in GraphPad Prism 8 to obtain EC₅₀ values.

Cell-surface ELISA

Membrane expression of APLNR was determined by enzyme-linked immunosorbent assay (ELISA) to detect the N-terminal Flag tag on the WT and mutants transiently expressed in HEK293T cells. After transfection, cells were seeded into 96-well flat-bottomed white microplates at a volume of 100 μl per well. After 24 h, cells were washed twice with PBS and fixed with 4% formaldehyde for 10 min followed by three times washing with PBS. Then cells were incubated with blocking buffer (1% BSA in PBS) for 1 h at room temperature. Afterwards, cells were incubated with anti-FLAG M2 HRP-conjugated monoclonal antibody (Sigma Aldrich) in blocking buffer for 30 min at room temperature. After washing three times with PBS, 80 μl/well diluent SuperSignal Elisa Femto Maximum Sensitivity Substrate (ThermoFisher Scientific) was added. Luminance signal was measured at 1-s intervals for 500 ms/per well.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Regulation of the GPCR activation by the monomer-oligomer transition is one of the important issues in GPCR biology. Apelin receptor is one of the well-studied examples that undergoes such regulation process. In previous studies, various structures of apelin receptor bound to small molecule, peptide and antibody agonists with or without G-protein have been determined. Structural analysis, single molecule and biochemical analyses revealed that agonists induce the dissociation of the apelin receptor dimer into monomers in the presence of G-protein, leading to full activation of the receptor. Several other features including cholesterol-mediated stabilization of the receptor dimer also have been revealed. In the present work, Ji et al determined twelve structures of apelin receptor in various states and proposed activation mechanism. The manuscript is well written and provides messages in activation mechanism of apelin receptor. However, most of messages are largely overlapped with the reported works, and the current work contains several issues that should be clarified. Q1. Line 245~250, Fig 5 and Supple Fig 5a ”.. the H8 from the active protB in AMG986–dimerINT2b bends inward and occupies the intracellular pocket, the G-protein instead prefers to couple to the inactive protA in order to minimize the potential energy barrier coupling to the protB,..” In the proposed model, the G-protein binds to the inactive protA of INT2b and converts it to the active form, whereas the partially active protB remains unchanged. Authors explained the reason in line 245~250. However, if protB is a partially (if not full) active form in the process of activation, the activation process must pass through this step. And, the G-protein should prefer to bind this partially active form and converts it into fully active conformation. If not, what is the next stage for this partially active conformation? In the aspects of energy barrier, protA also has TM6 in inactive form that should be rearranged to host G-protein. Because both protomers have energy barriers, G-protein must overcome these barriers in either case. Q2. There are many agonist-apelin receptor with/without Gi structures. Authors should compare their structures with the reported structures more completely (including rmsd values). At present, the reported structures are not properly compared and readers would not know any similarities (or differences) with the current ones and reported structures. Q3. Is it possible that each protomer present as a monomer in active or inactive conformation and simply associates to form a dimer rather than undergoes a ligand-induced conformational change in the dimeric form? Q4. Line 233~235 “The increasement of the proportions of INT1b and INT1c states further indicates that these two conformations are closer to the active state conformation in comparison with INT1a state (Fig. 3a, Supplementary Figs. 2a and 3a).” Is INT1c closer to the active state compared to 1a or 1b? What is the evidence? Comparison of all three structures (1a-1c and even 2a) reveals that all are virtually identical except the ligand orientation as described by authors. Authors should describe the rmsd values for INT1a-c, INT2a (and those from the reported structures). Also, if the rmsd value of the distances between protA and protB in different states are no larger than the rmsd of protA Ca (or protB), it is difficult to consider these as intermediates (or at least reaction intermediates). Q5. Many ICLs (ICL1 and ICL2) are overfitted in apo and some intermediates. These structures are compared and provides impression that there are noticeable structural differences in this region. However, in the map, these regions are difficult to build and inaccurate. Q6. Line 159; Distance between two F97 residues are longer in apo than those in intermediate 1a-c, 2a, so conclusion on the dissociation into monomer may not be appropriate. Q7. Line 163~185; 182 “.. AMG986 penetrates more deeply, driving the allosteric transition of F78, W261 and Y299, which could explain its capacity to induce the opening of TM6..” “More deeply” is not proper description because agonist in both prot of INT2a or prot A of INT2b is placed with similar depth compared to agonist in monomer-Gi; Rather, ligand AMG163 makes lateral shifts toward Y299. Please check. Q8. Line 185 “These findings indicate that the activation of APLNR is ligand-dependent, contradicting a recent report that proposed agonists alone cannot induce the outward movement of TM6…” In “CMPD644-ApelinR-Gi” structure (PDB 7W0L, NSMB, 2022), G-free protomer showed outward movement of TM6 in the presence of a small molecule agonist. Thus, it is hard to believe that same group (Xu group) would propose that an agonist alone cannot induce the outward movement of TM6. What they meant is likely that agonist alone cannot induce full activation, and G-protein would facilitate the full activation (& the specific agonist JN241-9 alone cannot induce the outward movement of TM6). Thus, authors should carefully interpret other’s work and cite properly. To this reviewer’s view, the concept of ligand-dependent activation of APLNR is already reported. It would be helpful to compare structures of INT2b with 7W0L (G-bound active protomer & G-free active protomer). Q9. No data for ACT2a and 2b are shown in Supple table. And authors should check if 12 structures are correct as this review has received 11 coordinates. Q10. In supple Fig 2 and 3; figure legends and Figures are different in a, b. Please check. Also Supple 5c is missing. Q11. line 195 – G-protein coupled dimer structure; comparison with aplein13 bound receptor with the current structures would be helpful (Gi orientation; etc) Q12. The alpha5 helix of protomer A in (967 and 989, active dimer) is not modeled well and authors should consider remodeling this region.", "reviewer_2": "This manuscript is potentially a very exciting study delving into the molecular interactions of Apelin receptor 1 dimerization and it's links to function. In many respects it is the first to successfully achieve so many structures of homo dimers in various intermediate, apo and ligand occupied states. The concept of GPCR homo and hetero dimers has been around for 20 years with lots of examples and both in vitro and in vivo data to support their existence and implications. However, advances in understanding them have stalled a) due to the fact that GPCRs can function as monomers and many structures of monomers exist, perpetuating a myth that dimers are artefacts and non significant. This study follows several previous studies in the last 2 years demonstrating that apelin receptors do form oligomers and structures can be obtained. The key in the current work is trying to understand the structural changes that occur between inactive and actives states and with different ligands that appear to have some bias and / or influence on oligomerization. Although I can not comment on the details of the structural work I can comment on the analysis and the functional aspects. In general the paper is exciting but there are some key aspects that need clarification and in my mind some important experiments performed to ensure the conclusions can be fully supported. Some thoughts: Can the authors mutate or delete H8 and see changes in the dimer behaviour with the ligand AMG986? Can they model the aN helices of other G -proteins and explain differences in the F101A mutants with AMG986 (Eg S1 B-d date)? In S1F they say Beta Arrestin 1 but in the legend and methods they say Beta Arrestin 2, which one is it? BRET assays depend on orientation of the donor and acceptor as the orientation impacts signal. Considering the fusions are on the C-terminus do the authors think that the change in H8 position is what is leading to the change in BRET rather then an actual difference in arrestin recruitment? Is it worth doing bystander BRET or complementation to confirm based arrestin recruitment to confirm the differences they are seeing? The beauty of realtime sensors like the GloSensor is they provide kinetic information, yet the assays were not performed to collect such information (Eg single time readings after 20 min incubation of ligand). Considering the importance of kinetics in receptor movement and oligomerization why not rerun the signalling assays to ascertain if there are kinetic differences in the mutants and between the ligands? It would be nice to see in this manuscript evidence that F101 mutants are monomers as I didn’t see that anywhere. From their analysis in S4 it looks like the NPxxY motif is the one that shows the biggest difference in terms of apo to active state, can the others comment on this as this appears to be away from any dimer interface. Why the big changes compared to some of the other key motifs? Homo dimers are reported to form for lots of class A receptors and B receptors, can they authors comment how common their findings might be?"}, "author_response": "We thank the referees for their valuable time in reviewing our manuscript and the constructive suggestions that they have provided. We have carefully taken these comments into consideration in preparing a revised version of our manuscript. Please find below a point-by-point response to the referees with our responses in blue and the reviewers’ comments in black. Reviewer #1: Regulation of the GPCR activation by the monomer-oligomer transition is one of the important issues in GPCR biology. Apelin receptor is one of the well-studied examples that undergoes such regulation process. In previous studies, various structures of apelin receptor bound to small molecule, peptide and antibody agonists with or without G-protein have been determined. Structural analysis, single molecule and biochemical analyses revealed that agonists induce the dissociation of the apelin receptor dimer into monomers in the presence of G-protein, leading to full activation of the receptor. Several other features including cholesterol-mediated stabilization of the receptor dimer also have been revealed. In the present work, Ji et al determined twelve structures of apelin receptor in various states and proposed activation mechanism. The manuscript is well written and provides messages in activation mechanism of apelin receptor. However, most of messages are largely overlapped with the reported works, and the current work contains several issues that should be clarified. Response: We are grateful for the reviewer’s positive comments on our study. 1. Line 245~250, Fig 5 and Supple Fig 5a ”.. the H8 from the active protB in AMG986–dimerINT2b bends inward and occupies the intracellular pocket, the G-protein instead prefers to couple to the inactive protA in order to minimize the potential energy barrier coupling to the protB,..” In the proposed model, the G-protein binds to the inactive protA of INT2b and converts it to the active form, whereas the partially active protB remains unchanged. Authors explained the reason in line 245~250. However, if protB is a partially (if not full) active form in the process of activation, the activation process must pass through this step. And, the G-protein should prefer to bind this partially active form and converts it into fully active conformation. If not, what is the next stage for this partially active conformation? In the aspects of energy barrier, protA also has TM6 in inactive form that should be rearranged to host G-protein. Because both protomers have energy barriers, G-protein must overcome these barriers in either case. Response: Thank you for your valuable comments. We totally concur with your insights that both protomers have energy barriers upon G-protein binding to either of them. Inspired by your suggestions, we have refined our model, as illustrated in Supplementary Fig. 6a (see below, Fig. R1), to reflect that G-protein can bind to either protA or protB in LBS2b. We have also revised the description in our manuscript as follows: “In the C2-symmetrical inactive LBS1a-1c and LBS2a complexes, G-protein binding to either protA or protB yields identical conformations (Scenario Ⅰ). For AMG986-bound LBS2b, G-protein binding to either protA or protB must overcome certain energy barriers. Binding to protB induces the assembly of the ACT2a complex (Scenario Ⅱ). In this instance, the α5 helix of Gα replaces the H8, driving protB from a partially active to a fully active conformation, while protA remains in its inactive state. On the other hand, the binding of G-protein to protA triggers the formation of the ACT2b complex (Scenario Ⅲ), wherein protA undergoes a classical activation mechanism characterized by an outward movement of TM6. Meanwhile, protB remains in a partially active state, with H8 bending inward to stabilize the open conformation of TM6, thereby preventing its transition to the fully active state (Scenario Ⅲ).” Reply figure 1. Conformational changes of APLNR dimerization are modulated by different agonists. Cartoon representations illustrate that G-protein binding to APLNR dimers with distinct conformations results in different dimer–G-protein complex conformations. 2. There are many agonist-apelin receptor with/without Gi structures. Authors should compare their structures with the reported structures more completely (including rmsd values). At present, the reported structures are not properly compared and readers would not know any similarities (or differences) with the current ones and reported structures. Response: Thank you for your constructive suggestions. In accordance with your suggestions, we compared previously reported and current dimeric APLNR structures in the presence or absence of G-protein and added the results in revised Supplementary Figs. 7a-b (see below, Fig. R2). Pairwise alignment of the entire dimeric assemblies revealed relatively small Cα root mean square deviation (RMSD) values (<1.0 Å) among G-protein-free dimers (Fig. R2a). In contrast, Cα RMSDs between structures with and without G-protein coupling exhibited larger deviations (1.0 to 2.5 Å), largely attributable to conformational shifts in TM6 and H8. Moreover, Cα RMSDs among G-protein-coupling dimers also showed relatively larger deviations (1.0 to 2.1 Å), suggesting that different ligands induce distinct dimer conformations in the active state (Fig. R2a). To assess the extent of dimer dissociation, we compared the Cα RMSDs of protB aligned on protA (Fig. R2b). Comparison between G-protein-bound and G-protein-free dimers revealed a remarkably wide range of Cα RMSDs (2.3 to 6.3 Å), particularly in comparisons involving the ELA–G complex (PDB: 7W0N), further indicating different ligands induce varying degrees of dimer dissociation during activation. We have also included the description in our manuscript. Reply figure 2. Heat map graphs showing Cα RMSDs of dimeric APLNR structures with or without G protein by pairwise alignments of these structures. i a, Pairwise structural alignments were performed across the full APLNR dimers to calculate Cα RMSDs and assess overall conformational differences. b, Pairwise alignments were performed using protA of the APLNR dimers to calculate Cα RMSDs of protB and assess its structural deviation relative to protA. *The reported structures, including ELA–G (PDB: 7W0N), cmpd644–G (PDB: 7W0L), apelin–G (PDB: 8XQE), JN241 (PDB: 8XQJ). All other structures were resolved in this study. i 3. Is it possible that each protomer present as a monomer in active or inactive conformation and simply associates to form a dimer rather than undergoes a ligand-induced conformational change in the dimeric form? Response: Thank you for your insightful comments. This question aims to discuss the formation mode of dimers with varying conformations, particularly whether the monomer in an active or inactive conformation simply associates to form a dimer (Fig. R3a) or a ligand-induced conformational change occurs within a pre-formed dimer (Fig. R3b)? The reviewer prefers the former perspective, which we also find reasonable given our observation of the G-free APLNR monomers. However, the latter viewpoint may hold greater probability, as we have determined that apo state APLNR can form a stable dimer through “FGTFF” motif and cholesterol-mediated hydrophobic interactions. Furthermore, the ligand-induce conformational change within the apo dimer may be more accessible compared to monomers transitioning to dimers, as fewer structural rearrangements are required (Fig. R3). Both viewpoints support the conclusion that distinct ligands may stabilize dimeric structures with different conformations. To avoid potential misunderstanding by readers, we have excluded discussion regarding the initial origin of distinct dimer states in the manuscript. Reply figure 3. Schematic representation illustrating the formation mode of dimers with varying conformations. a, The monomer in an active or inactive conformation simply associates to form a dimer. b, Ligand-induced conformational change occurs within a pre-formed dimer. 4. Line 233~235 “The increasement of the proportions of INT1b and INT1c states further indicates that these two conformations are closer to the active state conformation in comparison with INT1a state (Fig. 3a, Supplementary Figs. 2a and 3a).” Is INT1c closer to the active state compared to 1a or 1b? What is the evidence? Comparison of all three structures (1a-1c and even 2a) reveals that all are virtually identical except the ligand orientation as described by authors. Authors should describe the rmsd values for INT1a-c, INT2a (and those from the reported structures). Also, if the rmsd value of the distances between protA and protB in different states are no larger than the rmsd of protA Ca (or protB), it is difficult to consider these as intermediates (or at least reaction intermediates). Response: Thank you for pointing out this issue. Indeed, we have resolved three APLNR structures bound to JN241-9 without G-protein, of which conformational differences are limited to the ligand. Additionally, we have determined two AMG986-bound states of APLNR dimer in the absence of G-protein, exhibiting conformational variations in both the receptor and the ligand. Following the reviewer’s suggestion, we observed that the RMSD values for INT1a-c and INT2a are below 1.0 Å (Fig. R2a). Consequently, we acknowledge that it was improper to regard them as different intermediates. To prevent misinterpretation, we will describe these structures as “LBS” (Ligand-bound state) instead of “INT”, such as LBS1a, LBS1b and LBS1c. In the LBS1a structure, the residue Y105 of JN241-9 is oriented towards the crevice between TM4/5 and ECL2 of APLNR. This position is similar to the E104 of the antagonist JN241 in the JN241–APLNR structure (Fig. R4), indicating the ligands in LBS1a are closer to the inactive conformation. In contrast, the Y105 of JN241-9 in LBS1b points towards the intracellular region, adopting a conformation analogous to that observed in the fully active APLNR, in proximity to residues F782.53, W2616.48 and Y2997.43 (Fig. R4). Therefore, the ligands in LBS1b are closer to the active conformation. The ligands in LBS1c exhibit an activation level between that in LBS1a and LBS1b because their conformations in two protomers adopt that observed in LBS1a and LBS1b, respectively. Furthermore, the increasement of the proportions of LBS1b and LBS1c in the fully active state also indicates the ligands are closer to the active state conformation, laying the foundation for the full activation of APLNR. Reply figure 4. Comparison of the ligand binding pocket among the JN241–APLNR (PDB: 8XQJ), JN241–APLNR–G, JN241–APLNRLBS1a and JN241–APLNRLBS1b. i 5. Many ICLs (ICL1 and ICL2) are overfitted in apo and some intermediates. These structures are compared and provides impression that there are noticeable structural differences in this region. However, in the map, these regions are difficult to build and inaccurate. Response: We appreciate the reviewer’s careful evaluation and valuable comments. We have rebuilt the ICL regions of the apo dimer and all ligand-bound states of APLNR dimer without G-protein. In addition, residues that could not be reliably modeled due to poor density or ambiguity were removed to ensure structural accuracy. 6. Line 159; Distance between two F97 residues are longer in apo than those in intermediate 1a-c, 2a, so conclusion on the dissociation into monomer may not be appropriate. Response: Thank you for your comments. Structural analysis suggests that AMG986 can induce distinct dimeric conformations in the absence of G-protein, with the AMG986-bound LBS2b dimer showing a propensity to dissociate into monomers relative to the apo dimer. In contrast, JN241-9-bound states LBS1a, LBS1b, LBS1c and AMG986-bound state LBS2a do not display this dissociation tendency. To further investigate the pre-dissociation process, we examined the structural features of the dimeric interface and added the results in revised Supplementary Fig. 4 (see below, Fig. R5). We have also revised the description in our manuscript as follows: “While hydrophobic interactions involving the \"FGTFF\" motif (APLNR residues 97-101) contribute to dimeric stabilization in the extracellular half (Fig. R5a), the intracellular half of the dimer interface is primarily stabilized by more extensive interactions mediated by cholesterol clusters, particularly involving TM1, TM2, and TM4 (Fig. R5b). Cα distance measurements between TM1–TM4 residues of protB and nearby protA residues at the intracellular interface revealed a gradual increase from the apo state to LBS2b, with the most notable change at F782.53 (Fig. 2c and Figs. R5c-R5f).” These findings suggest that dimeric dissociation may initially occur at the interface formed by TM1-TM4, rather than at the “FGTFF” hydrophobic motif, which explains why the distance between the two F97 residues remains unchanged. Reply figure 5. Structural analysis of the dimeric interface and Cα distance measurement of residues within TM1-TM4 between protA and protB. a-b, Top view (a) and bottom view (b) of the APLNR dimeric interface in the apo state and AMG986-bound LBS2b. c-e, ProtB moves away from protA in the AMG986-bound LBS2b relative to the apo state, represented by residues in TM1 (c), TM2 (d) and TM4 (e). f, Cα distance measurement of residues within TM1-TM4 in different states. Each residue pair is presented with the first residue corresponding to protB and the second to protA, indicating their respective positions across the dimeric interface. 7. Line 163~185; 182 “.. AMG986 penetrates more deeply, driving the allosteric transition of F78, W261 and Y299, which could explain its capacity to induce the opening of TM6..” “More deeply” is not proper description because agonist in both prot of INT2a or prot A of INT2b is placed with similar depth compared to agonist in monomer-Gi; Rather, ligand AMG163 makes lateral shifts toward Y299. Please check. Response: Thank you for pointing out this issue. In investigating whether different ligands can promote APLNR dimer activation independently of G-protein coupling, we observed that AMG986 facilitates an outward movement of TM6 in protB, a conformational change not observed with JN241-9. This difference is attributed to AMG986 occupying a deeper binding pocket than JN241-9 in the G-free dimeric structures (Fig 2f). In the i AMG986-bound states of APLNR dimer, however, we fully agree with the reviewer’s insights. The ligand in protB of LBS2b displays a lateral shift toward Y2997.43 relative to its position in protA of LBS2b and both protomers of LBS2a (Fig 2d). Consequently, only protB of LBS2b undergoes outward movement of TM6 and adopts an active conformation. Accordingly, we have revised the description in our manuscript in line 174 as follows: “AMG986 in protB of LBS2b displays a lateral shift toward Y2997.43 relative to its position in LBS2a, thereby triggering a downward shift of key residues associated with activation, including F782.53, W2616.48 and Y2997.43, ultimately resulting the outward movement of TM6 in protB of LBS2b (Fig 2d).” Additionally, we have revised the description in line 191 as follows: “Furthermore, comparison of the ligand-binding pockets in protB showed that AMG986 penetrates more deeply than JN241-9, thereby driving the allosteric transition of F782.53, W2616.48 and Y2997.43, which could explain its capacity to induce the opening of TM6 (Fig. 2f).” 8. Line 185 “These findings indicate that the activation of APLNR is ligand-dependent, contradicting a recent report that proposed agonists alone cannot induce the outward movement of TM6…” In “CMPD644-ApelinR-Gi” structure (PDB 7W0L, NSMB, 2022), G-free protomer showed outward movement of TM6 in the presence of a small molecule agonist. Thus, it is hard to believe that same group (Xu group) would propose that an agonist alone cannot induce the outward movement of TM6. What they meant is likely that agonist alone cannot induce full activation, and G-protein would facilitate the full activation (& the specific agonist JN241-9 alone cannot induce the outward movement of TM6). Thus, authors should carefully interpret other’s work and cite properly. To this reviewer’s view, the concept of ligand-dependent activation of APLNR is already reported. It would be helpful to compare structures of INT2b with 7W0L (G-bound active protomer & G-free active protomer). Response: Thank you for your constructive suggestions. We have revised the description in our manuscript as follows: “These findings indicate that AMG986 can independently induce activation of a single protomer within the APLNR dimer, in contrast to JN241-9, which necessitates G-protein engagement for receptor activation.” Furthermore, structural comparison between the protB of LBS2b and both protomers of the cmpd644-bound APLNR–G complex (PDB: 7W0L) revealed that the i ligand in each 7W0L protomer undergoes a more substantial lateral displacement toward Y2997.43 than that in protB of LBS2b (Fig. R6), with the most pronounced shift observed in the G-bound active protomer (Fig. R6a). This enhanced shift correlates with a deeper downward movement of Y2997.43, thereby leading to full activation of the receptor. Reply figure 6. structural comparison between the protB of LBS2b and both protomers of the cmpd644-bound APLNR–G complex (PDB: 7W0L). i a, structural comparison between the protB of LBS2b and the G-bound active protomer of the cmpd644-bound APLNR–G complex. The binding modes of AMG986 and cmpd644 are presented. i b, structural comparison between the protB of LBS2b and the G-free active protomer of the cmpd644-bound APLNR–G complex. The binding modes of AMG986 and cmpd644 are presented. i 9. No data for ACT2a and 2b are shown in Supple table. And authors should check if 12 structures are correct as this review has received 11 coordinates. Response: We appreciate the reviewer’s careful evaluation and valuable comments. We have supplemented the cryo-EM data collection, model refinement and validation statistics for AMG986–dimerACT2a–G complex and AMG986–dimerACT2b–G complex in the i i Supplementary Table 2. In addition, the low-resolution map of the JN241-9-APLNR monomeric structure indicated the presence of monomers in the G-protein-free state. Regrettably, the map quality is too poor to build a corresponding structural model so there were only 11 coordinates. 10. In supple Fig 2 and 3; figure legends and Figures are different in a, b. Please check. Also Supple 5c is missing. Response: Thank you for pointing out this issue. The figure legends of supplementary Fig. 2 and 3 have been revised for clarity. In addition, supplementary Fig. 5 has been reorganized, and we have carefully verified the consistency of supplementary Fig. 5 between the manuscript and its corresponding figure legends. 11. line 195 – G-protein coupled dimer structure; comparison with aplein13 bound receptor with the current structures would be helpful (Gi orientation; etc) Response: Thank you for your insightful comments. In accordance with your suggestions, we performed structural comparison among the dimeric APLNR–G complexes aligned on i protA and added the results in revised Supplementary Fig. 7b (see below, Figs. R7a-R7c). These analyses demonstrated that protB shows varying degrees of pre-dissociation from protA, with the most pronounced dissociation observed in the ELA–dimer–G complex (Fig. R7b). Notably, TM6 of protB adopts an open conformation in i the cmpd644-, ELA-, and AMG986–ACT2b-bound complexes (Fig. R7b). Additionally, the αN helix of the G-protein in the apelin-, JN241-9-, and ELA-bound complexes undergoes a forward rotation of up to 27°, compared to its orientation in the AMG986–ACT2a-, AMG986–ACT2b-, and cmpd644-bound complexes (Fig. R7c). We have also included the description in our manuscript. Reply figure 7. structural comparison of apelin-bound dimeric APLNR–G complex with the i current structures. a-c, Structural comparison among the dimeric APLNR–G complexes aligned on protA. i Comparison of protB (b) reveals differences in the extent of its pre-dissociation from protA. The G i heterotrimer (c) undergoes a maximal rotation of 27°, as measured by αN helix. *The reported structures, including apelin–dimer–G (PDB: 8XQE), cmpd644–dimer–G (PDB: 7W0L) and ELA–G (PDB: 7W0N). All other structures were resolved in this study. i 12. The alpha5 helix of protomer A in (967 and 989, active dimer) is not modeled well and authors should consider remodeling this region. Response: We appreciate the reviewer’s careful evaluation and valuable comments. We have remodeled the alpha5 helix of G-protein in the AMG986-bound APLNR dimer–G i complexes as suggested by the reviewer. Reviewer #2: This manuscript is potentially a very exciting study delving into the molecular interactions of Apelin receptor 1 dimerization and it's links to function. In many respects it is the first to successfully achieve so many structures of homo dimers in various intermediate, apo and ligand occupied states. The concept of GPCR homo and hetero dimers has been around for 20 years with lots of examples and both in vitro and in vivo data to support their existence and implications. However, advances in understanding them have stalled a) due to the fact that GPCRs can function as monomers and many structures of monomers exist, perpetuating a myth that dimers are artefacts and non significant. This study follows several previous studies in the last 2 years demonstrating that apelin receptors do form oligomers and structures can be obtained. The key in the current work is trying to understand the structural changes that occur between inactive and actives states and with different ligands that appear to have some bias and / or influence on oligomerization. Although I can not comment on the details of the structural work I can comment on the analysis and the functional aspects. In general the paper is exciting but there are some key aspects that need clarification and in my mind some important experiments performed to ensure the conclusions can be fully supported. Response: We are immensely grateful to reviewer 2 for his/her strong support and appreciation of our work. Some thoughts: 1. Can the authors mutate or delete H8 and see changes in the dimer behaviour with the ligand AMG986? Response: Thank you for your valuable suggestions. To elucidate the role of H8 in APLNR function, we generated H8 deletion constructs based on both WT and F1013.24A. Surface expression of the H8-deleted receptor remained significantly diminished, even under 5-fold and 10-fold transfection levels relative to the full-length construct, highlighting the importance of H8 in stabilizing receptor conformation and ensuring proper membrane localization (Fig. R8c). Functional characterization using AMG986 revealed significantly diminished constitutive G-protein activity, as measured by the GloSensor cAMP assay, and a complete loss of β-arrestin recruitment, as detected by the BRET assay (Figs. R8a and R8b). This loss likely results from both reduced surface expression and the absence of the C-terminal region necessary for β-arrestin recruitment. From a structural perspective, H8 adopts an inward bend conformation in the AMG986-bound LBS2b structure, mimicking Gα engagement and contributing to stabilization of the active receptor conformation. Accordingly, deletion of H8 likely disrupts the formation of this conformation. Reply figure 8. Dose-response curves for AMG986-induced cAMP accumulation and β-arrestin2 recruitment and analysis of relative surface expression. a, cAMP accumulation was measured by GloSensor cAMP assay in HEK293T cells. b, β-arrestin2 recruitment was measured by BRET assay in HEK293T cells. c, Relative surface expression levels of mutants and truncations were measured by ELISA assay and normalized to the expression levels of WT. 2. Can they model the aN helices of other G -proteins and explain differences in the F101A mutants with AMG986 (Eg S1 B-d date)? Response: Thank you for your comments. It is challenging to obtain the experimental structural models probably due to the weak binding of G and G to APLNR. Additionally, the q 13 predicted dimeric and monomeric complex structures lack sufficient precision to reliably model the αN helix. Nevertheless, functional assay results (Fig. R9) show no significant differences in G, G , and G activation between AMG986-activated WT i q 13 and F1013.24A mutant APLNR, indicating a consistent G-protein binding mode, including the αN helix, across these G-protein subtypes. Reply figure 9. G-protein subtypes activation effects of AMG986 on WT and F101A. a-c, Dose-response curves show AMG986-induced G (a), G (b) and G (c) signaling were i q 13 measured by NanoBiT assay in HEK293T cells. d, Statistical analysis of Effects of the WT and F101A on AMG986-induced receptor activation. ns, no significance. P > 0.05; *P < 0.05; **P <0. 01; ***P < 0.001 by one-way ANOVA followed by Dunnett’s multiple comparisons test, compared with WT. 3. In S1F they say Beta Arrestin 1 but in the legend and methods they say Beta Arrestin 2, which one is it? Response: Thank you for pointing out this issue. The appropriate label in Fig. S1F should be “β-arrestin2”. We have made the necessary correction and revised the figure accordingly. 4. BRET assays depend on orientation of the donor and acceptor as the orientation impacts signal. Considering the fusions are on the C-terminus do the authors think that the change in H8 position is what is leading to the change in BRET rather then an actual difference in arrestin recruitment? Is it worth doing bystander BRET or complementation to confirm based arrestin recruitment to confirm the differences they are seeing? Response: Thank you for your careful evaluation and valuable comments. Structural comparison of the AMG986-dimerLBS2b and CB1-β-arrestin1 complex revealed that the conformational change of H8 in protB may influence the spatial orientation of the donor (Rluc) in the BRET assay (Fig. R10a), as mentioned by the reviewer. To further confirm the signal differences, we performed an additional experiment using the NanoBiT assay (Fig. R10b). The results showed that the dimer-reduced form (F1013.24A) exhibits a higher Emax than the dimeric form (WT) in the β-arrestin signaling (Figs. R10c and R10e), in agreement with the BRET results in our Supplementary Fig. 1f (Figs. R10d and R10e). Unlike the BRET assay, in which signal generation depends on the donor and acceptor being within a distance of 10 nm, the NanoBiT assay requires the complementary subunits LgBiT and SmBiT, present at a 1:1 ratio, to be brought into close proximity to form a functional luciferase enzyme that emits a luminescent signal. Therefore, the LgBiT fused to the C-terminus of protB does not interfere with the formation of the active luciferase complex. Consistently, both the BRET and NanoBiT assays demonstrate that the dimer-reduced form (F1013.24A) exhibits a higher Emax in β-arrestin signaling compared to the dimeric form (WT) receptor. Reply figure 10. β-arrestin2 recruitment in different aggregated forms were measured by NanoBiT and BRET assay. a, structural comparison of AMG986-dimerLBS2b and CB1-β-arrestin1 complex (PDB: 8WU1) was performed to analysis β-arrestin recruitment in APLNR. In this system, the donor (Rluc) is fused to the phosphorylated C- terminus of the receptor, while the acceptor (EYFP) is fused to the N-terminus of β-arrestin1. Conformational changes of H8 in protB may influence the orientation of the donor. b, Experimental principle of the NanoBiT assay. c, β-arrestin2 recruitment was measured by NanoBiT assay. d, β-arrestin2 recruitment was measured by BRET assay. e, Comparing the effects of the β-arrestin2 recruitment in NanoBiT and BRET assay. 5. The beauty of realtime sensors like the GloSensor is they provide kinetic information, yet the assays were not performed to collect such information (Eg single time readings after 20 min incubation of ligand). Considering the importance of kinetics in receptor movement and oligomerization why not rerun the signalling assays to ascertain if there are kinetic differences in the mutants and between the ligands? Response: Thank you for your careful evaluation and valuable comments. We performed kinetic detection using the GloSensor cAMP assay as suggested and added the results in revised Supplementary Fig. 1d (see below, Fig. R11). Baseline measurements prior to compounds addition indicated that the F1013.24A mutant exhibits higher constitutive G-protein activity compared to the wild-type (WT) receptor. Compounds were added at the second minute, Following compound addition, AMG986 induced a more rapid activation rate than JN241-9, as shown by comparison of the WT-AMG986 and WT-JN241-9 curves. Furthermore, the signaling response associated with monomeric activation (F101A) was greater than that of the dimeric form (WT) during the initial 15 minutes. However, both responses converged thereafter, further suggesting that most dimers transition into monomers upon receptor activation. We have also added the description in our manuscript. Reply figure 11. Dose-response curves show the duration of cAMP signaling response induced by 10 μM of each ligand. 6. It would be nice to see in this manuscript evidence that F101 mutants are monomers as I didn’t see that anywhere. Response: Thank you for pointing out this issue. Previous structural studies of APLNR have demonstrated that a π–π interaction network involving F1013.24 plays a central role in stabilizing the dimeric interface1. However, our current findings reveal that the F101A mutation does not completely disrupt dimer formation, due to the stabilizing effect of cholesterol clusters on the dimer interface of APLNR (Fig. R12). Therefore, the term “dimer-broken form” may be inaccurate in this context, and we have replaced it with “dimer-reduced form” throughout the manuscript. Reply figure 12. Analysis of interactions at the APLNR dimer interface. Beyond the hydrophobic interactions of the \"FGTFF\" motif (APLNR residues 97-101), the dimeric interface of APLNR also involves extensive interactions mediated by cholesterol clusters, which play a crucial role in stabilizing the dimer. 7. From their analysis in S4 it looks like the NPxxY motif is the one that shows the biggest difference in terms of apo to active state, can the others comment on this as this appears to be away from any dimer interface. Why the big changes compared to some of the other key motifs? Response: We appreciate the reviewer’s valuable comments. An analysis of 234 structures from 45 class A GPCRs in previous studies revealed a common activation mechanism that integrates key motifs (CWxP, PIF, Na⁺ pocket, NPxxY and DRY) into a unified conformational pathway2. This pathway operates through four hierarchical layers (Fig. R13): Layer 1: Signal Initiation. Agonist binding disrupts Na⁺-coordinated residues (D2.50, S3.39, N7.45), triggering inward movement of TM7, and residues at TM3 (3.40) and TM6 (6.44, 6.48) repack, initiating outward movement of TM6’s cytoplasmic end. Layer 2: Hydrophobic Lock Opening. Breaking of hydrophobic contacts (PIF motif) between TM3, TM5 and TM6, releasing TM6. Meanwhile, W6.48 rotation destabilizes TM6, facilitating its outward shift. Layer 3: Microswitch Rewiring. Y7.53 loses contacts with TM1/helix 8 and forms new bonds with TM3 (R3.50, I3.46), strengthening TM3-TM7 coupling. TM7 inward movement further displaces TM6 (NPxxY motif). Layer 4: G-Protein Coupling. Ionic lock (DRY motif) breaks, releasing R3.50. This residue engages Gα, while TM6’s outward shift creates a G-protein binding cavity. In summary, activation of GPCRs involves a conserved conformational rearrangement of key motifs, resulting in the outward displacement of TM6. Concurrently, TM7 moves inward to stabilize the active conformation of TM6. The NPxxY motif, situated at the intracellular end of TM7, exhibits the largest structural transition from the apo to the active state among all conserved motifs. Reply figure 13. Common activation pathway of class A GPCRs2. Four layers were qualitatively defined based on the topology of the pathway and their roles in activation: signal initiation (layer 1), signal propagation (layer 2), microswitches rewiring (layer 3) and G-protein coupling (layer 4). 8. Homo dimers are reported to form for lots of class A receptors and B receptors, can they authors comment how common their findings might be? Response: Thank you for your comments. While class C GPCRs are known to mediate signaling only in a dimeric form, class A and class B GPCRs were initially believed to function exclusively as monomers. However, evidence supporting dimerization in class A and class B receptors emerged with the development of advanced techniques such as photoaffinity labeling and cross-linking, which identified homodimers of β -adrenergic 2 receptor and heterodimers between cholecystokinin-8 (CCK-8) and dopamine (DA) receptors3,4. The existence of GPCR dimers is now well supported by a range of biochemical approaches, including epitope-tagging, immunoprecipitation, FRET and BRET assays. In class A GPCRs, several homodimer forms have been validated through these approaches: β₂-adrenergic receptor (β₂-AR), somatostatin receptor 5 (SSTR5), and δ-/κ-opioid receptors5; AT1R and A₂AR6; CCR2 and CCR57; apelin receptor8, and M₃ muscarinic receptor (M₃R)9. In addition, functionally relevant heterodimers have also been reported, such as the dopamine D₁ receptor and adenosine A₁ receptor complex10. Class B GPCRs also exhibit dimerization and oligomerization properties. GLP-1R has been confirmed to form homodimers through multiple resonance energy transfer assays, with TM4 identified as the interface of the dimeric interaction11. ECD-mediated oligomerization of PTH1R was similarly confirmed in living cells using BRET and FRET techniques12. Additionally, the GIPR has been shown to suppress PAC1 receptor-driven neuronal differentiation through heterocomplex formation13. Powerful advances in single-molecule microscopy have facilitated the measurements of the dynamic process of GPCR dimerization in real time. Homodimerization has been frequently reported for several class A GPCRs using various techniques: spot intensity analysis (APLNR, β AR, β AR, M R, FPR, D R)8,14- 1 2 1 3 17; photobleach step counting (μOR, PAFR, M R)18-20, and two-color coincidence 2 detection (S1P3R, mCannR2)21,22. These studies indicate that the dimeric formation of the receptor is transient and density-dependent. TIRFM is particularly suitable for assessing these dynamic membrane processes and quantifying oligomeric states. For example, at densities below 0.3 receptor particles/μm², ~60% of APLNR and ~44% of PAFR molecules formed dimers or oligomers. In class B GPCRs, CRF1R has shown a stable monomer/dimer equilibrium both at the plasma membrane and in the ER, as revealed by TIRFM23. The frequency and relevance of GPCR dimerization in vivo remains under debate. Some findings are derived from receptor overexpression systems, which may introduce experimental artifacts. Advanced imaging tools, such as super-resolution microscopy, can further surpasses the diffraction limit, offering quantitative insights into the stoichiometry of receptor oligomers. Given the limited structural understanding of class A and class B GPCR dimers, our study provides additional evidence supporting the existence of APLNR homodimer and elucidates a dynamic monomer–dimer transition mechanism that contributes to a deeper understanding of complex downstream signaling. The integration of complementary structural and functional techniques offers promising potential for accurately and dynamically monitoring GPCR dimerization events. 1 Yue, Y. et al. Structural insight into apelin receptor-G protein stoichiometry. Nature structural & molecular biology 29, 688-697, doi:10.1038/s41594-022-00797-5 (2022). 2 Zhou, Q. et al. Common activation mechanism of class A GPCRs. eLife 8, doi:10.7554/eLife.50279 (2019). 3 Fraser, C. M. & Venter, J. C. The size of the mammalian lung beta 2-adrenergic receptor as determined by target size analysis and immunoaffinity chromatography. Biochemical and biophysical research communications 109, 21-29, doi:10.1016/0006-291x(82)91560-1 (1982). 4 Agnati, L. F. et al. Differential modulation by CCK-8 and CCK-4 of [3H]spiperone binding sites linked to dopamine and 5-hydroxytryptamine receptors in the brain of the rat. Neuroscience letters 35, 179-183, doi:10.1016/0304-3940(83)90547-5 (1983). 5 Bader, J. E. & Beck-Sickinger, A. G. Fluorescence resonance energy transfer to study receptor dimerization in living cells. Methods in molecular biology (Clifton, N.J.) 259, 335-352, doi:10.1385/1-59259-754-8:335 (2004). 6 Oliveira, P. A. et al. Angiotensin II type 1/adenosine A (2A) receptor oligomers: a novel target for tardive dyskinesia. Scientific reports 7, 1857, doi:10.1038/s41598-017-02037-z (2017). 7 Mellado, M. et al. Chemokine receptor homo- or heterodimerization activates distinct signaling pathways. The EMBO journal 20, 2497-2507, doi:10.1093/emboj/20.10.2497 (2001). 8 Cai, X., Bai, B., Zhang, R., Wang, C. & Chen, J. Apelin receptor homodimer-oligomers revealed by single-molecule imaging and novel G protein-dependent signaling. Scientific reports 7, 40335, doi:10.1038/srep40335 (2017). 9 Hu, J. et al. Novel structural and functional insights into M3 muscarinic receptor dimer/oligomer formation. The Journal of biological chemistry 288, 34777-34790, doi:10.1074/jbc.M113.503714 (2013). 10 Ginés, S. et al. Dopamine D1 and adenosine A1 receptors form functionally interacting heteromeric complexes. Proceedings of the National Academy of Sciences of the United States of America 97, 8606-8611, doi:10.1073/pnas.150241097 (2000). 11 Harikumar, K. G. et al. Glucagon-like peptide-1 receptor dimerization differentially regulates agonist signaling but does not affect small molecule allostery. Proceedings of the National Academy of Sciences of the United States of America 109, 18607-18612, doi:10.1073/pnas.1205227109 (2012). 12 Pioszak, A. A., Harikumar, K. G., Parker, N. R., Miller, L. J. & Xu, H. E. Dimeric arrangement of the parathyroid hormone receptor and a structural mechanism for ligand-induced dissociation. The Journal of biological chemistry 285, 12435-12444, doi:10.1074/jbc.M109.093138 (2010). 13 Ke, R., Lok, S. I. S., Singh, K., Chow, B. K. C. & Lee, L. T. O. GIP receptor suppresses PAC1receptor-mediated neuronal differentiation via formation of a receptor heterocomplex. Journal of neurochemistry 157, 1850-1860, doi:10.1111/jnc.15220 (2021). 14 Calebiro, D. et al. Single-molecule analysis of fluorescently labeled G-protein-coupled receptors reveals complexes with distinct dynamics and organization. Proceedings of the National Academy of Sciences of the United States of America 110, 743-748, doi:10.1073/pnas.1205798110 (2013). 15 Hern, J. A. et al. Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules. Proceedings of the National Academy of Sciences of the United States of America 107, 2693-2698, doi:10.1073/pnas.0907915107 (2010). 16 Kasai, R. S. et al. Full characterization of GPCR monomer-dimer dynamic equilibrium by single molecule imaging. The Journal of cell biology 192, 463-480, doi:10.1083/jcb.201009128 (2011). 17 Tabor, A. et al. Visualization and ligand-induced modulation of dopamine receptor dimerization at the single molecule level. Scientific reports 6, 33233, doi:10.1038/srep33233 (2016). 18 Kuszak, A. J. et al. Purification and functional reconstitution of monomeric mu-opioid receptors: allosteric modulation of agonist binding by Gi2. The Journal of biological chemistry 284, 26732-26741, doi:10.1074/jbc.M109.026922 (2009). 19 Nenasheva, T. A. et al. Abundance, distribution, mobility and oligomeric state of M₂ muscarinic acetylcholine receptors in live cardiac muscle. Journal of molecular and cellular cardiology 57, 129-136, doi:10.1016/j.yjmcc.2013.01.009 (2013). 20 Liu, J. et al. Biased signaling due to oligomerization of the G protein-coupled platelet-activating factor receptor. Nature communications 13, 6365, doi:10.1038/s41467-022-34056-4 (2022). 21 Felce, J. H. et al. Receptor Quaternary Organization Explains G Protein-Coupled Receptor Family Structure. Cell reports 20, 2654-2665, doi:10.1016/j.celrep.2017.08.072 (2017). 22 Latty, S. L. et al. Referenced Single-Molecule Measurements Differentiate between GPCR Oligomerization States. Biophysical journal 109, 1798-1806, doi:10.1016/j.bpj.2015.09.004 (2015). 23 Teichmann, A. et al. The specific monomer/dimer equilibrium of the corticotropin-releasing factor receptor type 1 is established in the endoplasmic reticulum. The Journal of biological chemistry 289, 24250-24262, doi:10.1074/jbc.M114.553644 (2014)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Authors have clearly resolved all of my concerns in the revised manuscript. However, I strongly suggest that authors should add references to the revised line 272~288 (that correspond to Q#2) to provide clarity to the readers.", "reviewer_3": "I have been asked to review this manuscript after preliminary revision due to the withdrawal of review #2. The manuscript by Ji et al. describes twelve cryo-EM assemblies in detergent micelles of engineered apelin receptor with two different (non-physiological) agonists (AMG986 and JN241-9) with and without engineered Galphai/Beta1/gamma2 heterotrimer. The authors then use these twelve assemblies to construct a narrative on possible conformational transitions for the apelin receptor during activation. They use the molecular snapshots provided by these cryo-EM assemblies to argue for a particular reaction pathway. There is no way to know whether the proposed relationship between the reported assemblies represents the pathway that occurs in their highly reconstituted and engineered system, much less in either a recombinant cellular setting or physiologically. The supporting cell-based assay data is heavily normalized (minimum 2 normalization steps), which hampers independent assessment by the reader, and in several cases lacks appropriate controls to allow interpretation. Throughout, the findings from their cryo-EM assemblies are referred to as if they represent some physiological ground truth, which is a serious, unsupported, over-interpretation. I urge the authors to apply a much more conservative interpretation of their data. I also note that the novelty of the manuscript is undermined by existing publications of apelin receptor structures reported elsewhere, including by these authors. I would strongly suggest that the term “aggregation”, which the authors appear to use to mean dimerization / oligomerization should be replaced with either of these terms. Aggregation has a specific meaning in biochemistry, which is: the accumulation of misfolded or intrinsically disordered proteins into (typically) insoluble, non-functional complexes. Understanding whether GPCRs (outside Class C) do form dimers in physiological settings, and if they do, what the functional consequence is, remains an unresolved question in the field. Unfortunately, the field is largely divided into dogmatic camps of “GPCR dimerization is a general phenomenon with important physiological outcomes” and “GPCR dimerization is an artefact, with no relevance at all”. Despite the large amount of literature on the subject, conclusions remain equivocal, due to a fairly large body of literature that relies on transfected cell studies using various proximity measures that are not powered to detect the difference between close proximity and protein:protein interaction AND the difficulty in performing definitive experiments in physiologically meaningful settings. There is no teleological reason to believe GPCRs form dimers, unlike nuclear hormone receptors (dimerization required for DNA binding), receptor tyrosine kinases (dimerization required for transphosphorylation) or enzymes (dimerization provides positive or negative allostery to enable physiologically relevant sensitivity to changes in substrate availability). On the other hand, it is attractive to think that novel behaviors could emerge through the formation of GPCR heterodimers (although there is much evidence, particularly from the 1990’s to suggest signal crosstalk as a mechanism for new behaviors). In this setting, new publications on either side of this debate ought to be subject to careful critical analysis. Introduction: Page 2, lines 37-40 “GPCRs serve as central hubs in cellular signal transduction, ensuring the complexity and precision of downstream signaling through their structural versatility, characterized by diverse aggregated forms and dynamic conformational changes.” This is a meaningless word-salad, adding nothing to the paper. I would recommend removing. Page 2, lines 41-42 “Class A GPCRs exist in a dynamic equilibrium between monomeric and oligomeric forms”. Of the data cited none provide evidence for a dynamic equilibria between oligomeric states, a single figure in reference 6 (figure 3) has data that supports the contention that oxytocin receptors may form dimers in native tissue. The remainder of the citations rely on data that have alternative interpretations. I am concerned that there is an uncritical perpetuation of data over-interpretation supported by poor-quality and often careless experiments. Here are alternative interpretations for the common experimental types presented in these figures: • Co-immunoprecipitation. With the inclusion of all relevant controls (particularly for membrane proteins that have a propensity to aggregate), co-immunoprecipitation should only be used to conclude that 2 proteins are present as part of a complex, direct interaction cannot be concluded. Absence of data from native tissue should be viewed with scepticism. Antibodies for GPCRs are frequently un-specific. • Confocal microscopy showing “co-localisation”. Under standard techniques in an optimally adjusted system with a high NA objective the point-spread-function of GFP/Alexa488 is ~140 nm in the x-y and 380 nm in the z with a volume of about 4 x 10^6 nm^3 (obviously this gets bigger with longer wavelength). A typical GPCR occupies a volume of about 70 nm^3 , thus over 50,000 GPCRs can fit into a single PSF. Data is frequently reported where GPCRs do not appear to be localized to the plasma membrane, which is inconsistent with known properties. Antibodies for GPCRs are frequently un-specific. • BRET or FRET (resonance energy transfer techniques). Generally, only applicable in transfected cell studies. Relies on the assumption that direct interaction is required for resonance energy transfer. This is clearly not true, given the widespread use of bystander BRET to measure (for example) GPCR trafficking. Saturation BRET remains problematic as controls are difficult. If partner proteins for both donor and acceptor traffic to the same membrane subdomains, the readout for this assay will appear to indicate a specific interaction. Unless controls for collisional (random) BRET signals have equivalent expression (on a per-cell basis) to the test AND have comparable trafficking, interpretation is ambiguous. • Altered signalling output in the presence of a second GPCR. This could equally occur due to signal crosstalk. • Mutational analysis of putative dimerization interface (mostly TM bundle mutations, typically altering signalling output). Even if carefully controlled for expression level it is usually not possible to exclude the possibility that the mutation alters transmission of ligand binding event through the GPCR to its intracellular partner. Page 3 ,lines 52-55 “….supported by single-molecule imaging that confirms dimeric formation, and it is the first Class A GPCR whose stable dimeric structures have been captured by cryo-EM^8,15,16.” Reference 8 is provided to the contention that apelin receptor can interchange between dimers and monomers in cells (including using single molecule imaging). This reference is problematic, clustering (single molecule experiments, figure 1) lack appropriate controls (e.g. monomeric cell-surface receptor). If taken at face value, there is no equivalence between clustering and dimerization, this is below the resolution of the experiment. Even if dimerization is accepted there is not necessarily and equivalence between physical co-localisation and functional dimerization. Figure 2a, heavily cropped and manipulated western blots, non-equivalent loading of “mix” control, full blots not provided in supplementary information. Panels 2b&c are much more problematic, Oxytocin receptor is used as a ‘negative’ control in BRET experiments (i.e. as a receptor that does not dimerise), this is despite the fact that Reference 5 specifically demonstrates oxytocin receptor dimers by both FRET (weak evidence) but also by ligand allostery on native membranes (strong evidence), dimerization of oxytocin receptor is also reported in PMID: 27420737. Figure 3 & 4, no negative OR positive controls with both donor and acceptor included. Figure 5 uses interference peptides to infer TM1,2,3 & 4 as parts of the dimerization interface, this conflicts with data from PMID 35817871 (e.g. 7W0L) where TM4 plays no part and TM2 has only a single residue within a distance that could support an interaction (also noting that this is consistent with the present publication) bringing into question this methodology. The remaining figures also lack appropriate controls. I would suggest rephrasing to more accurately reflect the ambiguity of data: “APLNR is a model system that may be suitable for investigating the dynamic transition between monomeric and dimeric forms of class A GPCR, supported by transfected cell studies, and cryo-EM^8,15,16.” The clinical trial and published data on AMG986/Azelaprag is presented in a way that the reader might think that biased agonism and/or dimeric/monomeric receptor signalling profile is the reason for the clinical trial discontinuation (page 3, lines 71-74): “However, a Phase 2 clinical trial evaluating AMG986 was recently discontinued owing to potential adverse effects, highlighting the necessity to explore its molecular mechanism in stimulating various apelin receptor forms.” This does not seem to be the most parsimonious interpretation as the clinical trial was terminated due to ~5% of patients in the Azelaprag arm having blood markers consistent with liver toxicity (no patients in control arm). The size of the trial suggests that this is, in fact, an Azelaprag side effect, however the most likely cause is off-target liver toxicity, not an undesirable on-target effect, as implied by the authors. I suggest removing the second half of this sentence and replace with: ““However, a Phase 2 clinical trial evaluating AMG986 was recently discontinued owing to potential off-target liver toxicity.” No observation of transitions between different conformations is made in this manuscript. This would require NMR, single molecule FRET or HDX-MS (if the transitions are slow enough), or MD-simulations could be conducted to model transitions. None of these are performed in this manuscript, only static snapshots are determined with no way of knowing whether these occur in a particular order on a reaction pathway OR whether some are off-pathway conformations. Page 4, lines 85-87: “We therefore directly observed the stepwise transition and conformational changes of APLNR dimers towards monomers from the apo to the active state.” I suggest rephrasing to: “We therefore directly observed a range of conformational states of APLNR dimers and monomers, both apo and active states, which provide potential pathways for conformational transition between these states.” Results: Page 4, line 96 “…. network formed by F101^3.24 plays a …..” Here I assume the 3.24 refers to the Ballesteros-Weinstein numbering system for TM helices of GPCRs, please explain this to the reader and include the appropriate reference. I suggest “…. network formed by F101^3.24 (where the superscript refers to the Ballesteros-Weinstein transmembrane numbering system (ref)) plays a …..”. Reference is: Ballesteros, J.A., and Weinstein, H. (1995). Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors. Methods Neurosci. 25, 366–428. https://doi.org/10.1016/S1043-9471(05)80049-7. Page 4, lines 102-104: “AMG986 showed roughly 10-fold greater potency in activating various G-proteins, including Gi, Gq and G13, as well as β-arrestin, compared to JN241-9.” Please provide statistical analysis to support this difference. The appropriate analysis is to derive the pEC50 from each biological repeat, use this to derive a mean and error then test for difference between pEC50s (one-way ANOVA). It is more appropriate to display SD rather than SEM, since the former is an estimate of the biological variance. I note that supplemental file “583494_1_related_ms_11086941_sz33ll” contains individual columns that appear to sum to the total of technical replicates multiplied by biological repeats. Could the authors please confirm that they have no conflated technical and biological replicates? While Nature Communications makes no formal recommendation on use of error bars (see: https://www.nature.com/collections/qghhqm and https://www.nature.com/articles/nmeth.2659), best practice for this type of data would be SD. I also note that the double normalisation (individual baseline normalization, followed by conversion to fold over baseline, followed by conversion to % of WT-AMG896 Emax) has the potential to obscure differences in G protein activation whilst also potentially accentuating differences between ligands. I would prefer to see data where the final normalization step is removed. Page 4/5, lines 105-106. “Additionally, AMG986 induced a more rapid activation rate than JN241-9 (Supplementary Fig. 1d).” Firstly, please comment on the magnitude of difference, looking at the data this is very small and is hard to envision why one would think this is of any biological relevance. Secondly, please provide analysis to support that this difference is statistically significant. Third, as with the above comment, I suggest SD. The axis of supp fig 1d is labelled “cAMP accumulation %”, this is the % of what? For all cAMP data, are the authors confident that sensor has not been saturated? For example, was a cell permeant cAMP analoge (e.g. 8-CPT) included at the end of the assay? Page 5, lines 107-108. “Though negligible differences in EC50 or Emax from G-protein signaling, the dimer-reduced form (F1013.24A) showed a higher Emax than the dimeric form in the β-arrestin signaling (Supplementary Fig. 1f).” Please refer to the magnitude of change and also provide analysis supporting a statistically significant difference. Without experiments performed under parallel conditions to quantitate cell-surface expression this change in Emax could be as a result of altered receptor number. Elsewhere (supp fig 8) the authors report cell surface expression by ELISA, however it is not clear from the methods that this was performed under equivalent transfection conditions to the β-arrestin assay. It is not clear from the methods whether cell integrity would be maintained using the fixation technique used and no negative control is included. As this data stands, interpretation is ambiguous. I suggest a cautious interpretation of this result: “Though negligible differences in EC50 or Emax from G-protein signaling, the dimer-reduced form (F1013.24A) showed a higher Emax than the dimeric form in the β-arrestin signaling (Supplementary Fig. 1f), consistent with previous results^15, which may be indicative that the dimer exhibits G-protein bias compared to the monomer.” The apparent unity hill-slope of both cAMP and β-arrestin responses has no detectable change between the WT receptor and the F101A mutant (proposed monomeric form), nor does there appear to be a detectable change in pEC50. This suggests that in the dimeric form of the apelin receptor, there is no detectable co-operativity (negative or positive) for either ligand. Thermodynamically, it is hard to envision how dimerization would NOT alter ligand binding (see REF 6 and Birdsall, N.J.M. (2010). Class A GPCR heterodimers: evidence from binding studies. Trends Pharmacol. Sci. 31, 499–508. https://doi.org/10.1016/j.tips.2010.08.003.). A statement to this effect should be included immediately after the above sentence: “No detectable change in either Hill-slope or pEC50 is evident for either AMG986 or JN241-9 between WT and monomeric (F101A) receptors, suggesting that ligand binding allostery, if present, is not detectable.” Page 5 lines 110-112. “Moreover, cAMP accumulation results showed that the dimer-reduced form exhibits higher constitutive activity than that of dimeric form (Supplementary Figs. 1d-e).” Please refer to the magnitude of change and also provide analysis supporting a statistically significant difference. I would also suggest softening this statement, as without data showing that receptor expression is equivalent AND that the WT v’s F101A mutant do not alter (for example) adenylate cyclase or phosphodiesterase expression it is not possible to attribute the change in baseline to receptor constitutive activity. A solution would be to perform assays using an inverse agonist for Apelin receptor, although I am not aware of one. I would suggest the following: “Moreover, cAMP accumulation results showed that the dimer-reduced form appear to exhibit ~25% higher constitutive activity than that of dimeric form (Supplementary Figs. 1d-e, p<0.ABC).” The curve for used for these concentration-response curves is not stated, either in the results or in the methods. Please include the equation used in the methods section. If a 3 parameter logistic equation has been used, this should be justified as it would fail to take into account 2:2 stoichiometry. Page 5, line 120-122. “Here, we successfully achieved high-resolution cryo-EM structures of agonists-bound class A GPCR in various conformations without G-protein, delineating the dynamic process of activation.” Again, these are snapshots and there is no data that addresses whether they are all on the reaction pathway and if they are which order they occur. I suggest a more conservative interpretation of this data: “Here, we successfully achieved high-resolution cryo-EM structures of agonists-bound class A GPCR in various conformations without G-protein, providing potential insights into the dynamic process of activation.” Page 5, line 127. “Fortunately, we identified ….” Fortunately is unnecessary and pejorative, I suggest to start the sentence “We identified …..” Page 5, lines 133-135. “Additionally, the two AMG986-bound states of APLNR dimer without G-protein are distinguished by unique conformational changes of TM6 and helix 8 (H8) in one protomer (termed by LBS2a and LBS2b, respectively) (Supplementary Fig. 2b).” Conformation changes are assumed not measured, these are snapshots and there is no reason to believe they represent a particular reaction co-ordinate. I suggest: “Additionally, the two AMG986-bound states of APLNR dimer without G-protein are distinguished by unique conformations of TM6 and helix 8 (H8) in one protomer (termed by LBS2a and LBS2b, respectively) (Supplementary Fig. 2b).” Page 6, lines 136-137. “These results indicate that the conformations are in a ligand-dependent manner.” This sentence doesn’t make sense in English, I suggest: “These results indicate that the different ligands may select different conformational states.” Page 6, lines 143-146. “Data processing revealed that AMG986-bound dimeric APLNR–Gi1 complexes adopt two conformations in protB (termed by ACT2a and ACT2b, respectively), aligning with those structures without G-protein (Supplementary Fig. 3b).” is there meant to be an alignment between G-protein free and G-protein bound complexes in Supp Fig 3b? I don’t see one? Page 6, lines 149-151. “To elucidate the structural transition of APLNR from its apo to active state, we first compared two AMG986-bound states of APLNR dimer without G-protein (LBS2a and LBS2b) against the apo state to identify structural differences (Fig.2a)”. Implicit in this analysis is the assumption that the reaction co-ordinate for a GPCR to lower the energy barrier for nucleotide exchange at its cognate G protein involves binding of the agonist prior to binding of the G protein. What is the evidence that this is the case for the Apelin Receptor? The majority of Class A GPCRs where agonist binding has been studied such that G protein-coupling can be modulated by the addition of non-hydrolysable GTP analogues show that the high affinity state is one in which the G protein is engaged with the receptor. There are a range of Class A receptors where there is evidence of pre-coupling of G protein to receptor. Implicit in the ternary complex model is that G protein coupling may occur prior to agonist binding. Where is has been studied, apo GPCRs explore conformations that are either intermediate to activation or similar to fully active (ternary) conformations. Lastly, large scale MD simulations of many Class A GPCRs (including the Apelin receptor) supports the notion that these receptors explore active-like states in the absence of agonist binding. Taken together, and in the absence of data that supports agonist binding first, I think it is faulty logic to ascribe the agonist bound, G protein free state as a transition to the active state. It could be this, but it could also be a state that is not on the reaction pathway. Seed references are below: 1. Aranda-García, D., Stepniewski, T.M., Torrens-Fontanals, M. et al. Large scale investigation of GPCR molecular dynamics data uncovers allosteric sites and lateral gateways. Nat Commun 16, 2020 (2025). https://doi.org/10.1038/s41467-025-57034-y 2. Kim TH, Chung KY, Manglik A, Hansen AL, Dror RO, Mildorf TJ, Shaw DE, Kobilka BK, Prosser RS. The role of ligands on the equilibria between functional states of a G protein-coupled receptor. J Am Chem Soc. 2013 Jun 26;135(25):9465-74. doi: 10.1021/ja404305k. Epub 2013 Jun 14. PMID: 23721409; PMCID: PMC3763947. 3. Nobles M, Benians A, Tinker A. Heterotrimeric G proteins precouple with G protein-coupled receptors in living cells. Proc Natl Acad Sci U S A. 2005 Dec 20;102(51):18706-11. doi: 10.1073/pnas.0504778102. Epub 2005 Dec 13. PMID: 16352729; PMCID: PMC1317907. 4. De Lean A, Stadel JM, Lefkowitz RJ. A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase-coupled beta-adrenergic receptor. J Biol Chem. 1980 Aug 10;255(15):7108-17. PMID: 6248546. Figure 2 and supplementary figure 4. There are a number of relatively subtle changes in side chain position/rotation that are ascribed as being part of the activation pathway. The apparent resolution of the relevant parts of the receptor appears to be between 2 and 2.2A (supp fig 2 and 3), which should allow good confidence in assigning these. The maps on which these models are based and the models themselves are not provided (or perhaps I can’t find them?), so it is not possible as a reviewer to independently verify the side-chain movements being presented. I could not find an editorial policy on making these data (maps and models) available to reviewers, but this would be best-practice. Page 7, line 178. “Surprisingly, we observed ….”. No need for the pejorative surprising. Present the data and let the reader judge whether it is “surprising”. Page 7/8 lines 194-197. “These findings indicate that AMG986 can independently induce activation of a single protomer within the APLNR dimer, in contrast to JN241-9, which necessitates G-protein engagement for receptor activation.” These are snapshots of dynamic complexes and it is not clear that induced fit is in operation, I would encourage more cautious interpretation. I also note that if the authors interpretation is correct, one would predict that AMG986 would possess higher efficacy compared with JN241-9. I see no evidence of this. Pages 8 & 9. Section entitled: “G-protein coupled activation of APLNR facilitates the dimer-to-monomer transition” again, order of events is not known, it could be that dimer to monomer transition occurs first then G protein binding occurs then agonist binding occurs (or pretty much any other variation on this). Please apply a more cautious approach to interpretation OR provide evidence from an endogenous setting that supports one or other orders for the reaction pathway. Page 9, lines 236-240. “Furthermore, the kinetic GloSensor cAMP assay revealed that signaling response associated with monomeric activation (F101A) was greater than that of the dimeric form (WT) during the initial 15 minutes. However, both responses converged thereafter, further suggesting that most dimers transition into monomers upon receptor activation.” Please quantitate the difference in reaction rate and perform statistics to support that this. If this data quantitively and statistically supports faster cAMP inhibition kinetics if could be interpreted that the F101A mutant has higher propensity to pre-couple to its cognate G protein, thus increasing the agonist on-rate and subsequent Gi/o activation. Page 9, line 244. “Intriguingly, the ….” Please remove “intriguingly”. Page 10 & 11, section entitled “The extent of dimer dissociation upon APLNR activation is ligand-dependent”, once again, please apply a more conservative interpretation of the data. Page 10, lines 260-262. “These findings suggest that ligand-specific modulation of dimeric conformations may contributes to varying degrees of dimer dissociation upon activation.” Previously the authors imply that transition to the monomeric state enhances the rate of signalling, yet they do not assess whether the rate of signalling is differentially altered for the two ligands, which might be expected. Could the authors please comment? Figure 4c. The depletion of cholesterol from cells could conceivably alter availability of the G protein to the receptor and/or activity of adenylate cyclase (most isoforms are integral membrane proteins). As a minimum, the authors need to provide parallel experiments where the effect of cholesterol depletion on basal adenylate cyclase activity is assesses in cells that do not express the apelin receptor. Discussion should be rewritten with a more conservative interpretation of the data. I have been asked to comment on whether the authors have adequately addressed the comments by reviewer 2. On balance they have done a good job of this.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "I like to congratulate the authors for a very interesting and conclusive manuscript. All the concerns of Reviewer 2 have been addressed by the authors in their rebuttal letter. Except for the last point (how common might by the dimer/monomer transition observed for APLNR for class A and class B receptors?) the authors accordingly included changes in the manuscript. I would recommend adding a short statement in the discussion section addressing the above mentioned question. I enjoyed reading the manuscript and found only one additional point, not mentioned by the reviewers so far that in my opinion is so important that it needs clarification: To be sure that cholesterol depletion increases basal activity of APLNR it is necessary to show in the absence of APLNR how much cholesterol depletion affects forskolin-induced cAMP accumulation. Only if cholesterol depletion reduces cAMP accumulation to larger extent in APLNR expressing cells compared to non-APLNR expressing cells we can be sure that the results reflect an increase in basal activity of the receptor. Minor point: there is a little flaw in the figure legend of Fig. 4d: Dose-response curves for AMG986-induced cAMP accumulation in WT and F1013.24A….. If I’m correct, it should state: Concentration-response curves for the AMG986-mediated inhibition of forskolin-induced CAMP accumulations in WT and F1013.24A, ….."}, "author_response": "We thank the referees for their valuable time in reviewing our manuscript and the constructive suggestions that they have provided. We have carefully taken these comments into consideration in preparing a revised version of our manuscript. Please find below a point-by-point response to the referees with our responses in blue and the reviewers’ comments in black. Reviewer #1 (Remarks to the Author): Authors have clearly resolved all of my concerns in the revised manuscript. However, I strongly suggest that authors should add references to the revised line 272~288 (that correspond to Q#2) to provide clarity to the readers. Response: We thank the reviewer for the helpful comments, which have helped us improve the manuscript. We have added the relevant references in the corresponding sections as follows: “To date, multiple agonist-bound APLNR dimer structures in the presence or absence of G-protein have been resolved1,2. Accordingly, we compared the APLNR structures resolved in this study with previously reported structures. Pairwise alignment of the entire dimeric assemblies revealed relatively small Cα root mean square deviation (RMSD) values (<1.0 Å) among G-protein-free dimers (Supplementary Fig. 7a). In contrast, Cα RMSDs between structures with and without G-protein coupling exhibited larger deviations (1.0 to 2.5 Å), largely attributable to conformational shifts in TM6 and H8 (Supplementary Fig. 7a). Moreover, Cα RMSDs among G-protein-coupling dimers also showed relatively larger deviations (1.0 to 2.1 Å), suggesting that different ligands induce distinct dimer conformations in the active state (Supplementary Fig. 7a). To assess the extent of dimer dissociation, we compared the Cα RMSDs of protB aligned on protA (Supplementary Fig. 7b). Comparison between G-protein-bound and G-protein-free dimers revealed a remarkably wide range of Cα RMSDs (2.3 to 6.3 Å), particularly in comparisons involving the ELA–Gi complex (PDB: 7W0N)1, further indicating different ligands induce varying degrees of dimer dissociation during activation (Supplementary Figs. 7b-d).” Reviewer #3 (Remarks to the Author): I have been asked to review this manuscript after preliminary revision due to the withdrawal of review #2. The manuscript by Ji et al. describes twelve cryo-EM assemblies in detergent micelles of engineered apelin receptor with two different (non-physiological) agonists (AMG986 and JN241-9) with and without engineered Galphai/Beta1/gamma2 heterotrimer. The authors then use these twelve assemblies to construct a narrative on possible conformational transitions for the apelin receptor during activation. They use the molecular snapshots provided by these cryo-EM assemblies to argue for a particular reaction pathway. There is no way to know whether the proposed relationship between the reported assemblies represents the pathway that occurs in their highly reconstituted and engineered system, much less in either a recombinant cellular setting or physiologically. The supporting cell-based assay data is heavily normalized (minimum 2 normalization steps), which hampers independent assessment by the reader, and in several cases lacks appropriate controls to allow interpretation. Throughout, the findings from their cryo-EM assemblies are referred to as if they represent some physiological ground truth, which is a serious, unsupported, over-interpretation. I urge the authors to apply a much more conservative interpretation of their data. I also note that the novelty of the manuscript is undermined by existing publications of apelin receptor structures reported elsewhere, including by these authors. Response: We sincerely thank the reviewer for this thoughtful critique and agree that our structural findings should be interpreted with greater caution. In response, we have carefully revised the manuscript to moderate statements that may have overstated the physiological relevance of our results. Specifically, definitive statements regarding “the” activation pathway have been replaced with more conservative interpretation such as “potential conformational transitions” observed in our experimental system. While we acknowledge the inherent limitations of our engineered and reconstituted system, the twelve cryo-EM structures obtained under various ligand and G-protein conditions provide a unique series of molecular snapshots. Furthermore, these static snapshots are complemented by time-dependent signaling assays, which provide correlative evidence of the dynamic transition process. Within this in vitro framework, the combined data reveal a continuum of conformational states, allowing us to propose a plausible model for dynamic monomer-dimer transition in ligand-induced apelin receptor activation at atomic resolution. Rather than defining a universal physiological pathway, our objective is to generate testable hypotheses and mechanistic insights based on the structural and functional diversity we have observed. 1. I would strongly suggest that the term “aggregation”, which the authors appear to use to mean dimerization / oligomerization should be replaced with either of these terms. Aggregation has a specific meaning in biochemistry, which is: the accumulation of misfolded or intrinsically disordered proteins into (typically) insoluble, non-functional complexes. Response: The reviewer is correct to highlight this critical distinction in biochemical terminology. We sincerely appreciate the feedback and have revised the manuscript accordingly. The term “aggregation” has been replaced with “dimerization/oligomerization” to properly describe the specific protein-protein interactions observed in our study. Understanding whether GPCRs (outside Class C) do form dimers in physiological settings, and if they do, what the functional consequence is, remains an unresolved question in the field. Unfortunately, the field is largely divided into dogmatic camps of “GPCR dimerization is a general phenomenon with important physiological outcomes” and “GPCR dimerization is an artefact, with no relevance at all”. Despite the large amount of literature on the subject, conclusions remain equivocal, due to a fairly large body of literature that relies on transfected cell studies using various proximity measures that are not powered to detect the difference between close proximity and protein:protein interaction AND the difficulty in performing definitive experiments in physiologically meaningful settings. There is no teleological reason to believe GPCRs form dimers, unlike nuclear hormone receptors (dimerization required for DNA binding), receptor tyrosine kinases (dimerization required for transphosphorylation) or enzymes (dimerization provides positive or negative allostery to enable physiologically relevant sensitivity to changes in substrate availability). On the other hand, it is attractive to think that novel behaviors could emerge through the formation of GPCR heterodimers (although there is much evidence, particularly from the 1990’s to suggest signal crosstalk as a mechanism for new behaviors). In this setting, new publications on either side of this debate ought to be subject to careful critical analysis. 2. Introduction: Page 2, lines 37-40 “GPCRs serve as central hubs in cellular signal transduction, ensuring the complexity and precision of downstream signaling through their structural versatility, characterized by diverse aggregated forms and dynamic conformational changes.” This is a meaningless word-salad, adding nothing to the paper. I would recommend removing. Response: We thank the reviewer for this valuable comment. Following your suggestion, the manuscript has been revised to remove this sentence. 3. Page 2, lines 41-42 “Class A GPCRs exist in a dynamic equilibrium between monomeric and oligomeric forms”. Of the data cited none provide evidence for a dynamic equilibria between oligomeric states, a single figure in reference 6 (figure 3) has data that supports the contention that oxytocin receptors may form dimers in native tissue. The remainder of the citations rely on data that have alternative interpretations. I am concerned that there is an uncritical perpetuation of data over-interpretation supported by poor-quality and often careless experiments. Here are alternative interpretations for the common experimental types presented in these figures: • Co-immunoprecipitation. With the inclusion of all relevant controls (particularly for membrane proteins that have a propensity to aggregate), co-immunoprecipitation should only be used to conclude that 2 proteins are present as part of a complex, direct interaction cannot be concluded. Absence of data from native tissue should be viewed with scepticism. Antibodies for GPCRs are frequently un-specific. • Confocal microscopy showing “co-localisation”. Under standard techniques in an optimally adjusted system with a high NA objective the point-spread-function of GFP/Alexa488 is ~140 nm in the x-y and 380 nm in the z with a volume of about 4 x 10^6 nm^3 (obviously this gets bigger with longer wavelength). A typical GPCR occupies a volume of about 70 nm^3 , thus over 50,000 GPCRs can fit into a single PSF. Data is frequently reported where GPCRs do not appear to be localized to the plasma membrane, which is inconsistent with known properties. Antibodies for GPCRs are frequently un-specific. • BRET or FRET (resonance energy transfer techniques). Generally, only applicable in transfected cell studies. Relies on the assumption that direct interaction is required for resonance energy transfer. This is clearly not true, given the widespread use of bystander BRET to measure (for example) GPCR trafficking. Saturation BRET remains problematic as controls are difficult. If partner proteins for both donor and acceptor traffic to the same membrane subdomains, the readout for this assay will appear to indicate a specific interaction. Unless controls for collisional (random) BRET signals have equivalent expression (on a per-cell basis) to the test AND have comparable trafficking, interpretation is ambiguous. • Altered signalling output in the presence of a second GPCR. This could equally occur due to signal crosstalk. • Mutational analysis of putative dimerization interface (mostly TM bundle mutations, typically altering signalling output). Even if carefully controlled for expression level it is usually not possible to exclude the possibility that the mutation alters transmission of ligand binding event through the GPCR to its intracellular partner. Response: We thank the reviewer for their critical assessment, which has allowed us to significantly enhance our work. We agree that our original statement was an overgeneralization of the cited literature. Accordingly, we have revised the sentence to ensure greater precision and alignment with the published evidence as follows: “While Class C GPCRs are obligate dimers for their function, Class A GPCRs may exist as monomers or oligomers3-8, exhibiting distinct agonist affinity, efficacy, as well as the signaling profiles and pharmacological functions9-12.” 4. Page 3 ,lines 52-55 “….supported by single-molecule imaging that confirms dimeric formation, and it is the first Class A GPCR whose stable dimeric structures have been captured by cryo-EM^8,15,16.” Reference 8 is provided to the contention that apelin receptor can interchange between dimers and monomers in cells (including using single molecule imaging). This reference is problematic, clustering (single molecule experiments, figure 1) lack appropriate controls (e.g. monomeric cell-surface receptor). If taken at face value, there is no equivalence between clustering and dimerization, this is below the resolution of the experiment. Even if dimerization is accepted there is not necessarily and equivalence between physical co-localisation and functional dimerization. Figure 2a, heavily cropped and manipulated western blots, non-equivalent loading of “mix” control, full blots not provided in supplementary information. Panels 2b&c are much more problematic, Oxytocin receptor is used as a ‘negative’ control in BRET experiments (i.e. as a receptor that does not dimerise), this is despite the fact that Reference 5 specifically demonstrates oxytocin receptor dimers by both FRET (weak evidence) but also by ligand allostery on native membranes (strong evidence), dimerization of oxytocin receptor is also reported in PMID: 27420737. Figure 3 & 4, no negative OR positive controls with both donor and acceptor included. Figure 5 uses interference peptides to infer TM1,2,3 & 4 as parts of the dimerization interface, this conflicts with data from PMID 35817871 (e.g. 7W0L) where TM4 plays no part and TM2 has only a single residue within a distance that could support an interaction (also noting that this is consistent with the present publication) bringing into question this methodology. The remaining figures also lack appropriate controls. I would suggest rephrasing to more accurately reflect the ambiguity of data: “APLNR is a model system that may be suitable for investigating the dynamic transition between monomeric and dimeric forms of class A GPCR, supported by transfected cell studies, and cryo-EM^8,15,16.” Response: Thank you for your valuable suggestions. We have revised the manuscript accordingly as follows: “APLNR is a model system that may be suitable for investigating the dynamic transition between monomeric and dimeric forms of class A GPCR, supported by both transfected cell studies and cryo-EM structures1,2,6.” 5. The clinical trial and published data on AMG986/Azelaprag is presented in a way that the reader might think that biased agonism and/or dimeric/monomeric receptor signalling profile is the reason for the clinical trial discontinuation (page 3, lines 71-74): “However, a Phase 2 clinical trial evaluating AMG986 was recently discontinued owing to potential adverse effects, highlighting the necessity to explore its molecular mechanism in stimulating various apelin receptor forms.” This does not seem to be the most parsimonious interpretation as the clinical trial was terminated due to ~5% of patients in the Azelaprag arm having blood markers consistent with liver toxicity (no patients in control arm). The size of the trial suggests that this is, in fact, an Azelaprag side effect, however the most likely cause is off-target liver toxicity, not an undesirable on-target effect, as implied by the authors. I suggest removing the second half of this sentence and replace with: ““However, a Phase 2 clinical trial evaluating AMG986 was recently discontinued owing to potential off-target liver toxicity.” Response: We thank the reviewer for raising these important points. We have revised the manuscript accordingly as follows: “However, a Phase 2 clinical trial evaluating AMG986 was recently discontinued owing to potential off-target liver toxicity.” 6. No observation of transitions between different conformations is made in this manuscript. This would require NMR, single molecule FRET or HDX-MS (if the transitions are slow enough), or MD-simulations could be conducted to model transitions. None of these are performed in this manuscript, only static snapshots are determined with no way of knowing whether these occur in a particular order on a reaction pathway OR whether some are off-pathway conformations. Page 4, lines 85-87: “We therefore directly observed the stepwise transition and conformational changes of APLNR dimers towards monomers from the apo to the active state.” I suggest rephrasing to: “We therefore directly observed a range of conformational states of APLNR dimers and monomers, both apo and active states, which provide potential pathways for conformational transition between these states.” Response: We thank the reviewer for the insightful feedback. We have revised the manuscript accordingly as follows: “We therefore directly observed a range of conformational states of APLNR dimers and monomers, both apo and active states, which provide potential pathways for conformational transition between these states.” 7. Results: Page 4, line 96 “…. network formed by F101^3.24 plays a …..” Here I assume the 3.24 refers to the Ballesteros-Weinstein numbering system for TM helices of GPCRs, please explain this to the reader and include the appropriate reference. I suggest “…. network formed by F101^3.24 (where the superscript refers to the Ballesteros-Weinstein transmembrane numbering system (ref)) plays a …..”. Reference is: Ballesteros, J.A., and Weinstein, H. (1995). Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors. Methods Neurosci. 25, 366–428. https://doi.org/10.1016/S1043-9471(05)80049-7. Response: We appreciate the reviewer's careful reading and astute observations. In response, we have revised the manuscript as follows: “Previous structural studies of the APLNR have indicated that the π-π interactions network formed by F1013.24 (where the superscript refers to the Ballesteros-Weinstein transmembrane numbering system13) plays a central role in stabilizing the dimeric interface1.” 8. Page 4, lines 102-104: “AMG986 showed roughly 10-fold greater potency in activating various G-proteins, including Gi, Gq and G13, as well as β-arrestin, compared to JN241-9.” Please provide statistical analysis to support this difference. The appropriate analysis is to derive the pEC50 from each biological repeat, use this to derive a mean and error then test for difference between pEC50s (one-way ANOVA). It is more appropriate to display SD rather than SEM, since the former is an estimate of the biological variance. I note that supplemental file “583494_1_related_ms_11086941_sz33ll” contains individual columns that appear to sum to the total of technical replicates multiplied by biological repeats. Could the authors please confirm that they have no conflated technical and biological replicates? While Nature Communications makes no formal recommendation on use of error bars (see:https://www.nature.com/collections/qghhqmandhttps://www.nature.com/articles/nmeth.2659), best practice for this type of data would be SD. I also note that the double normalisation (individual baseline normalization, followed by conversion to fold over baseline, followed by conversion to % of WT-AMG896 Emax) has the potential to obscure differences in G protein activation whilst also potentially accentuating differences between ligands. I would prefer to see data where the final normalization step is removed. Response: We thank the reviewer for the insightful comments regarding the statistical analysis and data normalization for the potency comparison between AMG986 and JN241-9. We have carefully considered the points raised and provide the following clarifications: 1. The reviewer emphasizes the importance of robust statistical analysis. As suggested, we have now performed a formal statistical comparison of the pEC values derived 50 from individual biological replicates (n ≥ 3). This analysis, conducted using one-way ANOVA, confirms that the difference in potency between AMG986 and JN241-9 is statistically significant (p < 0.05), thereby quantitatively supporting the stated ~10-fold difference (Fig. R1). Furthermore, the source data have been provided as part of our submission, in full compliance with the Nature Communications policy on source data (https://www.nature.com/ncomms/submit/how-to-submit). 2. We appreciate the reviewer’s note on error bars. We chose to present the data as the mean ± SEM (Standard Error of the Mean) for the following reason: The primary goal of our experiment was to estimate and compare the mean potency (pEC ) of the 50 ligands across our biological replicates. The SEM is a direct measure of the precision of this estimated mean and is therefore most appropriate for making inferences about the population mean from our sample. While SD accurately describes the variability within our sample, our conclusion is focused on the difference between the mean potencies of the two compounds, for which the SEM is the conventional metric. 3. Regarding the normalization strategy, we agree that the final normalization step (to % of WT-AMG986 Emax) accentuates the difference in efficacy between the ligands. This was applied to directly visualize their relative maximal signaling capabilities, a key parameter in functional pharmacology. This approach is well-established for highlighting differences in efficacy and is consistent with methodologies used in other publications1,14. Reply figure 1. Dose-response curves and pEC analysis of ligands-triggered 50 signaling pathways of APLNR. a-c, The effects of two agonists on WT and partially dimer-disrupted mutant F101A. Dose-response curves show AMG986- and JN241-9-induced G (a), G (b) and G (c) i q 13 signaling were measured by NanoBiT assay in HEK293T cells. d, Dose-response curves show the duration of cAMP signaling response induced by 10 μM of each ligand. e, Dose-response curves for AMG986- and JN241-9-induced cAMP accumulation measured by GloSensor cAMP assay in HEK293T cells. f, β-arrestin2 recruitment were measured by BRET assay. Data are presented as mean ± SEM with a minimum of three technical replicates, which were performed in triplicates each. g, The pEC values corresponding to the dose-response curves in a-c and e, f are 50 summarized. Data are presented as pEC ± SEM with a minimum of three technical 50 replicates, which were performed in triplicates each. 9. Page 4/5, lines 105-106. “Additionally, AMG986 induced a more rapid activation rate than JN241-9 (Supplementary Fig. 1d).” Firstly, please comment on the magnitude of difference, looking at the data this is very small and is hard to envision why one would think this is of any biological relevance. Secondly, please provide analysis to support that this difference is statistically significant. Third, as with the above comment, I suggest SD. The axis of supp fig 1d is labelled “cAMP accumulation %”, this is the % of what? For all cAMP data, are the authors confident that sensor has not been saturated? For example, was a cell permeant cAMP analoge (e.g. 8-CPT) included at the end of the assay? Response: We thank the reviewer for these insightful questions regarding the cAMP kinetics data in Supplementary Fig. 1d. Please find our point-by-point responses below. 1. The “cAMP accumulation (%)” represents the values normalized to the cAMP level of WT-AMG986 at the 0-minute time point, set as 100%. While the absolute numerical difference may appear modest, AMG986 consistently demonstrated a lower percentage of cAMP accumulation than JN241-9 at every measured time point. This consistent and earlier separation of the curves indicates a faster activation rate for AMG986, a characteristic we believe is biologically relevant in the context of kinetic signaling profiles. 2. We appreciate the critical point regarding sensor saturation. We are confident that the cAMP sensor was operating within its linear dynamic range throughout our experiments for the following reasons: Our assay measures G-mediated inhibition of i cAMP. We first applied Forskolin to elevate cAMP to a stable plateau, creating a wide and robust window for observing inhibitory effects. This design prevents the issue of a limited “downward range” that could occur at basal cAMP levels and ensures the upper limit of the signal is within the sensor’s range. Furthermore, the cAMP accumulation traces show distinct and statistically significant steady-state plateaus between the WT and F101A groups following ligand application. These confirms that the sensor was not saturated at the lower end of the signal and was accurately reporting ligand-mediated cAMP changes. 10. Page 5, lines 107-108. “Though negligible differences in EC50 or Emax from G-protein signaling, the dimer-reduced form (F1013.24A) showed a higher Emax than the dimeric form in the β-arrestin signaling (Supplementary Fig. 1f).” Please refer to the magnitude of change and also provide analysis supporting a statistically significant difference. Without experiments performed under parallel conditions to quantitate cell-surface expression this change in Emax could be as a result of altered receptor number. Elsewhere (supp fig 8) the authors report cell surface expression by ELISA, however it is not clear from the methods that this was performed under equivalent transfection conditions to the β-arrestin assay. It is not clear from the methods whether cell integrity would be maintained using the fixation technique used and no negative control is included. As this data stands, interpretation is ambiguous. I suggest a cautious interpretation of this result: “Though negligible differences in EC50 or Emax from G-protein signaling, the dimer-reduced form (F1013.24A) showed a higher Emax than the dimeric form in the β-arrestin signaling (Supplementary Fig. 1f), consistent with previous results^15, which may be indicative that the dimer exhibits G-protein bias compared to the monomer.” Response: We thank the reviewer for raising these important points and providing valuable suggestions. The observed greater Emax for the F1013.24A than WT in the β-arrestin recruitment assay, while modest, was a consistent and reproducible effect. Additionally, the cell-surface ELISA was performed under equivalent transfection conditions to those used in the β-arrestin assays. This assay, involving formaldehyde fixation followed by detection of GPCRs on the cell surface using specific antibodies, is a well-established and widely used method for determining cell-surface expression of GPCRs15-17. This method has been validated to preserve cell integrity and specifically label plasma membrane-localized receptors. Furthermore, we have revised the manuscript accordingly as follows: “Though negligible differences in EC 50 or Emax from G-protein signaling, the dimer-reduced form (F1013.24A) showed a higher Emax than the dimeric form in the β-arrestin signaling (Supplementary Fig. 1f), consistent with previous results, which may be indicative that the dimer exhibits G-protein bias compared to the monomer.” 11. The apparent unity hill-slope of both cAMP and β-arrestin responses has no detectable change between the WT receptor and the F101A mutant (proposed monomeric form), nor does there appear to be a detectable change in pEC50. This suggests that in the dimeric form of the apelin receptor, there is no detectable co-operativity (negative or positive) for either ligand. Thermodynamically, it is hard to envision how dimerization would NOT alter ligand binding (see REF 6 and Birdsall, N.J.M. (2010). Class A GPCR heterodimers: evidence from binding studies. Trends Pharmacol. Sci. 31, 499–508. https://doi.org/10.1016/j.tips.2010.08.003.). A statement to this effect should be included immediately after the above sentence: “No detectable change in either Hill-slope or pEC50 is evident for either AMG986 or JN241-9 between WT and monomeric (F101A) receptors, suggesting that ligand binding allostery, if present, is not detectable.” Response: We thank you for your valuable perspective. We have revised the description in our manuscript as follows: “Though negligible differences in EC or Emax from G- 50 protein signaling, the dimer-reduced form (F1013.24A) showed a higher Emax than the dimeric form in the β-arrestin signaling (Supplementary Fig. 1f), consistent with previous results1, which may be indicative that the dimer exhibits G-protein bias compared to the monomer. No detectable change in either Hill-slope or pEC is 50 evident for either AMG986 or JN241-9 between dimers (WT) and the dimer-reduced form (F1013.24A), suggesting that ligand binding allostery, if present, is not detectable.” 12. Page 5 lines 110-112. “Moreover, cAMP accumulation results showed that the dimer-reduced form exhibits higher constitutive activity than that of dimeric form (Supplementary Figs. 1d-e).” Please refer to the magnitude of change and also provide analysis supporting a statistically significant difference. I would also suggest softening this statement, as without data showing that receptor expression is equivalent AND that the WT v’s F101A mutant do not alter (for example) adenylate cyclase or phosphodiesterase expression it is not possible to attribute the change in baseline to receptor constitutive activity. A solution would be to perform assays using an inverse agonist for Apelin receptor, although I am not aware of one. I would suggest the following: “Moreover, cAMP accumulation results showed that the dimer-reduced form appear to exhibit ~25% higher constitutive activity than that of dimeric form (Supplementary Figs. 1d-e, p<0.ABC).” Response: We thank you for your valuable comments We have revised the description in our manuscript as follows: “Moreover, cAMP accumulation results showed that the dimer-reduced form appear to exhibit ~25% higher constitutive activity than that of dimeric form (Supplementary Figs. 1d-e, p < 0.001).” The curve for used for these concentration-response curves is not stated, either in the results or in the methods. Please include the equation used in the methods section. If a 3 parameter logistic equation has been used, this should be justified as it would fail to take into account 2:2 stoichiometry. Response: We thank the reviewer for pointing out this omission. The following sentence has been added to the Methods section: “Concentration-response curves were fitted using the ‘log (agonist) vs. response (Three parameters)’ model in GraphPad Prism 8 to obtain EC₅₀ values.” In addition, the three-parameter logistic model was selected as it is a standard and reliable statistical method for characterizing agonist efficacy and obtaining EC₅₀ values in GPCR signaling assays. 13. Page 5, line 120-122. “Here, we successfully achieved high-resolution cryo-EM structures of agonists-bound class A GPCR in various conformations without G-protein, delineating the dynamic process of activation.” Again, these are snapshots and there is no data that addresses whether they are all on the reaction pathway and if they are which order they occur. I suggest a more conservative interpretation of this data: “Here, we successfully achieved high-resolution cryo-EM structures of agonists-bound class A GPCR in various conformations without G-protein, providing potential insights into the dynamic process of activation.” Response: We thank you for your valuable suggestion. We have revised the description in our manuscript as follows: “Here, we successfully achieved high-resolution cryo-EM structures of agonists-bound class A GPCR in various conformations without G-protein, providing potential insights into the dynamic process of activation.” 14. Page 5, line 127. “Fortunately, we identified ….” Fortunately is unnecessary and pejorative, I suggest to start the sentence “We identified …..” Response: We thank you for your helpful comments. We have removed this unnecessary word in the manuscript accordingly. 15. Page 5, lines 133-135. “Additionally, the two AMG986-bound states of APLNR dimer without G-protein are distinguished by unique conformational changes of TM6 and helix 8 (H8) in one protomer (termed by LBS2a and LBS2b, respectively) (Supplementary Fig. 2b).” Conformation changes are assumed not measured, these are snapshots and there is no reason to believe they represent a particular reaction co-ordinate. I suggest: “Additionally, the two AMG986-bound states of APLNR dimer without G-protein are distinguished by unique conformations of TM6 and helix 8 (H8) in one protomer (termed by LBS2a and LBS2b, respectively) (Supplementary Fig. 2b).” Response: We thank you for your thoughtful comments. We have revised the description in our manuscript as follows: “Additionally, the two AMG986-bound states of APLNR dimer without G-protein are distinguished by unique conformations of TM6 and helix 8 (H8) in one protomer (termed by LBS2a and LBS2b, respectively) (Supplementary Fig. 2b).” 16. Page 6, lines 136-137. “These results indicate that the conformations are in a ligand-dependent manner.” This sentence doesn’t make sense in English, I suggest: “These results indicate that the different ligands may select different conformational states.” Response: We thank the reviewer for pointing out this issue. We have revised the description in our manuscript as follows: “These results indicate that the different ligands may induce different conformational states.” 17. Page 6, lines 143-146. “Data processing revealed that AMG986-bound dimeric APLNR–Gi1 complexes adopt two conformations in protB (termed by ACT2a and ACT2b, respectively), aligning with those structures without G-protein (Supplementary Fig. 3b).” is there meant to be an alignment between G-protein free and G-protein bound complexes in Supp Fig 3b? I don’t see one? Response: We sincerely thank the reviewer for this careful observation. The original statement was indeed unclear. Supplementary Fig. 3b illustrates the two conformations (ACT2a and ACT2b) of the AMG986-bound dimeric APLNR–G complexes in protB, while i1 the corresponding two conformations (LBS2a and LBS2b) for the G-protein-free complexes are shown in Supplementary Fig. 2b. To prevent any misunderstanding, we have revised the description in the manuscript as follows: “Data processing revealed that AMG986-bound dimeric APLNR–G complexes adopt two conformations in i1 protB (termed by ACT2a and ACT2b, respectively), aligning with those structures without G-protein (Supplementary Figs. 2b and 3b).” 18. Page 6, lines 149-151. “To elucidate the structural transition of APLNR from its apo to active state, we first compared two AMG986-bound states of APLNR dimer without G-protein (LBS2a and LBS2b) against the apo state to identify structural differences (Fig.2a)”. Implicit in this analysis is the assumption that the reaction co-ordinate for a GPCR to lower the energy barrier for nucleotide exchange at its cognate G protein involves binding of the agonist prior to binding of the G protein. What is the evidence that this is the case for the Apelin Receptor? The majority of Class A GPCRs where agonist binding has been studied such that G protein-coupling can be modulated by the addition of non-hydrolysable GTP analogues show that the high affinity state is one in which the G protein is engaged with the receptor. There are a range of Class A receptors where there is evidence of pre-coupling of G protein to receptor. Implicit in the ternary complex model is that G protein coupling may occur prior to agonist binding. Where is has been studied, apo GPCRs explore conformations that are either intermediate to activation or similar to fully active (ternary) conformations. Lastly, large scale MD simulations of many Class A GPCRs (including the Apelin receptor) supports the notion that these receptors explore active-like states in the absence of agonist binding. Taken together, and in the absence of data that supports agonist binding first, I think it is faulty logic to ascribe the agonist bound, G protein free state as a transition to the active state. It could be this, but it could also be a state that is not on the reaction pathway. Seed references are below: 1. Aranda-García, D., Stepniewski, T.M., Torrens-Fontanals, M. et al. Large scale investigation of GPCR molecular dynamics data uncovers allosteric sites and lateral gateways. Nat Commun 16, 2020 (2025). https://doi.org/10.1038/s41467-025-57034-y 2. Kim TH, Chung KY, Manglik A, Hansen AL, Dror RO, Mildorf TJ, Shaw DE, Kobilka BK, Prosser RS. The role of ligands on the equilibria between functional states of a G protein-coupled receptor. J Am Chem Soc. 2013 Jun 26;135(25):9465-74. doi: 10.1021/ja404305k. Epub 2013 Jun 14. PMID: 23721409; PMCID: PMC3763947. 3. Nobles M, Benians A, Tinker A. Heterotrimeric G proteins precouple with G protein-coupled receptors in living cells. Proc Natl Acad Sci U S A. 2005 Dec 20;102(51):18706-11. doi: 10.1073/pnas.0504778102. Epub 2005 Dec 13. PMID: 16352729; PMCID: PMC1317907. 4. De Lean A, Stadel JM, Lefkowitz RJ. A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase-coupled beta-adrenergic receptor. J Biol Chem. 1980 Aug 10;255(15):7108-17. PMID: 6248546. Response: We sincerely thank the reviewer for this careful observation. Following the professional opinion, we change our statement to “We first compared two AMG986-bound states of APLNR dimer without G-protein (LBS2a and LBS2b) against the apo state to identify structural differences (Fig.2a)”. 19. Figure 2 and supplementary figure 4. There are a number of relatively subtle changes in side chain position/rotation that are ascribed as being part of the activation pathway. The apparent resolution of the relevant parts of the receptor appears to be between 2 and 2.2A (supp fig 2 and 3), which should allow good confidence in assigning these. The maps on which these models are based and the models themselves are not provided (or perhaps I can’t find them?), so it is not possible as a reviewer to independently verify the side-chain movements being presented. I could not find an editorial policy on making these data (maps and models) available to reviewers, but this would be best-practice. Response: We thank the reviewer for raising this point. The cryo-EM density maps and atomic models for all structures reported in this study have now been uploaded as supplementary files and are available for review. We hope this allows for a full and independent assessment of the side-chain conformations described. 20. Page 7, line 178. “Surprisingly, we observed ….”. No need for the pejorative surprising. Present the data and let the reader judge whether it is “surprising”. Response: We thank you for your helpful comments. We have removed this unnecessary word in the manuscript accordingly. 21. Page 7/8 lines 194-197. “These findings indicate that AMG986 can independently induce activation of a single protomer within the APLNR dimer, in contrast to JN241-9, which necessitates G-protein engagement for receptor activation.” These are snapshots of dynamic complexes and it is not clear that induced fit is in operation, I would encourage more cautious interpretation. I also note that if the authors interpretation is correct, one would predict that AMG986 would possess higher efficacy compared with JN241-9. I see no evidence of this. Response: Thanks for the cautious opinion. Based on amounts of publications, the TM6 outward movement is a clear indicator of GPCR activation18-20. Although the cryo-EM structures are snapshot results, as the reviewer accurate statement, the active conformation of a single protomer in the dimer induced by AMG986 is robust. Following the suggestions of the reviewer, we have revised the manuscript accordingly as follows: “These findings indicate that AMG986 can independently induce one protomer within the APLNR dimer to adopt an active conformation, in contrast to JN241-9, which requires G-protein engagement to promote receptor activation.” Moreover, following the guidance of reviewer, the G-protein and β-arrestin activation experiments measured by NanoBit and BRET assays fit for our “snapshot” results, further proving our conclusion. 22. Pages 8 & 9. Section entitled: “G-protein coupled activation of APLNR facilitates the dimer-to-monomer transition” again, order of events is not known, it could be that dimer to monomer transition occurs first then G protein binding occurs then agonist binding occurs (or pretty much any other variation on this). Please apply a more cautious approach to interpretation OR provide evidence from an endogenous setting that supports one or other orders for the reaction pathway. Response: We sincerely thank the reviewer for this cautious clarification. The order of G-protein coupling and dimer-to-monomer transition is not the main conclusion of this manuscript. To diminish the ambiguity, we switch the section title into “G-protein coupling promotes a dimer-to-monomer transition of APLNR.” 23. Page 9, lines 236-240. “Furthermore, the kinetic GloSensor cAMP assay revealed that signaling response associated with monomeric activation (F101A) was greater than that of the dimeric form (WT) during the initial 15 minutes. However, both responses converged thereafter, further suggesting that most dimers transition into monomers upon receptor activation.” Please quantitate the difference in reaction rate and perform statistics to support that this. If this data quantitively and statistically supports faster cAMP inhibition kinetics if could be interpreted that the F101A mutant has higher propensity to pre-couple to its cognate G protein, thus increasing the agonist on-rate and subsequent Gi/o activation. Response: We thank the reviewer for their insightful comments and the proposed mechanistic interpretation. We agree that the data support the conclusion that the F101A mutant may have a higher propensity for pre-coupling to its cognate G protein. The cAMP accumulation curves illustrate that F101A consistently demonstrated a lower percentage of cAMP accumulation than WT at every measured time point, suggesting its greater signaling response. We fully agree with the reviewer’s interpretation that this faster cAMP inhibition kinetics likely reflects a higher pre-coupling efficiency of the F101A mutant, leading to an increased agonist on-rate and subsequent Gi/o activation. 24. Page 9, line 244. “Intriguingly, the ….” Please remove “intriguingly”. Response: We thank you for your helpful comments. We have removed this unnecessary word in the manuscript accordingly. 25. Page 10 & 11, section entitled “The extent of dimer dissociation upon APLNR activation is ligand-dependent”, once again, please apply a more conservative interpretation of the data. Response: Thanks for the cautious opinion. First based on the cryo-EM structures, the activation of a single protomer in the dimer is only induced by AMG986 leading to greater extent of dimer dissociation. Moreover, the time-dependent cAMP accumulation assay result fits for the ligand-dependent dimer dissociation differences. 26. Page 10, lines 260-262. “These findings suggest that ligand-specific modulation of dimeric conformations may contributes to varying degrees of dimer dissociation upon activation.” Previously the authors imply that transition to the monomeric state enhances the rate of signalling, yet they do not assess whether the rate of signalling is differentially altered for the two ligands, which might be expected. Could the authors please comment? Response: As the reviewer mentioned, the rate of cAMP accumulation is shown in Supplementary Fig. 1d, AMG986 demonstrates a more rapid decline in cAMP levels compared to JN241-9, which supports the conclusion regarding ligand-dependent effects. 27. Figure 4c. The depletion of cholesterol from cells could conceivably alter availability of the G protein to the receptor and/or activity of adenylate cyclase (most isoforms are integral membrane proteins). As a minimum, the authors need to provide parallel experiments where the effect of cholesterol depletion on basal adenylate cyclase activity is assesses in cells that do not express the apelin receptor. Response: Thank you for your constructive suggestions. To determine whether cholesterol depletion specifically enhances the basal activity of APLNR, we performed additional experiments measuring cAMP levels in cells transfected with either an empty vector or wild-type APLNR, under both control conditions and after treatment with 3 mM methyl-β-cyclodextrin (MβCD) to deplete membrane cholesterol. Cholesterol depletion alone resulted in a reduction in forskolin-induced cAMP accumulation even in empty vector-transfected cells, likely because most isoforms of adenylate cyclase, being integral membrane proteins, exhibit decreased activity upon cholesterol removal. Notably, the decrease in cAMP levels was significantly greater in cells expressing APLNR compared to those harboring the empty vector (Fig. R2). This pronounced reduction supports the conclusion that cholesterol depletion specifically promotes APLNR dimers dissociation into monomers, thereby enhancing its constitutive activity. Reply Fig. 2 Cholesterol clusters in dimeric interface play a significant role in stabilizing dimeric formation. (a) Histogram depicting the effect of cholesterols removal by MβCD at 0 and 3 mM on the basal activity of cAMP accumulation in WT and empty vector, as measured by GloSensor cAMP assay in HEK293T cells. (b) cAMP levels were normalized to control (0 mM MβCD, set as 100%). Treatment with 3 mM MβCD caused a greater reduction in cAMP levels in WT APLNR–expressing cells than in empty vector controls, indicating that cholesterol depletion specifically enhances the basal activity of APLNR. Data are presented as mean ± SEM with a minimum of three technical replicates, which were performed in triplicates each. Data are presented as mean ± SEM. *P<0.05, **P<0.01 and ***P<0.001 by two-way ANOVA followed by Dunnett's multiple comparisons test. I have been asked to comment on whether the authors have adequately addressed the comments by reviewer 2. On balance they have done a good job of this. Reviewer #4 (Remarks to the Author): I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts. Reviewer #5 (Remarks to the Author): I like to congratulate the authors for a very interesting and conclusive manuscript. 1. All the concerns of Reviewer 2 have been addressed by the authors in their rebuttal letter. Except for the last point (how common might by the dimer/monomer transition observed for APLNR for class A and class B receptors?) the authors accordingly included changes in the manuscript. I would recommend adding a short statement in the discussion section addressing the above mentioned question. Response: Thanks for the insightful comments, we have incorporated a statement about dimer/monomer transition research in the discussion section, and we hope these revisions are helpful. 2. I enjoyed reading the manuscript and found only one additional point, not mentioned by the reviewers so far that in my opinion is so important that it needs clarification: To be sure that cholesterol depletion increases basal activity of APLNR it is necessary to show in the absence of APLNR how much cholesterol depletion affects forskolin-induced cAMP accumulation. Only if cholesterol depletion reduces cAMP accumulation to larger extent in APLNR expressing cells compared to non-APLNR expressing cells we can be sure that the results reflect an increase in basal activity of the receptor. Response: Thank you for your constructive suggestions. We have put detailed explanation for this concern in the question 27 of reviewer 3. 3. Minor point: there is a little flaw in the figure legend of Fig. 4d: Dose-response curves for AMG986-induced cAMP accumulation in WT and F1013.24A….. If I’m correct, it should state: Concentration-response curves for the AMG986-mediated inhibition of forskolin-induced CAMP accumulations in WT and F1013.24A, ….. Response: We thank the reviewer for pointing out this issue. We have revised the description in this figure legend as follows: “Concentration-response curves for the AMG986-mediated inhibition of forskolin-induced cAMP accumulations in WT and F1013.24A.” 1 Yue, Y. et al. Structural insight into apelin receptor-G protein stoichiometry. Nature structural & molecular biology 29, 688-697, doi:10.1038/s41594-022-00797-5 (2022). 2 Yue, Y. et al. Structural insights into the regulation of monomeric and dimeric apelin receptor. Nature communications 16, 310, doi:10.1038/s41467-024-55555-6 (2025). 3 Ginés, S. et al. Dopamine D1 and adenosine A1 receptors form functionally interacting heteromeric complexes. Proceedings of the National Academy of Sciences of the United States of America 97, 8606-8611, doi:10.1073/pnas.150241097 (2000). 4 Albizu, L. et al. Time-resolved FRET between GPCR ligands reveals oligomers in native tissues. Nature chemical biology 6, 587-594, doi:10.1038/nchembio.396 (2010). 5 Angers, S. et al. Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proceedings of the National Academy of Sciences of the United States of America 97, 3684-3689, doi:10.1073/pnas.97.7.3684 (2000). 6 Cai, X., Bai, B., Zhang, R., Wang, C. & Chen, J. Apelin receptor homodimer-oligomers revealed by single-molecule imaging and novel G protein-dependent signaling. Scientific reports 7, 40335, doi:10.1038/srep40335 (2017). 7 Liu, J. et al. Biased signaling due to oligomerization of the G protein-coupled platelet-activating factor receptor. Nature communications 13, 6365, doi:10.1038/s41467-022-34056-4 (2022). 8 Fricke, F., Beaudouin, J., Eils, R. & Heilemann, M. One, two or three? Probing the stoichiometry of membrane proteins by single-molecule localization microscopy. Scientific reports 5, 14072, doi:10.1038/srep14072 (2015). 9 Prinster, S. C., Hague, C. & Hall, R. A. Heterodimerization of g protein-coupled receptors: specificity and functional significance. Pharmacological reviews 57, 289-298, doi:10.1124/pr.57.3.1 (2005). 10 Jordan, B. A., Trapaidze, N., Gomes, I., Nivarthi, R. & Devi, L. A. Oligomerization of opioid receptors with beta 2-adrenergic receptors: a role in trafficking and mitogen-activated protein kinase activation. Proceedings of the National Academy of Sciences of the United States of America 98, 343-348, doi:10.1073/pnas.98.1.343 (2001). 11 Bellot, M. et al. Dual agonist occupancy of AT1-R-α2C-AR heterodimers results in atypical Gs-PKA signaling. Nature chemical biology 11, 271-279, doi:10.1038/nchembio.1766 (2015). 12 Callén, L. et al. Cannabinoid receptors CB1 and CB2 form functional heteromers in brain. The Journal of biological chemistry 287, 20851-20865, doi:10.1074/jbc.M111.335273 (2012). 13 Ballesteros, J. A. & Weinstein, H. in Methods in Neurosciences Vol. 25 (ed Stuart C. Sealfon) 366-428 (Academic Press, 1995). 14 Zhuang, Y. et al. Molecular recognition of morphine and fentanyl by the human μ-opioid receptor. Cell 185, 4361-4375.e4319, doi:10.1016/j.cell.2022.09.041 (2022). 15 Xiao, P. A.-O. et al. Tethered peptide activation mechanism of the adhesion GPCRs ADGRG2 and ADGRG4. 16 Lin, H. A.-O. et al. Metabolic signaling of ceramides through the FPR2 receptor inhibits adipocyte thermogenesis. 17 Cheng, J. et al. Autonomous sensing of the insulin peptide by an olfactory G protein-coupled receptor modulates glucose metabolism. 18 Weis, W. I. & Kobilka, B. K. The Molecular Basis of G Protein-Coupled Receptor Activation. 19 Zhou, Q. et al. Common activation mechanism of class A GPCRs. eLife 8, doi:10.7554/eLife.50279 (2019). 20 Zhang, M. et al. G protein-coupled receptors (GPCRs): advances in structures, mechanisms, and drug discovery. Signal transduction and targeted therapy 9, 88, doi:10.1038/s41392-024-01803-6 (2024)."}, {"round": 3, "reviewer_comments": {"reviewer_3": "I thank the authors for taking on board most of my comments and amending their manuscript accordingly. I am please that the authors performed the cholesterol depletion experiment on cells lacking the Apelin receptor (supp fig 8). Their conclusion, however, that the slightly smaller magnitude difference in basal cAMP for Aplein receptor containing cells v's control cells and between cholesterol replete and cholesterol depleted conditions (compared with a >2 fold effect of cholesterol depletion by itself) supports a role for cholesterol in Apelin signalling is, in my opinion, over-reach. Some data interpretation remains careless and unsystematic, including poor use of statistics, however I leave it in the editors hands to decide how this will be handled.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "The authors did a good job on revising the manuscript and most importantly showed the effect of cholesterol depletion on cAMP levels without the influence of receptors. Whereas I appreciate this additional information, I'm not supporting the way how the authors deal with this important additional results: The authors show the effect of cholesterol depletion on cAMP with and without Apelin receptors (Suppl. Figure 8d), where they see a reduction of cAMP levels to values of 38% without receptors. With expression of the receptors the decrease is enhanced to 30%. Even though this difference in 38% versus 30 % was significant, the wording in the text: \"We found that depletion of cholesterol led to a decrease in the value of cAMP accumulation without affecting the expression of receptors (Fig. 4c and Supplementary Fig. 8b). This decrease reflects an enhancement in basal activity, indicating that the cholesterol depletion promotes the dissociation of dimers into monomers.\" is misleading as it suggests that the major decrease in cAMP is due to increase in the constitutive APLNR activity, which is not the case, as cholesterol depletion causes a major decrease cAMP even in cells not expressing the receptor (reduction to 38 % of initial values). The authors need to rephrase it in a way that misinterpretation is avoided. The data of Fig 4c need to be shown together with those of supplemental Figure 8d in order to be meaningful. Looking carefully at the data, which show that the cAMP Signal most likely due to stronger amplification is quite a bit more sensitive to apelin receptor activation compared to the Gi protein dissociation assay, I can clearly see why this assay is well suited to detect constitutive receptor activity. However, I’m puzzled that given the higher amplification, why it does not level out equally at high agonist concentrations, whereas the Gi protein assay does. This is important, because mutations in the apelin receptor that reduce cholesterol binding induce a shift of the cAMP signals over the whole range of agonist concentrations (Figure 4d), which would indicate that the mutation would not only increase the constitutive activity of the apelin receptor but also the maximal agonist efficacy. This is something that I would not expect to see in the more sensitive cAMP glow-sensor assay but rather in the Gi dissociation assay (less amplification and overexpressed G proteins). What do the authors think about this and how do they explain and discuss this issue in their manuscript. specific newly identified issue: The legend of Y axis of Supplemetary Figurte 1 a-c in my opinion is wrong or at least misleading: It should be assoziation and not dissoziation as with high agonits they will see an increase in G protein dissoziation and not a decrease. Otherwise the axis needs to be inverted."}, "author_response": "We thank the referees for their valuable time in reviewing our manuscript and the constructive suggestions that they have provided. We have carefully taken these comments into consideration in preparing a revised version of our manuscript. Please find below a point-by-point response to the referees with our responses in blue and the reviewers’ comments in black. Reviewer #3 (Remarks to the Author): I thank the authors for taking on board most of my comments and amending their manuscript accordingly. I am please that the authors performed the cholesterol depletion experiment on cells lacking the Apelin receptor (supp fig 8). Their conclusion, however, that the slightly smaller magnitude difference in basal cAMP for Aplein receptor containing cells v's control cells and between cholesterol replete and cholesterol depleted conditions (compared with a >2 fold effect of cholesterol depletion by itself) supports a role for cholesterol in Apelin signalling is, in my opinion, over-reach. Some data interpretation remains careless and unsystematic, including poor use of statistics, however I leave it in the editors hands to decide how this will be handled. Response: We sincerely appreciate the reviewer’s careful evaluation and valuable comments. We agree with the reviewer’s point that the slightly smaller magnitude difference in basal cAMP between APLNR-expressing and control cells under cholesterol-depleted conditions, compared to the substantial effect of cholesterol depletion itself, should be interpreted with more caution. As shown in our Supplementary Figure 8d, cholesterol depletion led to a pronounced 70% reduction in cAMP levels in APLNR-expressing cells, while a similarly strong reduction (62%) was observed in non-transfected control cells. This indicates that the effect of cholesterol depletion on cAMP is not solely attributable to enhanced basal activity of APLNR. In response to the reviewer’s comment, we have revised the corresponding statement in the manuscript to reflect a more tempered interpretation. The original description: “This decrease reflects an enhancement in basal activity, indicating that the cholesterol depletion promotes the dissociation of dimers into monomers.” has been modified to: “This decrease suggests a possible enhancement in basal activity, which would further suggest that the cholesterol depletion may promote the dissociation of dimers into monomers.” Reviewer #4 (Remarks to the Author): I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts. Reviewer #5 (Remarks to the Author): The authors did a good job on revising the manuscript and most importantly showed the effect of cholesterol depletion on cAMP levels without the influence of receptors. Whereas I appreciate this additional information, I'm not supporting the way how the authors deal with this important additional results: The authors show the effect of cholesterol depletion on cAMP with and without Apelin receptors (Suppl. Figure 8d), where they see a reduction of cAMP levels to values of 38% without receptors. With expression of the receptors the decrease is enhanced to 30%. Even though this difference in 38% versus 30 % was significant, the wording in the text: \"We found that depletion of cholesterol led to a decrease in the value of cAMP accumulation without affecting the expression of receptors (Fig. 4c and Supplementary Fig. 8b). This decrease reflects an enhancement in basal activity, indicating that the cholesterol depletion promotes the dissociation of dimers into monomers.\" is misleading as it suggests that the major decrease in cAMP is due to increase in the constitutive APLNR activity, which is not the case, as cholesterol depletion causes a major decrease cAMP even in cells not expressing the receptor (reduction to 38 % of initial values). The authors need to rephrase it in a way that misinterpretation is avoided. The data of Fig 4c need to be shown together with those of supplemental Figure 8d in order to be meaningful. Response: Thank you for your constructive suggestions. We have put detailed explanation for this concern in the question of Reviewer #3. Furthermore, the data of Fig. 4c have been shown together with those of Supplementary Fig. 8d as suggested by the reviewer. Looking carefully at the data, which show that the cAMP Signal most likely due to stronger amplification is quite a bit more sensitive to apelin receptor activation compared to the Gi protein dissociation assay, I can clearly see why this assay is well suited to detect constitutive receptor activity. However, I’m puzzled that given the higher amplification, why it does not level out equally at high agonist concentrations, whereas the Gi protein assay does. This is important, because mutations in the apelin receptor that reduce cholesterol binding induce a shift of the cAMP signals over the whole range of agonist concentrations (Figure 4d), which would indicate that the mutation would not only increase the constitutive activity of the apelin receptor but also the maximal agonist efficacy. This is something that I would not expect to see in the more sensitive cAMP glow-sensor assay but rather in the Gi dissociation assay (less amplification and overexpressed G proteins). What do the authors think about this and how do they explain and discuss this issue in their manuscript. Response: Thank you for your thoughtful comment. The reviewer rightly noted that the cAMP accumulation assay, due to its signal amplification, is well suited for evaluating constitutive receptor activity under basal (ligand-free) conditions. We agree that the apparent shift in the raw cAMP accumulation curves could be misinterpreted as a change in both constitutive activity and maximal agonist efficacy. However, this is largely a matter of data normalization and interpretation. To assess constitutive activity, we compared absolute cAMP levels at the lowest ligand concentration, which clearly showed an increase in basal signaling for the cholesterol-binding mutant F101A (Fig. R1a). For evaluating maximal agonist efficacy, a different normalization approach is required. Specifically, we normalized the cAMP response at each agonist concentration to the basal (agonist-free) cAMP level for the same receptor variant, and then expressed the data relative to the response at the lowest agonist concentration (set as 100%) (Fig. R1b). This normalization aligns the dose-response curve baselines, permitting a direct assessment of maximal plateaus, which demonstrates near-identical maximal agonist efficacy between the F101A mutant and the wild-type receptor (WT). Consistent with the methodology of the NanoBiT Gi dissociation assay (Fig. R1c), this approach yields the same definitive finding: the cholesterol-binding mutation does not alter maximal agonist efficacy. Reply figure 1. Dose-response curves of ligands-triggered signaling pathways of APLNR. a, b, Dose-response curves for AMG986- and JN241-9-induced cAMP accumulation measured by GloSensor cAMP assay in HEK293T cells. Data are presented normalized to the cAMP response of WT at the lowest ligand concentration, which was set as 100% (a). Data are presented normalized to the basal (agonist-free) cAMP level for each agonist concentration, and then expressed the data relative to the response at the lowest agonist concentration (set as 100%) (b). c, Dose-response curves for agonists induced Gi signaling measured by NanoBiT assay in HEK293T cells. specific newly identified issue: The legend of Y axis of Supplemetary Figurte 1 a-c in my opinion is wrong or at least misleading: It should be assoziation and not dissoziation as with high agonits they will see an increase in G protein dissoziation and not a decrease. Otherwise the axis needs to be inverted. Response: Thank you for your careful attention to the labeling in Supplementary Figs. 1a-c. We have corrected this error accordingly. The Y-axis label has now been updated to “Gα-Gγ association”, which accurately describes that a decrease in the luminescence signal indicates the dissociation of the Gα and Gγ subunits upon receptor activation."}, {"round": 4, "reviewer_comments": {"reviewer_3": "The conclusion that the interaction of cholesterol with the Apelin receptor alters either basal activity or interconversion between dimer and monomer is not supported by these experiments. This conclusion remains overstated. The decision on rigor of statistical approaches (or lack thereof) lies with the editor.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "The authors have addressed all of my concerns. I have no further requests."}, "author_response": "We sincerely appreciate the reviewer’s careful evaluation and valuable comments. We agree with the reviewer’s point that the slightly smaller magnitude difference in basal cAMP between APLNR-expressing and control cells under cholesterol-depleted conditions, compared to the substantial effect of cholesterol depletion itself, should be interpreted with more caution. As shown in our Supplementary Figure 8d, cholesterol depletion led to a pronounced 70% reduction in cAMP levels in APLNR-expressing cells, while a similarly strong reduction (62%) was observed in non-transfected control cells. This indicates that the effect of cholesterol depletion on cAMP is not solely attributable to enhanced basal activity of APLNR. In response to the reviewer’s comment, we have revised the corresponding statement in the manuscript to reflect a more tempered interpretation. The original description: “This decrease reflects an enhancement in basal activity, indicating that the cholesterol depletion promotes the dissociation of dimers into monomers.” has been modified to: “This decrease suggests a possible enhancement in basal activity, which would further suggest that the cholesterol depletion may promote the dissociation of dimers into monomers.”"}]

Decision letter