
==== Front
Cell Discov
Cell Discov
Cell Discovery
2056-5968
Springer Nature Singapore Singapore

39223120
724
10.1038/s41421-024-00724-6
Article
Molecular mechanism of prolactin-releasing peptide recognition and signaling via its G protein-coupled receptor
Li Yang 12
Yuan Qingning 3
He Xinheng 12
Zhang Yumu 1
http://orcid.org/0000-0002-6425-3387
You Chongzhao 12
Wu Canrong 1
Li Jingru 13
http://orcid.org/0000-0002-6829-8144
Xu H. Eric eric.xu@simm.ac.cn

12
http://orcid.org/0000-0002-7175-5174
Zhao Li-Hua zhaolihuawendy@simm.ac.cn

124
1 grid.9227.e 0000000119573309 State Key Laboratory of Drug Research, Center for Structure and Function of Drug Targets, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
2 https://ror.org/05qbk4x57 grid.410726.6 0000 0004 1797 8419 University of Chinese Academy of Sciences, Beijing, China
3 https://ror.org/04523zj19 grid.410745.3 0000 0004 1765 1045 School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, Jiangsu China
4 grid.16821.3c 0000 0004 0368 8293 Translational Center for Medicinal Structural Biology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
3 9 2024
3 9 2024
2024
10 9125 1 2024
6 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by/4.0/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
Prolactin-releasing peptide (PrRP) is an RF-amide neuropeptide that binds and activates its cognate G protein-coupled receptor, prolactin-releasing peptide receptor (PrRPR), also known as GPR10. PrRP and PrRPR are highly conserved across mammals and involved in regulating a range of physiological processes, including stress response, appetite regulation, pain modulation, cardiovascular function, and potentially reproductive functions. Here we present cryo-electron microscopy structures of PrRP-bound PrRPR coupled to Gq or Gi heterotrimer, unveiling distinct molecular determinants underlying the specific recognition of the ligand’s C-terminal RF-amide motif. We identify a conserved polar pocket that accommodates the C-terminal amide shared by RF-amide peptides. Structural comparison with neuropeptide Y receptors reveals both similarities and differences in engaging the essential RF/RY-amide motifs. Our findings demonstrate the general mechanism governing RF-amide motif recognition by PrRPR and RF-amide peptide receptors, and provide a foundation for elucidating activation mechanisms and developing selective drugs targeting this important peptide–receptor system.

Subject terms

Cryoelectron microscopy
Molecular biology
National Natural Science Foundation of China (32371255, 32071203 to L.H.Z., 32130022 and 82121005 to H.E.X.); Natural Science Foundation of Shanghai (23ZR1475200 to L.H.Z.); the National Key R&D Program of China (2022YFC2703105 to H.E.X., 2019YFA0904200); CAS Strategic Priority Research Program (XDB37030103 to H.E.X.); Shanghai Municipal Science and Technology Major Project (2019SHZDZX02 to H.E.X.); Shanghai Municipal Science and Technology Major Project (H.E.X.); the Young Innovator Association of CAS (Y2022078 to L.H.Z.); the Lingang Laboratory (LG-GG-202204-01 to H.E.X.); State Key Laboratory of Drug Research (SKLDR-2023-TT-04 to H.E.X.).issue-copyright-statement© Center for Excellence in Molecular Cell Science, CAS 2024
==== Body
pmcIntroduction

Neuropeptides, with over 100 identified types, are the most abundant signaling molecules in the nervous system. Among these, RF-amide peptides, identifiable by their C-terminal Arg-Phe-NH2 (RF-amide) motif, play vital roles as neurotransmitters and neuromodulators. They are involved in a range of physiological processes, including metabolism, pain perception, and reproduction1. In mammals, the RF-amide peptide subfamily comprises prolactin-releasing peptide (PrRP), neuropeptide FF (NPFF), kisspeptin, RF-amide-related peptide (RFRP), and pyroglutamylated RF-amide peptide (QRFP). These neuropeptides interact with five G protein-coupled receptors (GPCRs): prolactin-releasing peptide receptor (PrRPR, GPR10)2, neuropeptide FF receptor 1/2 (NPFF1R/NPFF2R, GPR147/GPR74)3,4, kisspeptin receptor (KISS1R, GPR54)5, and pyroglutamylated RF-amide peptide receptor (QRFPR, GPR103)6. Notably, PrRP and PrRPR are highly conserved across mammals and are integral in regulating behaviors and physiological processes through the endocrine system.

PrRP, first isolated from bovine hypothalamus, has two biologically active isoforms, PrRP31 and PrRP20. PrRP20 is a 20-amino acid peptide with amidation at its C-terminus (Fig. 1a)2. Identified as the endogenous ligand for GPR10 through reverse pharmacology, PrRP20 shows a high affinity to this receptor2,7. PrRPR, primarily triggering Gq/11 signaling pathways, is also suggested to engage Gi/o pathways (Fig. 1b)8. PrRPR is abundantly expressed in the thalamic reticular nucleus, hypothalamic nuclei, area postrema, and nucleus of the solitary tract9,10. These regions regulate functions like stress, appetite, pain and reproduction. Though initially posited to govern prolactin release, prevailing evidence suggests that PrRP/PrRPR regulate food intake, energy metabolism11,12, pain perception13, stress response14, endocrine15, sleep and rhythm16,17, and confer neuroprotection18–20.Fig. 1 Overall structures of PrRP20–PrRPR–Gq and PrRP20–PrRPR–Gi complexes.

a Sequence of PrRP20. Residues are shown in green, magenta, light blue, and yellow, which represent polar, acidic, basic, and hydrophobic amino acids, respectively. b Schematic illustration of G protein coupling of PrRPR activated by PrRP20. c, d Cryo-EM density maps (left panel) and cartoon representation (right panel) of PrRP20–PrRPR–Gq–scFv16 (c) and PrRP20–PrRPR–Gi–scFv16 (d) complexes. Components of PrRPR complexes are colored as indicated.

Structure-activity relationship studies on PrRP analogs, focusing on the C-terminal RF-amide motif, have been crucial for understanding the interaction between PrRP and PrRPR2,9,21. These studies suggest that key amino acids for this interaction are located at the ligand’s C-terminus, and alterations in this region can significantly impact PrRP’s activity. This finding has important implications for the development of therapeutic applications targeting obesity, type 2 diabetes, sleep disorders, and epilepsy22,23.

However, despite their therapeutic potential, the complex interaction network between RF-amide peptides and their receptors24–26, along with a lack of structural information, has hindered a comprehensive understanding of their mechanisms. This study aims to bridge this gap by reporting cryo-electron microscopy (cryo-EM) structures of PrRP20-bound PrRPR coupled to Gq and Gi, respectively, shedding light on peptide agonist recognition and receptor activation. Our findings clarify the mechanism governing RF-amide motif recognition by PrRPR and provide a foundation for the rational design of selective drugs targeting this important peptide–receptor system.

Results

Cryo-EM analysis and overall structure

To facilitate the expression of these complexes, two maltose-binding protein (MBP) tags were introduced at the C-terminus of the wild-type (WT) full-length receptor, which also served as an affinity purification tag27. In addition, we employed the NanoBiT strategy to stabilize the GPCR–G protein complex, wherein the large subunit (LgBiT) and its complementary high-affinity peptide (HiBiT) (SmBiT or peptide 86) were fused to the C-terminus of the receptor and Gβ subunit, respectively28,29. Two types of Gα proteins were used to assemble the PrRPR complex: engineered Gαq and Gαi30. Incubation of the endogenous ligand PrRP20 with membranes from cells co-expressing PrRPR and Gαq or Gαi heterotrimers in the presence of scFv16 enables efficient assembly of the PrRP20–PrRPR–Gαq and PrRP20–PrRPR–Gαi complexes, producing highly homogeneous complex samples for structural studies31.

The PrRP20–PrRPR–Gq–scFv16 and PrRP20–PrRPR–Gi–scFv16 complex structures were determined using single-particle cryo-EM, achieving global resolutions of 2.96 Å and 2.97 Å, respectively (Fig. 1c, d; Supplementary Figs. S1, S2 and Table S1). The EM maps enabled accurate model building of the receptor residues Q54NTD to G2595.72 and A2666.23 to V3518.59 (superscripts indicate Ballesteros-Weinstein numbering) for the PrRP20–PrRPR–Gq–scFv16 complex, and Q54NTD to V2565.69 and A2666.23 to V3518.59 for the PrRP20–PrRPR–Gi–scFv16 complex (Supplementary Fig. S3). These maps were also clear for most of the residues in the ligand and Gq or Gi heterotrimers, providing detailed information about the ligand-binding pocket and the receptor–G protein coupling interface (Fig. 1c, d; Supplementary Fig. S3).

PrRPR adopts a seven-transmembrane folding conformation typical for G protein recruitment, and PrRP20 presents an L-shaped conformation (Fig. 2a, b). The two complex structures are highly similar, with root mean square deviation (RMSD) values of 0.501 Å for the complex and 0.581 Å for the receptor. In both structures, the EM maps showed clarity for PrRP20, except for the N-terminal 3 amino acids, with its C-terminus inserted into the ligand-binding pocket, consistent with the previous structure-activity relationship studies2,9,21. Unlike the previously reported α-helical structure for the C-terminal residues A10–G13 and R15–R19 of PrRP2032,33, these residues instead adopted a more relaxed, curled conformation in our structure (Fig. 2a, b). In the activated-state structure of the neuropeptide Y (NPY) receptor with the highest homology to PrRPR, the C-terminal region of its ligand NPY exhibited similar conformational changes34,35, indicating a common activation conformation for the two neuropeptides. Furthermore, an unexpected α-helical structure was found in the originally flexible N-terminal residues N5–Y9 of PrRP20, corresponding to the chemical environment of the sub-pocket at the receptor’s top.Fig. 2 Recognition of PrRP20 by PrRPR.

a, b Cross-section of the PrRP20-binding pocket in Gq-coupled PrRPR (a) and Gi-coupled PrRPR (b). The three-view drawing of density maps of PrRP20 are shown as grey meshes, and PrRP20 are displayed as orange (Gq-coupled) or pink (Gi-coupled) cartoons and sticks. c–h Detailed interactions of PrRP20 with residues in PrRPR. The binding sites of F20 (c), R19 (d), R15 (e), P16 and R15 (f), backbone of F20–V17 (g), R12, Y9 and W8 (h) are shown. Hydrogen bonds and salt bridges are depicted as red dashed lines. PrRP20 molecules are shown as cartoons and sticks. PrRPR is shown in blue (Gq-coupled) or green (Gi-coupled). i, j BRET2 assay to evaluate PrRP20-induced dissociation of heterotrimeric Gq protein (i) and Gi protein (j) (ΔpEC50 = pEC50 of PrRP20 to a specific PrRPR variant – pEC50 of PrRP20 to WT PrRPR; yellow column means ΔpEC50 ≤ –1; blue column means ΔpEC50 ≤ –2; red column means ΔpEC50 ≤ –3). Data are presented as means ± SEM; n = 3 independent samples, each consisting of triplicate measurements. Significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test. NA, no activity; ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001. Exact P values are provided in Supplementary Table S2. k Ligand RMSF of each residue in Gq-coupled (pink) and Gi-coupled (purple) PrRPR complexes.

Binding modes of PrRP20 for PrRPR

In the PrRP20–PrRPR–Gq–scFv16 and PrRP20–PrRPR–Gi–scFv16 structures, PrRP20 assumes a similar conformation within the ligand-binding pocket, defined by extracellular loops (ECLs) and transmembrane helices TM2, TM3, and TM5–TM7 of PrRPR (Fig. 2a, b). PrRP20 is organized into three distinct segments: the N-terminus (T1–I4), an α-helix (N5–Y9), and the extended C-terminus (A10–F20) (Fig. 1a). The C-terminus, forming an L-shaped arrangement with the other segments, inserts into the transmembrane helical bundle, crucial for receptor activation. At the bend located at R12, PrRP20 aligns its N-terminus and α-helix nearly parallel to the receptor’s extracellular surface. The α-helix interacts with ECL2, while the N-terminus faces the extracellular tip of TM4 (Fig. 2a, b).

At the base of the ligand-binding pocket, the conserved residues of RF-amide peptides, R19 and F20, create a sub-pocket (Fig. 2a, b). Here, the amide group of F20 engages in a polar network with C1132.57, T1172.61, Q1413.32, and H3217.39, indicating its importance in PrRP20’s affinity (Fig. 2c). The acidic nature of this network repels the negatively charged unacetylated C-terminus of PrRP202,9. On the opposite side, the hydrophobic pocket surrounding the side chain of F20 includes P1423.33, V1453.36, Y1463.37, L2295.42, L2946.51, and H2956.52 from TM3, TM5, and TM6, as confirmed by alanine mutagenesis analysis (Fig. 2c, i, j; Supplementary Table S2). In addition, R19 forms polar interactions with D3026.59, Y2255.38, and E21345.52. These interactions are crucial for peptide recognition (Fig. 2d), as indicated by a drastic reduction in PrRP20 potency upon mutation of these residues (Fig. 2i, j). Accordingly, our observations agree with the previous studies that demonstrated the importance of R19 and the acylated and bulky side chain of F20 for receptor activation9,21,36.

The interaction between PrRP20 and the upper sub-pocket of PrRPR, near the receptor’s extracellular surface, includes residues P16–W8 from PrRP20. In this interaction network, R15 is particularly essential9, as it interacts with receptor Y1202.64 (Fig. 2e). Y1202.64’s dual interaction with E21245.51 and π–π stacking with W12723.50 stabilizes the folding of the extracellular region (Fig. 2e). Mutations in these residues significantly reduce ligand efficacy in activating both Gq and Gi proteins (Fig. 2i, j). The upper sub-pocket is sealed off by residues including Y1202.64, E21245.51 and those surrounding P16 (R3016.58, F3137.31, and Q3177.35), with Q3177.35 anchoring PrRP20’s backbone through polar interactions (Fig. 2f, g). The Q3177.35A mutation most significantly reduced the potency of PrRP20-mediated Gq and Gi activation (Fig. 2i, j).

Deletion analysis of PrRP20’s N-terminal region revealed that the shortest effective analog is a C-terminal heptapeptide (I14–F20), but with substantially lower affinity9. This suggests that the α-helix and N-terminal region of PrRP20 may also contribute to receptor binding. In both structures, the intramolecular motif of W8, Y9, and R12 within PrRP20 stabilizes its α-helical structure (Fig. 2h). Additionally, hydrophobic interactions between W8 and the receptor’s ECL2 (L203ECL2, V208ECL2, L210ECL2) are pivotal, as alanine mutations in these residues diminish ligand efficacy (Fig. 2i). This finding aligns with the structure-activity relationship studies of the PrRP20 analog (W8–F20), which did not show reduced activity in vitro21.

We have developed molecular dynamics (MD) simulation systems for ligand–PrRPR–Gq and –Gi complexes, conducting three parallel simulations of 200 ns each to examine the dynamics. Initially, we assessed biased signaling through the estimation of binding free energies using MMGBSA. The binding free energies for Gq and Gi were calculated as –51.93 ± 6.99 kcal/mol and –44.15 ± 2.96 kcal/mol, respectively. These results support the notion of biased signaling towards Gq, demonstrating that the simulation trajectories are consistent with experimental findings. Further analysis involved calculating the root-mean-square fluctuation (RMSF) of all heavy atoms in the ligand for each residue, as depicted in Fig. 2k. The analysis revealed that Gi binding results in a more flexible ligand, particularly at residues T1, P2, and D3, consistent with the observed density loss. Notably, the regions beyond residue G13 displayed greater stability compared to the external segments (Fig. 2k).

Sequence alignment of PrRP across species shows the high conservation of the C-terminal tridecapeptide, especially in crucial ligand–receptor interaction residues, except for V17 (Supplementary Fig. S6a). This can be explained as V17 interacts with the receptor through its main chain peptide bond (Fig. 2g). Correspondingly, receptor residues interacting with this tridecapeptide are also highly conserved (Supplementary Fig. S6b, c), underscoring a conserved evolutionary mechanism for PrRP–PrRPR interaction across species.

Comparative analysis of the recognition mechanisms for RF/RY-amide motifs in PrRPR and other receptors

Our analysis expands to encompass other RF-amide peptides and the similar RY-amide peptides, examining their C-terminal motif interactions with respective receptors. PrRP20, part of the RF-amide family alongside NPFF, Kisspeptin, RFRP, and QRFP, shares the highly conserved C-terminal RF-amide (Supplementary Fig. S7a). We compared the amino acid residues of RF-amide peptide receptors (PrRPR, NPFF1R, NPFF2R, KISS1R, and QRFPR) to determine a common recognition mechanism for this motif (Supplementary Fig. S7b, c). Except for the substitution of H7.39 by the similarly polar residue Q7.39 in QRFPR, the residues C2.57, T2.61, Q3.32, and H7.39, key to the C-terminal amide recognition in PrRP20, are largely conserved, indicating a shared polar interaction network. However, variations exist around R19, with two hydrophobic residues mutated in KISS1R, but E45.52, Y5.38, and D/E6.59 remain conserved. The conservation around F20’s side chain is less stringent, but a general hydrophobic pocket was observed for all receptors, suggesting a common mechanism for recognizing the RF-amide motif. Therefore, our understanding of the interaction of PrRPR with the RF-amide may be applicable to other RF-amide peptide receptors.

The sequence homology between PrRPR and NPY receptors, specifically NPY1R, NPY2R, and NPY4R (Supplementary Fig. S8b), prompted a comparison between PrRP20 with their ligands, NPY and pancreatic polypeptide (PP), which possess a C-terminal RY-amide motif (Supplementary Fig. S8a). Structural comparisons of PrRP20–PrRPR complex with NPY–NPY1R, NPY–NPY2R, and PP–NPY4R complexes showed a high overlap in the binding posture of their ligands’ C-terminal R-F/Y residues (Fig. 3a). Sequence alignment indicates a certain level of conservation in the recognition patterns of NPYRs for the RY-amide motif, which are similar to the recognition mode of PrPRP for the RF-amide motif (Supplementary Fig. S8c).Fig. 3 Structural comparison of RF-amide motif and RY-amide motif interactions in PrRP20–PrRPR and NPY/PP–NPYRs complexes.

a Superimposition of structures of the PrRP20–PrRPR, NPY–NPY1R (PDB: 7X9A), NPY–NPY2R (PDB: 7X9B) and PP–NPY4R (PDB: 7X9C) complexes. b–e Binding mode of the RF-amide motif within the TMs of PrRPR (b) and RY-amide motif within the TMs of NPY1R (c), NPY2R (d), and NPY4R (e). Hydrogen bonds and salt bridges are depicted as red dashed lines.

Key residues like C2.57, T2.61, Q3.32, and H7.39, crucial for the activity of PrRP20 and NPY/PP, are conserved in both PrRPR and NPYRs, forming a polar network around the amide groups of F20 and Y36 (Fig. 3b–e; Supplementary Fig. S8c). However, differences emerge in the microenvironment around R19 and R35. While E45.52, Y5.38, and D6.59 remain highly conserved (Supplementary Fig. S8c), we observed that among NPYRs, only D6.59 forms a conserved polar interaction with R35 of NPY/PP (Fig. 3c–e). Notably, these interactions are disrupted by two key mutations in NPYRs: F/L4.60 and T5.39. The F/L4.60 mutation creates a steric clash with R35, pushing it away towards TM6. The T5.39 mutation enables R35 to form a new polar interaction with TM5 in NPY1R and NPY4R (Fig. 3c, e). Nevertheless, owing to the lesser steric hindrance of L4.60 in NPY2R, E20545.52 maintains its polar interaction with R35 (Fig. 3d).

The binding pockets for F20 of PrRP20 and Y36 of NPY/PP, though analogous, display different characteristics. In NPY1R and NPY4R, Y36’s hydroxyl group forms a polar interaction with Q5.46, critical for activity (Fig. 3c, e). However, this interaction is absent in NPY2R due to the L2275.46 mutation (Fig. 3d). In PrRPR, F20 lacks a hydroxyl group, preventing a similar interaction with T2335.46 (Fig. 3b). Interestingly, T2335.46 establishes a polar interaction with H2956.52, a feature absent in NPYRs. Additionally, Y3.37 in PrRPR, compared to T/Q3.37 in NPYRs, creates a more confined binding pocket for F20.

N6.55, highly conserved across PrRPR and NPYRs (Supplementary Fig. S8c), engages in different interactions. In PrRPR, it forms a polar interaction with the peptide bond between R19 and F20 of PrRP20 (Fig. 2g; Supplementary Fig. S9), while in NPYRs, it interacts with R33 of NPY/PP (Supplementary Fig. S9). At the equivalent position, the residue in PrRP20 is V17, which similarly forms an anchoring interaction with Q3177.35 in PrRPR (Fig. 2g). These discrepancies are due to sequence variations between PrRP20 and NPY/PP, excluding their RF/RY-amide motifs.

Activation mechanism of PrRPR

To analyze the activation mechanism, we performed structural comparisons of the PrRP20–PrRPR complexes with the antagonist-bound NPY1R, which affirms that PrRPR is in an activated state in both structures. This activation is highlighted by the outward displacement at the cytoplasmic end of TM6 (10.2 Å, measured at the Cα atom of D2696.26/R2546.26) and an inward movement at the cytoplasmic end of TM7 (1.6 Å, measured at the Cα atom of F3337.51/I3187.51), typical hallmarks of class A GPCR activation (Fig. 4a). Unlike the inactive conformation of NPY1R, PrRPR exhibits an outward shift at the extracellular end of TM3 (3.7 Å, measured at the Cα atom of F1383.29/P1173.29) and a lateral shift of TM6 towards TM5 (1.3 Å, measured at the Cα atom of D3026.59/D2876.59) (Fig. 4b, c). These structural changes are similar yet more pronounced than those in the activated NPY–NPY1R–Gi complex. Owing to the similarity of the conformations in both PrRP20–PrRPR complexes, we focus on the PrRP20–PrRPR–Gq complex to elucidate PrRPR’s activation mechanism.Fig. 4 Activation mechanism of PrRPR.

a–c Structure superposition of PrRPR–Gq and PrRPR–Gi complexes with the agonist (NPY)-bound NPY1R (PDB: 7X9A) and antagonist (BMS-193885)-bound NPY1R (PDB: 5ZBH). Side-view of the movement of TM6 and TM7 (a), TM3 (b) and top-view of the movement of TM6 (c) are shown. The movement directions of TMs in PrRPR relative to the inactive NPY1R are highlighted as red arrows. d–i Detailed interaction changes upon activation of PrRPR. Hydrogen bonds and salt bridges are depicted as red dashed lines. The movement orientations of amino acids and TMs are indicated as red arrows. Interactions between PrRP20 and residues located at the bottom of peptide-binding pocket (d). The possible clash is highlighted by a red dashed circle. The rearrangement of the residues in the PIF motif (e). The conformation comparison of PrRPR with antagonist-bound NPY1R (left panel) and PrRPR with agonist-bound NPY1R (right panel) (f, g). Conformational changes of the conserved “micro-switches” upon receptor activation, including ERY (h) and NPxxY (i) motifs.

At the base of the ligand-binding pocket, a hydrophobic lock consisting of V1453.36, Y1463.37, and H2956.52 prevents direct contact between PrRP20’s C-terminal residue F20 and the “Toggle Switch” residue W2916.48 (Fig. 4d). The interactions between F20 and TM3 residues in PrRPR, particularly the polar interaction of its amide group with Q1413.32, trigger an upward rotational change in Q1413.32, which initiate PrRPR activation. F20’s phenylmethyl group also forms extensive hydrophobic interactions with P1423.33, Y1463.37, and V1453.36 (Fig. 4d), causing an outward shift of TM3’s extracellular side and rearranging the side chains of the PIF motif (V1493.40, P2375.50, F2876.44) (Fig. 4e). This mechanism mirrors NPY1R’s primary activation pathway37.

Notably, we additionally revealed a unique activation pathway initiated by Y1463.37 in PrRPR. The substitution of NPY1R’s residues C1213.33 and T1253.37 by P1423.33 and Y1463.37 in PrRPR results in a larger displacement of TM3’s extracellular side. This shift facilitates an interaction between Y1463.37 with L2295.42 and T2335.46 on TM5 (Fig. 4f). Alanine substitution analysis of these amino acids, especially Y1463.37, confirms their crucial role in PrRPR activation (Fig. 2i, j). In contrast, the shorter side chain of T1253.37 in NPY1R is unable to interact with L2155.42 and Q2195.46, and Q2195.46 is firmly anchored within the helical bundle due to polar interactions with Y36 (Fig. 4f). The steric hindrance between TM3 and TM5 residues in PrRPR prompts TM5 to move horizontally towards TM6, allowing T2335.46 to interact with H2956.52 and induce a counter movement of TM6, which cannot be induced by Q2195.46 and T2806.52 in NPY1R (Fig. 4g). Moreover, the movement of H2956.52 may prompt conformational changes in W2916.48, leading to a significant outward displacement of TM6’s cytoplasmic end. Given the specificity of Y3.37 (Supplementary Figs. S7c and S8c), we propose that this activation mechanism is distinctive from those of the other RF/RY-amide peptide receptors.

Furthermore, other conserved residues in “micro-switches” (ERY and NPxxY motifs) in PrRPR undergo conformational changes, similar to those in the active-like state of NPY1R relative to the antagonist-bound NPY1R (Fig. 4h, i). These changes transmit the activation signal from PrRP20 to PrRPR’s cytoplasmic side for G protein coupling. Together, these structural insights reveal the intricate mechanism of PrRPR activation, emphasizing the nuanced differences and similarities in GPCR activation pathways.

PrRPR–Gq/Gi coupling mechanism

Our analysis reveals notable distinctions in the PrRPR–G protein complex structures, particularly in the conformations of TM6 and the α5 helix of the Gα subunit. In the PrRPR–Gi complex, TM6 exhibits an inward shift compared to PrRPR–Gq complex (2.0 Å, measured at the Cα atom of W2686.25), necessitating an adjustment of the α5 helix of the Gαi subunit into the transmembrane helical bundle (1.3 Å, measured at the Cα atom of E357/D357H5.22, where superscripts refer to the common Gα numbering system38) and towards intracellular loop 2 (ICL2, 5.4°) to avoid clashes (Fig. 5a). This adjustment is in contrast with other G protein-coupled class A GPCRs. For instance, TM6 in PrRPR–Gq complex shows a more significant outward shift (14.8°) compared to the 5HT2A–Gq complex, influencing the α5 helix orientation with a displacement of 1.2 Å (measured at the Cα atom of E357H5.22) at the C-terminus and a rotation of 3.5° at the N-terminus (Fig. 5b). Similarly, in PrRPR–Gi complex, an outward displacement (5.7°) of TM6 relative to NTSR1–Gi complex is accompanied by a tilt (10.0°) of the α5 helix towards ICL2 (Fig. 5c).Fig. 5 Gq/Gi coupling of PrRPR.

a An overall conformational comparison of Gq-coupled and Gi-coupled PrRPR. TM6, TM3, TM4 and ICL2 of receptors, as well as α5 helix of G proteins, are highlighted. b The conformational comparison of Gq-coupled PrRPR with Gq-coupled 5HT2A (PDB: 6WHA). c The conformational comparison of Gi-coupled PrRPR with Gi-coupled NTSR1 (PDB: 6OS9). d Alignment of the “wavy hook” in the extreme C-terminus (V/FH5.26–E/DH5.22) of Gαq and Gαi. Residues are shown in green, magenta, and yellow, which represent polar, acidic, and hydrophobic amino acids, respectively. e–h Distinct interaction patterns of residues from the “wavy hook” motif. Details of the interaction between PrRPR and V/FH5.26 (e), LH5.25 (f), N/GH5.24 (g), Y/CH5.23 and E/DH5.22 (h) of Gq and Gi subunits. Hydrogen bonds and salt bridges are depicted as red dashed lines. i, j BRET2 assay to evaluate PrRP20-induced dissociation of heterotrimeric Gq protein (i) and Gi protein (j) (Δspan = the span value of PrRP20 to a specific PrRPR variant – the span value of PrRP20 to WT PrRPR; yellow column means Δspan ≤ –50%; blue column means Δspan ≤ –80%). Data are presented as means ± SEM; n = 3 independent samples, each consisting of triplicate measurements; significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001. Exact span values are provided in Supplementary Table S3.

The interaction interface between the receptor cytoplasmic cavity and the extreme C-terminal “wavy hook” of the α5 helix (Fig. 5d), which thought to be one of the critical determinants of G protein selection39,40, varies between the PrRPR–Gq and PrRPR–Gi complexes. In the PrRPR–Gi structure, F361H5.26 faces spatial constraints from residues W2686.25 and R2726.29 of TM6 with its carbonyl interacting with H3398.47 in helix 8, while in the PrRPR–Gq structure, a more relaxed conformation exists due to the smaller V361H5.26 side chain and the rotated W2686.25 and R2726.29 (Fig. 5e). These conformational differences are attributed to the environmental variations surrounding V/FH5.26. However, L360H5.25, highly conserved across G protein families, occupies a similar hydrophobic pocket in both complexes, interacting with residues in TM3 and TM6 (Fig. 5f).

Additional contacts are observed between N359H5.24 of Gαq and H3398.47 and S3418.49 of PrRPR in the PrRPR–Gq complex, while only the interaction between the carbonyl of G359H5.24 in Gαi and H3398.47 of PrRPR is retained in the PrRPR–Gi complex (Fig. 5g). Similarly, the carbonyl groups of Y358H5.23 in PrRPR–Gq and C358H5.23 in PrRPR–Gi both interact with R1593.50. H3398.47A and R1593.50A mutations at the PrRPR–G protein interface significantly reduced the efficacy in activating both the Gq and Gi pathways (Fig. 5i, j). However, Y358H5.23 extends to TM2, TM3, and ICL2, forming a polar network with T952.39, D1583.49, and R170ICL2, which is absent in the case of C358H5.23 (Fig. 5h). In addition, only E357H5.22 in Gαq engages in a polar interaction with N932.37 of PrRPR, while D357H5.22 in Gαi does not (Fig. 5h), suggesting more extensive interactions between the receptor and Gq in the “wavy hook” region. Mutations of these residues in the receptor had a greater impact on Gq activation efficacy compared to Gi activation (Fig. 5i, j). These findings highlight the importance of the “wavy hook” in the selective coupling of PrRPR to G protein subtypes, particularly in its preference for Gq over Gi activation.

Discussion

PrRPR, the exclusive receptor in the PrRPR family, has no published crystal or cryo-EM structures to date. Our cryo-EM structures of PrRPR bound to PrRP20, coupled with heterotrimeric Gq and Gi proteins, respectively, provide an in-depth view of ligand recognition, receptor activation, and G protein coupling.

The ligand-binding studies highlight the critical role of the C-terminal RF-amide motif in PrRP20. Our findings show that this motif, especially the conserved residues R19 and F20, forms a unique polar and hydrophobic interaction network within PrRPR, crucial for ligand affinity and specificity. The conservation of this motif across different RF-amide peptide receptors suggests a universal mechanism for RF-amide recognition. The observed differences in the binding of the RY-amide motif between PrRPR and NPYRs provide structural insights into their selective ligand binding and functional diversity.

Our analysis of PrRPR’s activation mechanism reveals significant conformational changes in TM3, TM5, and TM6. The pivotal role of F20 in initiating these changes, particularly its interaction with Q1413.32 and Y1463.37, underscores a unique activation pathway in PrRPR, distinct from other class A GPCRs. The comparison with NPY1R highlights the receptor-specific nuances in GPCR activation.

The PrRPR–Gq/Gi coupling mechanisms, as revealed by our structures, underscore the importance of the Gα subunit’s α5 helix in determining G protein coupling specificity. The “wavy hook” region plays a critical role in the selective coupling of PrRPR to Gq and Gi. This selective coupling is crucial for the downstream signaling pathways mediated by PrRPR.

These findings have broad implications for our understanding of GPCR function and drug development. The detailed mechanism of ligand recognition and receptor activation could aid in the design of novel therapeutics targeting PrRPR-related physiological processes, including appetite regulation, stress response, and pain modulation. Moreover, our study contributes to the broader field of GPCR research by providing a structural template for understanding the interaction networks between RF-amide peptides and their receptors.

Materials and methods

Constructs

The full-length human PrRPR was cloned into the pFastBac (Thermo Fisher Scientific) vectors using the ClonExpress II One Step Cloning Kit (Vazyme Biotech), along with the N-terminal haemagglutinin (HA) signal peptide. LgBiT was added at the end of helix 8 with a 15-amino acid polypeptide linker in between, followed by a TEV protease cleavage site and an OMBP-MBP tag27. The engineered Gαq and Gαi chimeric proteins were designed as chimeras based on the mini-Gαs/q71 and mini-Gs/i143 skeletons30, respectively, with the N-terminal 1–18 amino acids and the GαAH domain of Gi1 replaced to facilitate binding to scFv1630,41–43. Human WT Gβ1, human Gγ2, and scFv16, as well as a Gβ1 fused with SmBiT at its C-terminus, were cloned into pFastBac vectors.

Insect cell expression

Human PrRPR, Gq chimera, Gi chimera, Gβ1, Gγ, and scFv16 were co-expressed in Sf9 insect cells using the baculovirus method (Expression Systems). Cell cultures were grown in ESF 921 serum-free medium (Expression Systems) to a density of 3 million cells per mL and then infected with six separate baculoviruses at a suitable ratio. The culture was collected by centrifugation 48 h after infection, and cell pellets were stored at −80 °C.

Complex purification

Cell pellets were thawed in 20 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM MgCl2, and CaCl2 supplemented with Protease Inhibitor Cocktail (TargetMol). For the PrRP20–PrRPR–Gq–scFv16 and PrRP20–PrRPR–Gi–scFv16 complexes, 10 μM PrRP20 (TGpeptide) and 25 mU/mL apyrase (Sigma) were added. The suspension was incubated for 1 h at room temperature, and the complex was solubilized from the membrane using 0.5% (w/v) lauryl maltose neopentylglycol (LMNG) (Anatrace) and 0.1% (w/v) cholesteryl hemisuccinate (CHS) (Anatrace) for 2 h at 4 °C. Insoluble material was removed by centrifugation at 70,000× g for 35 min, and the supernatant was purified by MBP affinity chromatography (Dextrin Beads 6FF, SMART Lifesciences). The resin was then packed and washed with 30 column volumes of 20 mM HEPES, pH 7.4, 100 mM NaCl, 0.01% (w/v) LMNG, 0.002% CHS, and 2 μM ligand. The complex sample was eluted in buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl, 0.01% (w/v) LMNG, 0.002% CHS, 2 μM ligand, and 10 mM maltose. Complex fractions were concentrated with a 100-kDa molecular weight cut-off (MWCO) Millipore concentrator for further purification. The complex was then subjected to size-exclusion chromatography on a Superdex 6 Increase 10/300 GL column (GE Healthcare) pre-equilibrated with size buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl, 0.00075% (w/v) LMNG, 0.00025% (w/v) GDN (Anatrace), 0.0002% CHS, and 2 μM ligand to separate complexes. Eluted fractions were evaluated by SDS-PAGE, and those consisting of receptor–Gq and receptor–Gi protein complexes were pooled and concentrated for cryo-EM experiments.

Cryo-EM grid preparation and data acquisition

Three microliters of the purified complexes at ~3 mg/mL and 11 mg/mL for PrRP20–PrRPR–Gq and PrRP20–PrRPR–Gi complexes, respectively, were applied onto a glow-discharged Quantifoil R1.2/1.3 300-mesh gold holy carbon grid. The sample was incubated for 5 s on the grids before blotting for 3 s under 100% humidity at 4 °C and then vitrified by plunging into liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). For both PrRPR complexes, cryo-EM data collection was performed on a Titan Krios G4 at a 300 kV accelerating voltage at the Advanced Center for Electron Microscopy, Shanghai Institute of Material Medica.

A total of 10,501 movies were collected for the PrRP20–PrRPR–Gq complex using a Falcon 4 detector in EER mode with a pixel size of 0.81 Å using the EPU. Movies were obtained at a dose rate of ~8 electrons per Å2 per second with a defocus ranging from –0.8 μm to −1.8 μm.

For PrRP20–PrRPR–Gi complex, a total of 7129 movies were collected by a Gatan K3 detector at a pixel size of 0.824 Å using the EPU. The micrographs were recorded in super-resolution mode at a dose rate of ~15 electrons per Å2 per second with a defocus ranging from −0.8 μm to −1.8 μm. The total exposure time was 2.35 s, and 36 frames were recorded per micrograph.

Cryo-EM data processing

MotionCor2 was used to perform the frame-based motion-correction algorithm to generate a drift-corrected micrograph for further processing, and CTFFIND4 provided the estimation of the contrast transfer function (CTF) parameters in CryoSPARC44,45.

For the PrRP20–PrRPR–Gq complex, template particle picker yielded 13,181,795 particle projections. The projections were subjected to reference-free 2D classification to discard poorly defined particles, producing 641,066 particle projections for further processing. This subset of particle projections was subjected to a round of Hetero Refinement with a pixel size of 0.81 Å. A selected subset containing 498,854 projections was used as a template for another round of template picking which yielded 5,616,578 particles in Relion. These projections were subjected to the same reference-free 2D classification and maximum likelihood-based three-dimensional classification, resulting in 826,485 and 429,903 particles, respectively. The particle projections generated by the two software were combined and duplicated particles were removed, resulting in 751,101 particles, which were used to obtain the final map using a pixel size of 0.81 Å. Further 3D classification without image alignment using a mask on the receptor produced one good subset accounting for 573,877 particles, which were subsequently subjected to 3D refinement and Bayesian polishing. The final refinement generated a map with an indicated global resolution of 2.96 Å and was subsequently post-processed by DeepEMhancer46.

For the PrRP20–PrRPR–Gi complex, template particle picker and blob picker yielded 10,203,281 and 5,594,186 particle projections, respectively. The two subsets of particle projections were subjected to reference-free 2D classification to discard poorly defined particles, producing 243,879 and 240,321 particle projections for further processing, respectively. These particle projections were combined and duplicated particles were removed, resulting in 401,694 particles in CryoSPARC. Further 3D classification without image alignment using a mask including only the receptor produced one good subset, which was subsequently subjected to 3D refinement, CTF refinement, and Bayesian polishing in Relion. The final refinement generated a map with an indicated global resolution of 2.97 Å and was subsequently post-processed by DeepEMhancer.

Model building

The initial templates of PrRPR were derived from Alphafold247. Models were docked into the EM density map using UCSF Chimera48. The initial models were then subjected to iterative rounds of manual adjustment based on the side-chain densities of bulky aromatic amino acids in Coot49 and automated refinement in PHENIX50. The final refinement statistics were validated using the module “comprehensive validation (cryo-EM)” in PHENIX50. The final refinement statistics are provided in Supplementary Table S1. All structural figures were prepared using Chimera48, Chimera X51, and PyMOL (https://pymol.org/2/).

BRET2 assays

AD293 cells (Agilent) were propagated in Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scientific), enriched with 10% fetal bovine serum (FBS, Thermo Fisher Scientific), and maintained at 37 °C in a 5% CO2 environment. Cells were then seeded in 6-well dishes at a density of 130,000–150,000 cells per mL and allowed to incubate overnight. On the next day, cells were transfected with plasmids at a 1:1:1:1 ratio for receptor:Gα-RLuc8:Gβ:Gγ-GFP252. PEI MAX 40 K (BIOHUB) was utilized to form complex with the plasmids at a ratio of 3 µL PEI per µg of plasmids, in OptiMEM (Gibco-Thermo Fisher Scientific) at a concentration of 10 ng plasmids per µL OptiMEM. After 24 h of incubation, the culture was harvested by centrifugation with PBS. The cell suspension was then allocated into a white 384-well plate at a volume of 30 µL per well, followed by the addition of 10 µL of freshly prepared 50 µM coelenterazine 400a (Yeasen). Following a 15-min equilibration period, cells were treated with 10 µL of ligand for an additional 5 min. Plates were subsequently read in a BioTek Synergy H1 microplate reader (BioTek) equipped with 395 nm (RLuc8-coelenterazine 400a) and 510 nm (GFP2) emission filters. BRET2 ratios were calculated as the ratio of the GFP2 emission to RLuc8 emission. Data were normalized to the baseline response of the ligand.

Receptor surface expression

The cell-surface expression levels of WT or mutant PrRPR were quantified using flow cytometry. AD293 cells were seeded at a density of 1.5 × 105 per well in 12-well culture plates and allowed to grow overnight. The cells were then transfected with 1.1 μg of the PrRPR construct using PEI MAX 40 K in each well and incubated for 24 h. Following this, the cells were rinsed once with PBS and detached using 0.2% (w/v) EDTA in PBS. The cells were blocked with PBS containing 5% (w/v) BSA for 15 min at room temperature, followed by incubation with the primary anti-Flag antibody (diluted with PBS containing 5% BSA at a ratio of 1:300, ABclonal) for 1 h at room temperature. The cells were then washed three times with PBS containing 1% (w/v) BSA and incubated with the anti-mouse Alexa-488-conjugated secondary antibody (diluted at a ratio of 1:1000, ABclonal) at 4 °C in the dark for 1 h. After three additional washes, the cells were collected, and the fluorescence intensity was quantified using a Luminex flow cytometer system (Guava® easyCyte) through Luminex guavaSoft 4.5 at an excitation of 488 nm and an emission of 519 nm. Approximately 10,000 cellular events per sample were collected, and the data were normalized to the WT PrRPR. Each experiment was performed at least three times, and the data are presented as means ± SEM.

MD simulation

The simulation systems were constructed using PrRPR–Gi and PrRPR–Gq protein complexes. Protonation states of residues were established utilizing Propka3 software53. Using CHARMM-GUI, these structures were embedded into a 155 Å × 155 Å POPC lipid bilayer54, which was subsequently enclosed by a 15 Å aqueous layer. The systems were then adjusted to a sodium chloride concentration of 0.15 mol/L and supplemented with counterions. The CHARMM36m force field was employed for both amino acids and lipids55. The systems underwent a comprehensive 7-step equilibration process as specified by CHARMM-GUI. For each system, three independent 200 ns production runs were conducted using pmemd.cuda in Amber2056 within the NPT ensemble at 303.15 K and 1 atm. Long-range electrostatic interactions were handled using the Particle Mesh Ewald method, and short-range electrostatic and van der Waals interactions were managed using a 12 Å cutoff, with a gradual transition between 10 Å and 12 Å. Hydrogen bonds were constrained using SHAKE, allowing for a timestep of 2 fs. The binding free energies of the G proteins were calculated using MMPBSA.py57. RMSF was determined by first aligning the PrRPR proteins using the rms command in CPPTRAJ, followed by the atomicfluct command in CPPTRAJ to calculate the RMSF of residues according to the coordinate of non-hydrogen atoms58.

Statistics

All functional study data were analyzed using GraphPad Prism 8.0 (Graphpad Software Inc.) and shown as means ± SEM from at least three independent experiments in triplicate. The significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test, and *P < 0.05 was considered statistically significant.

Supplementary information

Supplementary Information

Supplementary information

The online version contains supplementary material available at 10.1038/s41421-024-00724-6.

Acknowledgements

The cryo-EM data were collected at Advanced Center for Electron Microscopy at Shanghai Institute of Materia Medica, Chinese Academy of Sciences. We are grateful to Kai Wu and Wen Hu for collecting the cryo-EM data. We thank Zecai Chen, Xue Meng, and Jiuyin Xu for helping with experiments. This work was supported by the National Key R&D Program of China (2022YFC2703105 to H.E.X., 2019YFA0904200); the National Natural Science Foundation of China (32371255 and 32071203 to L.-H.Z., 32130022 and 82121005 to H.E.X.); the Natural Science Foundation of Shanghai (23ZR1475200 to L.-H.Z.); CAS Strategic Priority Research Program (XDB37030103 to H.E.X.); Shanghai Municipal Science and Technology Major Project (2019SHZDZX02 to H.E.X.); the Young Innovator Association of CAS (Y2022078 to L.-H.Z.); the Lingang Laboratory (LG-GG-202204-01 to H.E.X.); State Key Laboratory of Drug Research (SKLDR-2023-TT-04 to H.E.X.).

Author contributions

Y.L. designed the expression constructs and purified the protein complex under supervision of L.-H.Z. and H.E.X.; L.-H.Z. prepared the grids. Q.Y. performed cryo-EM data processing and model building. X.H. performed MD simulation. Y.L. constructed all the mutated plasmids and performed functional studies under supervision of L.-H.Z. and Y.Z.; J.L. helped with functional experiments. Y.L., C.Y. and Y.Z. participated in analyzing the structures. Y.L. prepared the figures and initial manuscript. C.Y. and Y.Z. participated in the preparation of the figures. C.W. participated in manuscript preparation. All authors discussed and commented on the manuscript. L.-H.Z. and H.E.X. revised the manuscript. L.-H.Z and H.E.X. conceived, designed, and supervised the overall project.

Data availability

The atomic coordinates and the electron microscopy maps have been deposited in the Protein Data Bank (PDB) and the Electron Microscopy Data Back (EMDB) under accession numbers: 8ZPT and EMD-60354 for the PrRP20–PrRPR–Gq complex, 8ZPS and EMD-60353 for the PrRP20–PrRPR–Gi complex.

Competing interests

The authors declare no competing interests.

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

These authors contributed equally: Yang Li, Qingning Yuan.
==== Refs
References

1. Quillet R RF-amide neuropeptides and their receptors in mammals: pharmacological properties, drug development and main physiological functions Pharmacol. Therap. 2016 160 84 132 10.1016/j.pharmthera.2016.02.005 26896564
Quillet, R. et al. RF-amide neuropeptides and their receptors in mammals: pharmacological properties, drug development and main physiological functions. Pharmacol. Therap. 160, 84–132 (2016).26896564 10.1016/j.pharmthera.2016.02.005
2. Hinuma S A prolactin-releasing peptide in the brain Nature 1998 393 272 276 10.1038/30515 9607765
Hinuma, S. et al. A prolactin-releasing peptide in the brain. Nature 393, 272–276 (1998).9607765 10.1038/30515
3. Bonini JA Identification and characterization of two G protein-coupled receptors for neuropeptide FF J. Biol. Chem. 2000 275 39324 39331 10.1074/jbc.M004385200 11024015
Bonini, J. A. et al. Identification and characterization of two G protein-coupled receptors for neuropeptide FF. J. Biol. Chem. 275, 39324–39331 (2000).11024015 10.1074/jbc.M004385200
4. Elshourbagy NA Receptor for the pain modulatory neuropeptides FF and AF is an orphan G protein-coupled receptor J. Biol. Chem. 2000 275 25965 25971 10.1074/jbc.M004515200 10851242
Elshourbagy, N. A. et al. Receptor for the pain modulatory neuropeptides FF and AF is an orphan G protein-coupled receptor. J. Biol. Chem. 275, 25965–25971 (2000).10851242 10.1074/jbc.M004515200
5. Kotani M The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54 J. Biol. Chem. 2001 276 34631 34636 10.1074/jbc.M104847200 11457843
Kotani, M. et al. The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54. J. Biol. Chem. 276, 34631–34636 (2001).11457843 10.1074/jbc.M104847200
6. Chartrel N Identification of 26RFa, a hypothalamic neuropeptide of the RFamide peptide family with orexigenic activity Proc. Natl. Acad. Sci. USA 2003 100 15247 15252 10.1073/pnas.2434676100 14657341
Chartrel, N. et al. Identification of 26RFa, a hypothalamic neuropeptide of the RFamide peptide family with orexigenic activity. Proc. Natl. Acad. Sci. USA 100, 15247–15252 (2003).14657341 10.1073/pnas.2434676100
7. Welch SK O’Hara BF Kilduff TS Heller HC Sequence and tissue distribution of a candidate G-coupled receptor cloned from rat hypothalamus Biochem. Biophys. Res. Commun. 1995 209 606 613 10.1006/bbrc.1995.1543 7733930
Welch, S. K., O’Hara, B. F., Kilduff, T. S. & Heller, H. C. Sequence and tissue distribution of a candidate G-coupled receptor cloned from rat hypothalamus. Biochem. Biophys. Res. Commun. 209, 606–613 (1995).7733930 10.1006/bbrc.1995.1543
8. Kimura A Prolactin-releasing peptide activation of the prolactin promoter is differentially mediated by extracellular signal-regulated protein kinase and c-Jun N-terminal protein kinase J. Biol. Chem. 2000 275 3667 3674 10.1074/jbc.275.5.3667 10652364
Kimura, A. et al. Prolactin-releasing peptide activation of the prolactin promoter is differentially mediated by extracellular signal-regulated protein kinase and c-Jun N-terminal protein kinase. J. Biol. Chem. 275, 3667–3674 (2000).10652364 10.1074/jbc.275.5.3667
9. Roland BL Anatomical distribution of prolactin-releasing peptide and its receptor suggests additional functions in the central nervous system and periphery Endocrinology 1999 140 5736 5745 10.1210/endo.140.12.7211 10579339
Roland, B. L. et al. Anatomical distribution of prolactin-releasing peptide and its receptor suggests additional functions in the central nervous system and periphery. Endocrinology 140, 5736–5745 (1999).10579339 10.1210/endo.140.12.7211
10. Ibata Y Morphological survey of prolactin-releasing peptide and its receptor with special reference to their functional roles in the brain Neurosci. Res. 2000 38 223 230 10.1016/S0168-0102(00)00182-6 11070188
Ibata, Y. et al. Morphological survey of prolactin-releasing peptide and its receptor with special reference to their functional roles in the brain. Neurosci. Res. 38, 223–230 (2000).11070188 10.1016/S0168-0102(00)00182-6
11. Lawrence CB Celsi F Brennand J Luckman SM Alternative role for prolactin-releasing peptide in the regulation of food intake Nat. Neurosci. 2000 3 645 646 10.1038/76597 10862694
Lawrence, C. B., Celsi, F., Brennand, J. & Luckman, S. M. Alternative role for prolactin-releasing peptide in the regulation of food intake. Nat. Neurosci. 3, 645–646 (2000).10862694 10.1038/76597
12. Lawrence CB Ellacott KL Luckman SM PRL-releasing peptide reduces food intake and may mediate satiety signaling Endocrinology 2002 143 360 367 10.1210/endo.143.2.8609 11796487
Lawrence, C. B., Ellacott, K. L. & Luckman, S. M. PRL-releasing peptide reduces food intake and may mediate satiety signaling. Endocrinology 143, 360–367 (2002).11796487 10.1210/endo.143.2.8609
13. Kalliomäki ML Prolactin-releasing peptide affects pain, allodynia and autonomic reflexes through medullary mechanisms Neuropharmacology 2004 46 412 424 10.1016/j.neuropharm.2003.09.021 14975697
Kalliomäki, M. L. et al. Prolactin-releasing peptide affects pain, allodynia and autonomic reflexes through medullary mechanisms. Neuropharmacology 46, 412–424 (2004).14975697 10.1016/j.neuropharm.2003.09.021
14. Maruyama M Prolactin-releasing peptide as a novel stress mediator in the central nervous system Endocrinology 2001 142 2032 2038 10.1210/endo.142.5.8118 11316770
Maruyama, M. et al. Prolactin-releasing peptide as a novel stress mediator in the central nervous system. Endocrinology 142, 2032–2038 (2001).11316770 10.1210/endo.142.5.8118
15. Iijima N A novel function of prolactin-releasing peptide in the control of growth hormone via secretion of somatostatin from the hypothalamus Endocrinology 2001 142 3239 3243 10.1210/endo.142.7.8257 11416047
Iijima, N. et al. A novel function of prolactin-releasing peptide in the control of growth hormone via secretion of somatostatin from the hypothalamus. Endocrinology 142, 3239–3243 (2001).11416047 10.1210/endo.142.7.8257
16. Zhang SQ Kimura M Inoué S Effects of prolactin-releasing peptide (PrRP) on sleep regulation in rats Psychiatry Clin. Neurosci. 2000 54 262 264 10.1046/j.1440-1819.2000.00670.x 11186069
Zhang, S. Q., Kimura, M. & Inoué, S. Effects of prolactin-releasing peptide (PrRP) on sleep regulation in rats. Psychiatry Clin. Neurosci. 54, 262–264 (2000).11186069 10.1046/j.1440-1819.2000.00670.x
17. Zhang SQ Inoué S Kimura M Sleep-promoting activity of prolactin-releasing peptide (PrRP) in the rat Neuroreport 2001 12 3173 3176 10.1097/00001756-200110290-00006 11711850
Zhang, S. Q., Inoué, S. & Kimura, M. Sleep-promoting activity of prolactin-releasing peptide (PrRP) in the rat. Neuroreport 12, 3173–3176 (2001).11711850 10.1097/00001756-200110290-00006
18. Špolcová A Anorexigenic lipopeptides ameliorate central insulin signaling and attenuate tau phosphorylation in hippocampi of mice with monosodium glutamate-induced obesity J. Alzheimer’s. Dis. 2015 45 823 835 10.3233/JAD-143150 25624414
Špolcová, A. et al. Anorexigenic lipopeptides ameliorate central insulin signaling and attenuate tau phosphorylation in hippocampi of mice with monosodium glutamate-induced obesity. J. Alzheimer’s. Dis. 45, 823–835 (2015).25624414 10.3233/JAD-143150
19. Popelová A Novel lipidized analog of prolactin-releasing peptide improves memory impairment and attenuates hyperphosphorylation of tau protein in a mouse model of tauopathy J. Alzheimer’s. Dis. 2018 62 1725 1736 10.3233/JAD-171041 29614684
Popelová, A. et al. Novel lipidized analog of prolactin-releasing peptide improves memory impairment and attenuates hyperphosphorylation of tau protein in a mouse model of tauopathy. J. Alzheimer’s. Dis. 62, 1725–1736 (2018).29614684 10.3233/JAD-171041
20. Holubová M Liraglutide and a lipidized analog of prolactin-releasing peptide show neuroprotective effects in a mouse model of β-amyloid pathology Neuropharmacology 2019 144 377 387 10.1016/j.neuropharm.2018.11.002 30428311
Holubová, M. et al. Liraglutide and a lipidized analog of prolactin-releasing peptide show neuroprotective effects in a mouse model of β-amyloid pathology. Neuropharmacology 144, 377–387 (2019).30428311 10.1016/j.neuropharm.2018.11.002
21. Boyle RG Structure-activity studies on prolactin-releasing peptide (PrRP). Analogues of PrRP-(19-31)-peptide J. Pept. Sci. 2005 11 161 165 10.1002/psc.612 15635649
Boyle, R. G. et al. Structure-activity studies on prolactin-releasing peptide (PrRP). Analogues of PrRP-(19-31)-peptide. J. Pept. Sci. 11, 161–165 (2005).15635649 10.1002/psc.612
22. Mráziková L Lipidized prolactin-releasing peptide as a new potential tool to treat obesity and type 2 diabetes mellitus: preclinical studies in rodent models Front. Pharmacol. 2021 12 779962 10.3389/fphar.2021.779962 34867411
Mráziková, L. et al. Lipidized prolactin-releasing peptide as a new potential tool to treat obesity and type 2 diabetes mellitus: preclinical studies in rodent models. Front. Pharmacol. 12, 779962 (2021).34867411 10.3389/fphar.2021.779962
23. Kuneš J Prolactin-releasing peptide: a new tool for obesity treatment J. Endocrinol. 2016 230 R51 R58 10.1530/JOE-16-0046 27418033
Kuneš, J. et al. Prolactin-releasing peptide: a new tool for obesity treatment. J. Endocrinol. 230, R51–R58 (2016).27418033 10.1530/JOE-16-0046
24. Engström M Brandt A Wurster S Savola JM Panula P Prolactin releasing peptide has high affinity and efficacy at neuropeptide FF2 receptors J. Pharmacol. Exp. Therap. 2003 305 825 832 10.1124/jpet.102.047118 12606605
Engström, M., Brandt, A., Wurster, S., Savola, J. M. & Panula, P. Prolactin releasing peptide has high affinity and efficacy at neuropeptide FF2 receptors. J. Pharmacol. Exp. Therap. 305, 825–832 (2003).12606605 10.1124/jpet.102.047118
25. Bruzzone F Distribution of 26RFa binding sites and GPR103 mRNA in the central nervous system of the rat J. Comp. Neurol. 2007 503 573 591 10.1002/cne.21400 17534937
Bruzzone, F. et al. Distribution of 26RFa binding sites and GPR103 mRNA in the central nervous system of the rat. J. Comp. Neurol. 503, 573–591 (2007).17534937 10.1002/cne.21400
26. Oishi S Activation of neuropeptide FF receptors by kisspeptin receptor ligands ACS Med. Chem. Lett. 2011 2 53 57 10.1021/ml1002053 24900254
Oishi, S. et al. Activation of neuropeptide FF receptors by kisspeptin receptor ligands. ACS Med. Chem. Lett. 2, 53–57 (2011).24900254 10.1021/ml1002053
27. Zhao F Structural insights into multiplexed pharmacological actions of tirzepatide and peptide 20 at the GIP, GLP-1 or glucagon receptors Nat. Commun. 2022 13 1057 10.1038/s41467-022-28683-0 35217653
Zhao, F. et al. Structural insights into multiplexed pharmacological actions of tirzepatide and peptide 20 at the GIP, GLP-1 or glucagon receptors. Nat. Commun. 13, 1057 (2022).35217653 10.1038/s41467-022-28683-0
28. Duan J Cryo-EM structure of an activated VIP1 receptor-G protein complex revealed by a NanoBiT tethering strategy Nat. Commun. 2020 11 4121 10.1038/s41467-020-17933-8 32807782
Duan, J. et al. Cryo-EM structure of an activated VIP1 receptor-G protein complex revealed by a NanoBiT tethering strategy. Nat. Commun. 11, 4121 (2020).32807782 10.1038/s41467-020-17933-8
29. Zhao LH Structure insights into selective coupling of G protein subtypes by a class B G protein-coupled receptor Nat. Commun. 2022 13 6670 10.1038/s41467-022-33851-3 36335102
Zhao, L. H. et al. Structure insights into selective coupling of G protein subtypes by a class B G protein-coupled receptor. Nat. Commun. 13, 6670 (2022).36335102 10.1038/s41467-022-33851-3
30. Nehmé R Mini-G proteins: Novel tools for studying GPCRs in their active conformation PLoS One 2017 12 e0175642 10.1371/journal.pone.0175642 28426733
Nehmé, R. et al. Mini-G proteins: Novel tools for studying GPCRs in their active conformation. PLoS One 12, e0175642 (2017).28426733 10.1371/journal.pone.0175642
31. Rasmussen SG Crystal structure of the β2 adrenergic receptor-Gs protein complex Nature 2011 477 549 555 10.1038/nature10361 21772288
Rasmussen, S. G. et al. Crystal structure of the β2 adrenergic receptor-Gs protein complex. Nature 477, 549–555 (2011).21772288 10.1038/nature10361
32. Deluca SH Rathmann D Beck-Sickinger AG Meiler J The activity of prolactin releasing peptide correlates with its helicity Biopolymers 2013 99 314 325 10.1002/bip.22162 23426574
Deluca, S. H., Rathmann, D., Beck-Sickinger, A. G. & Meiler, J. The activity of prolactin releasing peptide correlates with its helicity. Biopolymers 99, 314–325 (2013).23426574 10.1002/bip.22162
33. D’Ursi AM Structural studies on Hgr3 orphan receptor ligand prolactin-releasing peptide J. Med. Chem. 2002 45 5483 5491 10.1021/jm020975p 12459016
D’Ursi, A. M. et al. Structural studies on Hgr3 orphan receptor ligand prolactin-releasing peptide. J. Med. Chem. 45, 5483–5491 (2002).12459016 10.1021/jm020975p
34. Bader R Bettio A Beck-Sickinger AG Zerbe O Structure and dynamics of micelle-bound neuropeptide Y: comparison with unligated NPY and implications for receptor selection J. Mol. Biol. 2001 305 307 329 10.1006/jmbi.2000.4264 11124908
Bader, R., Bettio, A., Beck-Sickinger, A. G. & Zerbe, O. Structure and dynamics of micelle-bound neuropeptide Y: comparison with unligated NPY and implications for receptor selection. J. Mol. Biol. 305, 307–329 (2001).11124908 10.1006/jmbi.2000.4264
35. Tang T Receptor-specific recognition of NPY peptides revealed by structures of NPY receptors Sci. Adv. 2022 8 eabm1232 10.1126/sciadv.abm1232 35507650
Tang, T. et al. Receptor-specific recognition of NPY peptides revealed by structures of NPY receptors. Sci. Adv. 8, eabm1232 (2022).35507650 10.1126/sciadv.abm1232
36. Maletínská L Spolcová A Maixnerová J Blechová M Zelezná B Biological properties of prolactin-releasing peptide analogs with a modified aromatic ring of a C-terminal phenylalanine amide Peptides 2011 32 1887 1892 10.1016/j.peptides.2011.08.011 21872625
Maletínská, L., Spolcová, A., Maixnerová, J., Blechová, M. & Zelezná, B. Biological properties of prolactin-releasing peptide analogs with a modified aromatic ring of a C-terminal phenylalanine amide. Peptides 32, 1887–1892 (2011).21872625 10.1016/j.peptides.2011.08.011
37. Park C Structural basis of neuropeptide Y signaling through Y1 receptor Nat. Commun. 2022 13 853 10.1038/s41467-022-28510-6 35165283
Park, C. et al. Structural basis of neuropeptide Y signaling through Y1 receptor. Nat. Commun. 13, 853 (2022).35165283 10.1038/s41467-022-28510-6
38. Flock T Universal allosteric mechanism for Gα activation by GPCRs Nature 2015 524 173 179 10.1038/nature14663 26147082
Flock, T. et al. Universal allosteric mechanism for Gα activation by GPCRs. Nature 524, 173–179 (2015).26147082 10.1038/nature14663
39. García-Nafría J Nehmé R Edwards PC Tate CG Cryo-EM structure of the serotonin 5-HT(1B) receptor coupled to heterotrimeric G(o) Nature 2018 558 620 623 10.1038/s41586-018-0241-9 29925951
García-Nafría, J., Nehmé, R., Edwards, P. C. & Tate, C. G. Cryo-EM structure of the serotonin 5-HT(1B) receptor coupled to heterotrimeric G(o). Nature 558, 620–623 (2018).29925951 10.1038/s41586-018-0241-9
40. Kim HR Structural mechanism underlying primary and secondary coupling between GPCRs and the Gi/o family Nat. Commun. 2020 11 3160 10.1038/s41467-020-16975-2 32572026
Kim, H. R. et al. Structural mechanism underlying primary and secondary coupling between GPCRs and the Gi/o family. Nat. Commun. 11, 3160 (2020).32572026 10.1038/s41467-020-16975-2
41. Kim K Structure of a hallucinogen-activated Gq-coupled 5-HT(2A) serotonin receptor Cell 2020 182 1574 1588.e19 10.1016/j.cell.2020.08.024 32946782
Kim, K. et al. Structure of a hallucinogen-activated Gq-coupled 5-HT(2A) serotonin receptor. Cell 182, 1574–1588.e19 (2020).32946782 10.1016/j.cell.2020.08.024
42. Wang Y Molecular recognition of an acyl-peptide hormone and activation of ghrelin receptor Nat. Commun. 2021 12 5064 10.1038/s41467-021-25364-2 34417468
Wang, Y. et al. Molecular recognition of an acyl-peptide hormone and activation of ghrelin receptor. Nat. Commun. 12, 5064 (2021).34417468 10.1038/s41467-021-25364-2
43. Yin YL Molecular basis for kinin selectivity and activation of the human bradykinin receptors Nat. Struct. Mol. Biol. 2021 28 755 761 10.1038/s41594-021-00645-y 34518695
Yin, Y. L. et al. Molecular basis for kinin selectivity and activation of the human bradykinin receptors. Nat. Struct. Mol. Biol. 28, 755–761 (2021).34518695 10.1038/s41594-021-00645-y
44. Zheng SQ MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy Nat. Methods 2017 14 331 332 10.1038/nmeth.4193 28250466
Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).28250466 10.1038/nmeth.4193
45. Rohou A Grigorieff N CTFFIND4: fast and accurate defocus estimation from electron micrographs J. Struct. Biol. 2015 192 216 221 10.1016/j.jsb.2015.08.008 26278980
Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).26278980 10.1016/j.jsb.2015.08.008
46. Sanchez-Garcia R DeepEMhancer: a deep learning solution for cryo-EM volume post-processing Commun. Biol. 2021 4 874 10.1038/s42003-021-02399-1 34267316
Sanchez-Garcia, R. et al. DeepEMhancer: a deep learning solution for cryo-EM volume post-processing. Commun. Biol. 4, 874 (2021).34267316 10.1038/s42003-021-02399-1
47. Jumper J Highly accurate protein structure prediction with AlphaFold Nature 2021 596 583 589 10.1038/s41586-021-03819-2 34265844
Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).34265844 10.1038/s41586-021-03819-2
48. Pettersen EF UCSF Chimera-a visualization system for exploratory research and analysis J. Comput. Chem. 2004 25 1605 1612 10.1002/jcc.20084 15264254
Pettersen, E. F. et al. UCSF Chimera-a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).15264254 10.1002/jcc.20084
49. Emsley P Lohkamp B Scott WG Cowtan K Features and development of Coot Acta Crystallogr. Sect. D Biol. Crystallogr. 2010 66 486 501 10.1107/S0907444910007493 20383002
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. Sect. D Biol. Crystallogr. 66, 486–501 (2010).20383002 10.1107/S0907444910007493
50. Liebschner D Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix Acta Crystallogr. Sect. D Struct. Biol. 2019 75 861 877 10.1107/S2059798319011471 31588918
Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. Sect. D Struct. Biol. 75, 861–877 (2019).31588918 10.1107/S2059798319011471
51. Pettersen EF UCSF ChimeraX: structure visualization for researchers, educators, and developers Protein Sci. 2021 30 70 82 10.1002/pro.3943 32881101
Pettersen, E. F. et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021).32881101 10.1002/pro.3943
52. Olsen RHJ TRUPATH, an open-source biosensor platform for interrogating the GPCR transducerome Nat. Chem. Biol. 2020 16 841 849 10.1038/s41589-020-0535-8 32367019
Olsen, R. H. J. et al. TRUPATH, an open-source biosensor platform for interrogating the GPCR transducerome. Nat. Chem. Biol. 16, 841–849 (2020).32367019 10.1038/s41589-020-0535-8
53. Olsson MH Søndergaard CR Rostkowski M Jensen JH PROPKA3: consistent treatment of internal and surface residues in empirical pKa predictions J. Chem. Theory Comput. 2011 7 525 537 10.1021/ct100578z 26596171
Olsson, M. H., Søndergaard, C. R., Rostkowski, M. & Jensen, J. H. PROPKA3: consistent treatment of internal and surface residues in empirical pKa predictions. J. Chem. Theory Comput. 7, 525–537 (2011).26596171 10.1021/ct100578z
54. Wu EL CHARMM-GUI Membrane Builder toward realistic biological membrane simulations J. Comput. Chem. 2014 35 1997 2004 10.1002/jcc.23702 25130509
Wu, E. L. et al. CHARMM-GUI Membrane Builder toward realistic biological membrane simulations. J. Comput. Chem. 35, 1997–2004 (2014).25130509 10.1002/jcc.23702
55. Huang J CHARMM36m: an improved force field for folded and intrinsically disordered proteins Nat. Methods 2017 14 71 73 10.1038/nmeth.4067 27819658
Huang, J. et al. CHARMM36m: an improved force field for folded and intrinsically disordered proteins. Nat. Methods 14, 71–73 (2017).27819658 10.1038/nmeth.4067
56. Salomon-Ferrer R Götz AW Poole D Le Grand S Walker RC Routine microsecond molecular dynamics simulations with AMBER on GPUs. 2. Explicit solvent particle mesh Ewald J. Chem. Theory Comput. 2013 9 3878 3888 10.1021/ct400314y 26592383
Salomon-Ferrer, R., Götz, A. W., Poole, D., Le Grand, S. & Walker, R. C. Routine microsecond molecular dynamics simulations with AMBER on GPUs. 2. Explicit solvent particle mesh Ewald. J. Chem. Theory Comput. 9, 3878–3888 (2013).26592383 10.1021/ct400314y
57. Miller BR 3rd MMPBSA.py: an efficient program for end-state free energy calculations J. Chem. Theory Comput. 2012 8 3314 3321 10.1021/ct300418h 26605738
Miller, B. R. 3rd et al. MMPBSA.py: an efficient program for end-state free energy calculations. J. Chem. Theory Comput. 8, 3314–3321 (2012).26605738 10.1021/ct300418h
58. Roe DR Cheatham TE III PTRAJ and CPPTRAJ: software for processing and analysis of molecular dynamics trajectory data J. Chem. Theory Comput. 2013 9 3084 3095 10.1021/ct400341p 26583988
Roe, D. R. & Cheatham, T. E. III PTRAJ and CPPTRAJ: software for processing and analysis of molecular dynamics trajectory data. J. Chem. Theory Comput. 9, 3084–3095 (2013).26583988 10.1021/ct400341p
