Large-scale deorphanization of Nematostella vectensis neuropeptide G protein-coupled receptors supports the independent expansion of bilaterian and cnidarian peptidergic systems
- Living Systems Institute, University of Exeter, United Kingdom
- Animal Physiology & Neurobiology, Department of Biology, University of Leuven, Belgium
- Department of Neurosciences and Developmental Biology, Faculty of Life Sciences, University of Vienna, Austria
- Centre for Organismal Studies (COS), Heidelberg University, Germany
Figures
Figure 1 with 1 supplement
Identification of N. vectensis neuropeptides.
(A) Pipeline to identify neuropeptides and their receptors and to reconstruct the evolution of cnidarian peptidergic signaling. (B) Peptide sequence logos of N-terminal and C-terminal peptide cleavage sites based on peptides detected by LC-MS/MS. Cleavage occurs at the dashed lines. (C) N. vectensis neuropeptide precursor schemes of peptides for which we identified a receptor, with sequence logos of the encoded peptide(s) on the left and length of precursor on the right. a=amide.
Figure 1—figure supplement 1
Mass spectrometry pipeline.
(A) Pipeline for mass spectrometry identification of neuropeptide candidates. Created with BioRender.com. (B) Example spectra of detected HIRamide peptides with different lengths that originate from the same peptide on the HIRamide precursor. The shown spectra are from the encircled HIRamide peptide copy in (C). Figures within Panel B exported from PEAKS Studio X+ (v10.5 Build number 20200219, Bioinformatics Solutions, 2023).
© 2024, BioRender Inc. Figure 1—figure supplement 1 was created using BioRender.com, and is published under a CC BY-NC-ND license. Further reproductions must adhere to the terms of this license
Figure 2 with 1 supplement
Cluster map of selected class A G protein-coupled receptors (GPCRs).
(A) Number of class A GPCRs identified by HMMer search in the different investigated species. (B) Relationship of species used for cluster analysis in C. (C) Cluster analysis of major class A GPCR groups from cnidarian, bilaterian, and placozoan species. Each dot represents a GPCR sequence with color-coding and symbols according to the phylogeny in B. Connecting lines between single sequences show similarity with p-values indicated in the top right. Cluster annotations are based on deorphanized bilaterian class A GPCRs. Abbreviations in C: ACh = acetylcholine, lrrc = leucine-rich-repeat containing, P2Y=purinergic P2Y receptor. Silhouette images in B were taken from phylopic.org.
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Figure 2—source data 1
Raw cluster analysis CLANS file.
- https://cdn.elifesciences.org/articles/90674/elife-90674-fig2-data1-v1.txt
Figure 2—figure supplement 1
Cluster map of metazoan class A G protein-coupled receptors (GPCRs).
Each dot represents a GPCR sequence with color-coding according to species list on the left. Connecting lines between single sequences show similarity with p-values indicated in top right. Annotations are based on sequences of deorphanized bilaterian class A GPCRs. Abbreviations in C: ACh = acetylcholine, lrrc = leucine-rich-repeat containing, P2Y = purinergic P2Y receptor.
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Figure 2—figure supplement 1—source data 1
Raw cluster analysis CLANS file.
- https://cdn.elifesciences.org/articles/90674/elife-90674-fig2-figsupp1-data1-v1.txt
Figure 3 with 2 supplements
Dose-response curves of Nematostella neuropeptide G protein-coupled receptor (GPCR) pairs.
(A) Pharmacological assay and pipeline to identify peptide-GPCR pairs. (B) Dose-response curves of peptide-GPCR pairs with log peptide concentration plotted against normalized luminescence. GPCRs that are activated by the same peptide(s) are grouped together with peptide sequence shown above and peptide name highlighted in black. If several peptides activate the same receptor, peptide sequences are shown within the graph. Receptor identification number is encircled in the upper left of each curve, EC50 values are indicated in the lower right. Sample size per datapoint = 9. Error bars show distribution of datapoints with box indicating upper and lower quartile. (C) Histogram of EC50 values of peptide-GPCR pairs, showing only the lowest EC50 per GPCR. (D) Peptide-receptor pairings showing number of receptors activated by the different peptides. Connection strength indicates EC50 values.
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Figure 3—source data 1
Tibble with all data points used to calculate the dose-response curves and EC50 values, in .csv format.
- https://cdn.elifesciences.org/articles/90674/elife-90674-fig3-data1-v1.csv
Figure 3—figure supplement 1
Dose-response curves with EC50 values and peptide precursor of different HIRamide peptides.
(A) Dose-response curves of HIRamide peptide and GPR 21 + 29. Dose-response curves show activation of receptors GPR21 and GPR29 with different HIRamide peptide versions from the same precursor. Sequences and EC50 values for different HIRamides are shown below the dose-response curves. The graph in Figure 3 shows HIRamide3 as the peptide with the lowest EC50 values. GPR21 showed already at low concentrations a high base activation compared to the negative control, but did not show a clear increase in intensity at concentrations between 1E-13 and 1E-11. We therefore set the minimum to the value measured at a concentration of 1E-13 instead of the negative control and show a comparison of the corresponding graphs and EC50 values here. (B) HIRamide precursor. The N-terminal signal peptide of the precursors sequence is not highlighted, cleavage + amidation sites are highlighted in a darker gray. Tested peptides are underlined and colored as shown underneath the dose-response curves.
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Figure 3—figure supplement 1—source data 1
Tibble with all data points used to calculate the dose-response curves and EC50 values, in .csv format.
- https://cdn.elifesciences.org/articles/90674/elife-90674-fig3-figsupp1-data1-v1.csv
Figure 3—figure supplement 2
Dose-response curves with EC50 values and peptide precursor of different PRGamide peptide versions for two of the PRGamide receptors.
(A) Dose-response curves show activation of the receptors GPR28 and GPR32 with different PRGamide peptide versions from the same precursor. Sequences and EC50 values for different PRGamides are shown below the dose-response curves. The graph in Figure 3 shows the shortest PRGamide (GPRGamide) as the peptide with the lowest EC50 values, which is likely the fully processed version. (B) PRGamide precursor. The N-terminal signal peptide of the precursor sequence is not highlighted, cleavage and amidation sites are highlighted in a darker gray. The tested peptides are colored to match the color of the dose-response curves and the GPRG peptide is underlined.
Figure 4 with 2 supplements
Phylogeny of metazoan class A neuropeptide G protein-coupled receptors (GPCRs).
(A) Phylogeny of species used in B. (B) Phylogeny of neuropeptide GPCRs with names of ligands. Branches are color-coded according to A. Branches of deorphanized Nematostella GPCRs end in an asterisk. Alternating shades behind the tree branches highlight different monophyletic groups. Roman numbers 1–3 and Greek symbol gamma indicate approximate neuropeptide clusters shown in Figure 2. Left half circle of branch support indicates aBayes and the right half circle aLRT-SH-like support values. Detailed annotations in Supplementary file 11. (C) Table with number of receptors per group as highlighted in receptor phylogeny with a straight line indicating no receptor present. Two-letter abbreviations on top correspond to species in A. Abbreviations: a=amide, B=Bilateria, CCK = cholecystokinin, GnRH = gonadotropin releasing hormone, MIH = maturation-inducing hormone, Nm-U=neuromedin U, NpFF = neuropeptide FF, NpY/F=neuropeptide Y/neuropeptide F, P=Placozoa, PrP = prolactin releasing peptide, R.#=Nematostella GPCR number, sNpF = short neuropeptide F, t-FMRFa=trochozoan FMRFamide, TRH = thyrotropin releasing hormone.
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Figure 4—source data 1
Raw sequences used for tree building, .fasta format.
- https://cdn.elifesciences.org/articles/90674/elife-90674-fig4-data1-v1.txt
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Figure 4—source data 2
Aligned sequences used for tree building.
- https://cdn.elifesciences.org/articles/90674/elife-90674-fig4-data2-v1.txt
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Figure 4—source data 3
Trimmed sequence alignment used for tree building.
- https://cdn.elifesciences.org/articles/90674/elife-90674-fig4-data3-v1.txt
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Figure 4—source data 4
Tree file in nexus format.
- https://cdn.elifesciences.org/articles/90674/elife-90674-fig4-data4-v1.txt
Figure 4—figure supplement 1
Tree (FastTree) of neuropeptide G protein-coupled receptors (GPCRs) with bilaterian chemokine and related receptors.
Bilaterian sequences are shown in yellow, cnidarian sequences are shown in dark blue (Medusozoa) and magenta (Anthozoa), placozoan sequences are shown in light blue. Deorphanized N. vectensis sequences are shown in green. Sequences were aligned using muscle, alignment was trimmed with the gappyout option of trimal, tree was calculated using FastTree. Abbreviations: a = amide, CCK = cholecystokinin, GnRH = gonadotropn releasing hormone, GPR19 = G protein-coupled receptor 19, L11 = elevenin, MCH = melanin concentrating hormone, Nm-U = neuromedin U, NpFF = neuropeptide FF, NpS = neuropeptide S, NpY/F = neuropeptide Y/neuropeptide F, PrP = prolactin releasing peptide, sNpF = short neuropeptide F, TRH = thyrotropin releasing hormone.
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Figure 4—figure supplement 1—source data 1
Raw sequences used for tree building, .fasta format.
- https://cdn.elifesciences.org/articles/90674/elife-90674-fig4-figsupp1-data1-v1.zip
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Figure 4—figure supplement 1—source data 2
Aligned sequences used for tree building.
- https://cdn.elifesciences.org/articles/90674/elife-90674-fig4-figsupp1-data2-v1.zip
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Figure 4—figure supplement 1—source data 3
Trimmed sequence alignment used for tree building.
- https://cdn.elifesciences.org/articles/90674/elife-90674-fig4-figsupp1-data3-v1.zip
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Figure 4—figure supplement 1—source data 4
Tree file in nexus format.
- https://cdn.elifesciences.org/articles/90674/elife-90674-fig4-figsupp1-data4-v1.zip
Figure 4—figure supplement 2
Tree (IQtree) of neuropeptide G protein-coupled receptors (GPCRs) with bilaterian chemokine and related receptors.
Bilaterian sequences are shown in yellow, cnidarian sequences are shown in dark blue (Medusozoa) and magenta (Anthozoa), placozoan sequences are shown in light blue. Deorphanized N. vectensis sequences are shown in green. Sequences were aligned using mafft, alignment was trimmed with the gappyout option of trimal, tree was calculated using IQtree. Abbreviations: a = amide, CCK = cholecystokinin, GnRH = gonadotropin releasing hormone, GPR19 = G protein-coupled receptor 19, L11 = elevenin, MCH = melanin concentrating hormone, Nm-U = neuromedin U, NpB/W = neuropeptide B/neuropeptide W, NpFF = neuropeptide FF, NpS = neuropeptide S, NpY/F = neuropeptide Y/neuropeptide F, sNpF = short neuropeptide F.
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Figure 4—figure supplement 2—source data 1
Raw sequences used for tree building, .fasta format.
- https://cdn.elifesciences.org/articles/90674/elife-90674-fig4-figsupp2-data1-v1.txt
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Figure 4—figure supplement 2—source data 2
Aligned sequences used for tree building.
- https://cdn.elifesciences.org/articles/90674/elife-90674-fig4-figsupp2-data2-v1.txt
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Figure 4—figure supplement 2—source data 3
Trimmed sequence alignment used for tree building.
- https://cdn.elifesciences.org/articles/90674/elife-90674-fig4-figsupp2-data3-v1.txt
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Figure 4—figure supplement 2—source data 4
Tree file in nexus format.
- https://cdn.elifesciences.org/articles/90674/elife-90674-fig4-figsupp2-data4-v1.txt
Figure 5 with 3 supplements
Tissue-specific expression of neuropeptide precursors and receptors (G protein-coupled receptors [GPCRs]) in N. vectensis.
Dotplot for tissue-specific expression of peptide precursors and GPCRs. Red dots indicate expression in the developmental dataset, blue dots indicate expression in the adult dataset. Abbreviations: a=amide, e=embryonic, ect = ectoderm, endomes = endomesoderm, gland = glandular, muc = mucous, musc = muscle, neurogland = neuroglandular, PGCs = primary germ cells, pharyng = pharyngeal, pSC = putative stem cells, R=receptor (GPCR), retrac = retractor.
Figure 5—figure supplement 1
Tissue-specific expression of neuropeptide precursors and neuropeptide receptors (G protein-coupled receptors [GPCRs]) in N. vectensis.
Dotplot for tissue-specific expression of proneuropeptides and GPCRs. Proneuropeptides without a known receptor are also included. Red dots indicate expression in the developmental dataset, blue dots indicate expression in the adult dataset.
Figure 5—figure supplement 2
Expression of neuropeptide precursors and G protein-coupled receptors (GPCRs) in neuroglandular cell types in the developmental dataset.
Dot size indicates percentage of cells expressing the corresponding gene, color intensity indicates average expression.
Figure 5—figure supplement 3
Expression of neuropeptide precursors and G protein-coupled receptors (GPCRs) in neuroglandular cell types in the adult dataset.
Dot size indicates percentage of cells expressing the corresponding gene, color intensity indicates average expression.
Figure 6 with 1 supplement
Multilayer peptidergic connectomes in Nematostella.
Peptidergic networks in the (A) developmental and (B) adult subset. Nodes represent cell types, connections represent potential peptidergic signaling from neuropeptide-expressing cells to cells expressing one or more of the receptors for that neuropeptide. Colors represent different peptide-receptor signal channels (the different layers in the multilayer connectome).
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Figure 6—source data 1
Graph file of the multilayered peptidergic connectome in the developmental subset.
A serialized binary file in tbl_graph format, to be analyzed in R.
- https://cdn.elifesciences.org/articles/90674/elife-90674-fig6-data1-v1.zip
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Figure 6—source data 2
Graph file of the multilayered peptidergic connectome in the developmental subset.
In Gephi .gexf format, to be analyzed in Gephi.
- https://cdn.elifesciences.org/articles/90674/elife-90674-fig6-data2-v1.txt
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Figure 6—source data 3
Graph file of the multilayered peptidergic connectome in the adult subset.
A serialized binary file in tbl_graph format, to be analyzed in R.
- https://cdn.elifesciences.org/articles/90674/elife-90674-fig6-data3-v1.zip
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Figure 6—source data 4
Graph file of the multilayered peptidergic connectome in the adult subset.
In Gephi .gexf format, to be analyzed in Gephi.
- https://cdn.elifesciences.org/articles/90674/elife-90674-fig6-data4-v1.txt
Figure 6—figure supplement 1
Multilayer peptidergic connectomes in Nematostella.
(A) Network of all peptide-receptor pairs for the developmental subset, colored by network Leiden module. (B–D) Networks of LRWa (B), FLRNa (C), and PRGa (D) for the developmental subset. (E) Network of all peptide-receptor pairs for the adult subset, colored by network Leiden module. (F–L) Networks of LRWa (F), FLRNa (G), PRGa (H), QGRFa (I), HIRa (J), VRHa (K), and QWa (L) for the adult subset. Nodes represent cell types, connections represent potential peptidergic signaling from neuropeptide-expressing cells to cells expressing one or more of the receptors for that neuropeptide. For peptides with more receptors, different colors represent different peptide-receptor signal channels (the different layers in the multilayer connectome).
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Biological sample (N. vectensis) | Larval, juvenile, and adult N. vectensis | Specimens obtained form the Marine Invertebrate Culture Unit of the University of Exeter | N/A | N/A |
| Biological sample (cDNA) | cDNA obtained from N. vectensis | This study | N/A | RNA extracted with Trizol and cDNA synthesized with cDNA synthesis kit according to the manufacturer’s recommendation |
| Biological sample (peptide extract) | Peptide extracts obtained from N. vectensis | This study | N/A | Peptides extracted from N. vectensis according to protocol explained in Materials and methods |
| Genetic reagent (cDNA synthesis) | SuperScript III First-Strand Synthesis System | Invitrogen (from Thermo Fisher) | 18080051 | N/A |
| Genetic reagent (polymerase) | Q5 Hot Start High-Fidelity DNA Polymerase | New England Biolabs | M0493L | N/A |
| Genetic reagent (DNA assembly) | NEBuilder HiFi DNA Assembly Master Mix | New England Biolabs | E2621L | N/A |
| Genetic reagent (restriction enzyme) | EcoRV restriction enzyme | New England Biolabs | R3195L | N/A |
| Genetic reagent (restriction enzyme) | Afl2 restriction enzyme | New England Biolabs | R0520L | N/A |
| Genetic reagent (restriction enzyme) | Hind3 restriction enzyme | New England Biolabs | R3104L | N/A |
| Genetic reagent (restriction enzyme) | BamH1 restriction enzyme | New England Biolabs | R3136L | N/A |
| Genetic reagent (restriction enzyme) | EcoRI restriction enzyme | New England Biolabs | R3101L | N/A |
| Recombinant DNA reagent (plasmid) | pcDNA3.1(+) | Invitrogen (from Thermo Fisher) | V79020 | N/A |
| Recombinant DNA reagent (plasmid) | pRK5-Gqi9 | AddGene | 125711 | N/A |
| Recombinant DNA reagent (plasmid) | pcDNA3.1(+)-G5A | This study | N/A | Sequence information provided in Supplementary file 7 |
| Recombinant DNA reagent (plasmid) | pcDNA3.1(+)-Gqi5/9 | This study | N/A | Sequence information provided in Supplementary file 7 |
| Recombinant DNA reagent | Cloned N. vectensis GPCRs in pcDNA3.1(+) | Identified in this study | N/A | Full list of cloned GPCRs with sequences in Supplementary file 8 |
| Recombinant DNA reagent | Synthesized N. vectensis GPCRs in pcDNA3.1(+) | Identified in this study, codon optimized and synthesized by GenScript | N/A | Full list of synthesized GPCRs with original and codon-optimized sequences in Supplementary file 8 |
| Sequence-based reagent | Cloning primers to create Gqi5/9 | This study | N/A | Sequence information provided in Supplementary file 7 |
| Sequence-based reagent | GPCR cloning primers | This study | N/A | Full list of primers with sequences in Supplementary file 8 |
| Peptide, recombinant protein | Custom peptides | Identified in this study, synthesized by GenScript | N/A | Full list of peptides with sequences and purity in Supplementary files 2 and 3 |
| Commercial assay or kit (PCR clean-up) | Monarch PCR and DNA Cleanup Kit (5 μg) | New England Biolabs | T1030L | N/A |
| Commercial assay or kit (Miniprep) | GeneJET Plasmid Miniprep Kit | Thermo Fisher Scientific | K0503 | N/A |
| Strain, strain background (E. coli, TOP10) | One Shot TOP10 Chemically Competent E. coli | Thermo Fisher Scientific | C404010 | E. coli strain used for general cloning |
| Cell line (HEK293) | HEK293 Cells expressing GFP-AEQUORIN in Cytoplasma | Angio-Proteomie | cAP-0200GFP-AEQ-Cyto | N/A |
| Chemical compound, drug | DMEM | Gibco (from Thermo Fisher) | 10566016 | N/A |
| Chemical compound, drug | OptiMEM | Gibco (from Thermo Fisher) | 11058021 | N/A |
| Chemical compound, drug | FBS | Gibco (from Thermo Fisher) | 10500064 | N/A |
| Chemical compound, drug | PEI (polyethylenimine, 25k Mw) | Sigma-Aldrich | 408727 | Used 0.3 µl of a 1 mg/ml stock solution per 100 ng DNA |
| Chemical compound, drug | Transfectamine 5000 | AAT Bioquest | 60022 | Used 0.3 µl per 100 ng of DNA |
| Chemical compound, drug | Coelenterazine-H | Promega | S2011 | Diluted to 2 mM in ethanol and used at a final concentration of 4 µM in the assays |
| Chemical compound, drug | TRIzol Reagent | Invitrogen (from Thermo Fisher) | 15596026 | N/A |
| Other | Corning 96 Well White Polystyrene Microplate | Corning (from Sigma-Aldrich) | CLS3903 | Cell culture-treated assay plates |
| Other | Nunc EasYFlask T75 Cell Culture Flasks | Nunc (from Thermo Fisher) | 156499 | Cell culture flasks |
| Other | Flexstation 3 Multimode Microplate Reader | Molecular Devices | N/A | Plate reader |
| Software, algorithm | SoftMax Pro 7 | Molecular Devices | N/A | N/A |
| Software, algorithm | R | https://cran.rstudio.com/ | N/A | N/A |
| Software, algorithm | RStudio | https://posit.co/download/rstudio-desktop/ | N/A | N/A |
| Software, algorithm | SignalP4.1 | https://services.healthtech.dtu.dk/services/SignalP-6.0/ | N/A | N/A |
| Software, algorithm | NeuroPID | https://bio.tools/neuropid | N/A | N/A |
| Software, algorithm | HMMER3.1b2 | http://hmmer.org/download.html | N/A | N/A |
| Software, algorithm | CD-HIT | https://sites.google.com/view/cd-hit | N/A | N/A |
| Software, algorithm | TransDecoder v5.5.0 | https://github.com/TransDecoder/TransDecoder, Haas, 2024 | N/A | N/A |
| Software, algorithm | CLANS (desktop version) | https://mybiosoftware.com/clans-20101007-visualize-protein-families-based-pairwise-similarity.html | N/A | N/A |
| Software, algorithm | CLANS (online toolkit) | https://toolkit.tuebingen.mpg.de/tools/clans | N/A | N/A |
| Software, algorithm | Phobius | https://phobius.sbc.su.se/data.html | N/A | N/A |
| Software, algorithm | Muscle alignment tool | https://drive5.com/muscle5/ | N/A | N/A |
| Software, algorithm | MAFFT v7 | https://mafft.cbrc.jp/alignment/software/ | N/A | N/A |
| Software, algorithm | trimAl | http://trimal.cgenomics.org/trimal | N/A | N/A |
| Software, algorithm | IQ-tree2 | http://www.iqtree.org/ | N/A | N/A |
| Software, algorithm | Fasttree | http://www.microbesonline.org/fasttree/ | N/A | N/A |
Additional files
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Supplementary file 1
Mass spectrometry identification of N. vectensis neuropeptides.
The table contains the results of the mass spectrometry analysis including the name, sequence and identifier of the precursor sequences, N- and C-terminal flanking sequences, and peptide sequences.
- https://cdn.elifesciences.org/articles/90674/elife-90674-supp1-v1.xlsx
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Supplementary file 2
N. vectensis proneuropeptides.
The file contains a list of all N. vectensis proneuropeptide sequences identified in this study. Each precursor is provided with a list of alternative headers from the different resources, other studies that identified the same precursor, a list of peptides that were tested in this study, the receptors that were activated and cleavage site and signal peptides are indicated by different colors.
- https://cdn.elifesciences.org/articles/90674/elife-90674-supp2-v1.zip
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Supplementary file 3
Peptide mixes used for G protein-coupled receptor (GPCR) screening.
The file contains the peptide mixes that were used in the initial peptide mix-GPCR screening (with additional information about the peptides and dilution of stock solutions).
- https://cdn.elifesciences.org/articles/90674/elife-90674-supp3-v1.xlsx
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Supplementary file 4
G protein-coupled receptors (GPCR) screening with peptide mixes.
The file contains the data from the peptide mix-GPCR screening with the mixes (normalized as well as the original readings) and the readings to resolve those peptide mix-GPCR pairs that showed a positive signal and lead to the identification of the individual peptide-receptor pairs described in this study.
- https://cdn.elifesciences.org/articles/90674/elife-90674-supp4-v1.xlsx
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Supplementary file 5
Transcriptome resources.
The file contains a list of the transcriptomes used in this study, together with the sources and data from the BUSCO analysis including completeness of the transcriptomes with percentage of single, double, fragmented, and missing genes.
- https://cdn.elifesciences.org/articles/90674/elife-90674-supp5-v1.xlsx
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Supplementary file 6
Class A G protein-coupled receptor (GPCR) sequences.
The file contains all GPCR sequences that were identified from the different taxa in the HMMer screening. In .fasta format.
- https://cdn.elifesciences.org/articles/90674/elife-90674-supp6-v1.fasta
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Supplementary file 7
Gqi5/9 and G5A sequence.
The file contains the nucleotide sequences of the Gqi5/9 and G5A gene with primer sequences and information about the mutation of the original Gqi9 that we obtained and subcloned into the pcDNA3.1(+) vector.
- https://cdn.elifesciences.org/articles/90674/elife-90674-supp7-v1.txt
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Supplementary file 8
Tested N. vectensis G protein-coupled receptor (GPCR) sequences.
The file contains a list of all tested N. vectensis GPCRs, including original nucleotide and protein sequences, information about the cloning strategy and either the primer sequences of genes that we cloned ourselves or the codon-optimized sequences of genes that were ordered as synthetic constructs.
- https://cdn.elifesciences.org/articles/90674/elife-90674-supp8-v1.zip
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Supplementary file 9
Dose-response assay readings.
The file contains the original readings from our dose-response curves as well as additional measurements such as those for supplementary figures or of peptide-receptor pairs that initially gave a signal in our screen but did not produce confident dose-response curves, as well as negative controls from peptides that were tested at different concentrations on cells that were only transfected with G5A and Gqi5/9 containing plasmids.
- https://cdn.elifesciences.org/articles/90674/elife-90674-supp9-v1.zip
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Supplementary file 10
EC50 values and curve slopes.
The file contains the EC50 values and slopes of the dose-response curves for each peptide-receptor pair.
- https://cdn.elifesciences.org/articles/90674/elife-90674-supp10-v1.csv
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Supplementary file 11
Trees of sequences related to class A neuropeptide G protein-coupled receptors (GPCRs).
The file contains the detailed trees that are shown in Figure 4 and Figure 4—figure supplements 1 and 2, with exact support values and sequence identifiers of all branches.
- https://cdn.elifesciences.org/articles/90674/elife-90674-supp11-v1.pdf
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Supplementary file 12
Single-cell analysis of N. vectensis neuropeptide precursors and G protein-coupled receptors (GPCRs).
The file contains the detailed dotplots of Figure 5 with cell-type resolution of the developmental and the adult single-cell dataset.
- https://cdn.elifesciences.org/articles/90674/elife-90674-supp12-v1.pdf
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MDAR checklist
- https://cdn.elifesciences.org/articles/90674/elife-90674-mdarchecklist1-v1.pdf
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Large-scale deorphanization of Nematostella vectensis neuropeptide G protein-coupled receptors supports the independent expansion of bilaterian and cnidarian peptidergic systems
eLife 12:RP90674.
https://doi.org/10.7554/eLife.90674.3
