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Ancient coexistence of norepinephrine, tyramine, and octopamine signaling in
bilaterians
Philipp Bauknecht1 and Gáspár Jékely1*
1Max Planck Institute for Developmental Biology, Spemannstrasse 35, 72076 Tübingen, Germany *Correspondence to: [email protected] Abstract Norepinephrine/noradrenaline is a neurotransmitter implicated in arousal and other aspects of vertebrate behavior and physiology. In invertebrates, adrenergic signaling is considered absent and analogous functions are attributed to the biogenic amine octopamine. Here we describe the coexistence of signaling by norepinephrine, octopamine, and its precursor tyramine in representatives of the two major clades of Bilateria, the protostomes and the deuterostomes. Using phylogenetic analysis and receptor pharmacology we show that six receptors coexisted in the protostome-deuterostome last common ancestor, two each for the three ligands. All receptors were retained in the genomes of the deuterostome Saccoglossus kowalewskii (a hemichordate) and the protostomes Platynereis dumerilii (an annelid) and Priapulus caudatus (a priapulid). Adrenergic receptors were lost from most insects and nematodes and tyramine and octopamine receptors were lost from most deuterostomes. These results clarify the history of monoamine signaling in animals and highlight the importance of studying slowly evolving marine taxa.
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Introduction Norepinephrine is a major neurotransmitter in vertebrates with a variety of functions including roles in promoting wakefulness and arousal (Singh et al., 2015), regulating aggression (Marino et al., 2005), and autonomic functions such a heart beat (Kim et al., 2002). Signaling by the monoamine octopamine in protostome invertebrates is often considered equivalent to vertebrate adrenergic signaling (Roeder, 2005) with analogous roles in promoting aggression and wakefulness in flies (Crocker and Sehgal, 2008; Zhou et al., 2008), or the regulation of heart rate in annelids and arthropods (Crisp et al., 2010; Florey and Rathmayer, 1978). Octopamine is synthesized from tyramine (Figure 1A) which itself also acts as a neurotransmitter or neuromodulator in arthropods and nematodes (Jin et al., 2016; Kutsukake et al., 2000; Nagaya et al., 2002; Rex and Komuniecki, 2002; Roeder, 2005; Saudou et al., 1990; Selcho et al., 2012). Octopamine and norepinephrine are chemically similar, are synthesized by homologous enzymes (Monastirioti et al., 1996; Wallace, 1976), and signal by similar G-protein coupled receptors (GPCRs) (Evans and Maqueira, 2005; Roeder, 2005), further arguing for their equivalence. However, the precise evolutionary relationship of these transmitter systems is unclear.
The evolution of neurotransmitter systems has been analyzed by studying the distribution of monoamines or biosynthetic enzymes in different organisms (Gallo et al., 2016). This approach has limitations, however, because some of the biosynthetic enzymes are not specific to one substrate (Wallace, 1976) and because trace amounts of several monoamines are found across many organisms, even if specific receptors are often absent. For example, even if invertebrates can synthesize trace amounts of norepinephrine and vertebrates can synthesize tyramine and octopamine, these are not considered to be active neuronal signaling molecules, since the respective receptors are lacking. Consequently, the presence of specific neuronally expressed monoamine receptors is the best indicator that a particular monoamine is used in neuronal signaling (Arakawa et al., 1990; Saudou et al., 1990). We thus decided to analyze the evolution of adrenergic, octopamine, and tyramine receptors in bilaterians. Results Using database searches, sequence-similarity-based clustering, and phylogenetic analysis, we identified a few invertebrate GPCR sequences that were similar to vertebrate adrenergic α1 and α2 receptors (Figure 1B). An adrenergic α1 receptor ortholog is present in the sea urchin Strongylocentrotus purpuratus. Adrenergic α1 and α2 receptors were both present in Saccoglossus kowalewskii, a deuterostome hemichordate (Figure 1B and Supplementary figures 1-2), as previously reported (Krishnan et al., 2013). We also identified adrenergic α1 and α2 receptor orthologs in annelids and mollusks (members of the Lophotrochozoa), including Aplysia californica, and in the priapulid worm Priapulus caudatus (member of the Ecdysozoa)(Figure 1B). Adrenergic α receptors are also present in a few arthropods, including the crustacean Daphnia pulex and the moth Chilo suppressalis (the Chilo receptor was first described as an octopamine receptor (Wu et al., 2012)), but are absent from most other insects (Supplementary figures 1-2). We also identified α2 receptors in some nematodes. The identification of adrenergic α1 and α2 receptor orthologs in ambulacrarians, lophotrochozoans, and ecdysozoans indicates that both families were present in the protostome-deuterostome last common ancestor. We could not identify adrenergic β receptors outside chordates (Supplementary figure 3).
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Figure 1. Biosynthesis of monoamines and cluster map of GPCR sequences. (A) Biosynthesis of tyramine, octopamine, norepinephrine and epinephrine from tyrosine. The enzymes catalyzing the reaction steps are indicated. (B) Sequence-similarity-based cluster map of bilaterian octopamine, tyramine, and adrenergic GPCRs. Nodes correspond to individual GPCRs and are colored based on taxonomy. Edges correspond to BLAST connections of P value >1e-20.
To characterize the ligand specificities of these putative invertebrate adrenergic receptors we cloned them from S. kowalewskii, P. caudatus, and the marine annelid Platynereis dumerilii. We performed in vitro GPCR activation experiments using a Ca2+-mobilization assay (Bauknecht and Jékely, 2015; Tunaru et al., 2005). We found that norepinephrine activated both the adrenergic α1 and α2 receptors from all three species with EC50 values in the nanomolar range. In contrast, tyramine, octopamine, and dopamine were either inactive or only activated the receptors at higher concentrations (Figure 2, Table 1, and Supplementary figures 4-5). These phylogenetic and pharmacological results collectively establish these invertebrate receptors as bona fide adrenergic α receptors.
To investigate if adrenergic signaling coexists with octopamine and tyramine signaling in protostomes we searched for octopamine and tyramine receptors in P. dumerilii and P. caudatus. In phylogenetic and clustering analyses, we identified orthologs for tyramine type 1 and type 2 and octopamine α and β receptors in both species (Figure 1B and Supplementary figures 6-9). We performed activation assays with the P. dumerilii receptors. The tyramine type 1 and type 2 receptors orthologs were preferentially activated by tyramine with EC50 values in the nanomolar range (Figure 2, Table 1, and Supplementary figures 10-11). The P. dumerilii octopamine α receptor was activated by octopamine at a lower concentration than by tyramine and dopamine. The P. dumerilii octopamine β receptor was not active in our assay. These results show that specific receptor systems for norepinephrine, octopamine, and tyramine coexist in P. dumerilii and very likely also P. caudatus.
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Figure 2. Dose-Response curves of selected monoamine GPCRs from P. dumerilii, P. caudatus, and S. kowalevskii treated with varying concentrations of ligand. Data, representing luminescence units relative to the maximum of the fitted dose-response curves, are shown as mean ± SEM (n = 3) for selected monoamine GPCRs. All dose-response curves with diverse agonists and antagonists are shown in the Supplementary material. EC50 values are listed in Table 1.
When did tyramine and octopamine signaling originate? To answer this, we surveyed
available genome sequences for tyramine and octopamine receptors. As expected, we identified several receptors across the protostomes, including ecdysozoans and lophotrochozoans (Supplementary figures 6-9). However, chordate genomes lacked clear orthologs of these receptors. Strikingly, we identified tyramine type 1 and 2 and octopamine α and β receptor orthologs in the genome of the hemichordate S. kowalewskii (Figure 1B). In phylogenetic analyses, we recovered at least one S. kowalewskii sequence in each of the four receptor clades (one octopamine α, one octopamine β, two tyramine type 1, and two tyramine type 2 receptors), establishing these sequences as deuterostome orthologs of these predominantly protostome GPCR families (Supplementary figures 6-9).
We cloned the candidate S. kowalewskii tyramine and octopamine receptors and performed ligand activation experiments. The S. kowalewskii type 2 receptors were preferentially activated by tyramine in the nanomolar range. The type 1 receptor was only activated at very high ligand concentrations. The octopamine α and β receptors were preferentially activated by octopamine in the nanomolar, and low micromolar range, respectively (Figure 2, table 1, and Supplementary figures 10-11). These data show that octopamine and tyramine signaling also
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coexists with adrenergic signaling in this deuterostome, as in P. dumerilii and P. caudatus. The presence of tyramine signaling in S. kowalewskii is also supported by the phyletic distribution of tyrosine decarboxylase, a specific enzyme for tyramine synthesis (Alkema et al., 2005). Tyrosine decarboxylase is present in protostomes and S. kowalewskii but is absent from other deuterostomes (Supplementary figure 12).
We also tested the α adrenergic agonist clonidine and the GPCR antagonists mianserin and yohimbine on several receptors from all three species. These chemicals did not show specificity for any of the receptor types, suggesting these chemicals may not be useful for studying invertebrate neurotransmission (Table 1, and Supplementary figures 4-5, Supplementary figures 9-10). Discussion Our results established the coexistence of adrenergic, octopaminergic, and tyraminergic signaling in the deuterostome S. kowalewskii and the protostomes P. dumerilii and P. caudatus. The discovery of adrenergic signaling in some protostomes and octopamine and tyramine signaling in a deuterostome changes our view on the evolution of monoamine signaling in bilaterians (Figure 3). It is clear from the phyletic distribution of orthologous receptor systems that two families of receptors per each ligand were present in the protostome-deuterostome last common ancestor. These include the adrenergic α1 and α2 receptors, the tyramine type 1 and type 2 receptors, and the octopamine α and β receptors. From the six ancestral families, the octopamine and tyramine receptors were lost from most deuterostomes, and the adrenergic receptors were lost from most ecdysozoans.
Figure 3. Evolution of adrenergic, octopamine, and tyramine signaling in bilaterians. (A) Phylogenetic tree of major clades of bilaterian animals with the loss of specific GPCR families indicated. (B) Phyletic distribution of adrenergic, octopamine, and tyramine GPCR families across major bilaterian clades.
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We consider the measured in vitro ligand preferences for these receptors as indicative of their physiological ligands. In general, there is a two orders of magnitude difference in the EC50 values between the best ligand and other related ligands, indicating the high specificity of the receptors. The preferred ligand of all six orthologous receptor families is the same across protostomes and deuterostomes, indicating the evolutionary stability of ligand-receptor pairs, similar to the long-term stability of neuropeptide GPCR ligand-receptor pairs (Jékely, 2013; Mirabeau and Joly, 2013).
Understanding the ancestral role of these signaling systems and why they may have been lost differentially in different animal groups will require functional studies in organisms where all three neurotransmitter systems coexist. Signaling by norepinephrine in vertebrates has often been considered as equivalent to signaling by octopamine in invertebrates. Our results change this view and show that these signaling systems coexisted ancestrally and still coexist in some bilaterians, and thus cannot be equivalent. This has important implications for our interpretation of comparative studies of neural circuits and nervous system evolution.
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Acknowledgments We thank John Gerhart for Saccoglossus DNA and the image of Saccoglossus. We thank Mattias Hogvall for Priapulus DNA and the image of Priapulus. We also thank Elizabeth Williams for comments on the manuscript. The research leading to these results received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ European Research Council Grant Agreement 260821. P.B. is supported by the International Max Planck Research School (IMPRS) ‘‘From Molecules to Organisms.’’ Materials and Methods Gene identification and receptor cloning Platynereis protein sequences were collected from a Platynereis mixed stages transcriptome assembly (Conzelmann et al., 2013). The accession numbers of all newly identified GPCRs tested here are GenBank: KX372342, KX372343, KP293998, KU715093, KU530199, KU886229). GPCR sequences from other species were downloaded from NCBI. GPCRs were cloned into pcDNA3.1(+) (Thermo Fisher Scientific, Waltham, USA) as described before (Bauknecht and Jékely, 2015). Forward primers consisted of a spacer (ACAATA) followed by a
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BamHI or EcoRI restriction site, the Kozak consensus sequence (CGCCACC), the start codon (ATG) and a sequence corresponding to the target sequence. Reverse primers consisted of a spacer (ACAATA), a NotI restriction site, a STOP codon, and reverse complementary sequence to the target sequence. Primers were designed to end with a C or G with 72°C melting temperature. PCR was performed using Phusion polymerase (New England Biolabs GmbH, Frankfurt, Germany). The sequences of all Platynereis GPCRs tested here were deposited in GenBank (accession numbers: α1-adrenergic receptor, KX372342; α2-adrenergic receptor, KX372343 Tyramine-1 receptor, KP293998; Tyramine-2 receptor, KU715093; Octopamine α receptor, KU530199; Octopamine β receptor, KU886229). Tyramine receptor 1 has been previously published (Bauknecht and Jékely, 2015) as Pdu orphan GPCR 48. Cell culture and receptor deorphanization Cell culture assays were done as described before (Bauknecht and Jékely, 2015). Briefly, CHO-K1 cells were kept in Ham’s F12 Nut Mix medium (Thermo Fisher Scientific, Waltham, USA) with 10 % fetal bovine serum and penicillin-streptomycin (PenStrep, Invitrogen). Cells were seeded in 96-well plates (Thermo Fisher Scientific, Waltham, USA) at approximately 10,000 cells/well. After 1 day, cells were transfected with plasmids encoding a GPCR, the promiscuous Gα-16 protein (Offermanns and Simon, 1995), and a reporter construct GFP-apoaequorin (Baubet et al., 2000) (60 ng each) using 0.375 µl of the transfection reagent TurboFect (Thermo Fisher Scientific, Waltham, USA). After two days of expression, the medium was removed and replaced with Hank’s Balanced Salt Solution (HBSS) supplemented with 1.8 mM Ca2+, 10 mM glucose and 1 mM coelenterazine h (Promega, Madison, USA). After incubation at 37°C for 2 hours, cells were tested by adding synthetic monoamines (Sigma, St. Louis, USA) in HBSS supplemented with 1.8 mM Ca2+ and 10 mM glucose. Solutions containing norepinephrine, epinephrine or dopamine were supplemented with 100 µM ascorbic acid to prevent oxidation. Luminescence was recorded for 45 seconds in a plate reader (BioTek Synergy Mx or Synergy H4, BioTek, Winooski, USA). For inhibitor testing, the cells were incubated with yohimbine or mianserin (Sigma, St. Louis, USA) for 1 hour. Then, synthetic monoamines were added to yield in each case the smallest final concentration expected to elicit the maximal response in the absence of inhibitor and luminescence was recorded for 45 seconds. Data were integrated over the 45-second measurement period. Data for dose-response curves were recorded in triplicate for each concentration. Dose-response curves were fitted with a four-parameter curve using Prism 6 (GraphPad, La Jolla, USA). The curves were normalized to the calculated upper plateau values (100% activation). Bioinformatics Protein sequences were downloaded from the NCBI. Redundant sequences were removed from the collection using CD-HIT (Li and Godzik, 2006) with an identity cutoff of 95%. Sequence cluster maps were created with CLANS2 (Frickey and Lupas, 2004) using the BLOSUM62 matrix and a P-value cutoff of 1E-70. For phylogenetic trees, protein sequences were aligned with MUSCLE (Edgar, 2004). Alignments were trimmed with TrimAI (Capella-Gutiérrez et al., 2009) in “Automated 1” mode. The best amino acid substitution model was selected using ProtTest 3 (Darriba et al., 2011). Maximum likelihood trees were calculated with RAxML (Stamatakis, 2014) using the CIPRES Science Gateway (Miller et al., 2010). Bootstrap analysis was done and automatically stopped (Pattengale et al., 2010) when the Majority Rule Criterion (autoMRE) was met. The resulting trees were visualized with FigTree
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(http://tree.bio.ed.ac.uk/software/figtree/). The identifiers of deorphanized octopamine and tyramine receptors (Balfanz et al., 2014; Blais et al., 2010; Chang et al., 2000; Chen et al., 2010; Gerhardt et al., 1997; Gross et al., 2015; Huang et al., 2012; Jezzini et al., 2014; Kastner et al., 2014; Lind et al., 2010; Rex and Komuniecki, 2002; Verlinden et al., 2010; Wu et al., 2012; Wu et al., 2014; Wu et al., 2015) were tagged with _Oa, _Ob, _T1, or _T2.
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EC50 (M)/IC50 (M) Tyramine Octopami
ne Norepinephrine
Epinephrine
Dopamine
Clonidine Yohimbine
Mianserin
P. dumerilii α1-adrenergic KX372342
inactive inactive 2.12E-07 3.79E-07 1.25E-04 inactive 4.41E-06 3.79E-06
P. dumerilii α2-adrenergic KX372343
8.36E-05 2.67E-06 5.25E-09 1.06E-08 1.63E-06 2.57E-06 5.71E-06 2.53E-05
S. kowalevskii α1-adrenergic ALR88680
inactive n/a 1.67E-08 1.94E-08 3.79E-06 inactive 1.32E-05 4.46E-06
S. kovalewskii α2-adrenergic XP_002734932
3.75E-06 1.94E-06 1.16E-13 2.31E-09 5.60E-09 3.61E-08 7.90E-05 n/a
P. caudatus α1-adrenergic XP_014662992
inactive inactive 7.49E-09 inactive inactive inactive n/a n/a
P. caudatus α2-adrenergic XP_014681069
inactive inactive 2.20E-06 4.52E-07 inactive 1.02E-07 n/a 9.85E-07
P. dumerilii Tyramine-1 KP293998
1.12E-08 2.70E-06 n/a n/a 7.79E-06 n/a 2.06E-06 1.05E-05
P. dumerilii Tyramine-2 KU715093
7.02E-09 7.82E-07 n/a n/a 3.89E-06 n/a 5.38E-05 6.36E-06
S. kovalewskii Tyramine-1 XP_002742354
8.40E-05 n/a inactive inactive n/a 2.86E-04 1.68E-06 1.70E-05
S. kovalewskii Tyramine-2A XP_002734062
1.22E-09 6.24E-08 inactive inactive 7.18E-08 1.39E-06 n/a 1.61E-04
S. kovalewskii Tyramine-2B XP_006812999
3.71E-09 1.57E-06 1.15E-04 2.85E-05 1.44E-06 1.60E-05 2.06E-05 1.88E-05
P. dumerilii Octopamine α KU530199
1.34E-05 2.56E-07 n/a n/a inactive n/a 9.02E-09 1.62E-06
S. kowalevskii Octopamine α XP_006823182
1.72E-05 6.89E-07 5.30E-05 1.85E-05 2.65E-04 1.64E-07 7.78E-06 2.16E-05
S. kowalevskii Octopamine β XP_002733926
inactive 6.37E-08 3.49E-06 inactive inactive inactive 1.57E-04 6.45E-06
Table 1. EC50 (M)/IC50 (M) values of all tested GPCRs with the indicated ligands or inhibitors. The most effective ligand for each receptor is shown in bold.
All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.. https://doi.org/10.1101/063743doi: bioRxiv preprint
13
Supplementary figure 1. Maximum likelihood tree of α1-adrenergic receptors. Bootstrap support values are shown for selected nodes. This tree is part of a larger tree containing all investigated GPCRs.
0.5
EFX64650.1_Daphnia_pulex
XP_010205582.1_Colius_striatus
XP_009050255.1_Lottia_gigantea
XP_014662992.1_Priapulus_caudatus
XP_008279073.1_Stegastes_partitus
CDQ72256.1_Oncorhynchus_mykiss
XP_005285240.1_Chrysemys_picta_bellii
XP_003970475.1_Takifugu_rubripes
XP_014743771.1_Sturnus_vulgaris
XP_008334310.1_Cynoglossus_semilaevis
XP_014822169.1_Calidris_pugnax
XP_014051077.1_Salmo_salar
CDQ56739.1_Oncorhynchus_mykiss
KTG32062.1_Cyprinus_carpio
XP_010788374.1_Notothenia_coriiceps
XP_004460877.1_Dasypus_novemcinctus
XP_013133031.1_Oreochromis_niloticus
NP_001007359.1_Danio_rerio
XP_012687224_Clupea_harengus
XP_003227101.1_Anolis_carolinensis
XP_013867515.1_Austrofundulus_limnaeus
XP_003782039.1_Otolemur_garnettii
CDQ56384.1_Oncorhynchus_mykiss
KTF87842.1_Cyprinus_carpio
XP_014068076.1_Salmo_salar
XP_007430469.1_Python_bivittatus
XP_007247270.1_Astyanax_mexicanus
XP_010154880.1_Eurypyga_helias
XP_009993014.1_Chaetura_pelagica
XP_011355023.1_Pteropus_vampyrus
XP_008630655.1_Corvus_brachyrhynchos
NP_001118147.1_Oncorhynchus_mykiss
XP_013091628_Biomphalaria_glabrata
XP_006138206_Pelodiscus_sinensis
XP_008489604.1_Calypte_anna
XP_007896221.1_Callorhinchus_milii
XP_015268171.1_Gekko_japonicus
XP_002935531.2_Xenopus_tropicalis
XP_697043.2_Danio_rerio
XP_007548430.1_Poecilia_formosa
XP_003726237.1_Strongylocentrotus_purpuratus
KTG02316.1_Cyprinus_carpio
XP_006211986.1_Vicugna_pacos
XP_007477871.1_Monodelphis_domestica
XP_005142671.1_Melopsittacus_undulatus
XP_005719861.1_Pundamilia_nyererei
XP_012959596.1_Anas_platyrhynchos
XP_006625863.1_Lepisosteus_oculatus
XP_013787844.1_Limulus_polyphemus
XP_004665892.1_Jaculus_jaculus
AAA35496.1_Homo_sapiens_AA1A
XP_001922013.3_Danio_rerio
XP_012697555.1_Clupea_harengus
XP_003748403.1_Metaseiulus_occidentalis
XP_009566450.1_Cuculus_canorus
ALR88680.1_Saccoglossus_kowalevskii_AA1
XP_010795800.1_Notothenia_coriiceps
XP_015827734.1_Nothobranchius_furzeri
XP_008059269.1_Tarsius_syrichta
XP_010886834.1_Esox_lucius
XP_006276442.2_Alligator_mississippiensis
XP_004460873.1_Dasypus_novemcinctus
KX372342_Platynereis_dumerilii_AA1
KFM77668.1_Stegodyphus_mimosarum
ETE57756.1_Ophiophagus_hannah
XP_010795024.1_Notothenia_coriiceps
XP_002197113.1_Taeniopygia_guttata
XP_007424603.1_Python_bivittatus
XP_015598849.1_Cephus_cinctus
XP_014663408.1_Priapulus_caudatus
XP_015233524.1_Cyprinodon_variegatus
ADK98420.1_Meriones_unguiculatus
XP_008325595.1_Cynoglossus_semilaevis
XP_010143615.1_Buceros_rhinoceros_silvestris
CDQ95487.1_Oncorhynchus_mykiss
XP_012709980.1_Fundulus_heteroclitus
XP_006629253.1_Lepisosteus_oculatus
XP_002933012.2_Xenopus_tropicalis
CDQ73039.1_Oncorhynchus_mykiss
KPP75495.1_Scleropages_formosus
XP_004065916.1_Oryzias_latipes
XP_008921792.1_Manacus_vitellinus
KTG31243.1_Cyprinus_carpio
XP_009899896.1_Picoides_pubescens
XP_007235606.1_Astyanax_mexicanus
XP_004073535.1_Oryzias_latipes
XP_006805960.1_Neolamprologus_brichardi
XP_015152983.1_Gallus_gallus
XP_003793998.1_Otolemur_garnettii
XP_009951638.1_Leptosomus_discolor
XP_008282667.1_Stegastes_partitus
XP_011422115.1_Crassostrea_gigasELU14162.1_Capitella_teleta
XP_010177730.1_Mesitornis_unicolor
XP_006114239.1_Pelodiscus_sinensis
XP_012736728.1_Fundulus_heteroclitus
XP_013917984.1_Thamnophis_sirtalis
XP_008499885.1_Calypte_anna
XP_013419311.1_Lingula_anatina
XP_015255918.1_Cyprinodon_variegatus
XP_013864145.1_Austrofundulus_limnaeus
XP_006252170.1_Rattus_norvegicus
XP_011478573.1_Oryzias_latipes
XP_008417330.1_Poecilia_reticulata
XP_010900547.1_Esox_lucius
XP_012936806.1_Aplysia_californica
XP_014777674.1_Octopus_bimaculoides
XP_006010360.1_Latimeria_chalumnae
XP_010853649.1_Bison_bison_bison
1
100
100
85
83
71
99
80
93
96
95
63
α1-adrenergic receptors
All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.. https://doi.org/10.1101/063743doi: bioRxiv preprint
14
Supplementary figure 2. Maximum likelihood tree of α2-adrenergic receptors. Bootstrap support values are shown for selected nodes. This tree is part of a larger tree containing all investigated GPCRs.
0.5
XP_010784224.1_Notothenia_coriiceps
CDJ80122.1_Haemonchus_contortus
XP_012722422.1_Fundulus_heteroclitus
KPP70140.1_Scleropages_formosus
XP_013875010.1_Austrofundulus_limnaeus
Q90WY4.1_Danio_rerio
CDQ90138.1_Oncorhynchus_mykiss
XP_014026328.1_Salmo_salar
KTF91499.1_Cyprinus_carpio
KX372343_Platynereis_dumerilii_AA2
KTG06372.1_Cyprinus_carpio
XP_008315824.1_Cynoglossus_semilaevis
XP_010968209.1_Camelus_bactrianus
CDQ73934.1_Oncorhynchus_mykiss
XP_014938420.1_Acinonyx_jubatus
CAD11981.1_Rhogeessa_tumida
EDL01717.1_Mus_musculus
XP_006778809.1_Myotis_davidii
XP_015267985.1_Gekko_japonicus
XP_011573649.1_Aquila_chrysaetos_canadensis
XP_012692326.1_Clupea_harengus
XP_012730184.1_Fundulus_heteroclitus
XP_014333406.1_Bos_mutus
XP_010732944.1_Larimichthys_crocea
CAG05052.1_Tetraodon_nigroviridis
XP_008296024.1_Stegastes_partitus
KQK80321.1_Amazona_aestiva
XP_010981560.1_Camelus_dromedarius
XP_015350355.1_Marmota_marmota_marmota
XP_014055357.1_Salmo_salar
XP_015262427.1_Gekko_japonicus
XP_014274544.1_Halyomorpha_halys
Q8JG70.1_Danio_rerio
XP_014390455.1_Myotis_brandtii
CAG00720.1_Tetraodon_nigroviridis
XP_002734932.1_Saccoglossus_kowalevskii_AA2
XP_013920993.1_Thamnophis_sirtalis
CDQ75057.1_Oncorhynchus_mykiss
XP_012873058.1_Dipodomys_ordii
XP_014242340.1_Cimex_lectularius
XP_009911888.1_Haliaeetus_albicilla
XP_007905605.1_Callorhinchus_milii
XP_005813740.2_Xiphophorus_maculatus
XP_013986063.1_Salmo_salar
XP_002644716.1_Caenorhabditis_briggsae
XP_007173405.1_Balaenoptera_acutorostrata_scammoni
XP_009067029.1_Lottia_gigantea
XP_011433775.1_Crassostrea_gigas
CAL36767.1_Branchiostoma_floridae
XP_002595000.1_Branchiostoma_floridae
XP_015718435.1_Coturnix_japonica
XP_005308213.1_Chrysemys_picta_bellii
XP_009806348.1_Gavia_stellata
EHH14168.1_Macaca_mulatta
XP_010778794.1_Notothenia_coriiceps
CEF71092.1_Strongyloides_ratti
XP_010733243.1_Larimichthys_crocea
EFX76926.1_Daphnia_pulex
XP_008296631.1_Stegastes_partitus
XP_015200296.1_Lepisosteus_oculatus
XP_015244492.1_Cyprinodon_variegatus
CDQ85392.1_Oncorhynchus_mykiss
XP_014046094.1_Salmo_salar
XP_004539207_Maylandia_zebra
XP_003966929.1_Takifugu_rubripes
XP_012953671.1_Anas_platyrhynchos
XP_008334393.1_Cynoglossus_semilaevis
CAG01293.1_Tetraodon_nigroviridis
XP_013156970.1_Falco_peregrinus
XP_014062277.1_Salmo_salar
P32251.1_Carassius_auratus
KTG45995.1_Cyprinus_carpio
XP_007420380.1_Python_bivittatus
XP_007236033.1_Astyanax_mexicanus
XP_006135062.1_Pelodiscus_sinensis
XP_003787216.1_Otolemur_garnettii
XP_005446496.1_Falco_cherrug
CAD20302.1_Tachyoryctes_sp.
XP_012685854.1_Clupea_harengus
XP_015463451.1_Astyanax_mexicanus
XP_008538974.1_Equus_przewalskii
XP_015252539.1_Cyprinodon_variegatus
XP_012675626.1_Clupea_harengus
XP_015673604.1_Protobothrops_mucrosquamatus
XP_012879340.1_Dipodomys_ordii
XP_014010145.1_Salmo_salar
EDL94448.1_Rattus_norvegicus
XP_003755431.1_Sarcophilus_harrisii
XP_003218553.1_Anolis_carolinensis
XP_014958903.1_Ovis_aries
XP_010777801.1_Notothenia_coriiceps
XP_008320162.1_Cynoglossus_semilaevis
XP_014767930.1_Octopus_bimaculoides
XP_015202895.1_Lepisosteus_oculatus
CDQ94371.1_Oncorhynchus_mykiss
XP_012579513.1_Condylura_cristata
XP_008198464.2_Tribolium_castaneum
XP_013030003.1_Anser_cygnoides_domesticus
XP_003972567.1_Takifugu_rubripes
XP_006022896.1_Alligator_sinensis
XP_013857577.1_Austrofundulus_limnaeus
XP_008315656.1_Cynoglossus_semilaevis
XP_006750950.1_Leptonychotes_weddellii
XP_012626815.1_Microcebus_murinus
XP_005874627.1_Myotis_brandtii
ERE78076.1_Cricetulus_griseus
XP_008142634.1_Eptesicus_fuscus
XP_005485737.1_Zonotrichia_albicollis
XP_004066404.1_Oryzias_latipes
XP_006880000.1_Elephantulus_edwardii
XP_004265893.1_Orcinus_orca
XP_013383118.1_Lingula_anatina
XP_006609424.1_Apis_dorsata
XP_015262234.1_Gekko_japonicus
CAC87884.1_Takifugu_rubripes
XP_015390193.1_Panthera_tigris_altaica
KKF18259.1_Larimichthys_crocea
NP_997522.1_Danio_rerio
XP_013802685.1_Apteryx_australis_mantelli
XP_010350316.1_Saimiri_boliviensis_boliviensis
NP_001072843.1_Xenopus_tropicalis
XP_005998869.1_Latimeria_chalumnae
XP_010587294.1_Loxodonta_africana
XP_006011954.1_Latimeria_chalumnae
XP_008280550.1_Stegastes_partitus
XP_015228090.1_Cyprinodon_variegatus
XP_005416433.1_Geospiza_fortis
CAI70426.1_Cricetomys_gambianus
XP_005983264.1_Pantholops_hodgsonii
XP_007120031.1_Physeter_catodon
XP_010886433.1_Esox_lucius
Q91081.1_Labrus_ossifagus
XP_010886159.1_Esox_lucius
XP_004942333.2_Gallus_gallus
EPB80586_Ancylostoma_ceylanicum
ELT99717.1_Capitella_teleta
XP_009881752.1_Charadrius_vociferus
XP_013820290.1_Capra_hircus
NP_000672.3_Homo_sapiens_AA2A
XP_007234270.1_Astyanax_mexicanus
ETE69512.1_Ophiophagus_hannah
KPP71495.1_Scleropages_formosus
AEP17845.1_Anomalurus_beecrofti
XP_014681069.1_Priapulus_caudatus
XP_002605761.1_Branchiostoma_floridae
KF460458.1_Chilo_suppressalis_Oa
XP_010895107.1_Esox_lucius
XP_012943687.1_Aplysia_californica
KPP60422.1_Scleropages_formosus
XP_004073361.1_Oryzias_latipes
XP_007903477.1_Callorhinchus_milii
XP_005235799.1_Falco_peregrinus
XP_012697346.1_Clupea_harengus
XP_008325312.1_Cynoglossus_semilaevis
AAN78417.1_Tupaia_belangeri
XP_013868957.1_Austrofundulus_limnaeus
XP_004912925.1_Xenopus_tropicalis
XP_008276148.1_Stegastes_partitus
XP_002663705.3_Danio_rerio
XP_007437437.1_Python_bivittatus
XP_008626884.1_Corvus_brachyrhynchos
XP_014011189.1_Salmo_salar
XP_003217630.1_Anolis_carolinensis
XP_013096124.1_Biomphalaria_glabrata
XP_015738423.1_Coturnix_japonica
XP_004631588.1_Octodon_degus
XP_014059545.1_Salmo_salar
96
77
95
99
96
100
α2-adrenergic receptors
All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.. https://doi.org/10.1101/063743doi: bioRxiv preprint
15
Supplementary figure 3. Maximum likelihood tree of β-adrenergic receptors. Bootstrap support values are shown for some nodes of interest. This tree is part of a larger tree containing all investigated GPCRs.
Supplementary figure 4. Maximum likelihood tree of Tyramine-1 receptors. Bootstrap support values are shown for selected nodes. This tree is part of a larger tree containing all investigated GPCRs. The identifiers of deorphanized tyramine receptors were tagged with _T1.
XP_004587615.1_Ochotona_princeps
CAA06540.1_Petromyzon_marinus
XP_005324245.1_Ictidomys_tridecemlineatus
XP_002122923.1_Ciona_intestinalis
XP_006002873.1_Latimeria_chalumnae
KTG34400.1_Cyprinus_carpio
XP_003796342.1_Otolemur_garnettii
KTG06855.1_Cyprinus_carpio
XP_006994457.1_Peromyscus_maniculatus_bairdii
XP_004696769.1_Echinops_telfairi
XP_015202883.1_Lepisosteus_oculatus
NP_001093397.1_Sus_scrofa
XP_007232946.1_Astyanax_mexicanus
AAQ91625.1_Branchiostoma_floridae
XP_014326866.1_Xiphophorus_maculatus
XP_006000436.1_Latimeria_chalumnae
XP_003217407.2_Anolis_carolinensis
XP_008417789.1_Poecilia_reticulata
XP_012880977.1_Dipodomys_ordii
XP_008325872.1_Cynoglossus_semilaevis
XP_014746696.1_Sturnus_vulgaris
AGU01500.1_Sus_scrofa
XP_003404888.1_Loxodonta_africana
XP_014708070.1_Equus_asinus
KKF21487.1_Larimichthys_crocea
XP_006766281.1_Myotis_davidii
AAA89068.1_Rattus_norvegicus
XP_012707242.1_Fundulus_heteroclitus
XP_014054344.1_Salmo_salar
XP_005795585.2_Xiphophorus_maculatus
XP_004620654.1_Sorex_araneus
XP_015262106.1_Gekko_japonicus
XP_015678290.1_Protobothrops_mucrosquamatus
XP_002710343.1_Oryctolagus_cuniculus
XP_006784977.1_Neolamprologus_brichardi
XP_004073575.1_Oryzias_latipes
CBN81445.1_Dicentrarchus_labrax
XP_013873112.1_Austrofundulus_limnaeus
XP_008586147.1_Galeopterus_variegatus
XP_006893779.1_Elephantulus_edwardii
XP_009284014.1_Aptenodytes_forsteri
XP_014803719.1_Calidris_pugnax
XP_004384975.1_Trichechus_manatus_latirostris
XP_010895941.1_Esox_lucius
KTG42897.1_Cyprinus_carpio
XP_007906212.1_Callorhinchus_milii
NP_001122161.1_Danio_rerio
XP_009474370.1_Nipponia_nippon
XP_015306903.1_Macaca_fascicularis
XP_013870150.1_Austrofundulus_limnaeus
XP_011609112.1_Takifugu_rubripes
XP_519708.2_Pan_troglodytes
NP_001117912.1_Oncorhynchus_mykiss
XP_013863035.1_Austrofundulus_limnaeus
XP_008930097.1_Manacus_vitellinus
XP_010869486.2_Esox_lucius
XP_008690966.1_Ursus_maritimus
KKF11668.1_Larimichthys_crocea
XP_015356717.1_Marmota_marmota_marmota
AAF20199.1_Homo_sapiens
XP_008150402.1_Eptesicus_fuscus
XP_004460730.1_Dasypus_novemcinctus
NP_001082940.1_Danio_rerio
XP_003782105.1_Otolemur_garnettii
ACX46713.1_Pimephales_promelas
XP_007903898.1_Callorhinchus_milii
XP_009470511.1_Nipponia_nippon
XP_006277924.1_Alligator_mississippiensis
NP_001083418.1_Xenopus_laevis
XP_008488388.1_Calypte_anna
XP_005719651.1_Pundamilia_nyererei
XP_006985221.1_Peromyscus_maniculatus_bairdii
AAG31164.1_Ovis_aries
KPP58190.1_Scleropages_formosus
KPP77020.1_Scleropages_formosus
CAG12607.1_Tetraodon_nigroviridis
XP_015731815.1_Coturnix_japonica
XP_008836112.1_Nannospalax_galili
NP_001116897.2_Xenopus_tropicalis
XP_007260201.1_Astyanax_mexicanus
CAA06541.1_Petromyzon_marinus
XP_012669957.1_Clupea_harengus
CAA06539.1_Myxine_glutinosa
XP_008977573.1_Callithrix_jacchus
XP_009069256.1_Acanthisitta_chloris
Q28927.2_sheep
XP_005468045.1_Oreochromis_niloticus
NP_031446.2_Mus_musculus
XP_005614864.1_Equus_caballus
XP_006879982.1_Elephantulus_edwardii
AAA35550.1_Homo_sapiens
XP_007447551.1_Lipotes_vexillifer
XP_015232667.1_Cyprinodon_variegatusXP_014901064.1_Poecilia_latipinna
XP_007421173.1_Python_bivittatus
XP_004664808.1_Jaculus_jaculus
ELW64306.1_Tupaia_chinensis
XP_007553424.1_Poecilia_formosa
XP_007516440.1_Erinaceus_europaeus
XP_007188760.1_Balaenoptera_acutorostrata_scammoni
NP_001085791.1_Xenopus_laevis
XP_006052758.1_Bubalus_bubalis
XP_003934046.2_Saimiri_boliviensis_boliviensis
ETE71498.1_Ophiophagus_hannah
XP_008062588.1_Tarsius_syrichta
XP_007231763.1_Astyanax_mexicanus
XP_015245990.1_Cyprinodon_variegatus
XP_015229372.1_Cyprinodon_variegatus
XP_008275795.1_Stegastes_partitus
ABG56138.1_Bos_taurus
XP_008277801.1_Stegastes_partitus
XP_007435758.1_Python_bivittatus
XP_007574343.1_Poecilia_formosa
XP_010383600.1_Rhinopithecus_roxellana
XP_009645348.1_Egretta_garzetta
XP_013912817.1_Thamnophis_sirtalis
NP_000675.1_Homo_sapiens
ERE85201.1_Cricetulus_griseus
XP_004080293.1_Oryzias_latipes
XP_015818613.1_Nothobranchius_furzeri
XP_012691165.1_Clupea_harengus
XP_012597080.1_Microcebus_murinus
NP_001096122.1_Danio_rerio
XP_004428726.1_Ceratotherium_simum_simum
XP_015204860.1_Lepisosteus_oculatus
XP_003477383.1_Cavia_porcellus
AAI60515.1_Xenopus_tropicalis
XP_006863914.1_Chrysochloris_asiatica
XP_013918020.1_Thamnophis_sirtalis
XP_004686925.1_Condylura_cristata
XP_010625409.1_Fukomys_damarensis
72
100
30
100
90
0.5
β-adrenergic receptors
0.5
XP_312420.3_Anopheles_gambiae_str._PEST
XP_011339818.1_Cerapachys_biroi
XP_002426131.1_Pediculus_humanus_corporis
XP_014359277.1_Papilio_machaon
KRT86051.1_Oryctes_borbonicus
KOB69231.1_Operophtera_brumata
KFB35059.1_Anopheles_sinensisKFB51134.1_Anopheles_sinensis
XP_013197949.1_Amyelois_transitellaAFG26689.1_Chilo_suppressalis
NP_001164311.1_Tribolium_castaneum
ACJ06651.1_Spodoptera_littoralis
XP_003392960.1_Bombus_terrestris
XP_002742354.2_Saccoglossus_kowalevskii_T1
XP_014278335.1_Halyomorpha_halys
XP_014776496.1_Octopus_bimaculoides
XP_001863342.1_Culex_quinquefasciatus
XP_012227598.1_Linepithema_humile
XP_013190483.1_Amyelois_transitella
XP_008472990.1_Diaphorina_citri
KPJ14377.1_Papilio_machaon
XP_002415939.1_Ixodes_scapularis
XP_013791379.1_Limulus_polyphemus
XP_014481134.1_Dinoponera_quadriceps
Q25321.1_Locusta_migratoria_T1
XP_012259361.1_Athalia_rosae
BAB71788.1_Drosophila_melanogaster_T1
XP_013784920.1_Limulus_polyphemus
XP_014099568.1_Bactrocera_oleae
AKQ63052.1_Platynereis_dumerilii_T1
XP_011501106.1_Ceratosolen_solmsi_marchali
KPI95840.1_Papilio_xuthus
XP_011451309.1_Crassostrea_gigas
XP_004526236.1_Ceratitis_capitata
XP_001652255.2_Aedes_aegypti
XP_011156273.1_Solenopsis_invicta
EF490687.1_Rhipicephalus_microplus_T1
XP_001958478.1_Drosophila_ananassae
XP_002008573.2_Drosophila_mojavensis
EHJ68870.1_Danaus_plexippus
ELT94407.1_Capitella_teleta
BAL72847.1_Phormia_regina
NP_001024335.1_Caenorhabitis_elegans_T1
ENN74962.1_Dendroctonus_ponderosae
XP_014672793.1_Priapulus_caudatus
CAQ48240.1_Periplaneta_americana_T1
ABY71758.1_Macrobrachium_rosenbergii
XP_001352884.2_Drosophila_pseudoobscura_pseudoobscura
KMQ93013.1_Lasius_niger
XP_008206976.1_Nasonia_vitripennis
AHN85845.1_Nicrophorus_vespilloides
XP_014614240.1_Polistes_canadensis
AFX62896.1_Pieris_rapae
ALM55746.1_Sitophilus_oryzae
CAB76374.1_Apis_mellifera_T1
XP_011562493.1_Plutella_xylostella
EGT40715_Caenorhabditis_brenneri
XP_011202418.1_Bactrocera_dorsalis
XP_013116014.1_Stomoxys_calcitrans
XP_011194697.1_Bactrocera_cucurbitae
XP_001985558.1_Drosophila_grimshawi
KOB71731.1_Operophtera_brumata
XP_002068046.2_Drosophila_willistoni
XP_011312300.1_Fopius_arisanus
KOB67833.1_Operophtera_brumata
CAA64865.1_Bombyx_mori_T1
XP_014240675.1_Cimex_lectularius
ALC43118.1_Drosophila_busckii
KDR16545.1_Zootermopsis_nevadensis
ETN58983.1_Anopheles_darlingi
XP_006822832.1_Saccoglossus_kowalevskii
XP_014218105.1_Copidosoma_floridanum
XP_005185605.2_Musca_domesticaKNC24708.1_Lucilia_cuprina
ENN72328.1_Dendroctonus_ponderosae
XP_014235818.1_Trichogramma_pretiosum
KDR16544.1_Zootermopsis_nevadensis
81
73
98
74
95
Tyramine-1 receptors
All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.. https://doi.org/10.1101/063743doi: bioRxiv preprint
16
Supplementary figure 5. Maximum likelihood tree of Tyramine-2 receptors. Bootstrap support values are shown for selected nodes. This tree is part of a larger tree containing all investigated GPCRs. The identifiers of deorphanized tyramine receptors were tagged with _T2.
Supplementary figure 6. Maximum likelihood tree of Octopamine-α receptors. Bootstrap support values are shown for selected nodes. This tree is part of a larger tree containing all investigated GPCRs. The identifiers of deorphanized octopamine receptors were tagged with _Oa.
0.5
DAA64505.1_Tribolium_castaneum
XP_002056769.2_Drosophila_virilis
XP_013142077.1_Papilio_polytes
XP_001649033.2_Aedes_aegypti
KPJ03809.1_Papilio_xuthus
KU715093_Platynereis_dumerilii_T2
XP_004529356.1_Ceratitis_capitata
XP_011154023.1_Harpegnathos_saltator
XP_001998427.2_Drosophila_mojavensis
XP_015174118.1_Polistes_dominula
XP_011069002.1_Acromyrmex_echinatior
XP_011184892.1_Bactrocera_cucurbitae
XP_012281361.1_Orussus_abietinus
KDR18992.1_Zootermopsis_nevadensis
AHN85846.1_Nicrophorus_vespilloides
XP_011184896.1_Bactrocera_cucurbitae
ETN64745.1_Anopheles_darlingi
XP_014097299.1_Bactrocera_oleae
XP_011875691.1_Vollenhovia_emeryi
KNC22325.1_Lucilia_cuprina
XP_001954230.1_Drosophila_ananassaeNP_650652.1_Drosophila_melanogaster_T2
XP_003426713.1_Nasonia_vitripennis
XP_008475339.1_Diaphorina_citri
XP_005185145.1_Musca_domestica
XP_011555966.1_Plutella_xylostella
ETN64509.1_Anopheles_darlingi
XP_001994504.1_Drosophila_grimshawi
XP_014356598.1_Papilio_machaon
XP_012224475.1_Linepithema_humile
XP_011500363.1_Ceratosolen_solmsi_marchali
XP_309588.5_Anopheles_gambiae_str._PEST
XP_011293189.1_Musca_domestica
ALC46687.1_Drosophila_busckii
XP_014097290.1_Bactrocera_oleae
KNC22330.1_Lucilia_cuprina
XP_001998428.2_Drosophila_mojavensis
XP_014488846.1_Dinoponera_quadriceps
XP_003702699.1_Megachile_rotundata
ERL83362.1_Dendroctonus_ponderosae
XP_001649034.2_Aedes_aegypti
EHJ64085.1_Danaus_plexippus
XP_004529353.2_Ceratitis_capitata
XP_008188216.1_Acyrthosiphon_pisum
XP_014235895.1_Trichogramma_pretiosum
XP_013165484.1_Papilio_xuthus
XP_013188063.1_Amyelois_transitella
XP_006608318.1_Apis_dorsata
XP_013786964.1_Limulus_polyphemus
KOF79848.1_Octopus_bimaculoides
XP_006812999.1_Saccoglossus_kowalevskii_T2B
KFB41294.1_Anopheles_sinensis
XP_011203887.1_Bactrocera_dorsalis
XP_002734062.1_Saccoglossus_kowalevskii_T2A
BAI52937.1_Bombyx_mori_T2
XP_011633420.1_Pogonomyrmex_barbatus
ALC46686.1_Drosophila_busckii
ADK91078.1_Chilo_suppressalis_T2
XP_001359694.4_Drosophila_pseudoobscura_pseudoobscura
XP_014678717.1_Priapulus_caudatus
KOC70478.1_Habropoda_laboriosa
XP_002429476.1_Pediculus_humanus_corporis
XP_014778476.1_Octopus_bimaculoides
XP_011184895.1_Bactrocera_cucurbitaeXP_013112132.1_Stomoxys_calcitrans
XP_001994503.1_Drosophila_grimshawi
XP_014207104.1_Copidosoma_floridanum
XP_013785118.1_Limulus_polyphemus
ELU14919.1_Capitella_teleta
XP_002096110.1_Drosophila_yakuba
XP_004529355.2_Ceratitis_capitata
KOX76145.1_Melipona_quadrifasciata
KPJ21156.1_Papilio_machaon
XP_015026222.1_Drosophila_virilis
XP_013112135.1_Stomoxys_calcitrans
XP_015368899.1_Diuraphis_noxia
XP_011203890.1_Bactrocera_dorsalis
XP_003393336.1_Bombus_terrestris
XP_002072092.2_Drosophila_willistoni
XP_008197781.1_Tribolium_castaneum
XP_012257334.1_Athalia_rosae98
77
92
100
100
98
Tyramine-2 receptors
0.5
XP_012156222.1_Ceratitis_capitata
XP_002429940.1_Pediculus_humanus_corporis
XP_011872000.1_Vollenhovia_emeryi
AFG22597.1_Mythimna_separata
EHJ74631.1_Danaus_plexippus
XP_001869401.1_Culex_quinquefasciatus
XP_002408812.1_Ixodes_scapularis
XP_013110682.1_Stomoxys_calcitrans
AAP93817.1_Periplaneta_americana_Oa
XP_011264281.1_Camponotus_floridanus
XP_015116423.1_Diachasma_alloeum
XP_014225936.1_Trichogramma_pretiosum
XP_012223081.1_Linepithema_humile
XP_003736341.2_Drosophila_pseudoobscura_pseudoobscura
XP_008558339.1_Microplitis_demolitor
XP_013772246.1_Limulus_polyphemus
XP_011147506.1_Harpegnathos_saltator
XP_014291339.1_Halyomorpha_halys
XP_001648244.2_Aedes_aegypti
XP_014772693.1_Octopus_bimaculoides
XP_011291813.1_Musca_domestica
XP_011185898.1_Bactrocera_cucurbitae
XP_001994470.1_Drosophila_grimshawi
XP_012136296.1_Megachile_rotundata
XP_009058244.1_Lottia_gigantea
KOX67468.1_Melipona_quadrifasciata
GU074418.1_Balanus_improvisus_Oa
XP_004523572.1_Ceratitis_capitata
KOC61009.1_Habropoda_laboriosa
EGK96547.1_Anopheles_gambiae_Oa
XP_012261366.1_Athalia_rosae
XP_012937854.1_Aplysia_californica
XP_013079946.1_Biomphalaria_glabrata
XP_014091610.1_Bactrocera_oleae
XP_311113.4_Anopheles_gambiae_str._PEST
XP_002096452.2_Drosophila_yakuba
JN641302.1_Chilo_suppressalis_Oa
XP_002072172.2_Drosophila_willistoni
XP_014675454.1_Priapulus_caudatus
XP_014613083.1_Polistes_canadensis
U62771.1_Lymnaea_stagnalis_Oa
XP_011303410.1_Fopius_arisanus
XP_013790352.1_Limulus_polyphemus
XP_014362386.1_Papilio_machaon
XP_014486016.1_Dinoponera_quadriceps
XP_011185894.1_Bactrocera_cucurbitae
NP_001280520.1_Tribolium_castaneum
XP_011555307.1_Plutella_xylostella
AHN85844.1_Nicrophorus_vespilloides
XP_011702891.1_Wasmannia_auropunctata
ERL88449.1_Dendroctonus_ponderosae
XP_014253839.1_Cimex_lectularius
KU530199_Platynereis_dumerilii_Oa
CAD67999.1_Apis_mellifera_Oa
ABI33825.1_Manduca_sexta
AAC17442.1_Drosophila_melanogaster_Oa
XP_011505609.1_Ceratosolen_solmsi_marchali
XM_013530280.1_Lingula_anatina
XP_006823182.1_Saccoglossus_kowalevskii_OA1
BAF33393.1_Bombyx_mori_OaXP_013183250.1_Amyelois_transitella
84
9883
77
Octopamine−α receptors
All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.. https://doi.org/10.1101/063743doi: bioRxiv preprint
17
Supplementary figure 7. Maximum likelihood tree of Octopamine-β receptors. Bootstrap support values are shown for selected nodes. This tree is part of a larger tree containing all investigated GPCRs. The identifiers of deorphanized octopamine receptors were tagged with _Ob.
0.5
FC563777.1_Lottia_gigantea
XP_011140150.1_Harpegnathos_saltator
AF117654.1_Aplysia_kurodai_Ob
HF548211.1_Apis_mellifera_Ob
XP_396348.4_Apis_mellifera
XP_014241423.1_Cimex_lectularius
XP_011702778.1_Wasmannia_auropunctata
XP_011557485.1_Plutella_xylostella
XP_002054844.1_Drosophila_virilis
XP_012215458.1_Linepithema_humile
XP_011344277.1_Cerapachys_biroi
XP_003701983.1_Megachile_rotundata
XP_002733926.1_Saccoglossus_kowalevskii_Ob
XP_014467276.1_Dinoponera_quadriceps
XP_009052442.1_Lottia_gigantea
XP_014207204.1_Copidosoma_floridanum
JN620367.1_Chilo_suppressalis_Ob
XP_008548361.1_Microplitis_demolitor
ELT86969.1_Capitella_teleta
NP_001280514.1_Tribolium_castaneum
HF548212.1_Apis_mellifera_Ob
KNC25005.1_Lucilia_cuprina
GU074422.1_Balanus_improvisus_Ob
AEO17899.1_Drosophila_melanogaster
XP_014611330.1_Polistes_canadensis
CCO13922.1_Apis_mellifera_Ob
XP_012281474.1_Orussus_abietinusAB470228.1_Bombyx_mori_Ob
EHJ66744.1_Danaus_plexippus
XP_011876313.1_Vollenhovia_emeryi
XP_014088159.1_Bactrocera_oleae
XP_002074057.1_Drosophila_willistoni
XP_011294329.1_Musca_domestica
XP_011166275.1_Solenopsis_invicta
XP_012269188.1_Athalia_rosae
XP_011504152.1_Ceratosolen_solmsi_marchali
XP_012261826.1_Athalia_rosae
XP_011630965.1_Pogonomyrmex_barbatus
XP_013165427.1_Papilio_xuthus
XP_008207151.1_Nasonia_vitripennis
XP_002422997.1_Pediculus_humanus_corporis
XP_013096128.1_Biomphalaria_glabrata
XP_015180910.1_Polistes_dominula
XP_011349656.1_Cerapachys_biroi
NP_001280505.1_Tribolium_castaneum
XP_001994735.1_Drosophila_grimshawi
XP_013389322.1_Lingula_anatina
AY055377.1_Spisula_solidissima_Ob
AGV79326.1_Chilo_suppressalis
XP_014221613.1_Trichogramma_pretiosum
XP_011263631.1_Camponotus_floridanus
KDR19125.1_Zootermopsis_nevadensis
XP_001357862.3_Drosophila_pseudoobscura_pseudoobscura
XP_012281328.1_Orussus_abietinus
AHN85842.1_Nicrophorus_vespilloides
GU074421.1_Balanus_improvisus_Ob
XP_001947781.1_Acyrthosiphon_pisum
XP_001948521.2_Acyrthosiphon_pisum
XP_003706543.1_Megachile_rotundata
GU074419.1_Balanus_improvisus_Ob
AHN85841.1_Nicrophorus_vespilloides
XP_011053483.1_Acromyrmex_echinatiorXP_011870420.1_Vollenhovia_emeryi
XP_013099911.1_Stomoxys_calcitrans
XP_012166217.1_Bombus_terrestris
XP_014771877.1_Octopus_bimaculoides
XP_004922133.2_Bombyx_mori
XP_012343074.1_Apis_florea
GU074420.1_Balanus_improvisus_Ob
EFX87996.1_Daphnia_pulexXP_014671143.1_Priapulus_caudatus
XP_011448928.1_Crassostrea_gigas
XP_015118835.1_Diachasma_alloeum
XP_013185050.1_Amyelois_transitella
KNC25006.1_Lucilia_cuprinaXP_014254146.1_Cimex_lectularius
XP_001955623.2_Drosophila_ananassae
XP_011312392.1_Fopius_arisanus
Q4LBB9.2_Drosophila_melanogaster_Ob
AF222978.1_Aplysia_californica_Ob
XP_003402376.1_Bombus_terrestris
XP_004523493.1_Ceratitis_capitata
KU886229_Platynereis_dumerilii
XP_002000086.1_Drosophila_mojavensis
87
80
86
71
99
73
74100
Octopamine−β receptors
All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.. https://doi.org/10.1101/063743doi: bioRxiv preprint
18
Supplementary figure 8. Dose-response curves of α1-adrenergic receptors from P. dumerilii, P. caudatus, and S. kowalevskii treated with varying concentrations of ligand or inhibitor. Data, representing luminescence units relative to the maximum of the fitted dose-response curves, are shown as mean ± SEM (n = 3). EC50 and IC50 values are listed in Table 1.
-12 -10 -8 -6 -4 -20
50
100
150
log conc (log[M])
activ
atio
n [%
]
-12 -10 -8 -6 -4 -20
50
100
150
log conc (log[M])
activ
atio
n [%
]P. dumerilii α1-adrenergic
-12 -10 -8 -6 -4 -20
50
100
150
log conc (log[M])
activ
atio
n [%
]
S. kowalevskii α1-adrenergic
-12 -10 -8 -6 -4 -20
50
100
150
log conc (log[M])
activ
atio
n [%
]
-12 -10 -8 -6 -4 -20
50
100
150
P. caudatus α1-adrenergic
log conc (log[M])
activ
atio
n [%
]
Norepinephrine
Dopamine
Epinephrine
Yohimbine
Mianserin
-12 -10 -8 -6 -4 -20
50
100
150
log conc (log[M])
activ
atio
n [%
]
-12 -10 -8 -6 -4 -20
50
100
150
log conc (log[M])
activ
atio
n [%
]Yohimbine
Mianserin
Yohimbine
Mianserin
Octopamine
Norepinephrine
Dopamine
Epinephrine
Norepinephrine
A
B
C
All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.. https://doi.org/10.1101/063743doi: bioRxiv preprint
19
Supplementary figure 9. Dose-response curves of α2-adrenergic receptors from P. dumerilii, P. caudatus, and S. kowalevskii treated with varying concentrations of ligand or inhibitor. Data, representing luminescence units relative to the maximum of the fitted dose-response curves, are shown as mean ± SEM (n = 3). EC50 and IC50 values are listed in Table 1.
-12 -10 -8 -6 -4 -20
50
100
150
log conc (log[M])
activ
atio
n [%
]
-12 -10 -8 -6 -4 -20
50
100
150
log conc (log[M])
activ
atio
n [%
]
-20 -15 -10 -5 00
50
100
150
log conc (log[M])
activ
atio
n [%
]
-15 -10 -5 00
50
100
150
log conc (log[M])
activ
atio
n [%
]
-12 -10 -8 -6 -4 -20
50
100
150
log conc (log[M])
activ
atio
n [%
]
-12 -10 -8 -6 -4 -20
50
100
150
log conc (log[M])
activ
atio
n [%
]
-12 -10 -8 -6 -4 -20
50
100
150
log conc (log[M])
activ
atio
n [%
]
-12 -10 -8 -6 -4 -20
50
100
150
log conc (log[M])
activ
atio
n [%
]
Tyramine
Octopamine
Norepinephrine
Epinephrine
Yohimbine
Mianserin
Yohimbine
Mianserin
Yohimbine
Mianserin
Octopamine
Norepinephrine
Epinephrine
Tyramine
Clonidine
Dopamine
Clonidine
Norepinephrine
Epinephrine
Clonidine
Dopamine
P. dumerilii α2-adrenergic
S. kowalevskii α2-adrenergic
P. caudatus α2-adrenergic
A
B
C
All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.. https://doi.org/10.1101/063743doi: bioRxiv preprint
20
Supplementary figure 10. Dose-response curves of tyramine receptors from P. dumerilii and S. kowalevskii treated with varying concentrations of ligand or inhibitor. Data, representing luminescence units relative to the maximum of the fitted dose-response curves, are shown as mean ± SEM (n = 3). EC50 and IC50 values are listed in Table 1.
-12 -10 -8 -6 -4 -20
50
100
150
log conc (log[M])
activ
atio
n [%
]
-12 -10 -8 -6 -4 -20
50
100
150
log conc (log[M])
activ
atio
n [%
]
-12 -10 -8 -6 -4 -20
50
100
150
log conc (log[M])
activ
atio
n [%
]
-12 -10 -8 -6 -4 -20
50
100
150
log conc (log[M])
activ
atio
n [%
]
-12 -10 -8 -6 -4 -20
50
100
150
log conc (log[M])
activ
atio
n [%
]
-12 -10 -8 -6 -4 -20
50
100
150
log conc (log[M])
activ
atio
n [%
]
-12 -10 -8 -6 -4 -20
50
100
150
log conc (log[M])
activ
atio
n [%
]
-12 -10 -8 -6 -4 -2 00
50
100
150
log conc (log[M])
activ
atio
n [%
]
-14 -12 -10 -8 -6 -4 -20
50
100
150
log conc (log[M])
activ
atio
n [%
]
-14 -12 -10 -8 -6 -4 -20
50
100
150
log conc (log[M])
activ
atio
n [%
]
-12 -10 -8 -6 -4 -20
50
100
150
log conc (log[M])
activ
atio
n [%
]
-12 -10 -8 -6 -4 -20
50
100
150
log conc (log[M])
activ
atio
n [%
]
Tyramine
Octopamine
Dopamine
Yohimbine
Mianserin
Yohimbine
Mianserin
Tyramine
Octopamine
Dopamine
Tyramine
Octopamine
Clonidine
Dopamine
Tyramine
Octopamine
Tyramine
Octopamine
Norepinephrine
Epinephrine
Clonidine
Dopamine
Clonidine
Dopamine
Yohimbine
Mianserin
Yohimbine
Mianserin
Yohimbine
Mianserin
P. dumerilii Tyramine-1
P. dumerilii Tyramine-2
S. kowalevskii Tyramine-1
A
B
C
S. kowalevskii Tyramine-2AD
S. kowalevskii Tyramine-2BE
All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.. https://doi.org/10.1101/063743doi: bioRxiv preprint
21
Supplementary figure 11. Dose-response curves of octopamine receptors from P. dumerilii and S. kowalevskii treated with varying concentrations of ligand or inhibitor. Data, representing luminescence units relative to the maximum of the fitted dose-response curves, are shown as mean ± SEM (n = 3). EC50 and IC50 values are listed in Table 1.
-12 -10 -8 -6 -4 -2 00
50
100
150
log conc (log[M])
activ
atio
n [%
]
-12 -10 -8 -6 -4 -20
50
100
150
log conc (log[M])
activ
atio
n [%
]
-12 -10 -8 -6 -4 -20
50
100
150
log conc (log[M])
activ
atio
n [%
]
-12 -10 -8 -6 -4 -20
50
100
150
log conc (log[M])
activ
atio
n [%
]
-12 -10 -8 -6 -4 -20
50
100
150
log conc (log[M])
activ
atio
n [%
]
-12 -10 -8 -6 -4 -20
50
100
150
log conc (log[M])
activ
atio
n [%
]
-12 -10 -8 -6 -4 -20
50
100
150
log conc (log[M])
activ
atio
n [%
]
Tyramine
Octopamine
Yohimbine
Mianserin
Yohimbine
Mianserin
Yohimbine
Mianserin
Tyramine
Octopamine
Norepinephrine
Dopamine
Clonidine
Epinephrine
Octopamine
Norepinephrine
P. dumerilii Octopamine α
S. kowalevskii Octopamine α
S. kowalevskii Octopamine β
A
B
C
All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.. https://doi.org/10.1101/063743doi: bioRxiv preprint
22
Supplementary figure 12. Maximum likelihood tree of tyrosine decarboxylase and aromatic amino acid decarboxylase enzymes. Bootstrap support values are shown for selected nodes. P. dumerilii, P. caudatus, and S. kowalevskii sequences are highlighted in color. The Caenorhabditis elegans tyrosine decarboxylase was experimentally shown to be required for tyramine biosynthesis (Alkema et al., 2005).
0.2
XP_010219192.1_Tinamus_guttatus
XP_001899866.1_Brugia_malayi
KNC26593.1_Lucilia_cuprina
XP_014605048.1_Polistes_canadensis
XP_012680382.1_Clupea_harengus
AHN85839.1_Nicrophorus_vespilloides
XP_003407186.1_Loxodonta_africana
XP_001870804.1_Culex_quinquefasciatus
AAI30528.1_Homo_sapiens
lcl|Contig8616_Platynereis_dumerilii
XP_004739444.1_Mustela_putorius_furo
XP_008193582.1_Tribolium_castaneum
XP_006875343.1_Chrysochloris_asiatica
XP_012534953.1_Monomorium_pharaonis
KDR12158.1_Zootermopsis_nevadensis
XP_008548824.1_Microplitis_demolitor
XP_308519.3_Anopheles_gambiae_str._PEST
XP_012506091.1_Propithecus_coquereli
XP_012226027.1_Linepithema_humile
XP_003896028.1_Papio_anubisELV13924.1_Tupaia_chinensis
XP_010006646.1_Chaetura_pelagica
XP_011297267.1_Fopius_arisanus
XP_006621116.1_Apis_dorsata
XP_014671545.1_Priapulus_caudatus
XP_014291341.1_Halyomorpha_halys
CEF60746.1_Strongyloides_ratti
NP_001161568.1_Saccoglossus_kowalevskii
BAO52000.1_Gryllus_bimaculatus
XP_014227242.1_Trichogramma_pretiosum
XP_004630583.1_Octodon_degus
NP_001191536.1_Aplysia_californica
NP_495744.1_Caenorhabditis_elegans
XP_009028665.1_Helobdella_robusta
XP_014769443.1_Octopus_bimaculoides
XP_013070812.1_Biomphalaria_glabrata
XP_014774406.1_Octopus_bimaculoides
XP_015245016.1_Cyprinodon_variegatus
XP_012262153.1_Athalia_rosae
BAP16218.1_Gallus_gallus
XP_009048193.1_Lottia_gigantea
ELU11164.1_Capitella_teleta
XP_014649145.1_Ceratotherium_simum_simum
XP_013419956.1_Lingula_anatina
NP_058712.2_Rattus_norvegicus
XP_003762585.1_Sarcophilus_harrisii
XP_002074575.1_Drosophila_willistoni
XP_014666248.1_Priapulus_caudatus
XP_004526583.1_Ceratitis_capitata
XP_005396658.1_Chinchilla_lanigera
XP_010138453.1_Buceros_rhinoceros_silvestris
XP_848285.2_Canis_lupus_familiaris
EKC41301.1_Crassostrea_gigas
XP_003797782.1_Otolemur_garnettii
CAJ38793.1_Platynereis_dumerilii
XP_001950555.2_Acyrthosiphon_pisum
XP_012265736.1_Athalia_rosae
EFX81779.1_Daphnia_pulex
XP_972728.2_Tribolium_castaneum
BAM35936.1_Lymnaea_stagnalis
XP_011196622.1_Bactrocera_cucurbitae
XP_009050823.1_Lottia_gigantea
XP_012147325.1_Megachile_rotundata
XP_012879949.1_Dipodomys_ordii
XP_013401543.1_Lingula_anatina
XP_013789180.1_Limulus_polyphemus
XP_014775466.1_Octopus_bimaculoides
XP_015508789.1_Parus_major
XP_015345826.1_Marmota_marmota_marmota
XP_005108337.1_Aplysia_californica
KRT84890.1_Oryctes_borbonicus
XP_008489540.1_Calypte_anna
XP_014241491.1_Cimex_lectularius
XP_013088909.1_Biomphalaria_glabrata
XP_010339695.1_Saimiri_boliviensis_boliviensis
XP_004839911.1_Heterocephalus_glaber
XP_003704683.1_Megachile_rotundata
XP_001656857.1_Aedes_aegypti
XP_006000049.1_Latimeria_chalumnae
XP_011441559.1_Crassostrea_gigas
XP_014489121.1_Dinoponera_quadriceps
XP_013772406.1_Limulus_polyphemus
XP_005180269.1_Musca_domestica
XP_011449820.1_Crassostrea_gigas
BAQ94593.1_Ambigolimax_valentianus
XP_012265735.1_Athalia_rosae
XP_014667849.1_Priapulus_caudatus
XP_005434516.1_Falco_cherrug
EFO20782.2_Loa_loa
XP_012025784.1_Ovis_aries_musimon
XP_014230308.1_Trichogramma_pretiosum
XP_004010670.1_Ovis_aries
KOX68436.1_Melipona_quadrifasciata
XP_002002172.1_Drosophila_mojavensis
XP_002731843.1_Saccoglossus_kowalevskii
XP_006973100.1_Peromyscus_maniculatus_bairdii
ADP08788.1_Azumapecten_farreri
XP_012942331.1_Aplysia_californica
XP_008847491.1_Nannospalax_galili
XP_010022195.1_Nestor_notabilis
XP_008548921.1_Microplitis_demolitor
XP_013000851.1_Cavia_porcellus
EKC37654.1_Crassostrea_gigas
KHN71251.1_Toxocara_canis
XP_011297268.1_Fopius_arisanus
XP_005949669.1_Haplochromis_burtoni
XP_006635648.1_Lepisosteus_oculatus
ELU05968.1_Capitella_teleta
XP_015054436.1_Drosophila_yakuba
XP_013421253.1_Lingula_anatina
XP_003699867.1_Megachile_rotundata
XP_002735383.1_Saccoglossus_kowalevskii
XP_014291344.1_Halyomorpha_halys
XP_008553493.1_Microplitis_demolitor
XP_009056307.1_Lottia_gigantea
XP_001379620.3_Monodelphis_domestica
XP_014245782.1_Cimex_lectularius
KTG31909.1_Cyprinus_carpio
XP_008202389.1_Nasonia_vitripennis
XP_005107794.1_Aplysia_californica
XP_004415117.1_Odobenus_rosmarus_divergens
XP_004694560.1_Condylura_cristata
XP_004582273.2_Ochotona_princeps
XP_003744997.1_Metaseiulus_occidentalis
XP_013781988.1_Limulus_polyphemus
XP_011443238.1_Crassostrea_gigas
XP_006030466.1_Alligator_sinensis
XP_002426339.1_Pediculus_humanus_corporis
ELU18925.1_Capitella_teleta
XP_012055250.1_Atta_cephalotes
XP_002597913.1_Branchiostoma_floridae
lcl|comp422145_c0_seq4_Platynereis_dumerilii
XP_009680213.1_Struthio_camelus_australis
XP_012940696.1_Aplysia_californica
XP_012272741.1_Orussus_abietinus
XP_008198031.1_Tribolium_castaneum
XP_007942142.1_Orycteropus_afer_afer
XP_012274121.1_Orussus_abietinus
XP_009023368.1_Helobdella_robusta
ACJ13262.1_Drosophila_melanogaster
XP_015122001.1_Diachasma_alloeum
XP_011290217.1_Musca_domestica
XP_011061414.1_Acromyrmex_echinatior
XP_005278593.1_Chrysemys_picta_belliiXP_004708097.1_Echinops_telfairi
XP_002050467.1_Drosophila_virilisXP_001360141.2_Drosophila_pseudoobscura_pseudoobscura
ENN79134.1_Dendroctonus_ponderosae
XP_012942336.1_Aplysia_californica
XP_003966032.1_Takifugu_rubripes
XP_013404119.1_Lingula_anatina
EKC25403.1_Crassostrea_gigas
KFB44891.1_Anopheles_sinensis
XP_008768473.1_Rattus_norvegicus
XP_002426536.1_Pediculus_humanus_corporis
XP_007061422.1_Chelonia_mydas
ELU12210.1_Capitella_teleta
XP_006570903.1_Apis_mellifera
XP_544676.3_Canis_lupus_familiaris
XP_010895280.1_Esox_lucius
XP_002630249.1_Caenorhabditis_briggsae
XP_007895676.1_Callorhinchus_milii
96
100
100
52100
100
95
100
91
arom
atic
am
ino
acid
dec
arbo
xyla
sety
rosi
ne d
ecar
boxy
lase
All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.. https://doi.org/10.1101/063743doi: bioRxiv preprint