Huang W et al. (2025), Discovery and functional characterization of a ...

ECB-ART-53811

Proc Natl Acad Sci U S A 2025 Apr 28;12213:e2420966122. doi: 10.1073/pnas.2420966122.

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Discovery and functional characterization of a bombesin-type neuropeptide signaling system in an invertebrate.

Huang W , Zhong X , Zampronio CG , Bottrill AR , Jones KGE , Tinoco AB , Guo L , Egertová M , Mirabeau O , Elphick MR .

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Neuropeptide signaling systems are key regulators of physiological and behavioral processes in animals. However, the evolutionary history of some neuropeptides originally discovered in vertebrates is unknown. The peptide bombesin (BN) was first isolated from the skin of the toad Bombina bombina and subsequently BN-related neuropeptides have been identified in other chordates, including gastrin-releasing peptide (GRP) and neuromedin B (NMB) in mammals, and a GRP-like peptide in the cephalochordate Branchiostoma japonicum. However, BN-type neuropeptides have hitherto not been identified in any nonchordate animals. Here, we report the discovery and functional characterization of a BN-type neuropeptide signaling system in an echinoderm-the starfish Asterias rubens. BN-type precursor proteins were identified in several echinoderm species based on their amino acid sequences and gene structures, and the mature structure of the A. rubens BN-type neuropeptide ArBN was determined using mass spectrometry. A protein related to vertebrate GRP/NMB-type G protein-coupled receptors was identified experimentally as the receptor for ArBN in A. rubens. Analysis of the distribution of the ArBN precursor in A. rubens using mRNA in situ hybridization and immunohistochemistry revealed a widespread pattern of expression in the central nervous system, digestive system, and locomotory organs. Moreover, effects of ArBN in A. rubens included contraction and retraction of the evertible stomach and inhibition of feeding behavior. Our findings show that the evolutionary history of BN-type neuropeptide signaling can be traced back to the deuterostome common ancestor of echinoderms and chordates. Furthermore, an ancient role of BN-type neuropeptides as regulators of feeding behavior has been revealed.

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	Fig. 1. Sequence alignment and phylogenetic analysis of putative echinoderm and chordate BN-type peptides/precursors. (A) Alignment of putative echinoderm BN-type neuropeptides with chordate BN-type neuropeptides. Conserved residues that occur in at least one echinoderm species and several chordate species are highlighted in yellow. Species and peptide names are highlighted in taxon-specific colors: yellow (Echinodermata), purple (Cephalochordata), blue (Vertebrata). Species name abbreviations: Ajap (A. japonicus), Asol (Acanthaster cf. solaris), Arub (A. rubens), Bbom (B. bombina), Bflo (B. floridae), Ggal (Gallus gallus), Hsap (Homo sapiens), Locu (Lepisosteus oculatus), Ovic (Ophionotus victoriae), Psau (Phyllomedusa sauvagii), Rpip (R. pipiens), Spur (Strongylocentrotus purpuratus). Peptide abbreviations: BN (Bombesin), RN (Ranatensin), PN (Phyllolitorin), GRP (Gastrin-releasing peptide), NMB (Neuromedin B). Accession numbers of the precursor sequences used for the neuropeptide alignment in this figure are listed in SI Appendix, Table S1. (B) Phylogenetic tree generated using the maximum likelihood method (1,000 bootstrap replicates, JTT+I+G4 substitution model) and showing that the putative A. rubens BN-type precursor (ArubBNP) (arrow) and related proteins in other echinoderms are positioned in the same clade as chordate BN-type precursors. This clade is distinct from other clades that comprise protostome CCHa/EP-type precursors and vertebrate ET-type precursors. Elevenin-type precursors are included as an outgroup. Bootstrap support for each node is represented by colored squares and the colored backgrounds highlight different taxonomic groups (see key). The scale bar represents the average residue substitution per site. Abbreviations for species names not shown in part A of this figure: Bmor (Bombyx mori), Dmel (D. melanogaster), Ecor (Eupeodes corollae), Gaeg (Gigantopelta aegis), Hruf (Haliotis rufescens), Lcha (Latimeria chalumnae), Llon (Lineus longissimus), Mcal (Mytilus californianus), Pvan (Perinereis vancaurica), Rtem (Rana temporaria), Tcas (Tribolium castaneum). Accession numbers of precursor sequences used to generate this phylogenetic tree are listed in SI Appendix, Table S2.
	Fig. 2. Comparison of the exon/intron structure of genes encoding precursors of putative BN-type peptides in echinoderms and BN-type peptides in chordates. Exons are color-coded to show the N-terminal signal peptide (blue), BN-type peptide (red), C-terminal glycine predicted to be a substrate for amidation (orange), cleavage sites (green), and other regions (gray). Protein-coding exons are shown as rectangles and introns as lines (with length stated underneath). A conserved phase 1 intron (intron 1) interrupts the neuropeptide-encoding sequences and a conserved phase 0 intron (intron 3 in echinoderms, intron 2 in chordates) interrupts the coding sequences for the C-terminal region of the precursor proteins. Intron 2 (phase 0) in echinoderms is unique to this phylum. Full names of species: Ajap (A. japonicus), Arub (A. rubens), Asol (A. cf. solaris), Bflo (B. floridae), Ggal (G. gallus), Hsap (H. sapiens), Locu (L. oculatus), Spur (S. purpuratus). Full names of peptide precursor genes: BNP (Bombesin precursor), GRPP (Gastrin-releasing peptide precursor), NMBP (Neuromedin B precursor). Accession numbers of the precursor cDNAs and corresponding genomic sequences shown in Fig. 2 are listed in SI Appendix, Table S3. An alignment that shows the positions of introns with respect to the precursor amino acid sequences is shown in SI Appendix, Fig. S2.
	Fig. 3. Phylogenetic and functional identification of the A. rubens receptor ArBNR1 as a BN-type receptor. The phylogenetic tree was generated using the maximum likelihood method (1,000 bootstrap replicates, LG+F+G4 model) and it shows that ArBNR1 (arrow) is positioned in a clade that contains chordate BN-type receptors and that is distinct from clades comprising chordate ET-type receptors, protostome CCHa/EP-type receptors, and protostome elevenin-type receptors. Orexin-type receptors were included as an outgroup to root the tree. The colored squares represent bootstrap support for clades and colored backgrounds highlight different taxonomic groups (see key). The scale bar represents the average residue substitution per site. Abbreviations for species names: Acal (Aplysia californica), Ajap (A. japonicus), Arub (A. rubens), Asol (A. cf. solaris), Bflo (B. floridae), Bmor (B. mori), Ctel (Capitella teleta), Dmel (D. melanogaster), Dpul (Daphnia pulex), Gaeg (G. aegis), Ggal (G. gallus), Hsap (H. sapiens), Lgig (Lottia gigantea), Locu (L. oculatus), Pdum (Platynereis dumerilii), Rtem (R. temporaria), Skow (S. kowalevskii), Spur (S. purpuratus). Accession numbers for the receptor sequences used to generate this tree are listed in SI Appendix, Table S4. The inset shows that the peptide ArBN (EPRRNYNRVFGPTY-NH2) acts as a ligand for ArBNR1, triggering dose-dependent luminescence in Chinese hamster ovary (CHO)-K1 cells coexpressing ArBNR1 and Gqs5, with half-maximal response concentration (EC50) of 1.69 × 10−11 M. No ArBN-induced luminescence is observed in control experiments where cells were cotransfected with empty pcDNA3.1(+) vector and Gqs5. Each point represents mean values ± SEM from at least four independent experiments performed in triplicate (Dataset S1). Luminescence is expressed as a percentage of the maximal response observed in each experiment.
	Fig. 4. Localization of ArBN precursor expression in A. rubens using mRNA in situ hybridization. (A) Transverse section of a radial nerve cord incubated with antisense probes revealing stained cells in the hyponeural region and the ectoneural region. The Inset shows absence of staining in an adjacent section of the radial nerve cord incubated with sense probes, demonstrating the specificity of staining observed with antisense probes. (B) Higher magnification image of the upper boxed region in panel A, showing stained cells in the hyponeural region (arrow) of the radial nerve cord. (C) Higher magnification image of the lower boxed region in panel A, showing stained cells in the ectoneural region (arrowhead) of the radial nerve cord. (D) Longitudinal section of a tube foot revealing stained cells concentrated at one side of the subepithelial layer along the length of the stem and in the basal nerve ring of the disk region. (E) Higher magnification image of the boxed region in panel D, showing stained cells (arrow) at the junction between subepithelial layer of the stem and the basal nerve ring of the disk region. (F) Transverse section of a circumoral nerve ring showing stained cells in the hyponeural region (arrow) and the ectoneural region. (G) Higher magnification image of the boxed region in panel F, showing stained cells in the ectoneural region (arrowhead) of the circumoral nerve ring. (H) Longitudinal section of the disk region of a tube foot revealing abundant stained cells in the basal nerve ring (arrowhead). (I) Transverse section of the central disk region of the starfish body revealing sparsely distributed stained cells in the cardiac stomach. (J) Higher magnification image of the boxed region in panel I showing a stained cell (arrow). (K) Transverse section of the central disk region revealing stained cells in the pyloric stomach. (L) Higher magnification image of the boxed region in panel K showing a stained cell (arrow). Abbreviations: BNP, basi-epithelial nerve plexus; BNR, basal nerve ring; CONR, circumoral nerve ring; CS, cardiac stomach; CT, collagenous tissue; Di, tube foot disk; Ec, ectoneural region; Hy, hyponeural region; Lu, lumen; Mu, mucosa; PS, pyloric stomach; RHS, radial hemal strand; RNC, radial nerve cord; TF, tube foot. (Scale bars, 100 μm in A, D, F, H, I, and K, and Inset in A; 10 μm in B, C, E, G, J, and L.)
	Fig. 5. Localization of ArBN precursor expression in A. rubens using immunohistochemistry. (A) Transverse section of a radial nerve cord showing ArBNP-immunoreactivity (ir) in the hyponeural and ectoneural regions. (B) Higher magnification image of the upper boxed region in panel A showing stained cells in the hyponeural region (arrow) and stained fibers in the neuropile of the ectoneural region. (C) Higher magnification image of the lower boxed region in panel A showing stained cells (arrowhead) in the epithelial layer of the ectoneural region. (D) Longitudinal section of a tube foot showing ArBNP-ir in the subepithelial nerve plexus along the length of the stem and in the basal nerve ring of the disk region. (E) Higher magnification image of the boxed region in panel D, showing stained cells (arrow) and stained fibers (asterisk) associated with the basal nerve ring of the disk region. (F) Transverse section of the circumoral nerve ring showing ArBNP-ir in the hyponeural and ectoneural regions. (G) Higher magnification image of the upper boxed region of panel F, showing stained cells (arrow) in the hyponeural region. (H) Higher magnification image of the lower boxed region of panel F, showing stained cells (arrowhead) in the epithelium of the ectoneural region. (I) ArBNP-ir in marginal nerve (arrow) and lateral motor nerve (arrowhead). (J) Transverse section of an arm tip, showing ArBNP-ir in the terminal tentacle, lateral lappet, and optic cushion. (K) High magnification image of the lower boxed region of panel J, showing stained cells (arrowhead) in the optic cushion. (L) High magnification image of the upper boxed region of panel J, showing stained cells (arrow) in a lateral lappet. (M) Transverse section of the central disk region showing ArBNP-ir in the peristomial membrane (arrow). (N) Transverse section of the central disk region showing ArBNP-ir in the esophagus (arrow). (O) Transverse section of the central disk region revealing ArBNP-ir in the cardiac stomach, including stained fibers in the basi-epithelial nerve plexus (arrowhead) and a stained cell in the mucosa (arrow). (P) Transverse section of the central disk region showing ArBNP-ir in the pyloric stomach. (Q) Higher magnification image of the boxed region of panel P, showing staining in the basi-epithelial nerve plexus (arrowhead) and in a cell located in the mucosa (arrow). (R) Longitudinal section of an arm showing ArBNP-ir in the basi-epithelial nerve plexus on the oral side of a pyloric duct (arrow). (S) Transverse section of the aboral body wall revealing ArBN-ir in a pedicellaria (arrow). (T) Transverse section of the aboral body wall showing ArBN-ir in a papula. Abbreviations: BNP, basi-epithelial nerve plexus; BW, body wall; CONR, circumoral nerve ring; CS, cardiac stomach; Ec, ectoneural region; Ep, epithelium; Hy, hyponeural region; LL, lateral lappet; LMN, lateral motor nerve; Lu, lumen; MN, marginal nerve; Mu, mucosa; OC, optic cushion; OES, esophagus; Pa, papula; PC, pyloric cecum; PD, pyloric duct; Pe, pedicellaria; PM, peristomial membrane; PS, pyloric stomach; RHS, radial hemal strand; RNC: radial nerve cord; SNP, subepithelial nerve plexus; TF, tube foot; TT, terminal tentacle. (Scale bars, 100 μm in A, D, F, I, J, M–O, and R; 20 μm in B, C, E, G, H, K, and L; 40 μm in Q; and 200 μm in P, S, and T.)
	Fig. 6. ArBN causes dose-dependent contraction of cardiac stomach and tube foot preparations from A. rubens. (A) Representative recording showing the effects of NGFFYamide (10−7 M) and ArBN (10−10 to 10−6 M) on a cardiac stomach preparation, with washing between each application of the test agent. (B) Graph showing that ArBN causes dose-dependent contraction of cardiac stomach preparations (n = 10) at concentrations ranging from 10−9 M to 10−6 M, using the method shown in A. (C) Representative recording showing the effects of NGFFYamide (10−7 M) and ArBN (10−10 to 10−6 M) on a cardiac stomach preparation, without washing between each application of the test agent. (D) Graph showing the cumulative contracting effect of ArBN (10−10 to 10−6 M) on cardiac stomach preparations (n = 8), using the method shown in C. The graphs in B and D show mean percentage (±SEM) of the contraction induced by 10−7 M NGFFYamide. (E) Representative recording showing the effects of acetylcholine (ACh, 10−5 M) and ArBN (10−10 to 10−6 M) on a tube foot preparation, with washing between each application of the test agent. (F) Graph showing that ArBN causes dose-dependent contraction of tube foot preparations (n = 10) at concentrations ranging from 10−9 M to 10−6 M, using the method shown in E. (G) Representative recording showing the effect of ACh (10−5 M) and ArBN (10−10 to 10−6 M) on a tube foot preparation, without washing between each application of the test agent. (H) Graph showing the cumulative contracting effect of ArBN (10−10 to 10−6 M) on tube foot preparations (n = 7), using the method shown in G. The graphs in F and H show mean percentage (±SEM) of the contraction induced by 10−5 M ACh. In A, C, E, and G, the gray upward pointing arrowheads show when 10−7 M NGFFYamide was added, the green upward pointing arrowheads show when 10−5 M ACh was added, the pink upward pointing arrowheads show when ArBN was added (left to right, from 10−10 to 10−6 M) and the blue downward pointing arrowheads show when the preparation was washed with seawater. The raw data for the graphs shown in this figure are available in Dataset S2.
	Fig. 7. ArBN causes cardiac stomach retraction and inhibits feeding behavior in A. rubens. (A and B) ArBN causes cardiac stomach retraction in A. rubens. (A) Photographs from an experiment showing that the two-dimensional area (marked by white dashed lines) of a cardiac stomach is progressively reduced at 120, 240, and 360 s after injection (0 s) of 10 μL of ArBN (10−4 M). (B) Graph comparing the everted cardiac stomach area in starfish injected with water (blue; 10 µL; n = 7), ArBN (yellow; 10 µL of 10−4 M; n = 7), and NGFFYamide (gray; 10 µL of 10−4 M; n = 7). The two-dimensional area of cardiac stomach everted at each time point was normalized to the area of cardiac stomach everted just before injection. Each point represents means (±SEM) from seven separate experiments. (C and D) ArBN inhibits feeding behavior in A. rubens. (C) For starfish that successfully fed, injection of ArBN (10 μL of 10−4 M) had no effect on the time taken for starfish to first touch a mussel. (D) For starfish that successfully fed, the time taken to enclose a mussel was significantly longer (P = 0.0324) in the group that was injected with 10 μL of 10−4 M ArBN (yellow, n = 16) than in the group injected with 10 μL of water (blue, n = 16). Data were analyzed statistically using a two-tailed Student’s t-test in Prism 10 and are shown as scatter plots. The raw data for the graphs shown in this figure are available in Datasets S3 and S4.