Urbilaterian origin of paralogous GnRH and corazonin neuropeptide signalling pathways

Abstract

Gonadotropin-releasing hormone (GnRH) is a key regulator of reproductive maturation in humans and other vertebrates. Homologs of GnRH and its cognate receptor have been identified in invertebrates-for example, the adipokinetic hormone (AKH) and corazonin (CRZ) neuropeptide pathways in arthropods. However, the precise evolutionary relationships and origins of these signalling systems remain unknown. Here we have addressed this issue with the first identification of both GnRH-type and CRZ-type signalling systems in a deuterostome-the echinoderm (starfish) Asterias rubens. We have identified a GnRH-like neuropeptide (pQIHYKNPGWGPG-NH2) that specifically activates an A. rubens GnRH-type receptor and a novel neuropeptide (HNTFTMGGQNRWKAG-NH2) that specifically activates an A. rubens CRZ-type receptor. With the discovery of these ligand-receptor pairs, we demonstrate that the vertebrate/deuterostomian GnRH-type and the protostomian AKH systems are orthologous and the origin of a paralogous CRZ-type signalling system can be traced to the common ancestor of the Bilateria (Urbilateria).

Figure 1. Phylogenetic analysis of GnRH/AKH/ACP/CRZ-type receptors using a Bayesian method reveals two distinct clades–a GnRH/AKH/ACP-type receptor clade and a CRZ-type receptor clade.

Single representatives of both clades are present in the starfish A. rubens (A. rub., black boxes). GnRH-type receptors are labelled using red squares, AKH-type receptors using orange squares, ACP-type receptors using pink squares and CRZ-type receptors using purple circles. Neuropeptide S and CCAP receptors were used as an outgroup (condensed). The stars represent posterior probabilities and the pastel coloured backgrounds represent different groups of animals (see legend). The scale bar indicates amino acid substitutions per site. Species for which receptor-ligand interactions have been experimentally characterized are coloured in green, including the A. rubens receptors characterized in this study (boxed). Species names are as follows: A. rub, Asterias rubens; S. pur, Strongylocentrotus purpuratus; B. flo, Branchiostoma floridae; H. sap, Homo sapiens; D. rer, Danio rerio; G. gal, Gallus gallus; C. tel, Capitella teleta, C. gig, Crassostrea gigas; L. gig, Lottia gigantea; S. mar, Strigamia maritima; D. pul, Daphnia pulex; B. mor, Bombyx mori; R. pro, Rhodnius prolixus; A. gam, Anopheles gambiae; I. sca, Ixodes scapularis; S. kow, Saccoglossus kowalevskii; O. vul, Octopus vulgaris. [accession numbers and references for the receptor sequences are included the legend of Supplementary Figure S3].

Figure 2. Identification of GnRH-type and Corazonin-type (CRZ)-type signalling systems in the starfish Asterias rubens.

(a) Amino acid sequences of two A. rubens GnRH/CRZ-type neuropeptide precursor proteins–precursor 1 and precursor 2. Signal peptides are highlighted in blue, putative neuropeptides (without post-translational modifications) are highlighted in red (peptide 1) or purple (peptide 2) and dibasic cleavage sites are highlighted in green. Peptides 1 and 2 with post-translational N-and C-terminal modifications, determined by mass spectrometry, are shown below the precursor sequences. (b) Peptide 1 causes dose-dependent stimulation of a bioluminescence response in CHO-K1 cells stably expressing aequorin and Gα16 and transfected with ArGnRHR; EC₅₀ = 6.03 × 10⁻¹⁰M. Peptide 2 has no effect when tested over the same concentration range as peptide 1, demonstrating the specificity of the activation of ArGnRHR by peptide 1, which is therefore designated as “ArGnRH”. (c) Comparison of the total bioluminescent responses of ArGnRHR-expressing cells for 30 seconds after the addition of BSA media (control), peptide 1 (10⁻⁵M) or peptide 2 (10⁻⁵M). (d) Peptide 2 causes dose-dependent stimulation of a bioluminescence response in CHO-K1 cells stably expressing aequorin and Gα16 and transfected with ArCRZR; EC₅₀ = 1.15 × 10⁻⁷M. Peptide 1 has no effect when tested over a similar concentration range as peptide 2, demonstrating the specificity of the activation of ArCRZR by peptide 2, which is therefore designated as “ArCRZ”. (e) Comparison of the total bioluminescent responses of ArCRZR-expressing cells for 30 seconds after the addition of BSA media (control), peptide 1 (10⁻⁵M) or peptide 2 (10⁻⁵M).

Figure 3. Schematic showing the evolution of GnRH-type and CRZ-type receptors in the Bilateria.

GnRH-type receptors (red) and CRZ-type receptors (purple) arose by gene duplication in a common ancestor of the Bilateria. A second gene duplication of a GnRH-type receptor in a common ancestor of the Arthropoda gave rise to AKH-type receptors (orange) and ACP-type receptors (pink). CRZ-type receptors have been lost in multiple lineages (purple crosses), including vertebrates, and the ACP-type receptor has been lost in Drosophila (pink cross). The occurrence of each receptor type in species belonging to different phyla is shown on the right (white box denotes loss of a receptor). Species where neuropeptide ligands for receptors have been identified are labelled with a yellow asterisk. Note that, as reported in this paper, the starfish Asterias rubens is the first and only deuterostome in which the neuropeptide ligands for a GnRH-type receptor and a CRZ-type receptor have been identified. The “?” in the CRZR box for Branchiostoma floridae indicates uncertainty regarding the structure of a candidate ligand, as discussed in the main text of this paper. Images of representative animals from each phylum were created by the authors, with the exception of the sea urchin image, which was obtained from https://openclipart.org/detail/170807/sea-urchin-silhouette.