Abstract
Neuropeptides play a central role as neurotransmitters, neuromodulators and hormones in orchestrating arthropod physiology. The post-genomic surge in identified neuropeptides and their putative receptors has not been matched by functional characterization of ligand-receptor pairs. Indeed, until very recently no G protein-coupled receptors (GPCRs) had been functionally defined in any crustacean. Here we explore the structurally-related, functionally-diverse gonadotropin-releasing hormone paralogs, corazonin (CRZ) and red-pigment concentrating hormone (RPCH) and their G-protein coupled receptors (GPCRs) in the crab, Carcinus maenas. Using aequorin luminescence to measure in vitro Ca2+ mobilization we demonstrated receptor-ligand pairings of CRZ and RPCH. CRZR-activated cell signaling in a dose-dependent manner (EC50 0.75 nM) and comparative studies with insect CRZ peptides suggest that the C-terminus of this peptide is important in receptor-ligand interaction. RPCH interacted with RPCHR with extremely high sensitivity (EC50 20 pM). Neither receptor bound GnRH, nor the AKH/CRZ-related peptide. Transcript distributions of both receptors indicate that CRZR expression was, unexpectedly, restricted to the Y-organs (YO). Application of CRZ peptide to YO had no effect on ecdysteroid biosynthesis, excepting a modest stimulation in early post-molt. CRZ had no effect on heart activity, blood glucose levels, lipid mobilization or pigment distribution in chromatophores, a scenario that reflected the distribution of its mRNA. Apart from the well-known activity of RPCH as a chromatophorotropin, it also indirectly elicited hyperglycemia (which was eyestalk-dependent). RPCHR mRNA was also expressed in the ovary, indicating possible roles in reproduction. The anatomy of CRZ and RPCH neurons in the nervous system is described in detail by immunohistochemistry and in situ hybridization. Each peptide has extensive but non-overlapping distribution in the CNS, and neuroanatomy suggests that both are possibly released from the post-commissural organs. This study is one of the first to deorphanize a GPCR in a crustacean and to provide evidence for hitherto unknown and diverse functions of these evolutionarily-related neuropeptides.
Highlights
Cellular signaling, which pervades and integrates all aspects of metazoan life, involves a seemingly bewildering variety of messenger molecules
The complete cDNA sequences encoding G protein-coupled receptor (GPCR) were identified from tBLASTn searches of the Carcinus transcriptomes (TSA accession code: GFX F00000000, BioProject No PRJNA400568, Oliphant et al, under review) and functionally identified as the corazonin receptor (CRZR) and red pigment concentrating hormone receptor (RPCHR)
Full length sequences encoding a putative adipokinetic hormone/corazonin-related peptide receptor (ACPR) and its cognate ligand were identified from our transcriptome analysis, but this GPCR did not bind C. maenas ACP, and further investigations into some other incomplete candidate ACPR sequences were abandoned
Summary
Cellular signaling, which pervades and integrates all aspects of metazoan life, involves a seemingly bewildering variety of messenger molecules. The first step in signal transduction, for the majority of neuropeptides, involves interaction with plasmamembrane bound G protein-coupled receptors (GPCRs). With the advent of large scale genome and transcriptome sequencing and analysis, a huge variety of neuropeptides and their putative cognate GPCRs have been identified, and leading on from the sequencing of the Drosophila genome over 15 years ago (Adams et al, 2000), more than 250 genomes have been sequenced for insects. Genomic and transcriptomic data on the identity and function of crustacean neuropeptides and cognate receptors has lagged behind those of insects, despite their huge economic importance in aquaculture. In silico genome and transcriptome mining has led to the discovery of a large number of peptide and putative peptide receptors in crustaceans (See Christie et al, 2015 for a comprehensive list). Correlating sequence identity with functionality is perhaps meaningless given the inevitable sequence similarities within the five subfamilies of GPCRs; i.e. rhodopsin, secretin, glutamate, adhesion and frizzled-tastes-2 (Fredriksson et al, 2003)
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call