Deorphanization of biogenic amine-responsive G protein-coupled receptors in various model protozoa and platyhelminthes (original) (raw)
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PLoS ONE, 2012
G protein-coupled receptors (GPCRs) represent the largest known superfamily of membrane proteins extending throughout the Metazoa. There exists ample motivation to elucidate the functional properties of GPCRs given their role in signal transduction and their prominence as drug targets. In many target organisms, these efforts are hampered by the unreliable nature of heterologous receptor expression platforms. We validate and describe an alternative loss-of-function approach for ascertaining the ligand and G protein coupling properties of GPCRs in their native cell membrane environment. Our efforts are focused on the phylum Platyhelminthes, given the heavy health burden exacted by pathogenic flatworms, as well as the role of free-living flatworms as model organisms for the study of developmental biology. RNA interference (RNAi) was used in conjunction with a biochemical endpoint assay to monitor cAMP modulation in response to the translational suppression of individual receptors. As proof of principle, this approach was used to confirm the neuropeptide GYIRFamide as the cognate ligand for the planarian neuropeptide receptor GtNPR-1, while revealing its endogenous coupling to Ga i/o. The method was then extended to deorphanize a novel Ga s-coupled planarian serotonin receptor, DtSER-1. A bioinformatics protocol guided the selection of receptor candidates mediating 5-HT-evoked responses. These results provide functional data on a neurotransmitter central to flatworm biology, while establishing the great potential of an RNAi-based deorphanization protocol. Future work can help optimize and adapt this protocol for higher-throughput platforms as well as other phyla.
Identification of a platyhelminth neuropeptide receptor
International Journal for Parasitology, 2007
We report the characterisation of the first neuropeptide receptor from the phylum Platyhelminthes, an early-diverging phylum which includes a number of important human and veterinary parasites. The G protein-coupled receptor (GPCR) was identified from the model flatworm Girardia tigrina (Tricladida: Dugesiidae) based on the presence of motifs widely conserved amongst GPCRs. In two different assays utilising heterologous expression in Chinese hamster ovary cells, the Girardia GPCR was most potently activated by neuropeptides from the FMRFamide-like peptide class. The most potent platyhelminth neuropeptide in both assays was GYIRFamide, a FMRFamide-like peptide known to be present in G. tigrina. There was no activation by neuropeptide Fs, another class of flatworm neuropeptides. Also active were FMRFamide-like peptides derived from other phyla but not known to be present in any platyhelminth. Most potent among these were nematode neuropeptides encoded by the Caenorhabditis elegans flp-1 gene which share a PNFLRFamide carboxy terminal motif. The ability of nematode peptides to stimulate a platyhelminth receptor demonstrates a degree of structural conservation between FMRFamide-like peptide receptors from these two distinct, distant phyla which contain parasitic worms.
G Protein-Coupled Receptors in Invertebrates: A State of the Art
International Review of Cytology, 2003
G protein-coupled receptors (GPCRs) constitute one of the largest and most ancient superfamilies of membrane-spanning proteins. We focus on neuropeptide GPCRs, in particular on those of invertebrates. In general, such receptors mediate the responses of signaling molecules that constitute the highest hierarchical position in the regulation of physiological processes. Until recently, only a few of these receptors were identified in invertebrates. However, the availability of a plethora of genomic information has boosted the discovery of novel members in several invertebrate species, such as Drosophila, in which 18 neuropeptide GPCRs have been characterized. The finalization of genomic projects in other invertebrates will lead to a similar expansion of GPCR understanding. Many new insights regarding neuropeptide regulation have followed from the discovery of their cognate receptors. Furthermore, information on GPCR signaling is still fragmentary and the elucidation of these pathways in model insects such as Drosophila will lead to further insights in other species, including mammals. In this review we present the current status of what is known about invertebrate GPCRs, discuss some novel perceptions that follow from the identified members, and, finally, present some future prospects.
Journal of Neurochemistry, 2003
Biogenic amine receptors mediate wide-ranging hormonal and modulatory functions in vertebrates, but are largely unknown in primitive invertebrates. In a representative of the most basal multicellular animals possessing a nervous system, the cnidarian Renilla koellikeri, aminergic-like receptors were previously characterized pharmacologically and found to engender control of the animal's bioluminescent and peristaltic reactions. Using degenerate oligonucleotides in a RT-PCR strategy, we obtained a full-length cDNA encoding a polypeptide with typical G protein-coupled receptor (GPCR) characteristics and which displayed a significant degree of sequence similarity (up to 45%) to biogenic amine receptors, particularly dopamine and adrenergic receptors. The new receptor, named Ren1, did not resemble any one specific type of amine GPCR and thus could not be identified on the basis of sequence. Ren1 was expressed transiently and stably in cultured mammalian cells, as demonstrated by immunocyto-chemistry and western blotting. Functional analysis of transfected HEK293, LTK-and COS-7 cells, based on both cAMP and Ca 2+ signalling assays, revealed that Ren1 was not activated by any of the known biogenic amines tested and several related metabolites. The results indicated, however, that cells stably expressing Ren1 contained, on average, an 11-fold higher level of cAMP than the controls, in the absence of agonist stimulation. The high basal cAMP levels were shown to be specific for Ren1 and to vary proportionally with the level of Ren1 expressed in the transfected cells. Taken together, the data suggested that Ren1 was expressed as a constitutively active receptor. Its identification provides a basis for examination of the early evolutionary emergence of GPCRs and their functional properties.
Neuropeptide signalling systems in flatworms
Parasitology, 2006
Two distinct families of neuropeptides are known to endow platyhelminth nervous systems-the FMRFamide-like peptides (FLPs) and the neuropeptide Fs (NPFs). Flatworm FLPs are structurally simple, each 4-6 amino acids in length with a carboxy terminal aromatic-hydrophobic-Arg-Phe-amide motif. Thus far, four distinct flatworm FLPs have been characterized, with only one of these from a parasite. They have a widespread distribution within the central and peripheral nervous system of every flatworm examined, including neurones serving the attachment organs, the somatic musculature and the reproductive system. The only physiological role that has been identified for flatworm FLPs is myoexcitation. Flatworm NPFs are believed to be invertebrate homologues of the vertebrate neuropeptide Y (NPY) family of peptides. Flatworm NPFs are 36-39 amino acids in length and are characterized by a caboxy terminal GRPRFamide signature and conserved tyrosine residues at positions 10 and 17 from the carboxy terminal. Like FLPs, NPF occurs throughout flatworm nervous systems, although less is known about its biological role. While there is some evidence for a myoexcitatory action in cestodes and flukes, more compelling physiological data indicate that flatworm NPF inhibits cAMP levels in a manner that is characteristic of NPY action in vertebrates. The widespread expression of these neuropeptides in flatworm parasites highlights the potential of these signalling systems to yield new targets for novel anthelmintics. Although platyhelminth FLP and NPF receptors await identification, other molecules that play pivotal roles in neuropeptide signalling have been uncovered. These enzymes, involved in the biosynthesis and processing of flatworm neuropeptides, have recently been described and offer other distinct and attractive targets for therapeutic interference.
Frontiers in Endocrinology, 2021
Nematode parasites undermine human health and global food security. The frontline anthelmintic portfolio used to treat parasitic nematodes is threatened by the escalation of anthelmintic resistance, resulting in a demand for new drug targets for parasite control. Nematode neuropeptide signalling pathways represent an attractive source of novel drug targets which currently remain unexploited. The complexity of the nematode neuropeptidergic system challenges the discovery of new targets for parasite control, however recent advances in parasite ‘omics’ offers an opportunity for the in silico identification and prioritization of targets to seed anthelmintic discovery pipelines. In this study we employed Hidden Markov Model-based searches to identify ~1059 Caenorhabditis elegans neuropeptide G-protein coupled receptor (Ce-NP-GPCR) encoding gene homologs in the predicted protein datasets of 10 key parasitic nematodes that span several phylogenetic clades and lifestyles. We show that, whil...
Discovery of multiple neuropeptide families in the phylum Platyhelminthes
International Journal for Parasitology, 2009
Available evidence shows that short amidated neuropeptides are widespread and have important functions within the nervous systems of all flatworms (phylum Platyhelminthes) examined, and could therefore represent a starting point for new lead drug compounds with which to combat parasitic helminth infections. However, only a handful of these peptides have been characterised, the rigorous exploration of the flatworm peptide signalling repertoire having been hindered by the dearth of flatworm genomic data. Through searches of both expressed sequence tags and genomic resources using the basic local alignment search tool (BLAST), we describe 96 neuropeptides on 60 precursors from 10 flatworm species. Most of these (51 predicted peptides on 14 precursors) are novel and are apparently restricted to flatworms; the remainder comprise nine recognised peptide families including FMRFamide-like (FLPs), neuropeptide F (NPF)-like, myomodulin-like, buccalin-like and neuropeptide FF (NPFF)-like peptides; notably, the latter have only previously been reported in vertebrates. Selected peptides were localised immunocytochemically to the Schistosoma mansoni nervous system. We also describe several novel flatworm NPFs with structural features characteristic of the vertebrate neuropeptide Y (NPY) superfamily, previously unreported characteristics which support the common ancestry of flatworm NPFs with the NPY-superfamily. Our dataset provides a springboard for investigation of the functional biology and therapeutic potential of neuropeptides in flatworms, simultaneously launching flatworm neurobiology into the post-genomic era.
Parasite, 2022
Genomic resources for Platyhelminthes of the class Monogenea are scarce, despite the diversity of these parasites, some species of which are highly pathogenic to their fish hosts. This work aimed to generate de novo-assembled transcriptomes of two monogenean species, Scutogyrus longicornis (Dactylogyridae) and Rhabdosynochus viridisi (Diplectanidae), providing a protocol for cDNA library preparation with low input samples used in single cell transcriptomics. This allowed us to work with sub-microgram amounts of total RNA with success. These transcriptomes consist of 25,696 and 47,187 putative proteins, respectively, which were further annotated according to the Swiss-Prot, Pfam, GO, KEGG, and COG databases. The completeness values of these transcriptomes evaluated with BUSCO against Metazoa databases were 54.1% and 73%, respectively, which is in the range of other monogenean species. Among the annotations, a large number of terms related to G-protein-coupled receptors (GPCRs) were found. We identified 109 GPCR-like sequences in R. viridisi, and 102 in S. longicornis, including family members specific for Platyhelminthes. Rhodopsin was the largest family according to GRAFS classification. Two putative melatonin receptors found in S. longicornis represent the first record of this group of proteins in parasitic Platyhelminthes. Forty GPCRs of R. viridisi and 32 of S. longicornis that were absent in Vertebrata might be potential drug targets. The present study provides the first publicly available transcriptomes for monogeneans of the subclass Monopisthocotylea, which can serve as useful genomic datasets for functional genomic research of this important group of parasites.
Gene, 2004
Biogenic amine receptors mediate wide-ranging hormonal and modulatory functions in vertebrates, but are largely unknown in primitive invertebrates. In a representative of the most basal multicellular animals possessing a nervous system, the cnidarian Renilla koellikeri, aminergic-like receptors were previously characterized pharmacologically and found to engender control of the animal's bioluminescent and peristaltic reactions. Using degenerate oligonucleotides in a RT-PCR strategy, we obtained a full-length cDNA encoding a polypeptide with typical G protein-coupled receptor (GPCR) characteristics and which displayed a significant degree of sequence similarity (up to 45%) to biogenic amine receptors, particularly dopamine and adrenergic receptors. The new receptor, named Ren1, did not resemble any one specific type of amine GPCR and thus could not be identified on the basis of sequence. Ren1 was expressed transiently and stably in cultured mammalian cells, as demonstrated by immunocyto-chemistry and western blotting. Functional analysis of transfected HEK293, LTK-and COS-7 cells, based on both cAMP and Ca 2+ signalling assays, revealed that Ren1 was not activated by any of the known biogenic amines tested and several related metabolites. The results indicated, however, that cells stably expressing Ren1 contained, on average, an 11-fold higher level of cAMP than the controls, in the absence of agonist stimulation. The high basal cAMP levels were shown to be specific for Ren1 and to vary proportionally with the level of Ren1 expressed in the transfected cells. Taken together, the data suggested that Ren1 was expressed as a constitutively active receptor. Its identification provides a basis for examination of the early evolutionary emergence of GPCRs and their functional properties.