Putative G Protein-coupled Receptor Research Articles

Comparative transcriptomics supports the presence of G protein‐coupled receptor‐based signaling in unicellular marine eukaryotes

AbstractBeing abundant primary producers, single‐celled eukaryotic diatoms and dinoflagellates dominate marine food webs and significantly impact the ecology of the oceans. These organisms face competition and predation which have contributed to the evolution of chemical defenses and their ability to sense chemical cues. Elucidating how these chemical cues are perceived by organisms requires identification of proteins involved in relaying chemical information to cells. The functions of G protein‐coupled receptor (GPCR) signaling diversified over evolutionary time in multicellular organisms, but the role that GPCR signaling plays in unicellular eukaryotes remains largely unknown. Using comparative transcriptomic analysis, we identified repertoires of GPCR signaling proteins from 81 diatom and 36 dinoflagellate transcriptomes. Diatoms and dinoflagellates express putative GPCRs that are highly diverged from known metazoan GPCRs along with well‐conserved core components of GPCR signaling pathways. Unlike other eukaryotes that express multiple classes of GPCRs, diatoms and dinoflagellates were each found to express a single, major class of putative GPCRs with greatest similarity to class C and rhodopsin‐like GPCRs, respectively. Furthermore, only 4.2% of the GPCR‐like transcripts from dinoflagellates were predicted to be true GPCRs compared to 48% in diatoms. These results suggest that further expansion of GPCRs did not take place within each of these two major phytoplankton groups, and that dinoflagellate GPCRs are more divergent from known GPCRs than are diatom GPCRs. Our results provide additional evidence for the global existence of relatively ancestral GPCR signaling machinery in marine phytoplankton.

Identification of G protein-coupled receptor signaling pathway proteins in marine diatoms using comparative genomics

BackgroundThe G protein-coupled receptor (GPCR) signaling pathway plays an essential role in signal transmission and response to external stimuli in mammalian cells. Protein components of this pathway have been characterized in plants and simpler eukaryotes such as yeast, but their presence and role in unicellular photosynthetic eukaryotes have not been determined. We use a comparative genomics approach using whole genome sequences and gene expression libraries of four diatoms (Pseudo-nitzschia multiseries, Thalassiosira pseudonana, Phaeodactylum tricornutum and Fragilariopsis cylindrus) to search for evidence of GPCR signaling pathway proteins that share sequence conservation to known GPCR pathway proteins.ResultsThe majority of the core components of GPCR signaling were well conserved in all four diatoms, with protein sequence similarity to GPCRs, human G protein α- and β-subunits and downstream effectors. There was evidence for the Gγ-subunit and thus a full heterotrimeric G protein only in T. pseudonana. Phylogenetic analysis of putative diatom GPCRs indicated similarity but deep divergence to the class C GPCRs, with branches basal to the GABAB receptor subfamily. The extracellular and intracellular regions of these putative diatom GPCR sequences exhibited large variation in sequence length, and seven of these sequences contained the necessary ligand binding domain for class C GPCR activation. Transcriptional data indicated that a number of the putative GPCR sequences are expressed in diatoms under various stress conditions in culture, and that many of the GPCR-activated signaling proteins, including the G protein, are also expressed.ConclusionsThe presence of sequences in all four diatoms that code for the proteins required for a functional mammalian GPCR pathway highlights the highly conserved nature of this pathway and suggests a complex signaling machinery related to environmental perception and response in these unicellular organisms. The lack of evidence for some GPCR pathway proteins in one or more of the diatoms, such as the Gγ-subunit, may be due to differences in genome completeness and genome coverage for the four diatoms. The high divergence of putative diatom GPCR sequences to known class C GPCRs suggests these sequences may represent another, potentially ancestral, subfamily of class C GPCRs.

A putative G protein-coupled receptor involved in innate immune defense of Procambarus clarkii against bacterial infection

The immune functions of G protein-coupled receptor (GPCR) were widely investigated in mammals. However, limited researches on immune function of GPCRs were reported in invertebrates. In the present study, the immune functions of HP1R gene, a putative GPCR identified from red swamp crayfish Procambarus clarkii were reported. Expression of HP1R gene was significant up-regulated in response to heat-killed Aeromonas hydrophila challenge. HP1R gene silencing mediated by RNA interference significantly enhanced the susceptibility of red swamp crayfish to A. hydrophila and Vibrio alginolyticus, indicating that HP1R was required for red swamp crayfish to defend against bacterial challenge. In HP1R-silenced crayfish, increased bacterial burden and decreased THC in response to bacterial challenge were observed when compared with control crayfish. No significant difference of proPO gene expression was observed between HP1R-silenced and control crayfish after challenge with heat-killed A. hydrophila. However, PO activity in response to bacterial challenge was significantly reduced in HP1R-silenced crayfish. The results collectively indicated that HP1R was an important immune molecule which was required for red swamp crayfish to defend against bacterial infection.

Functional Characterization of Three G Protein-coupled Receptors for Pigment Dispersing Factors in Caenorhabditis elegans

Here, we report the identification, cloning, and functional characterization of three Caenorhabditis elegans G protein-coupled pigment dispersing factor (PDF) receptors, which we designated as Ce_PDFR-1a, -b, and -c. They represent three splice isoforms of the same gene (C13B9.4), which share a high degree of similarity with the Drosophila PDF receptor and are distantly related to the mammalian vasoactive intestinal peptide receptors (VPAC2) and calcitonin receptors. In a reverse pharmacological screen, three bioactive C. elegans neuropeptides, which were recently identified as the Drosophila PDF orthologues, were able to activate these receptors in a dose-dependent manner with nanomolar potency (isoforms a and b). Integrated green fluorescent protein reporter constructs reveal the expression of these PDF receptors in all body wall muscle cells and many head and tail neurons involved in the integration of environmental stimuli and the control of locomotion. Using a custom data analysis system, we demonstrate the involvement of this newly discovered neuropeptide signaling system in the regulation of locomotor behavior. Overexpression of PDF-2 phenocopies the locomotor defects of a PDF-1 null mutant, suggesting that they elicit opposite effects on locomotion through the identified PDF receptors. Our findings strengthen the hypothesis that the PDF signaling system, which imposes the circadian clock rhythm on behavior in Drosophila, has been functionally conserved throughout the protostomian evolutionary lineage.

Neuropeptide G-protein-coupled receptors, their cognate ligands and behavior in Caenorhabditis elegans

Despite a simple nervous system, the free-living nematode Caenorhabditis elegans exhibits complex behaviors. The identification of peptide ligands for a G-protein-coupled receptor (GPCR) has provided insight into the neuronal circuitry involved in the regulation of feeding behavior in this worm. Progress in this regard has been accelerated by the discovery that functional expression of worm GPCRs in mammalian cells can be highly temperature dependent. Gene silencing and behavioral analysis has further identified several putative peptide GPCRs that are implicated in reproduction and locomotion. These studies suggest that these peptide GPCRs are legitimate targets for the discovery of novel anthelmintic agents.

Cloning and Expression of a cDNA for a Putative G Protein-Coupled Receptor from the Hepatopancreas of the Crayfish, Procambarus clarkii

The authors cloned a novel cDNA encoding a putative G protein-coupled receptor (GPCR) from the hepatopancreas of the crayfish, Procambarus clarkii, using a screening approach with synthetic oligonucleotides. The oligonucleotides were designed homologous to the transmembrane spanning domain III of previously cloned receptors from various living organisms. Sequence analysis revealed that one of the positive clones contained a cDNA insertion of 3489 bp representing the mRNA coding for a part of a putative GPCR (termed HP1R). The clone was truncated at the 5′ end. The long 3′-untranslated regions (UTR) of 2446 bp contained three typical AATAAA censensus sequences for mRNA polyadenylation followed by the poly(A) tail at the 3′ end. To obtain a full-length cDNA clone, rapid amplification of the 5′ cDNA ends (5′ RACE) technique was then applied. Sequence analysis revealed that the full-length cDNA clone had an open-reading frame of 1116 bp with a 103-bp 5′-UTR. The predicted amino acid sequence of HP1R was 372 residues long. Hydropathicity analysis of HP1R suggested the presence of seven transmembrane domains. The database search revealed that the predicted sequence is most closely related to probable GPCR AH9.1 from Caenorhabditis elegans (27% identity, 48% homology). In addition, HP1R has lower homologies with receptors for somatostatin, opioid, dopamine, adrenalin, and so on. The similarity implied that HP1R is a new member of putative GPCRs whose endogenous ligands were unknown. In addition, RT–PCR analysis suggested that the transcript was expressed predominantly in the hepatopancreas but poorly in the muscle or brain.

Cloning Genes Encoding Receptors Related to Chemoattractant Receptors

We report the cloning of a novel human gene (GPR32) encoding a putative G-protein-coupled receptor (GPCR) of 356 amino acids and a related pseudogene ψGPR32. The deduced amino acid sequence of GPR32 shares 35–39% identity with members of the chemoattractant receptor family. ψGPR32 shares 93% nucleotide identity with GPR32. We identified a mouse EST encoding a putative GPCR (GPR33) of 309 amino acids. The deduced amino acid sequence of GPR33 shares 30–35% identity with members of the chemoattractant receptor family and 36% identity with the receptor encoded by GPR32. The human orthologue of GPR33 contains a single basepair substitution with respect to the mouse, resulting in the presence of an in-frame stop codon within the predicted second intracellular loop, demonstrating that it is a pseudogene. Through fluorescencein situhybridization and physical mapping of YACs, both GPR32 and ψGPR32 were mapped to chromosomal 19, region q13.3, while ψGPR33 was mapped to chromosome 14q12.

Ventricular zone gene-1 (vzg-1) encodes a lysophosphatidic acid receptor expressed in neurogenic regions of the developing cerebral cortex.

Neocortical neuroblast cell lines were used to clone G-protein-coupled receptor (GPCR) genes to study signaling mechanisms regulating cortical neurogenesis. One putative GPCR gene displayed an in situ expression pattern enriched in cortical neurogenic regions and was therefore named ventricular zone gene-1 (vzg-1). The vzg-1 cDNA hybridized to a 3.8-kb mRNA transcript and encoded a protein with a predicted molecular mass of 41-42 kD, confirmed by Western blot analysis. To assess its function, vzg-1 was overexpressed in a cell line from which it was cloned, inducing serum-dependent "cell rounding." Lysophosphatidic acid (LPA), a bioactive lipid present in high concentrations in serum, reproduced the effect seen with serum alone. Morphological responses to other related phospholipids or to thrombin, another agent that induces cell rounding through a GPCR, were not observed in vzg-1 overexpressing cells. Vzg-1 overexpression decreased the EC50 of both cell rounding and Gi activation in response to LPA. Pertussis toxin treatment inhibited vzg-1-dependent LPA-mediated Gi activation, but had no effect on cell rounding. Membrane binding studies indicated that vzg-1 overexpression increased specific LPA binding. These analyses identify the vzg-1 gene product as a receptor for LPA, suggesting the operation of LPA signaling mechanisms in cortical neurogenesis. Vzg-1 therefore provides a link between extracellular LPA and the activation of LPA-mediated signaling pathways through a single receptor and will allow new investigations into LPA signaling both in neural and nonneural systems.