characterization-g-protein-coupled-receptors-fluorescence-based

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

KU Leuven

Members of the cholecystokinin (CCK)/gastrin family of peptides, including the arthropod sulfakinins, and their cognate receptors, play an important role in the regulation of feeding behavior and energy homeostasis. Despite many efforts after the discovery of CCK/gastrin immunoreactivity in nematodes 23 yr ago, the identity of these nematode CCK/gastrin-related peptides has remained a mystery ever since. The Caenorhabditis elegans genome contains two genes with high identity to the mammalian CCK receptors and their invertebrate counterparts, the sulfakinin receptors. By using the potential C. elegans CCK receptors as a fishing hook, we have isolated and identified two CCK-like neuropeptides encoded by neuropeptide-like protein-12 (nlp-12) as the endogenous ligands of these receptors. The neuropeptide-like protein-12 peptides have a very limited neuronal expression pattern, seem to occur in vivo in the unsulfated form, and react specifically with a human CCK-8 antibody. Both receptors and ligands share a high degree of structural similarity with their vertebrate and arthropod counterparts, and also display similar biological activities with respect to digestive enzyme secretion and fat storage. Our data indicate that the gastrin-CCK signaling system was already well established before the divergence of protostomes and deuterostomes.

Discovery of a cholecystokinin-gastrin-like signaling system in nematodes.

Neuromedin U (NMU) in vertebrates is a structurally highly conserved neuropeptide of which highest levels are found in the pituitary and gastrointestinal tract. In Drosophila, two neuropeptide genes encoding pyrokinins (PKs), capability (capa) and hugin, are possible insect homologs of vertebrate NMU. Here, the ligand for an orphan G protein-coupled receptor in the nematode Caenorhabditis elegans (Ce-PK-R) was found using a bioinformatics approach. After cloning and expressing Ce-PK-R in HEK293T cells, we found that it was activated by a neuropeptide from the C. elegans NLP-44 precursor (EC(50)=18nM). This neuropeptide precursor is reminiscent of insect CAPA precursors since it encodes a PK-like peptide and two periviscerokinin-like peptides (PVKs). Analogous to CAPA peptides in insects and NMUs in vertebrates, whole mount immunostaining in C. elegans revealed that the CAPA precursor is expressed in the nervous system. The present data also suggest that the ancestral CAPA precursor was already present in the common ancestor of Protostomians and Deuterostomians and that it might have been duplicated into CAPA and HUGIN in insects. In vertebrates, NMU is the putative homolog of a protostomian CAPA-PK.

A neuromedin-pyrokinin-like neuropeptide signaling system in Caenorhabditis elegans.

To obtain reliable quantitative RT-PCR data, normalization relative to stable housekeeping genes is required. However, in practice, expression levels of 'typical' housekeeping genes have been found to vary between tissues and under different experimental conditions. To date, validation studies of reference genes in insects are extremely rare and have never been performed in locusts. In this study, putative housekeeping genes were identified in the desert locust, Schistocerca gregaria and two different software programs (geNorm and Normfinder) were applied to assess the stability of these genes.

Identification and validation of housekeeping genes in brains of the desert locust Schistocerca gregaria under different developmental conditions.

This study addresses the biological function of CG18594, a Drosophila melanogaster phosphatidylethanolamine-binding protein (PEBP) that we named PEBP1, by combining fly genetics, survival experiments and differential proteomics. We demonstrate that transgenic flies overexpressing PEBP1 are highly protected against bacterial infection due to the release of immunity-related proteins in their hemolymph. Apart from proteins that have been reported earlier to participate in insect immunity, we also identify proteins involved in metabolism and signaling, and, in addition, twelve (hypothetical) proteins with unknown function. This is the first report demonstrating an immune function for a Drosophila PEBP protein.

Unraveling the protective effect of a Drosophila phosphatidylethanolamine-binding protein upon bacterial infection by means of proteomics.

The neuropeptides pigment dispersing factor (PDF) and vasoactive intestinal peptide (VIP) are known as key players in the circadian clock system of insects and mammals, respectively. In this study, we report the discovery and characterization of a widely conserved PDF-like neuropeptide precursor pathway in nematodes. Using a combinatorial approach of biochemistry and peptidomics, we have biochemically isolated, identified and characterized three PDF-like neuropeptides in the free-living nematode Caenorhabditis elegans. The two PDF encoding genes, which were designated pdf-1 and pdf-2, display a very strong conservation within the phylum of nematodes. Many of the PDF expressing cells in C. elegans play a role in the control of locomotion and the integration of environmental stimuli, among which light. Our real-time PCR analysis indicates that both PDF genes are consistently expressed during the day and do not affect each other's expression. The transcription of both PDF genes seems to be regulated by atf-2 and ces-2, which encode bZIP transcription factors homologous to Drosophila vrille and par domain protein 1 (Pdp1epsilon), respectively. Together, our data suggest that the PDF neuropeptide pathway, which seems to be conserved throughout the protostomian evolutionary lineage, might be more complex than previously assumed.

Discovery and characterization of a conserved pigment dispersing factor-like neuropeptide pathway in Caenorhabditis elegans.

The striking similarities between the innate defences of vertebrates and invertebrates as well as the amenability of Caenorhabditis elegans for genetic analysis, have made this free-living ground nematode a popular model system in the study of bacterial pathogenesis. Although genetic studies have brought new insights, showing the inducibility and pathogen-specificity of the immune response, there is still much to be discovered about the exact mechanisms underlying resistance to infection. In this paper a different angle was adopted to study host-pathogen interactions in C. elegans. We report the application of differential gel electrophoresis (DIGE), combined with mass spectrometry to search for proteins that are differentially synthesised in the worm after infection with the gram-negative bacterium Aeromonas hydrophila. Given the dynamic nature of an immune response, the proteome of C. elegans was investigated at three different time-points after infection. A total of 65 differential proteins were identified. This study confirms the involvement of galectins, C-type lectins and lipid binding proteins in the immunity of C. elegans. In addition a number of unknown proteins, which might represent important players of the worm's defence system, were isolated and identified. This work gives a first indication of the complex changes that occur at the protein level during infection.

A differential proteomics study of Caenorhabditis elegans infected with Aeromonas hydrophila.

The success of invertebrates throughout evolution is an excellent illustration of the efficiency of their defence strategies. Caenorhabditis elegans has proven to be an appropriate model for transcriptome studies of host-pathogen interactions. The aim of this paper is to complement this knowledge by investigating the worm's response to a Staphylococcus aureus infection through a 2-dimensional differential proteomics approach.

Proteome changes of Caenorhabditis elegans upon a Staphylococcus aureus infection.

Despite the general knowledge and repeated predictions of peptide G protein-coupled receptors following the elucidation of the Caenorhabditis elegans genome in 1998, only a few have been deorphanized so far. This was attributed to the apparent lack of coevolution between (neuro)peptides and their cognate receptors. To resolve this issue, we have used an in silico genomic data mining tool to identify the real putative peptide GPCRs in the C. elegans genome and then made a well-considered selection of orphan peptide GPCRs. To maximize our chances of a successful deorphanization, we adopted a combined reverse pharmacology approach. At this moment, we have successfully uncovered four C. elegans neuropeptide signaling systems that support the theory of receptor-ligand coevolution. All four systems are extremely well conserved within nematodes and show a high degree of similarity with their vertebrate and arthropod counterparts. Our data indicate that these four neuropeptide signaling systems have been well conserved during the course of evolution and that they were already well established prior to the divergence of protostomes and deuterostomes.

Coevolution of neuropeptidergic signaling systems: from worm to man.

As a consequence of the Earth's axial rotation, organisms display daily recurring rhythms in behavior and biochemical properties, such as hormone titers. The neuronal system controlling such changes is best studied in the fruit fly Drosophila melanogaster. In the nematode worm Caenorhabditis elegans, most homologs of these genes function in the heterochronic pathway controlling the (timing of) developmental events. Recent data indicate that in the worm at least one of the genes involved in developmental timing is also active in circadian rhythm control, thereby opening up new perspectives on a central (neuronal) timer interfering with many processes. Also, new neuropeptidergic clock homologs have been identified in nematodes, supporting the idea of a broad range of clock-regulated targets. We will describe the current knowledge on homologous clock genes in C. elegans with a focus on the recently discovered pigment dispersing factor gene homologs. Similarities between developmental and daily timing are discussed.

C. elegans homologs of insect clock proteins: a tale of many stories.

In Caenorhabditis elegans, pdfr-1 encodes three receptors of the secretin receptor family. These G protein-coupled receptors are activated by three neuropeptides, pigment dispersing factors 1a, 1b and 2, which are encoded by pdf-1 and pdf-2. We isolated a PDF receptor loss-of-function allele (lst34) by means of a mutagenesis screen and show that the PDF signaling system is involved in locomotion and egg-laying. We demonstrate that the pdfr-1 mutant phenocopies the defective locomotor behavior of the pdf-1 mutant and that pdf-1 and pdf-2 behave antagonistically. All three PDF receptor splice variants are involved in the regulation of locomotor behavior. Cell specific rescue experiments show that this pdf mediated behavior is regulated by neurons rather than body wall muscles. We also show that egg-laying patterns of pdf-1 and pdf-2 mutants are affected, but not those of pdfr-1 mutants, pointing to a novel role for the PDF-system in the regulation of egg-laying.

PDF receptor signaling in Caenorhabditis elegans modulates locomotion and egg-laying.

Gonadotropin-releasing hormone (GnRH) is a critical and central hormone that regulates vertebrate reproduction. The high conservation of GnRH signaling within the chordates (deuterostomians) raises the important question as to whether its appearance might date back prior to the divergence of protostomian and deuterostomian lineages, about 700 million years ago. This leads to several important questions regarding the evolution of the GnRH family. Has GnRH been retained in most protostomian lineages? And was regulation of reproduction already a function of ancestral GnRH? The first question can undoubtedly be answered affirmatively since several GnRH-like sequences have been found in wide variety of protostomian and deuterostomian phyla. However, based on their different primary functions in different phyla - which implies a less unanimous answer on the second question - consistency in the nomenclature of this peptide family has been lost. A comparative and phylogenetic approach shows that the ecdysozoan adipokinetic hormones (AKHs), lophotrochozoan GnRHs and chordate GnRHs are structurally related and suggests that they all originate from a common ancestor. This review supports the view that the AKH-GnRH signaling system probably arose very early in metazoan evolution, prior to the divergence of protostomians and deuterostomians.

Gonadotropin-releasing hormone and adipokinetic hormone signaling systems share a common evolutionary origin.

Vasopressin- and oxytocin-related neuropeptides are key regulators of animal physiology, including water balance and reproduction. Although these neuropeptides also modulate social behavior and cognition in mammals, the mechanism for influencing behavioral plasticity and the evolutionary origin of these effects are not well understood. Here, we present a functional vasopressin- and oxytocin-like signaling system in the nematode Caenorhabditis elegans. Through activation of its receptor NTR-1, a vasopressin/oxytocin-related neuropeptide, designated nematocin, facilitates the experience-driven modulation of salt chemotaxis, a type of gustatory associative learning in C. elegans. Our study suggests that vasopressin and oxytocin neuropeptides have ancient roles in modulating sensory processing in neural circuits that underlie behavioral plasticity.

Vasopressin/oxytocin-related signaling regulates gustatory associative learning in C. elegans.

Like most organisms, the nematode Caenorhabditis elegans relies heavily on neuropeptidergic signaling. This tiny animal represents a suitable model system to study neuropeptidergic signaling networks with single cell resolution due to the availability of powerful molecular and genetic tools. The availability of the worm's complete genome sequence allows researchers to browse through it, uncovering putative neuropeptides and their cognate G protein-coupled receptors (GPCRs). Many predictions have been made about the number of C. elegans neuropeptide GPCRs. In this review, we report the state of the art of both verified as well as predicted C. elegans neuropeptide GPCRs. The predicted neuropeptide GPCRs are incorporated into the receptor classification system based on their resemblance to orthologous GPCRs in insects and vertebrates. Appointing the natural ligand(s) to each predicted neuropeptide GPCR (receptor deorphanization) is a crucial step during characterization. The development of deorphanization strategies resulted in a significant increase in the knowledge of neuropeptidergic signaling in C. elegans. Complementary localization and functional studies demonstrate that neuropeptides and their GPCRs represent a rich potential source of behavioral variability in C. elegans. Here, we review all neuropeptidergic signaling pathways that so far have been functionally characterized in C. elegans.

Neuropeptide GPCRs in C. elegans.

Neuropeptidergic signaling is widely adopted by animals for the regulation of physiology and behavior in a rapidly changing environment. The vasopressin/oxytocin neuropeptide family originates from an ancestral peptide precursor in the antecedent of protostomian and deuterostomian animals. In vertebrates, vasopressin and oxytocin have both hormonal effects on peripheral target tissues, such as in the regulation of reproduction and water balance, and neuromodulatory actions in the central nervous system controlling social behavior and cognition. The recent identification of vasopressin/oxytocin-related signaling in C. elegans reveals that this peptidergic system is widespread among nematodes. Genetic analysis of the C. elegans nematocin system denotes vasopressin/oxytocin-like peptides as ancient neuromodulators of neuronal circuits involved in reproductive behavior and associative learning, whereas former invertebrate studies focused on conserved peripheral actions of this peptide family. Nematocin provides neuromodulatory input into the gustatory plasticity circuit as well as into distinct male mating circuits to generate a coherent mating behavior. Molecular interactions are comparable to those underlying vasopressin- and oxytocin-mediated effects in the mammalian brain. Understanding how the vasopressin/oxytocin family fine-tunes neuronal circuits for social behavior, learning and memory poses a major challenge. Functional conservation of these effects in nematodes and most likely in other invertebrates enables the development of future models to help answering this question.

Ancient neuromodulation by vasopressin/oxytocin-related peptides.

The antiglycemic drug metformin, widely prescribed as first-line treatment of type II diabetes mellitus, has lifespan-extending properties. Precisely how this is achieved remains unclear. Via a quantitative proteomics approach using the model organism Caenorhabditis elegans, we gained molecular understanding of the physiological changes elicited by metformin exposure, including changes in branched-chain amino acid catabolism and cuticle maintenance. We show that metformin extends lifespan through the process of mitohormesis and propose a signaling cascade in which metformin-induced production of reactive oxygen species increases overall life expectancy. We further address an important issue in aging research, wherein so far, the key molecular link that translates the reactive oxygen species signal into a prolongevity cue remained elusive. We show that this beneficial signal of the mitohormetic pathway is propagated by the peroxiredoxin PRDX-2. Because of its evolutionary conservation, peroxiredoxin signaling might underlie a general principle of prolongevity signaling.

Liesbet Temmerman

Department of Biology

KU Leuven

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

Liesbet Temmerman

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biology

KU Leuven

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

Department of Biology