Endocrine archeology: do insects retain ancestrally inherited counterparts of the vertebrate releasing hormones GnRH, GHRH, TRH, and CRF?

Vertebrate releasing hormones include gonadotropin releasing hormone (GnRH), growth hormone releasing hormone (GHRH), corticotropin releasing hormone (CRF), and thyrotropin-releasing hormone (TRH). They are synthesized in the hypothalamus and stimulate the release of pituitary hormones. Here we review the knowledge on hormone releasing systems in the protostomian lineage. We address the question: do insects have peptides that may be phylogenetically related to an ancestral GnRH, GHRH, TRH, and CRF? Such endocrine archeology has become possible thanks to the growing list of fully sequenced genomes as well as to the continuously improving bioinformatic tool set. It has recently been shown that the ecdysozoan (nematodes and arthropods) adipokinetic hormones (AKHs), the lophotrochozoan (annelids and mollusks) GnRHs as well as the protochordate GnRHs are structurally related. The adipokinetic hormone precursor-related peptides (APRPs), in locusts encoded by the same gene that contains the AKH-coding region, have been forwarded as the structural counterpart of GHRH of vertebrates. CRF is relatively well conserved in insects, in which it functions as a diuretic hormone. Members of TRH-receptor family seem to have been conserved in some arthropods, but other elements of the thyroid hormone signaling system are not. A challenging idea is that in insects the functions of the thyroid hormones were taken over by juvenile hormone (JH). Our reconstruction suggests that, perhaps, the ancestral releasing hormone precursors played a role in controlling energy metabolism and water balance, and that releasing hormone functions as present in extant vertebrates were probably secondarily acquired.

Adipokinetic hormone signaling through the gonadotropin-releasing hormone receptor modulates egg-laying in Caenorhabditis elegans.

In mammals, hypothalamic gonadotropin-releasing hormone (GnRH) is a neuropeptide that stimulates the release of gonadotropins from the anterior pituitary. The existence of a putative functional equivalent of this reproduction axis in protostomian invertebrates has been a matter of debate. In this study, the ligand for the GnRH receptor in the nematode Caenorhabditis elegans (Ce-GnRHR) was found using a bioinformatics approach. The peptide and its precursor are reminiscent of both insect adipokinetic hormones and GnRH-preprohormone precursors from tunicates and higher vertebrates. We cloned the AKH-GnRH-like preprohormone and the Ce-GnRHR and expressed the GPCR in HEK293T cells. The GnRHR was activated by the C. elegans AKH-GnRH-like peptide (EC(50) = 150 nM) and by Drosophila AKH and other nematode AKH-GnRHs that we found in EST databases. Analogous to both insect AKH receptor and vertebrate GnRH receptor signaling, Ce-AKH-GnRH activated its receptor through a Galpha(q) protein with Ca(2+) as a second messenger. Gene silencing of Ce-GnRHR, Ce-AKH-GnRH, or both resulted in a delay in the egg-laying process, comparable to a delay in puberty in mammals lacking a normal dose of GnRH peptide or with a mutated GnRH precursor or receptor gene. The present data support the view that the AKH-GnRH signaling system probably arose very early in metazoan evolution and that its role in reproduction might have been developed before the divergence of protostomians and deuterostomians.

Comparison of Caenorhabditis elegans NLP peptides with arthropod neuropeptides.

Neuropeptides are small messenger molecules that can be found in all metazoans, where they govern a diverse array of physiological processes. Because neuropeptides seem to be conserved among pest species, selected peptides can be considered as attractive targets for drug discovery. Much can be learned from the model system Caenorhabditis elegans because of the availability of a sequenced genome and state-of-the-art postgenomic technologies that enable characterization of endogenous peptides derived from neuropeptide-like protein (NLP) precursors. Here, we provide an overview of the NLP peptide family in C. elegans and discuss their resemblance with arthropod neuropeptides and their relevance for anthelmintic discovery.

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

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.

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

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.

Comparative peptidomics of Caenorhabditis elegans versus C. briggsae by LC-MALDI-TOF MS.

Neuropeptides are important signaling molecules that function in cell-cell communication as neurotransmitters or hormones to orchestrate a wide variety of physiological conditions and behaviors. These endogenous peptides can be monitored by high throughput peptidomics technologies from virtually any tissue or organism. The neuropeptide complement of the soil nematode Caenorhabditis elegans has been characterized by on-line two-dimensional liquid chromatography and quadrupole time-of-flight tandem mass spectrometry (2D-nanoLC Q-TOF MS/MS). Here, we use an alternative peptidomics approach combining liquid chromatography (LC) with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry to map the peptide content of C. elegans and another Caenorhabditis species, Caenorhabditis briggsae. This study allows a better annotation of neuropeptide-encoding genes from the C. briggsae genome and provides a promising basis for further evolutionary comparisons.

Neuropeptidergic signaling in the nematode Caenorhabditis elegans.

The nematode Caenorhabditis elegans joins the menagerie of behavioral model systems next to the fruit fly Drosophila melanogaster, the marine snail Aplysia californica and the mouse. In contrast to Aplysia, which contains 20,000 neurons having cell bodies of hundreds of microns in diameter, C. elegans harbors only 302 tiny neurons from which the cell lineage is completely described, as is the case for all the other somatic cells. As such, this nervous system appears at first sight incommensurable with those of higher organisms, although genome-wide comparison of predicted C. elegans genes with their counterparts in vertebrates revealed many parallels. Together with its short lifespan and ease of cultivation, suitability for high-throughput genetic screenings and genome-wide RNA interference approaches, access to an advanced genetic toolkit and cell-ablation techniques, it seems that this tiny transparent organism of only 1mm in length has nothing to hide. Recently, highly exciting developments have occurred within the field of neuropeptidergic signaling in C. elegans, not only because of the availability of a sequenced genome since 1998, but especially because of state of the art post genomic technologies, that allow for molecular characterization of the signaling molecules. Here, we will focus on endogenous, bioactive (neuro)peptides and mainly discuss biosynthesis, peptide sequence information, localization and G-protein coupled receptors of the three major peptide families in C. elegans.

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

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.

PACAP and PDF signaling in the regulation of mammalian and insect circadian rhythms.

Endogenous circadian clocks are inherent to all living organisms. They are needed to guarantee successful life since they regulate very important biological processes such as behavior and reproduction. Secretin-like G-protein coupled receptors are very important factors in the signal transduction pathways of circadian clocks. In this review, we will focus on the role of two secretin-like signaling pathways that play an important role in the regulation of the mammalian and the insect clock, respectively: the pituitary adenylate cyclase-activating polypeptide (PACAP) and pigment dispersing factor (PDF) signaling pathways. Both pathways are most likely related although their function in the biological clock differs.

Deorphanizing g protein-coupled receptors by a calcium mobilization assay.

G protein-coupled receptors (GPCRs) comprise one of the largest families of transmembrane proteins involved in signal transduction of diverse external stimuli and represent the most successful target class in drug discovery. The availability of genome sequences in the postgenomic era has paved the way for in silico identification of novel GPCR family members based upon sequence similarity. Consequently, newly discovered receptors are by definition orphan GPCRs with no known ligand, and their functional characterization now poses a major challenge. Over the years, advances in understanding of GPCR biology have led to the development of cell-based assay systems that link orphan GPCRs to their activating ligand(s) in high-throughput format (reverse pharmacology). Many of these technologies monitor important steps in the GPCR activation cycle such as the accumulation of secondary messenger molecules (e.g., cAMP, calcium). In this chapter, we present a calcium mobilization assay in mammalian cells to detect changes in intracellular calcium concentration upon receptor activation by the use of a fluorescent probe. This is currently one of the most frequently used assay systems for GPCR deorphanization.

Characterization of an allatotropin-like peptide receptor in the red flour beetle, Tribolium castaneum.

Following a reverse pharmacology approach, we identified an allatotropin-like peptide receptor in Tribolium castaneum. Allatotropins are multifunctional neuropeptides initially isolated from the tabacco hornworm, Manduca sexta. They have been shown to be myoactive, to be cardio-acceleratory, to inhibit active ion transport, to stimulate juvenile hormone production and release and to be involved in the photic entrainment of the circadian clock. A tissue distribution analysis of the T. castaneum allatotropin-like peptide receptor by means of qRT-PCR revealed a prominent sexual dimorphism, the transcript levels being significantly higher in the male fat body and reproductive system. The endogenous ligand of the receptor, Trica-ATL, is able to increase the frequency and tonus of contractions in the gut and in the reproductive tract of mature red flour beetles.

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

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.

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

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.

Coevolution of neuropeptidergic signaling systems: from worm to man.

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.

Approaches to identify endogenous peptides in the soil nematode Caenorhabditis elegans.

The transparent soil nematode Caenorhabditis elegans can be considered an important model organism due to its ease of cultivation, suitability for high-throughput genetic screens, and extremely well-defined anatomy. C. elegans contains exactly 959 cells that are ordered in defined differentiated tissues. Although C. elegans only possesses 302 neurons, a large number of similarities among the neuropeptidergic signaling pathways can be observed with other metazoans. Neuropeptides are important messenger molecules that regulate a wide variety of physiological processes. These peptidergic signaling molecules can therefore be considered important drug targets or biomarkers. Neuropeptide signaling is in the nanomolar range, and biochemical elucidation of individual peptide sequences in the past without the genomic information was challenging. Since the rise of many genome-sequencing projects and the significant boost of mass spectrometry instrumentation, many hyphenated techniques can be used to explore the "peptidome" of individual species, organs, or even cell cultures. The peptidomic approach aims to identify endogenously present (neuro)peptides by using liquid chromatography and mass spectrometry in a high-throughput way. Here we outline the basic procedures for the maintenance of C. elegans nematodes and describe in detail the peptide extraction procedures. Two peptidomics strategies (off-line HPLC-MALDI-TOF MS and on-line 2D-nanoLC-Q-TOF MS/MS) and the necessary instrumentation are described.

APRP, the second peptide encoded by the adipokinetic hormone gene(s), is highly conserved in evolution: a role in control of ecdysteroidogenesis?

Since the early days of cloning the first adipokinetic hormone (AKH) gene, researchers recognized that this gene also codes for a joining region and for a second peptide called adipokinetic hormone precursor related peptide (APRP). In species with more than one AKH gene, such as locusts, APRPs can form both homodimers and heterodimers. Database analysis showed that APRPs might belong to the ancient family of growth hormone releasing factor but they still are functionally orphan. We investigated whether some of the APRP forms play a role in control of reproduction or/and growth via stimulation of ecdysteroidogenesis.

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.