Evolutionary Origin of GnIH and NPFF in Chordates: Insights from Novel Amphioxus RFamide Peptides

Gonadotropin-inhibitory hormone (GnIH) is a newly identified hypothalamic neuropeptide that inhibits pituitary hormone secretion in vertebrates. GnIH has an LPXRFamide (X = L or Q) motif at the C-terminal in representative species of gnathostomes. On the other hand, neuropeptide FF (NPFF), a neuropeptide characterized as a pain-modulatory neuropeptide, in vertebrates has a PQRFamide motif similar to the C-terminal of GnIH, suggesting that GnIH and NPFF have diverged from a common ancestor. Because GnIH and NPFF belong to the RFamide peptide family in vertebrates, protochordate RFamide peptides may provide important insights into the evolutionary origin of GnIH and NPFF. In this study, we identified a novel gene encoding RFamide peptides and two genes of their putative receptors in the amphioxus Branchiostoma japonicum. Molecular phylogenetic analysis and synteny analysis indicated that these genes are closely related to the genes of GnIH and NPFF and their receptors of vertebrates. We further identified mature RFamide peptides and their receptors in protochordates. The identified amphioxus RFamide peptides inhibited forskolin induced cAMP signaling in the COS-7 cells with one of the identified amphioxus RFamide peptide receptors expressed. These results indicate that the identified protochordate RFamide peptide gene is a common ancestral form of GnIH and NPFF genes, suggesting that the origin of GnIH and NPFF may date back to the time of the emergence of early chordates. GnIH gene and NPFF gene may have diverged by whole-genome duplication in the course of vertebrate evolution.     

Driving reproduction: RFamide peptides behind the wheel

The availability of tools for probing the genome and proteome more efficiently has allowed for the rapid discovery of novel genes and peptides that play important, previously uncharacterized roles in neuroendocrine regulation. In this review, the role of a class of neuropeptides containing the C-terminal Arg-Phe-NH(2) (RFamide) in regulating the reproductive axis will be highlighted. Neuropeptides containing the C-terminal Phe-Met-Arg-Phe-NH(2) (FMRFamide) were first identified as cardioregulatory elements in the bi-valve mollusk Macrocallista nimbosa. During the past two decades, numerous studies have shown the presence of structurally similar peptides sharing the RFamide motif across taxa. In vertebrates, RFamide peptides have pronounced influences on opiatergic regulation and neuroendocrine function. Two key peptides in this family are emerging as important regulators of the reproductive axis, kisspeptin and gonadotropin-inhibitory hormone (GnIH). Kisspeptin acts as the accelerator, directly driving gonadotropin-releasing hormone (GnRH) neurons, whereas GnIH acts as the restraint. Recent evidence suggests that both peptides play a role in mediating the negative feedback effects of sex steroids. This review presents the hypothesis that these peptides share complementary roles by responding to internal and external stimuli with opposing actions to precisely regulate the reproductive axis.     

Behavioral effects of 26RFamide and related peptides

A novel 26-amino acid peptide possessing the Arg-Phe-NH(2) motif at its C-terminal extremity has been recently characterized and named 26RFamide (26RFa). The 26RFa precursor encompasses several potential cleavage sites and thus may generate various mature peptides including an N-terminally extended form of 26RFa (termed 43RFa), two fragments of 26RFa (26RFa(1-16) and 26RFa(20-26)), and a 9-amino acid peptide (9RFa) located in tandem in the human 26RFa precursor. In the present study, we have investigated the central effects of 26RFa and related peptides on food intake and locomotor activity in mice. We observed that i.c.v. injection of 26RFa, 43RFa, 26RFa(20-26) and 9RFa stimulated food consumption while 26RFa(1-16) and 26RFa(8-16) had no effect. A dose-dependent stimulation of locomotor activity was observed after i.c.v. administration of 26RFa, 43RFa and 26RFa(1-16), but not 26RFa(20-26), 26RFa(8-16) or 9RFa. These data indicate that the novel neuropeptides 26RFa and 43RFa act centrally to stimulate feeding and locomotor activities but the domains of the peptide involved in each of these responses are different suggesting that the two behavioral effects may be mediated through distinct receptors.     

Recent advances in mammalian RFamide peptides: the discovery and functional analyses of PrRP, RFRPs and QRFP

Since the first discovery of a peptide with RFamide structure at its C-terminus (i.e., an RFamide peptide) from an invertebrate in 1977, numerous studies on RFamide peptides have been conducted, and a variety have been identified in various phyla throughout the animal kingdom. The first reported mammalian RFamide peptides were neuropeptide FF (NPFF) and neuropeptide AF (NPAF) in 1985. However, for many years after this, no new novel RFamide peptides were identified in mammals. A breakthrough in discovering mammalian RFamide peptides was made possible by reverse pharmacology on the basis of orphan G protein-coupled receptor (GPCR) research. The first report of an RFamide peptide identified from orphan GPCR research was prolactin (PRL)-releasing peptide (PrRP) in 1998. To date, a total of five RFamide peptide genes have been discovered in mammals. Orphan GPCR research has contributed considerably to the identification of these peptides and their receptor genes. This paper examines these mammalian RFamide peptides focusing especially on PrRP, RFamide-related peptides (RFRPs) and, the most recently identified, pyroglutamylated RFamide peptide (QRFP), the discovery of all of which the authors were at least partly involved in. We review here the strategies employed for the identification of these peptides and examine their characteristics, tissue distribution, receptors and functions.     

Structure and functions of the novel hypothalamic RFamide neuropeptides R-RFa and 26RFa in vertebrates

A number of RFamide peptides have been characterized in invertebrate species and these peptides have been found to exert a broad spectrum of biological activities. In contrast, in vertebrates, our knowledge on RFamide peptides is far more limited and only a few members of the RFamide peptide family have been identified in various vertebrate classes during the last years. The present review focuses on two novel RFamide peptides, Rana RFamide (R-RFa) and 26RFa, that have been recently isolated from the amphibian brain. R-RFa shares the C-terminal LPLRFamide motif with other RFamide peptides previously identified in mammals, birds and fish. The distribution of R-RFa in the frog brain exhibits strong similarities with those of other LPLRFamide peptides, notably in the periventricular region of the hypothalamus. There is also evidence that the physiological functions of R-RFa and other LPLRFamide peptides have been conserved from fish to mammals; in particular, all these peptides appear to be involved in the control of pituitary hormone secretion. 26RFa does not exhibit any significant structural identity with other RFamide peptides and this peptide is the only member of the family that possesses an FRFamide motif at its C-terminus. The strong conservation of the primary structure of 26RFa from amphibians to mammals suggests that this RFamide peptide is involved in important biological functions in vertebrates. As for several other RFamide peptides, 26RFa-containing neurons are present in the hypothalamus, notably in two nuclei involved in the control of feeding behavior. Indeed, 26RFa is a potent stimulator of appetite in mammals. Concurrently, recent data suggest that 26RFa exerts various neuroendocrine regulatory activities at the pituitary and adrenal level.     

Roles of prolactin-releasing peptide and RFamide related peptides in the control of stress and food intake

Subsequent to the isolation of the first recognized RFamide neuropeptide, FMRFamide, from the clam, a large number of these peptides have been identified. There are now five groups of RFamide peptides identified in mammals. RFamide peptides show diversity with respect to their N-terminal sequence and biological activity. RFamide peptides have been implicated in a variety of roles, including energy metabolism, stress and pain modulation, as well as effects in the neuroendocrine and cardiovascular systems. In the present minireview, we focus on prolactin-releasing peptide (PrRP) and RFamide related peptide (RFRP) with respect to their roles in the control of energy metabolism and stress responses. Both food intake and stressful stimuli activate PrRP neurons. The administration of PrRP affects energy metabolism and neuroendocrine systems. PrRP-deficient or PrRP receptor-deficient mice show abnormal energy metabolism and/or stress responses. On the other hand, RFRP neurons are activated by stressful stimuli and the administration of RFRP induces neuroendocrine and behavioral stress responses. Taken together, these data suggests that PrRP and RFRP neurons play a role in the control of energy metabolism and/or stress responses.     

Molecular properties of endogenous RFamide-related peptide-3 and its interaction with receptors

Based on database searches of DNA sequences, we previously reported a gene encoding peptides possessing Arg-Phe-NH(2) (RFamide) at their C termini. This gene, RFamide-related peptide (RFRP), was expected to encode several different peptides (i.e., RFRP-1, -2, and -3). In the present study, we purified endogenous RFRP-3 from bovine hypothalamus, and demonstrated that it consisted of 28 amino acid residues. After constructing a sandwich enzyme immunoassay for RFRP-3, we analyzed the tissue distribution of endogenous RFRP-3 in rats and found its concentration to be highest in the hypothalamus. In binding assays, [125I]-labeled RFRP-3 bound to OT7T022 with high affinity, but its binding affinity to HLWAR77 was low. On the other hand, [125I]-labeled neuropeptide FF (NPFF) bound to both OT7T022 and HLWAR77 with high affinity. By serial deletion in the N-terminal portions of RFRP-3 and NPFF, we found that four C-terminal amino acid residues (i.e., PQRFamide), which were common between the two peptides, comprised a core sequence responsible for binding with the receptors, whereas three amino acid residues (i.e., PNL in RFRP-3 and LFQ in NPFF) added to the N terminus of PQRFamide played crucial roles in the agonistic activities of RFRP-3 and NPFF for OT7T022 and HLWAR77, respectively.     

RFamide neuropeptide actions on the molluscan ventricle: Interactions with primary neurotransmitters

Different RFamide neuropeptides, some of non-molluscan origin, were examined for their effect on the ventricles of Buccinum undatum and Busycon canaliculatum. None of the peptides tested were inhibitory on these ventricles. All the peptides were extremely active, causing excitation of the preparations at low concentrations. The neuropeptides were then tested with the primary neurotransmitters. In the case of serotonin, the excitatory primary neurotransmitter, the RFamide neuropeptides induced a response, which was greatly enhanced by serotonin. Acetylcholine, the inhibitory neurotransmitter, induced relaxation whenever added, following a neuropeptide. The neuropeptides seemed to be independent of external Ca(2+), since in Ca(2+)-free media tension was induced. On the contrary, serotonin was dependent on external Ca(2+). These findings indicate that the neuropeptides generated tension via a different receptor to that of the primary neurotransmitters, using a different 2nd messenger and activating different Ca(2+) sources. Finally, the parent neuropeptide Phe-Leu-Arg-Phe-NH(2), when added following a different RFamide peptide, excited the preparation further, thus indicating the presence of a receptor that has higher affinity for some structures than others. When Phe-Met-Arg-Phe-NH(2) followed Phe-Leu-Arg-Phe-NH(2), no such response was recorded since the latter is of higher potency than the former.     

Identification of a novel frog RFamide and its effect on the latency of the tail-flick response of the newt

Neuropeptide FF, one of the mammalian PQRFamides, has been reported to affect the latency of the tail-flick response in rat. We intended to examine the nociceptive effect by the peptide PQRFamides from the comparative aspect. Using the dot immunoblot method with antiserum to FMRFamide as an assay system, a peptide (frog's nociception-related peptide, fNRP) which has the C-terminal sequence PQRFamide was isolated from the brain of the frog, Rana catesbeiana. The determined sequence, SIPNLPQRF-NH(2), is the same as that named first (frog growth hormone-releasing peptide-gene-related peptide-1: fGRP-RP-1, which is encoded in the cDNA of the fGRP precursor. Since the peptide was isolated from the frog brain, we tested another amphibian, the newt, which has a tail, by the hot beam tail-flick test. Intraperitoneal injection of fNRP significantly increased the latency of the pain response (tail-flick) 90 min after administration. The effect was blocked by simultaneous administration of 5 mM naloxone. The result provides evidence for the interaction of fNRP and opioid steps in the analgesia pathways in the newt.     

Identification of the novel bioactive peptides dRYamide-1 and dRYamide-2, ligands for a neuropeptide Y-like receptor in Drosophila

A number of bioactive peptides are involved in regulating a wide range of animal behaviors, including food consumption. Vertebrate neuropeptide Y (NPY) is a potent stimulator of appetitive behavior. Recently, Drosophila neuropeptide F (dNPF) and short NPF (sNPF), the Drosophila homologs of the vertebrate NPY, were identified to characterize the functions of NPFs in the feeding behaviors of this insect. Dm-NPFR1 and NPFR76F are the receptors for dNPF and sNPF, respectively; both receptors are G protein-coupled receptors (GPCRs). Another GPCR (CG5811; NepYR) was indentified in Drosophila as a neuropeptide Y-like receptor. Here, we identified 2 ligands of CG5811, dRYamide-1 and dRYamide-2. Both peptides are derived from the same precursor (CG40733) and have no significant structural similarities to known bioactive peptides. The C-terminal sequence RYamide of dRYamides is identical to that of NPY family peptides; on the other hand, dNPF and sNPF have C-terminal RFamide. When administered to blowflies, dRYamide-1 suppressed feeding motivation. We propose that dRYamides are related to the NPY family in vertebrates, similar to dNPF and sNPF.     

Hypothalamic LPXRF-amide peptides in vertebrates: identification, localization and hypophysiotropic activity

Probing undiscovered neuropeptides that play important roles in the regulation of pituitary function in vertebrates is essential for the progress of neuroendocrinology. Recently, we identified a novel hypothalamic neuropeptide with a C-terminal LPLRF-amide sequence in the quail brain. This avian neuropeptide was shown to be located in the hypothalamo-hypophysial system and to decrease gonadotropin release from cultured anterior pituitary. We, therefore, designated this novel neuropeptide as gonadotropin-inhibitory hormone (GnIH). We further identified novel hypothalamic neuropeptides closely related to GnIH in the brains of other vertebrates, such as mammals, amphibians, and fish. The identified neuropeptides possessed a LPXRF-amide (X = L or Q) motif at their C-termini. These LPXRF-amide peptides also were localized in the hypothalamus and other brainstem areas and regulated pituitary hormone release. Subsequently, cDNAs that encode LPXRF-amide peptides were characterized in vertebrate brains. In this review, we summarize the identification, localization, and hypophysiotropic activity of these newly identified hypothalamic LPXRF-amide peptides in vertebrates.     

Molecular evolution and functional characterization of the orexigenic peptide 26RFa and its receptor in vertebrates

Several neuropeptides possessing the RFamide motif at their C-termini (designated RFamide peptides) have been characterized in the hypothalamus of a variety of vertebrates. To date, five groups of the RFamide peptide family have been shown to exert several important neuroendocrine, behavioral, sensory, and autonomic functions. Since the discovery of the 26-amino acid RFamide peptide (termed 26RFa) from the frog brain, 26RFa has been shown to exert orexigenic activity in mammals and to be a ligand of the previously identified orphan G-protein-coupled receptor GPR103. Recently, 26RFa and its cognate receptor GPR103 have been identified in the brain of birds. This mini-review summarizes the advances in the identification, localization, and functions of 26RFa and its cognate receptor GPR103 in vertebrates and highlights recent progress made in birds.     

RFamide neuropeptide actions on the molluscan heart

FMRFamide and the related tetrapeptide FLRFamide are highly excitatory in molluscan non-cardiac smooth muscle. They are also exceptionally excitatory in the atrium and internally perfused ventricle of Busycon canaliculatum. These two peptides, usually thought of as classic molluscan cardio-acceleratory agents are in fact simply two members of a large and ever growing superfamily, the RFamide family, whose phylogenetic distribution has been so elegantly mapped by Walker. Members of this family, often with extended peptide chains (e.g. penta, hepta and decapeptides), stretch in their known distribution from the cnidaria to the chordates. The effects of some of the members of this superfamily (FMRFamide. FLRFamide, YMRFamide, TNRNFLRFamide, SDPFLRFamide, LMS) were examined. The neuropeptides were found to be very potent at very low concentrations (10(-9) M) in the ventricle of both Buccinium and Busycon. Other neuropeptides (HFMRdFamide, SCPb, NLERFamide and pEGRFamide) were found to be without any effect. The Ca2+ dependency of these neuropeptides was also tested. The peptides appear to induce contraction of the ventricles by release of Ca2+ from internal pools. The neuropeptides appear to stimulate contraction in these cardiac muscles through a completely different pathway to Serotonin (the main excitatory neurotransmitter for the cardiac muscle). When the peptides were applied together with Serotonin an additive effect was observed clearly indicating the release of Ca2+ through different pathways. The nature of the RFamide receptor was also tested. It appears that the RFamide neuropeptides mobilize the 2nd messenger IP3 (Inositol trisphosphate), since the IP3 blocker Neomycin Sulphate inhibited the response of the neuropeptides.     

Cloning and characterization of Xen-dorphin prohormone from Xenopus laevis: a new opioid-like prohormone distinct from proenkephalin and prodynorphin

Opioid-like peptides mediate analgesia and induce behavioral effects such as tolerance and dependence by ligand-receptor-mediated mechanisms. The classical opioid prohormones can generate several bioactive peptides, and these divergent families of prohormones share a common well conserved ancestral opioid motif (Tyr-Gly-Gly-Phe). Evidence from pharmacological and molecular cloning studies indicates the presence of multiple isoforms of opioid ligands and receptors that are as yet uncharacterized. To identify potential new members we used the opioid motif as an anchor sequence and isolated two distinct isoforms (Xen-dorphins A and B) of an opioid prohormone from Xenopus laevis brain cDNA library. Xen-dorphin prohormones can generate multiple novel opioid ligands distinct from the known members of this family. Both isoforms are present in a wide variety of tissues including the brain. Two potential bioactive peptides, Xen-dorphin-1A and -1B, that were chemically synthesized showed opioid agonist activity in frog and rat brain membranes using a [35S]GTPgammaS assay. Initial radioligand binding experiments demonstrated that Xen-dorphin-1B binds with high affinity to opioid receptor(s) and with potential preference to the kappa-opioid receptor subtype. Cloning of the Xen-dorphin prohormone provides new evidence for the potential presence of other members in the opioid peptide superfamily.     

Insight into tachykinin-related peptides,their receptors,and invertebrate tachykinins: a review

Tachykinins (TKs) constitute the largest vertebrate neuropeptide family with multifunctions in central and peripheral tissues. In several invertebrate species, two types of structurally related peptides, 'tachykinin-related peptides (TKRPs)' and 'invertebrate tachykinins (inv-TKs)' have been identified. TKRPs, isolated from the nerve and/or gut tissues, contain the common C-terminal sequence -Phe-X-Gly-Y-Arg-NH(2) (X and Y are variable) analogous to the vertebrate TK consensus -Phe-X-Gly-Leu-Met-NH(2), and exhibit vertebrate TK-like contractile activity on invertebrate gut tissues. Inv-TKs have been shown to be present exclusively in the salivary gland of several species, to share vertebrate TK consensus motif, and to possess TK-like potencies on vertebrate, not invertebrate tissues. However, the functional and evolutionary relevance of TKRPs and inv-TKs to vertebrate TKs remains to be understood. Recent studies have revealed that TKRP precursors dramatically differ from vertebrate preprotachykinins in structural organization and that TKRP receptors share structural and functional properties with vertebrate TK receptors. Moreover, the C-terminal arginine in TKRPs has been shown to play an essential role in discriminating their receptors from vertebrate TK receptors. Such recent marked progress is expected to enhance further investigation of biological roles of TKRPs. This review provides an overview of the basic findings obtained previously and a buildup of new knowledge regarding TKRPs and inv-TKs. We also compare TKRPs and inv-TKs to vertebrate TKs with regard to evolutionary relationships in structure and function among these structurally related peptides.     

Characterization of a cDNA encoding a precursor of Carassius RFamide, structurally related to a mammalian prolactin-releasing peptide

We have characterized the cDNA encoding Carassius RFamide (C-RFa), which is structurally related to mammalian prolactin-releasing peptides (PrRPs), from the brain of the crucian carp. The deduced C-RFa precursor has been shown to comprise 117 amino acids, encoding a single C-RFa sequence. A comparative study of amino acid sequences has revealed that several sequences conserved in preproPrRPs are also found in the C-RFa precursor. Furthermore, the abundant presence of the C-RFa mRNA in the telencephalon, optic tectum, medulla oblongata, and proximal half eye ball was demonstrated by Southern blot analysis of RT-PCR products.     

Anatomical distribution and biochemical characterization of the novel RFamide peptide 26RFa in the human hypothalamus and spinal cord

26RFa is a novel RFamide peptide originally isolated in the amphibian brain. The 26RFa precursor has been subsequently characterized in various mammalian species but, until now, the anatomical distribution and the molecular forms of 26RFa produced in the CNS of mammals, in particular in human, are unknown. In the present study, we have investigated the localization and the biochemical characteristics of 26RFa-like immunoreactivity (LI) in two regions of the human CNS--the hypothalamus and the spinal cord. Immunohistochemical labeling using specific antibodies against human 26RFa and in situ hybridization histochemistry revealed that in the human hypothalamus 26RFa-expressing neurons are located in the paraventricular and ventromedial nuclei. In the spinal cord, 26RFa-expressing neurons were observed in the dorsal and lateral horns. Characterization of 26RFa-related peptides showed that two distinct molecular forms of 26RFa are present in the human hypothalamus and spinal cord, i.e. 26RFa and an N-terminally elongated form of 43 amino acids designated 43RFa. These data provide the first evidence that 26RFa and 43RFa are actually produced in the human CNS. The distribution of 26RF-LI suggests that 26RFa and/or 43RFa may modulate feeding, sexual behavior and transmission of nociceptive stimuli.     

Central administration of the RFamide peptides, QRFP-26 and QRFP-43, increases high fat food intake in rats

Pyrogultamylated arginine-phenylalanineamide peptide (QRFP) is strongly conserved across species and is a member of the family of RFamide-related peptides, with the motif Arg-Phe-NH(2) at the C-terminal end. The precursor peptide for QRFP generates a 26-amino acid peptide (QRFP-26) and a 43-amino acid peptide (QRFP-43), both of which bind to the G protein-coupled receptor, GPR103. Recently, QRFP has been characterized in rats, mice and humans and has been reported to have orexigenic properties. In rodents, prepro-QRFP mRNA is expressed in localized regions of the mediobasal hypothalamus, a region implicated in feeding behavior. Increased intake of a high fat diet contributes to increased weight gain and obesity. Therefore, the current experiments investigated the effects of QRFP administration in rats and the effects of a high fat diet on prepro-QRFP mRNA and GPR103 receptor mRNA levels. Intracerebroventricular administration of QRFP-26 (3.0nM, 5.0nM) and QRFP-43 (1.0nM, 3.0nM) dose-dependently increased 1h, 2h, and 4h cumulative intake of high fat (55% fat), but not low fat (10% fat) diet. In Experiment 2, hypothalamic prepro-QRFP mRNA levels and GPR103 receptor mRNA levels were measured in rats fed a high fat or a low fat diet for 21 days. Prepro-QRFP mRNA was significantly increased in the ventromedial nucleus/arcuate nucleus of the hypothalamus of rats fed a high fat diet compared to those fed a low fat diet, while GPR103 mRNA levels were unchanged. These findings suggest that QRFP is a regulator of dietary fat intake and is influenced by the intake of a high fat diet.     

Identification of kinin-related peptides in the disease vector, Rhodnius prolixus

We have used an in silico approach to identify a gene from the blood-gorging vector, Rhodnius prolixus, that is predicted to produce an insect kinin prepropeptide. The prepropeptide is 398 amino acids in length and can potentially produce a large number of kinin-related peptides following post-translational processing. A comparison with other insect kinin precursor sequences demonstrates greatest conservation at the C-terminal region of the kinin peptides. Multiple peptides predicted from the kinin gene are phenotypically expressed in R. prolixus, as revealed by MALDI-TOF MS MS, including 12 kinins and one kinin precursor peptide (KPP). Six of these peptides are characterized by the typical insect kinin C-terminal motif FX₁X₂WGamide and five of these are also found as truncated forms. Five peptides were identified with an atypical, though similar, FX₁X₂WAamide C-terminus. There is also peptide with a C-terminal DDNGamide motif and a number of non-amidated peptides.