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The novel RFamide peptide 26RFa, the endogenous ligand of the orphan receptor GPR103, affects food intake, locomotion, and activity of the gonadotropic axis. However, little is known regarding the localization of 26RFa receptors. The present report provides the first detailed mapping of 26RFa binding sites and GPR103 mRNA in the rat central nervous system (CNS). 26RFa binding sites were widely distributed in the brain and spinal cord, whereas the expression of GPR103 mRNA was more discrete, notably in the midbrain, the pons, and the medulla oblongata, suggesting that 26RFa can bind to a receptor(s) other than GPR103. Competition experiments confirmed that 26RFa interacts with an RFamide peptide receptor distinct from GPR103 that may be NPFF2. High densities of 26RFa binding sites were observed in olfactory, hypothalamic, and brainstem nuclei involved in the control of feeding behavior, including the piriform cortex, the ventromedial and dorsomedial hypothalamic nuclei, the paraventricular nucleus, the arcuate nucleus, the lateral hypothalamic area, and the nucleus of the solitary tract. The preoptic and anterior hypothalamic areas were also enriched with 26RFa recognition sites, supporting a physiological role of the neuropeptide in the regulation of the gonadotropic axis. A high density of 26RFa binding sites was detected in regions of the CNS involved in the processing of pain, such as the dorsal horn of the spinal cord and the parafascicular thalamic nucleus. The wide distribution of 26RFa binding sites suggests that 26RFa has multiple functions in the CNS that are mediated by at least two distinct receptors., ((c) 2007 Wiley-Liss, Inc.)

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.

The octadecaneuropeptide ODN (QATVGDVNTDRPGLLDLK), a biologically active fragment of diazepam-binding inhibitor, exerts a number of behavioral and neurophysiological activities. The presence of a proline residue in the sequence of ODN led us to investigate the role of proline endopeptidase (PEP) in the catabolism of this neuropeptide. The effect of PEP on the breakdown of ODN and related analogs was studied by combining RP-HPLC analysis and MALDI-TOF MS characterization. Incubation of ODN with PEP generated two products, i.e. ODN3-18 and ODN5-18 which resulted from cleavage of the Ala-Thr and Val-Gly peptide bonds. S 17092, a specific PEP inhibitor, significantly reduced the PEP-induced cleavages of ODN. Similarly, [Ala2]OP showed S 17092-sensitive post-alanine cleavage, while [pGlu1]ODN and OP (ODN11-18) were not catabolized by the enzyme. For all these peptides, cleavage of the Pro-Gly peptide bond by PEP was never observed, even after prolonged incubation times. In contrast, PEP hydrolyzed human urotensin II at the canonical post-proline site. Collectively, these data suggest that the Ala2 residue is the preferential cleavage site of ODN and that the Pro-Gly bond of ODN is not hydrolyzed by PEP. In addition, this study reveals for the first time that the endoproteolytic activity of PEP can specifically take place after a valine moiety.

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.

In the present study, we report the identification, in the frog brain, of a novel neuropeptide, termed 26RFa, that belongs to the RFamide peptide family. The cDNAs encoding the precursors for 26RFa have been characterized in human and rats. In rats, prepro-26RFa mRNA is expressed exclusively in two hypothalamic nuclei involved in the control of feeding behavior. Intracerebroventricular injection of 26RFa in mice induced a dose-dependent increase in food consumption. Taken together, these data indicate that 26RFa is a novel neuropeptide that may have important biological functions in vertebrates.

The human urotensin II (UII) precursor encompasses several potential cleavage sites and thus, processing of pro-UII may generate various forms of mature UII including the peptides of 11 (UII11), 16 (UII16) and 19 (UII19) residues. Until now, the native form of human UII had not been characterized. Here, we show that the major UII peptide occurring in the human spinal cord corresponds to UII11. In contrast, neither the UII16 nor the UII19 forms could be detected. In 50% of the brainstem and in all the spinal cord extracts analysed, a second minor UII-immunoreactive peptide was resolved. Immunohistochemical labelling of the cervical segment of the human spinal cord revealed that the UII-immunoreactive material was confined to a subset of ventral horn motoneurones. These data provide the first evidence that in the human, the UII precursor, expressed in motoneurones, is processed at the tribasic KKR93 cleavage site to generate a mature form of UII of 11 amino acids. The absence of N-terminally elongated forms of UII of 16 and 19 residues indicates that pro-UII is not cleaved at the R85 or K88 monobasic sites. Finally, the minor UII-immunoreactive peptide detected in several tissue extracts might correspond to an extended form of UII resulting from the processing of the UII precursor at the basic RK50 or RK66 doublets.

Trout POMC-A exhibits a unique C-terminal extension of 25-amino acids which is processed in the pituitary and hypothalamus to generate two novel decapeptides, EQWGREEGEE and ALGERKYHFQ-NH(2). The fibers containing these two decapeptides are widely distributed in the brain, suggesting that they may exert neurotransmitter or neuromodulator activities. In the present study, we have investigated the ontogeny of the decapeptide EQWGREEGEE in the trout pituitary and brain. In the pituitary of 29-day embryos and 33-day alevins, EQWGREEGEE-immunoreactive material was observed in a cluster of cells located in the central and rostral region of the gland, respectively. In 47-day alevins, a second group of cells exhibiting EQWGREEGEE-like immunoreactivity was detected in the caudal region of the pituitary and the intensity of labeling in these cells increased in 61-day fry. In the brain, EQWGREEGEE immunoreactivity was detected in 47-day alevins. In 47- and 61-day larvae, immunoreactive elements were mainly detected in the diencephalon. Characterization of the immunoreactive material by reversed-phase high-performance liquid chromatographic analysis combined with radioimmunoassay detection revealed the existence of two major forms which exhibited different retention times than synthetic EQWGREEGEE. The present study indicates that EQWGREEGEE-related peptides are present in the trout pituitary early during ontogeny and appear in the brain only later, and that processing of the C-terminal extension of POMC-A generates distinct molecular species at different developmental stages. These data suggest that alternative processing of the C-terminal domain of POMC-A gives rise to various peptide products that may exert specific activities during trout development.

The trout possesses two POMC genes as a result of duplication of its genome some 25-100 million years ago. One of the POMC molecules exhibits a unique C-terminal extension of 25 amino acid residues which is not found in any other POMC characterized so far. In order to isolate possible novel peptides derived from trout POMC-A, we have raised antibodies against two synthetic epitopes derived from the C-terminal region of the precursor. Two native decapeptides were isolated in pure form from an extract of trout pituitary. The primary structures of these peptides were established as Glu-Gln-Trp-Gly-Arg-Glu-Glu-Gly-Glu-Glu and Ala-Leu-Gly-Glu-Arg-Lys-Tyr-His-Phe-Gln-NH2. The structure of the trout POMC-A cDNA reveals that both peptides are flanked by pairs of basic amino acids or a glycine residue, indicating that they can actually be generated during post-translational processing of POMC-A., (Copyright 1997 Academic Press.)

Two complementary DNAs encoding distinct forms of POMC have been characterized in the trout pituitary. One of the POMC variants (POMC-A) possesses a C-terminal extension of 25 amino acids, which has no equivalent in other POMCs described to date. This C-terminal peptide contains three pairs of basic amino acids, suggesting that it may be the precursor of multiple processed peptides. In addition, the presence of a C-terminal glycine residue suggests that some of the processing products may be alpha-amidated. To characterize the molecular forms of the peptides generated from the C-terminal domain of trout POMC-A, we have developed specific antibodies against the C-terminal pentapeptide YHFQG and its alpha-amidated derivative YHFQ-NH2. Immunocytochemical labeling of pituitary sections with antibodies against YHFQ-NH2 revealed the presence of numerous immunoreactive cells in the pars intermedia and the rostral pars distalis. In contrast, the antibodies against YHFQG produced only weak immunostaining. HPLC analysis combined with RIA detection revealed that extracts of the pars intermedia and pars distalis contain several peptides derived from the C-terminal extension of trout POMC-A, with the predominant molecular form exhibiting the same retention time as ALGERKYHFQ-NH2. Tryptic digestion of this major form produced a peptide that coeluted with YHFQ-NH2. These data indicate that the processing of the C-terminal extension of trout POMC-A generates several novel peptides including the decapeptide amide ALGERKYHFQ-NH2.