Identification of 26Rfa, a Hypothalamic Neuropeptide of the Rfamide Peptide Family with Orexigenic Activity
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Identification of 26RFa, a hypothalamic neuropeptide of the RFamide peptide family with orexigenic activity Nicolas Chartrel*, Cynthia Dujardin*, Youssef Anouar*, Je´ roˆ me Leprince*, Annick Decker*, Stefan Clerens†, Jean-Claude Do-Re´ go‡, Frans Vandesande†, Catherine Llorens-Cortes§, Jean Costentin‡, Jean-Claude Beauvillain¶, and Hubert Vaudry*ʈ *European Institute for Peptide Research (IFRMP23), Laboratory of Cellular and Molecular Neuroendocrinology, Institut National de la Sante´ etdela Recherche Me´dicale, U 413, University of Rouen, 76821 Mont-Saint-Aignan, France; †Laboratory of Neuroendocrinology and Immunological Biotechnology, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium; ‡European Institute for Peptide Research (IFRMP23), Centre National de la Recherche Scientifique, Unite´Mixte de Recherche 6036, Laboratory of Experimental Neuropsychopharmacology, University of Rouen, 76183 Rouen, France; §Institut National de la Sante´et de la Recherche Me´dicale, U 36, Colle`ge de France, 75005 Paris, France; and ¶Federative Research Institute 22, Laboratory of Neuroendocrinology and Neuronal Physiopathology, Institut National de la Sante´et de la Recherche Me´dicale, U 422, 59045 Lille, France Edited by Tomas Ho¨kfelt, Karolinska Institute, Stockholm, Sweden, and approved October 10, 2003 (received for review July 24, 2003) A neuropeptide was isolated from a frog brain extract by HPLC ligand of the orphan G protein-coupled receptor hGR3 (11). purification and characterized by mass spectrometry. This 26-aa PrRP has been initially named because of its ability to stimulate neuropeptide, which belongs to the RFamide peptide family, was prolactin release (11), but recent studies suggest that this peptide designated 26RFa, and its primary structure was established as plays a role in the control of food intake (12), stress response, VGTALGSLAEELNGYNRKKGGFSFRF-NH2. Research in databases re- and nociception (13). Finally, database search has led to the vealed the presence of sequences homologous to frog 26RFa in the identification of a third human gene that encodes three putative human genome and in rat ESTs. On the basis of this sequence RFRPs (14), and one of these peptides, RFRP-1, has been found information, the cDNAs encoding the human and rat 26RFa pre- to stimulate prolactin secretion in vivo (14). The avian counter- cursors were cloned. The two preproteins show a similar organi- part of RFRP-1 has been recently isolated in the Japanese quail; zation, with the 26RFa sequence located in the C-terminal region of this peptide, which inhibits gonadotropin release, has been called the precursor. Human preprotein (prepro)-26RFa encodes an addi- GnIH (15). tional putative RFamide peptide that is not found in the rat The concentration of neuropeptides in the brain of cold- precursor. The primary structures of human, rat, and frog 26RFa blooded vertebrates is several orders of magnitude higher than exhibit Ϸ80% identity, and the C-terminal octapeptide has been in the brain in mammmals (16, 17). Taking advantage of this fully conserved from amphibians to mammals. In situ hybridization particularity, we have been able to isolate, from the frog brain, histochemistry revealed that, in the rat brain, the 26RFa gene is several neuropeptides, including secretoneurin (18), urotensin II exclusively expressed in the ventromedial hypothalamic nucleus (19), and cortistatin (20), which have all been subsequently and in the lateral hypothalamic area. 26RFa induced a dose- identified in humans (21–23). In the course of these studies, we dependent stimulation in cAMP production by rat pituitary cells in have found that the frog brain contains several RFRPs, and we vitro and markedly increased food intake in mice. The conservation have characterized one of them, the dodecapeptide R-RFa (2), of the primary structure of 26RFa during vertebrate evolution, the which turned out to be the orthologue of mammalian RFRP-1 discrete localization of the mRNA encoding its precursor in hypo- (14) and avian GnIH (15). thalamic nuclei involved in the control of feeding behavior, and the Here, we report the identification of 26RFa, an RFRP isolated PHYSIOLOGY observation that 26RFa possesses orexigenic properties indicate from the frog brain that exhibits no meaningful sequence that this neuropeptide may play important biological functions. similarity with any known vertebrate RFRPs. We provide the sequences of the cDNAs encoding the 26RFa precursors in rat he tetrapeptide Phe-Met-Arg-Phe-NH2 (FMRFamide) was and human. We describe the distribution of preprotein (prepro)- Toriginally isolated from the ganglia of the venus clam (1). 26RFa mRNA in the rat brain, and we show that 26RFa is a Since then, several FMRFamide-related peptides (RFRPs), potent orexigenic peptide in mice. which all possess the Arg-Phe-NH motif at their C terminus, 2 Materials and Methods have been isolated in various groups of invertebrates (see ref. 2 for review), and it has been found that a single species may Purification of 26RFa from Frog Brain. Adult male frogs (Rana express multiple members of RFRPs. In particular, in Caeno- esculenta) were purchased from a commercial source (Coue´tard, rhabditis elegans, 22 genes encoding 59 distinct RFRPs have been St-Hilaire de Riez, France). Whole brains from 2,500 animals identified (3). Although genomic data predict that the human (235 g wet weight) were collected on dry ice and kept frozen at Ϫ genome encodes a number of proteins encompassing RFamide- 80°C. The tissues were boiled in 0.5 M acetic acid for 15 min, ϫ related sequences, only a few RFRPs have been isolated so far homogenized, and centrifuged (4,000 g, 4°C, 30 min). Purifi- in vertebrates. Notably, in human, only three genes encoding RFRPs precursors have been characterized to date. One of these genes encodes the precursor for two RFRPs, i.e., neuropeptide This paper was submitted directly (Track II) to the PNAS office. FF (NPFF) and neuropeptide AF (4, 5). These two neuropep- Abbreviations: GnIH, gonadotropin-inhibiting hormone; LH, lateral hypothalamic area; NPFF, neuropeptide FF; NPFF-LI, NPFF-like immunoreactivity; PrRP, prolactin-releasing tides modulate the action of morphine (6) and are thus thought peptide; RFRP, FMRFamide-related peptide; R-RFa, Rana RFamide; VMH, ventromedial to be involved in the transmission of pain stimuli. However, hypothalamic nucleus; prepro, preprotein. NPFF is probably implicated in other physiological processes, Data deposition: The sequences reported in this paper have been deposited in the SWISS- such as regulation of energy homeostasis (7), arterial blood PROT͞TrEMBL database [accession no. P83683 (frog 26RFa)], and in the GenBank database pressure (8), and hormone secretion (9, 10). A second RFRP [accession nos. AY438326 (human prepro-26RFa) and AY438327 (rat prepro-26RFa)]. gene encodes the precursor for prolactin-releasing peptide ʈTo whom correspondence should be addressed. E-mail: [email protected]. (PrRP), which has been originally identified as the endogenous © 2003 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.2434676100 PNAS ͉ December 9, 2003 ͉ vol. 100 ͉ no. 25 ͉ 15247–15252 Downloaded by guest on September 26, 2021 Fig. 1. Purification of 26RFa from frog brain. (A) After prepurification on Sep-Pak cartridges, the brain extract was analyzed on a semipreparative reverse-phase HPLC column. The fractions denoted by the bars contained NPFF-LI, and fraction 61 was selected for further purification. (B) Final purification step of 26RFa on an analytical Vydac C18 reverse-phase HPLC column. The column was eluted with a linear gradient of increasing acetonitrile concentration (denoted by the dashed line), and individual peaks were collected by hand. The arrow shows the peak containing NPFF-LI. The arrowheads show where peak collection began and ended. cation of the peptide was performed as described (16). Briefly, GeneAmp PCR system 9700. To amplify the DNA sequences the supernatant was pumped at a flow rate of 2 ml͞min through encoding human 26RFa, two successive PCR reactions were 10 Sep-Pak C18 cartridges (Waters) connected in series. Bound performed by using oligonucleotides whose sequences were material was eluted with 70% (vol͞vol) acetonitrile in water, and deduced from the human hypothetical mRNAXM071001 in acetonitrile was partially evaporated under reduced pressure. GenBank. In the first PCR amplification, the sense primer The solution was diluted with 10 ml of water͞trifluoroacetic acid 5Ј-ATGCCTCCTGGACGAGGA-3Ј and the antisense primer (TFA) (99.9:0.1, vol͞vol) and pumped onto a 1 ϫ 25-cm Vydac 5Ј-ATCCTTCCAAGGCGTCCTG-3Ј were used. The products 218TP1010 C18 reverse-phase HPLC column (Touzart et obtained were used in a second PCR performed with the sense Matignon, Courtaboeuf, France) equilibrated with 0.12% TFA primer 5Ј-AGCCTGTCCCGGGGAAACA-3Ј and the antisense at a flow rate of 2 ml͞min by using the gradient indicated in Fig. primer 5Ј-TCACCGCCGACCGAAGCG-3Ј, to amplify the full- 1A. Absorbance was measured at 215 and 280 nm, and eluting length ORF encoding human 26RFa. To amplify the rat 26RFa fractions were assayed for NPFF-like immunoreactivity (NPFF- cDNA, a first PCR was performed on reverse-transcribed hy- LI) as described (2). Fraction 61 was selected for further pothalamic RNA by using oligonucleotides designed based on a purification and successively chromatographed on a 0.46 ϫ genomic clone (CH230-239D4) identified in the rat High ϫ 25-cm Shodex C8P-50 4E C8 column, a 0.46