MOLECULAR EVOLUTION OF GPCRS: 26Rfa/GPR103

in Journal of Molecular Endocrinology

Neuropeptides possessing the Arg-Phe-NH2 (RFamide) motif at their C-termini (designated as RFamide peptides) have been characterized in a variety of animals. Among these, neuropeptide 26RFa (also termed QRFP) is the latest member of the RFamide peptide family to be discovered in the hypothalamus of vertebrates. The neuropeptide 26RFa/QRFP is a 26-amino acid residue peptide that was originally identified in the frog brain. It has been shown to exert orexigenic activity in mammals and to be a ligand for the previously identified orphan G protein-coupled receptor, GPR103 (QRFPR). The cDNAs encoding 26RFa/QRFP and QRFPR have now been characterized in representative species of mammals, birds, and fish. Functional studies have shown that, in mammals, the 26RFa/QRFP–QRFPR system may regulate various functions, including food intake, energy homeostasis, bone formation, pituitary hormone secretion, steroidogenesis, nociceptive transmission, and blood pressure. Several biological actions have also been reported in birds and fish. This review summarizes the current state of identification, localization, and understanding of the functions of 26RFaQRFP and its cognate receptor, QRFPR, in vertebrates.

Abstract

Neuropeptides possessing the Arg-Phe-NH2 (RFamide) motif at their C-termini (designated as RFamide peptides) have been characterized in a variety of animals. Among these, neuropeptide 26RFa (also termed QRFP) is the latest member of the RFamide peptide family to be discovered in the hypothalamus of vertebrates. The neuropeptide 26RFa/QRFP is a 26-amino acid residue peptide that was originally identified in the frog brain. It has been shown to exert orexigenic activity in mammals and to be a ligand for the previously identified orphan G protein-coupled receptor, GPR103 (QRFPR). The cDNAs encoding 26RFa/QRFP and QRFPR have now been characterized in representative species of mammals, birds, and fish. Functional studies have shown that, in mammals, the 26RFa/QRFP–QRFPR system may regulate various functions, including food intake, energy homeostasis, bone formation, pituitary hormone secretion, steroidogenesis, nociceptive transmission, and blood pressure. Several biological actions have also been reported in birds and fish. This review summarizes the current state of identification, localization, and understanding of the functions of 26RFaQRFP and its cognate receptor, QRFPR, in vertebrates.

Introduction

Neuropeptides that possess the Arg-Phe-NH2 motif at their C-termini (i.e., RFamide peptides) have been characterized both in invertebrates and vertebrates. The first RFamide peptide to be identified was the cardioexcitatory peptide Phe-Met-Arg-Phe-NH2 (FMRFamide), which was isolated from the ganglia of the Venus clam Macrocallista nimbosa (Price & Greenberg 1977). Since then, a number of RFamide peptides have been identified in invertebrates, where these peptides seem to act as neurotransmitters and neuromodulators (for review, see Walker et al. (2009)).

A number of immunohistochemical studies that used antisera against FMRFamide suggested that the nervous system of vertebrates also contained neuropeptides immunologically related to FMRFamide (Raffa 1988, Vallarino et al. 1991, 1994, 1995, Rastogi et al. 2001). In fact, several neuropeptides harboring the RFamide sequence at their C-terminal end have been characterized in the brain of various vertebrates. In the past, the existence of five groups within the RFamide peptide family has been recognized in vertebrates, namely the neuropeptide FF (NPFF) group, the prolactin-releasing peptide (PrRP) group, the gonadotropin-inhibitory hormone (GnIH) group, the kisspeptin group, and the 26RFa/QRFP group (for reviews, see Ukena & Tsutsui (2005), Bruzzone et al. (2006), Osugi et al. (2006), Tsutsui & Ukena (2006), Tsutsui (2009), Tsutsui et al. (2010a,b), Chartrel et al. (2011), Leprince et al. (2013); Fig. 1). These RFamide peptides have been shown to exert important neuroendocrine, behavioral, sensory, and autonomic functions (for reviews, see Chartrel et al. 2002, 2006a, Ukena & Tsutsui 2005, Tsutsui & Ukena 2006). Among these vertebrate RFamide peptides, NPFF is well documented as a morphine modulatory peptide (Panula et al. 1999). In addition, GnIH and kisspeptin appear to play key roles in the regulation of the reproductive axis (Tsutsui et al. 2010b). This review summarizes the current state of knowledge on the molecular evolution and functions of 26RFa/QRFP, the latest member of the RFamide peptide family to be discovered in vertebrates, and of its cognate receptor, QRFPR. This review also indicates future directions in this research field.

Figure 1
Figure 1

Phylogenetic tree of the RFamide peptide family in vertebrates. Studies over the past decade have demonstrated that the brain of vertebrates produces a variety of RFamide peptides. To date, five groups have been identified within this family: the neuropeptide FF (NPFF) group, the prolactin-releasing peptide (PrRP) group, the gonadotropin-inhibitory hormone (GnIH) group, the kisspeptin group, and the 26RFa/QRFP group.

Citation: Journal of Molecular Endocrinology 52, 3; 10.1530/JME-13-0207

Unity and diversity of the structure of 26RFa/QRFP in vertebrates

The 26-amino acid residue RFamide peptide, 26RFa/QRFP, was identified for the first time in the brain of an amphibian species (Chartrel et al. 2003). An antibody against the RFamide motif was used to screen, by RIA, peptide fractions purified from a brain extract of the European green frog (Rana esculenta). After HPLC purification, the sequence of the isolated substance was analyzed by mass spectrometry MS/MS fragmentation; it turned out to be a 26-amino acid peptide possessing the RFamide motif at its C-terminus, namely VGTALGSLAEELNGYNRKKGGFSFRFamide. This neuropeptide had not been reported in any animals previously and was designated as 26RFa (Fig. 2A; Chartrel et al. 2003).

Figure 2
Figure 2

Alignments of the amino acid sequences of identified 26RFa/QRFP peptides (A) and their precursor proteins (B) deduced from mammalian (human, bovine, rat, and mouse), avian (chicken, quail, and zebra finch), amphibian (Xenopus), and fish (goldfish) cDNAs. The predicted signal peptide sequences are underlined with a dashed line. <E represents pyroglutamic acid. The positions of identified mature peptides in the precursor proteins are underlined with solid lines. The human and Xenopus 26RFa/QRFP precursors may also generate a nine-amino acid peptide, termed 9RFa (boxed). Fully conserved amino acids are highlighted with red boxes and highly conserved amino acids with gray boxes respectively. The Lys (K)-Arg (R) dibasic processing sites in birds and Xenopus, the single Arg (R) putative processing sites in mammals and fish, and the Gly (G) C-terminal amidation signals are shown in bold. Gaps marked by hyphens were inserted to optimize homology. The GenBank accession numbers of these sequences are as follows: human 26RFa/QRFP, NP_937823; bovine 26RFa/QRFP, NP_937865; rat 26RFa/QRFP, NP_937843; mouse 26RFa/QRFP, NP_906269; chicken 26RFa/QRFP, XP_001235089; quail 26RFa/QRFP, BAI81890; zebra finch 26RFa/QRFP, BAK32798; Xenopus tropicalis 26RFa/QRFP, XP_002936227; and goldfish 26RFa/Qrfp, ACI46681.

Citation: Journal of Molecular Endocrinology 52, 3; 10.1530/JME-13-0207

The amino acid sequence of frog 26RFa was employed to identify the cDNA encoding the counterpart of 26RFa in rat and humans (Chartrel et al. 2003). Concurrently, two other research groups independently identified 26RFa/QRFP precursors using a bioinformatic approach in the rat, mouse, bovine, and human genomes and paired 26RFa/QRFP with a previously identified orphan G protein-coupled receptor (GPCR), GPR103, also known as AQ27 or SP9155 (Fukusumi et al. 2003, Jiang et al. 2003; Fig. 2B). GPR103 has thus been renamed QRFPR by the HUGO Gene Nomenclature Committee (http://www.genenames.org/). The mature 43-amino acid residue RFamide peptide was identified from the culture medium of CHO cells that expressed the human peptide precursor (Fukusumi et al. 2003). As the N-terminal amino acid was pyroglutamic acid, this RFamide peptide was also named pyroglutamylated RFamide peptide (QRFP; Fukusumi et al. 2003). Subsequently, the cDNAs encoding the 26RFa/QRFP precursors have been characterized in goldfish (Liu et al. 2009), quail (Ukena et al. 2010), chicken (Ukena et al. 2010), and zebra finch (Tobari et al. 2011) (Fig. 2B). Although the 26RFa/qrfp cDNA has not been characterized in the European green frog, the corresponding sequence in the African clawed frog (Xenopus tropicalis) is present in the database (Fig. 2B). Furthermore, homologous sequences have been listed in the genome database of reptilian (lizard) and fish (stickleback, medaka, fugu, and zebrafish) species (Liu et al. 2009). These data have revealed the existence of the 26RFa/QRFP-encoding gene in representative species of the whole vertebrate phyla, including fish, amphibians, reptilians, birds, and mammals (Chartrel et al. 2011, Ukena et al. 2011).

As there are several monobasic processing sites in the 26RFa/QRFP precursor protein, alternative cleavage may yield various N-terminally elongated forms of 26RFa/QRFP (Chartrel et al. 2006b, 2011). HPLC analysis combined with RIAs indicated the existence of both 26- and 43-amino acid residue RFamide peptide-like immunoreactivities in the hypothalamus and spinal cord of humans (Bruzzone et al. 2006). Indeed, an N-terminally extended peptide of 43 residues, called 43RFa or QRFP, has been characterized in rat brain extracts, as well as in PC12 cells and the culture medium of CHO cells that express the human precursor, as described above (Fukusumi et al. 2003, Bruzzone et al. 2006, Takayasu et al. 2006; Fig. 2A). The human and Xenopus 26RFa/QRFP precursors may also generate a nine-amino acid peptide, termed 9RFa, located upstream of 26RFa/QRFP (Fig. 2B). However, 9RFa has not been detected in tissue extracts to date. Structure–activity relationship studies have revealed that the synthetic C-terminal heptapeptide (26RFa2026; GGFSFRFamide) is responsible for the biological activity of 26RFa/QRFP (Le Marec et al. 2011, Neveu et al. 2012). A reverse pharmacological study has demonstrated that 26RFa/QRFP is a natural ligand for the previously identified orphan receptor, GPR103 (QRFPR), as described below (Fukusumi et al. 2003, Jiang et al. 2003, Takayasu et al. 2006).

As reported above, the mature forms of 26RFa/QRFP have been identified in the brains of amphibians and mammals (Chartrel et al. 2003, Bruzzone et al. 2006, Takayasu et al. 2006), but, until recently, the existence of 26RFa/QRFP has not been investigated in birds. Among the RFamide peptide family, only the GnIH group had been found in avian at the time we started this study (for reviews, see Ukena & Tsutsui 2005, Tsutsui & Ukena 2006, Tsutsui 2009, Tsutsui et al. 2010a,b). We therefore looked for 26RFa/QRFP in the avian brain and found the presence of a gene encoding the 26RFa/QRFP precursor in chicken after searching the genomic database. Subsequently, the cDNA of the 26RFa/QRFP precursor was sequenced in the quail hypothalamus (Ukena et al. 2010). The quail precursor protein demonstrates 88% overall similarity with the chicken sequence, 47% with the corresponding human sequence, and 40% with the rat sequence (Ukena et al. 2011). A Lys-Arg dibasic cleavage site is present in the C-terminal region of the quail and chicken precursor sequences, but not in that of mammalian sequences (Fig. 2B). This indicates that the mature peptide consists of 27 amino acid residues in quail and chicken, unlike the 26 residues in the amphibian 26RFa/QRFP sequence (Chartrel et al. 2003). In fact, MS analysis combined with immunoaffinity purification has revealed that the 27-amino acid sequence corresponds to the mature form of the peptide in the quail hypothalamus, indicating that the peptide is actually produced from the precursor in the hypothalamus (Ukena et al. 2010; Fig. 2A). More recently, a 26RFa/QRFP ortholog, consisting of 25 amino acids, and the related cDNA have been characterized in the brain of zebra finch (Tobari et al. 2011; Fig. 2).

Synteny analysis of the 26RFa/QRFP gene revealed that the chromosomal region encompassing the 26RFa/QRFP gene is highly conserved from amphibians to human. Indeed, all these regions contain paralogs of several other genes and thus clearly constitute a paralogon (Fig. 3). However, this paralogon has not been preserved in fish (Fig. 3), possibly because of the specific genome duplication and rearrangements that have occurred during the evolution in the fish lineage. To date, the existence of 26RFa/qrfp gene in coelacanth and lamprey is still unclear (Fig. 3).

Figure 3
Figure 3

Synteny analysis around 26RFa/QRFP gene loci. Orthologous or paralogous genes are linked by horizontal lines. The 26RFa/QRFP genes are shown white in black boxes. The nucleotide position of each gene on the chromosome is shown under each gene. The GenBank accession numbers of 26RFa/QRFP genes are as follows: human 26RFa/QRFP, AB109625.1; mouse 26RFa/Qrfp, AB109628.1; chicken 26RFa/QRFP, XM_001235088.2; Xenopus tropicalis 26RFa/qrfp, XM_004916673.1; zebrafish 26RFa/qrfp, XP_00133883; medaka 26RFa/qrfp, XP_004073955.1.

Citation: Journal of Molecular Endocrinology 52, 3; 10.1530/JME-13-0207

Comparative aspects of biological actions of 26RFa/QRFP in vertebrates

Mammals

The mRNAs encoding 26RFa/QRFP and its cognate receptor QRFPR are highly expressed in the dorsolateral and mediobasal hypothalamic areas of rodents (Chartrel et al. 2003, Takayasu et al. 2006, Bruzzone et al. 2007). These two areas are known to be involved in the regulation of energy homeostasis. In a similar way, in human, 26RFa/QRFP-producing cells are localized in the paraventricular and ventromedial nuclei of the hypothalamus (Bruzzone et al. 2006), which are also known to regulate food intake. Indeed, i.c.v. injection of 26RFa/QRFP has been demonstrated to stimulate food intake in rodents (Chartrel et al. 2003, Do Régo et al. 2006, Moriya et al. 2006, Takayasu et al. 2006, Primeaux et al. 2008, 2013, Lectez et al. 2009, Primeaux 2011).

In addition to its orexigenic effects, 26RFa/QRFP has been reported to exert a wide range of biological actions (Fig. 4). In an earlier report, i.v. administration of 26RFa/QRFP was found to increase plasma aldosterone levels in a dose-dependent manner in rats (Fukusumi et al. 2003). Recently, it has been reported that 26RFa/QRFP and QRFPR are present in the human and rat adrenal gland and that 26RFa/QRFP stimulates corticosteroid secretion by human adrenocortical cells (Ramanjaneya et al. 2013). In the rat pancreas, glucose-evoked insulin secretion is reduced by perfusion of 26RFa/QRFP (Egido et al. 2007). In the adipocyte cell line 3T3-L1, 26RFa/QRFP inhibits isoproterenol-induced lipolysis (Malumba et al. 2010). As 3T3-L1 cells express the QRFPR-encoding gene, it appears that 26RFa/QRFP may act in an autocrine/paracrine manner to regulate adipogenesis (Malumba et al. 2010). According to Alonzeau et al. (2013), 26RFa/QRFP is also expressed in human prostate cancer and stimulates the neuroendocrine differentiation and migration of cancer cells. Administration of 26RFa/QRFP in the brain increases plasma luteinizing hormone (LH) levels in both sexes in rat (Navarro et al. 2006, Patel et al. 2008) and stimulates prolactin and growth hormone secretion in male rhesus monkeys (Qaiser et al. 2012, Wahab et al. 2012). Intrathecally administered 26RFa/QRFP induces analgesic effects in rat under formalin and carrageenan tests (Yamamoto et al. 2008). Central injection of 26RFa/QRFP in mice causes a rise in blood pressure and heart rate (Takayasu et al. 2006). Mice deficient in the receptor for 26RFa/QRFP (Qrfpr) suffer from osteopenia (Baribault et al. 2006). This observation indicates that 26RFa/QRFP plays a major role in bone formation, via QRFPR that is expressed in bone (Baribault et al. 2006).

Figure 4
Figure 4

Demonstrated biological actions of 26RFa/QRFP–QRFPR system in vertebrates. Both 26RFa/QRFP and QRFPR have been found to exert a wide array of biological activities.

Citation: Journal of Molecular Endocrinology 52, 3; 10.1530/JME-13-0207

Non-mammalian vertebrates

In goldfish, quantitative RT-PCR analysis demonstrated high expression of 26RFa/qrfp mRNA in the hypothalamus, optic tectum–thalamus, and testis. The expression of 26RFa/qrfp mRNA in the hypothalamus is augmented at 4 days after food deprivation (Liu et al. 2009). In addition, serum LH levels are significantly increased at 1 h, but not at 3 and 6 h after i.p. injection of 26RFa/QRFP (Liu et al. 2009). As 26RFa/Qrfp has no effect on LH release from pituitary cells in primary culture, it is thought that, in fish, the peptide may stimulate the gonadotropic axis by acting exclusively at the hypothalamic level. These results suggest that 26RFa/Qrfp regulates energy homeostasis and the hypothalamic–pituitary–gonadal axis in fish, as also observed in mammals.

In birds, the expression of 26RFa/QRFP mRNA in the quail brain has been investigated in different brain regions, i.e., the cerebrum, diencephalon, mesencephalon, and cerebellum, by quantitative PCR analysis. A high level of expression of 26RFa/QRFP mRNA is present in the diencephalon, including the hypothalamus, while 26RFa/QRFP mRNA is almost undetectable in other brain regions (Ukena et al. 2010). In colchicine-treated birds (quail and chicken), 26RFa/QRFP-immunoreactive cell bodies were found only in the anterior hypothalamic nucleus in the diencephalon (Ukena et al. 2010). Furthermore, in situ hybridization has shown specific expression of 26RFa/QRFP mRNA in the anterior hypothalamic nucleus in the chick brain, and the distribution of 26RFa/QRFP mRNA-containing perikarya clearly matches with that of 26RFa/QRFP-immunoreactive neurons (Ukena et al. 2010). In the zebra finch, in situ hybridization analysis has revealed that expression of 26RFa/QRFP mRNA is localized to the anterior–medial hypothalamic area, the ventromedial nucleus of the hypothalamus, and the lateral hypothalamic area (Tobari et al. 2011). These neuroanatomical data suggest that, in birds, 26RFa/QRFP produced in the hypothalamus participates in the control of feeding behavior, as shown previously in rodents (Chartrel et al. 2006b, Do Régo et al. 2006, Moriya et al. 2006, Primeaux et al. 2008).

To assess the above speculation, the effect of central injection of 26RFa/QRFP has been surveyed in both broiler and layer chick lines. I.c.v. injection of 26RFa/QRFP stimulates feeding behavior in broiler chicks, but not in layer chicks (Ukena et al. 2010). It is likely that the different effects in these two chick lines can be explained by the following reports. It has been demonstrated that the effect of 26RFa/QRFP on feeding behavior in rodents differs according to the energy status and/or the species (Primeaux et al. 2013). Although 26RFa/QRFP hardly affects food intake in normally fed rats (Fukusumi et al. 2003, Kampe et al. 2006, Patel et al. 2008), at least under a low-fat diet (Primeaux et al. 2008), 26RFa/QRFP induces a marked orexigenic effect in mice and food-restricted rats (Chartrel et al. 2003, 2005, Do Régo et al. 2006, Moriya et al. 2006, Takayasu et al. 2006, Lectez et al. 2009). In addition, it has been demonstrated that 26RFa/QRFP selectively increases the intake of a high-fat diet in rats (Primeaux et al. 2008, 2013, Primeaux 2011). On the other hand, to determine the biologically active core of 26RFa/QRFP, the effect of a synthetic C-terminal octapeptide (26RFa-8; KGGFAFRFamide) of 26RFa/QRFP has been tested on feeding behavior of chicken. This C-terminal sequence is highly conserved from fish to mammals (Fig. 2). The synthetic C-terminal octapeptide, 26RFa-8, stimulates food intake in broiler chicks, but not in layer chicks, in much the same manner as the full-length peptide (Ukena et al. 2010). Consistent with this observation, a synthetic C-terminal heptapeptide of 26RFa/QRFP (26RFa2026; GGFSFRFamide) exerts an orexigenic effect in mice (Do Régo et al. 2006). In addition, 26RFa2026 evokes a significant increase in serum LH levels in female rats (Navarro et al. 2006). Taken together, it appears that the C-terminal region of 26RFa/QRFP is responsible for the biological activity of the peptide. In addition to the chick data, it has been reported that central injection of 26RFa/QRFP in free-feeding male zebra finches stimulates food intake for 24 h, without a change in body mass (Tobari et al. 2011). These results also indicate that 26RFa/QRFP exerts an orexigenic activity in various avian species.

Comparative aspects of QRFPR in vertebrates

Mammals

In humans, 26RFa/QRFP has been found to be an endogenous ligand for the orphan receptor, GPR103 (QRFPR), which is a class A GPCR (Fukusumi et al. 2003, Jiang et al. 2003). QRFPR shares relatively high sequence similarity with other RFamide receptors, notably those for NPFF, PrRP, kisspeptin, and GnIH, and to a lesser extent with the other peptidergic receptors for neuropeptide Y (NPY), galanin, orexin, and cholecystokinin (Lee et al. 2001, Jiang et al. 2003). Surprisingly, 26RFa/QRFP displays a moderate affinity for NPFF2 (NPFFR2, the receptor for NPFF) and a low affinity for NPFF1 (NPFFR1, the receptor for GnIH) (Gouardères et al. 2007). In addition, QRFPR possesses several characteristic features of class A GPCRs, such as i) a disulfide bridge between the two Cys (C) residues located in the first and second extracellular loops (EL1 and EL2), ii) the existence of an Asp (D) residue within the second transmembrane domain (TM2) that seems to play a pivotal role in G protein coupling, iii) a conserved Glu (E)-Arg (R) doublet sequence at the N-terminal end of the second intracellular loop (IL2), and iv) three conserved residues, i.e., Phe (F), Pro (P) and Asn (N), within TM6 and TM7, which are crucial for receptor activation (Fig. 5). QRFPs with 26 and 43 amino acid residues bind to QRFPR with high affinity (EC50=3.2 and 0.52 nM respectively) (Fukusumi et al. 2003, Jiang et al. 2003). It has also been demonstrated that 26- and 43-amino acid residue QRFPs inhibit cAMP formation with similar efficacy in QRFPR-transfected CHO cells (Fukusumi et al. 2003). Furthermore, 26RFa/QRFP markedly increases intracellular Ca2+ concentration ([Ca2+]i) in a pertussis toxin-independent manner. These results suggest that QRFPR is coupled to a Gi/0 and/or to a Gq protein (Fukusumi et al. 2003). The affinity and potency of the C-terminal heptapeptide 26RFa2026 (GGFSFRFamide) have been investigated and were found to be lower than those of 26RFa/QRFP. These data indicate that this heptapeptide is a relatively weak ligand for QRFPR (Fukusumi et al. 2003, Le Marec et al. 2011). Furthermore, it has been reported that 26RFa/QRFP enhances corticosteroid secretion in human adrenocortical cells by regulating key steroidogenic enzymes involving MAPK/PKC and Ca2+ signaling pathways via QRFPR (Ramanjaneya et al. 2013).

Figure 5
Figure 5

Alignment of the amino acid sequences of the G protein-coupled receptor for 26RFa/QRFP, QRFPR, in mammals (human and rat), birds (chicken and zebra finch), frog (Xenopus), and fish (zebrafish). Fully conserved amino acids are highlighted with green boxes and highly conserved amino acids with gray boxes. Putative transmembrane domains (TMD) are underlined. The disulfide bridge between the two Cys (C) residues located in the first and second extracellular loops is indicated by a line. The Asp (D) residue in TMD2 involved in G protein coupling, the conserved Glu (E)-Arg (R) residues in the second intracellular loop, and the conserved Phe (F), Pro (P), and Asn (N) residues in TMD6 and TMD7 are represented by colored letters. A hyphen has been inserted to obtain optimal homology. The GenBank accession numbers of these sequences are as follows: human QRFPR, NP_937822; rat QRFPR, NP_937842; chicken QRFPR, NP_001120642; zebra finch QRFPR, NP_001243137; Xenopus tropicalis QRFPR, NP_001072295; and zebrafish Qrfpr, XP_001920042.

Citation: Journal of Molecular Endocrinology 52, 3; 10.1530/JME-13-0207

In contrast to humans, who only have a single QRFPR-encoding gene, two isoforms of the receptor for 26RFa/QRFP have been characterized in rodents. These 26RFa/QRFP receptor isoforms have been designated as QRFPR1 and QRFPR2 in rat and mouse (Kampe et al. 2006, Takayasu et al. 2006); 26RFa/QRFP stimulates inositol trisphosphate in rat QRFPR1 and QRFPR2 with similar efficacy (Kampe et al. 2006) and binds to mouse QRFPR1 and QRFPR2 with similar affinity (Takayasu et al. 2006).

The distribution of QRFPR mRNA and its peptide binding sites have been studied by in situ hybridization and autoradiography respectively. In rat, Qrfpr mRNA-containing cells are notably expressed in the midbrain, the pons, and the medulla oblongata, while 26RFa/QRFP-binding sites are widely distributed throughout the brain and spinal cord (Bruzzone et al. 2007). These results suggest that 26RFa/QRFP can bind to a receptor(s) other than QRFPR. Indeed, it has been found by competition experiments that 26RFa/QRFP interacts with NPFF2, the cognate receptor for NPFF (Bruzzone et al. 2007). The widespread distribution of 26RFa/QRFP-binding sites suggests that 26RFa/QRFP exerts multiple functions in the brain and spinal cord that are mediated by at least two distinct receptors, QRFPR and NPFF2 (Bruzzone et al. 2007).

Non-mammalian vertebrates

In birds, the cDNAs encoding QRFPR have been characterized in the brain of chicken and zebra finch (Ukena et al. 2010, Tobari et al. 2011). The sequence of chicken QRFPR is highly similar to those of human and rat QRFPR (Fig. 5). The action of 26RFa/QRFP on chicken QRFPR has been studied by measuring [Ca2+]i in HEK293T cells that had been transiently transfected with chicken QRFPR. In these cells, 26RFa/QRFP increases [Ca2+]i in a dose-dependent manner, with an EC50 value of around 40 nM (Ukena et al. 2010). The mRNA of QRFPR is widely expressed in chicken and zebra finch brains and the highest concentration of mRNA is observed in the diencephalon (Ukena et al. 2010, Tobari et al. 2011). As the mRNA of QRFPR is expressed in the brain outside the diencephalon in chicken, as it is in rat (Bruzzone et al. 2007), 26RFa/QRFP may exert multiple functions in addition to regulating food intake (Ukena et al. 2010).

Synteny analysis has revealed the existence of species-specific paralogous genes of QRFPR in mouse, zebrafish and coelacanth (Fig. 6). These paralogous genes may have emerged along with the species-specific gene or genome duplications that occurred during the course of vertebrate evolution. Phylogenetic analysis data are consistent with synteny analysis (Fig. 7). Although there are homologous sequences to QRFPR in the genome database of Xenopus, zebrafish, coelacanth and lamprey (Figs 5 and 6), Qrfpr has been studied only in mammals and birds. Further characterization of QRFPR is thus needed to determine the functional significance of the 26RFa/QRFP–QRFPR system in other vertebrate phyla, such as reptilians, amphibians, and fish.

Figure 6
Figure 6

Synteny analysis around QRFPR gene loci. Orthologous or paralogous genes are linked by horizontal lines. The QRFPR genes are shown white in black boxes. The nucleotide position of each gene on the chromosome is shown under each gene. The GenBank accession numbers of QRFPR genes are as follows: human QRFPR, JF810892.1; mouse Qrfpr1, BC096610.1; chicken QRFPR, NM_001127170.1; Xenopus tropicalis qrfpr, NM_001078827.1; and medaka qrfpr, XP_004080459.1. Ensembl genome database accession numbers are as follows: mouse Qrfpr2, ENSMUSG00000029917; zebrafish qrfpr1, ENSDARG00000039349; zebrafish qrfpr2, ENSDARG00000068422; zebrafish qrfpr3, ENSDARG00000092652; coelacanth qrfpr, ENSLACG00000016226; and sea lamprey qrfpr, ENSPMAG00000005451. GENSCAN (http://genes.mit.edu/GENSCAN.html) was used to predict putative coelacanth Qrfpr3 precursor protein.

Citation: Journal of Molecular Endocrinology 52, 3; 10.1530/JME-13-0207

Figure 7
Figure 7

Phylogenetic analysis of QRFPR precursor proteins. Drosophila melanogaster peptide GPCR was used as an outgroup. NPY receptors are included in the phylogenetic tree as a reference group of vertebrate GPCR. Scale bar refers to a phylogenetic distance of 0.1 nucleotide substitutions per site. Numbers on the branches indicate bootstrap percentage following 1000 replications in constructing the tree. The GenBank accession numbers of the NPY1R genes are as follows: human NPY1R, NM_000909; mouse Npy1r, NM_010934; chicken NPY1R, NM_001031535; anole lizard NPY1R, XM_003221700; Xenopus laevisnpy1r, NM_001085879; zebrafish npy1r, NM_001102391; and Drosophila melanogaster peptide GPCR, AY217746.1.

Citation: Journal of Molecular Endocrinology 52, 3; 10.1530/JME-13-0207

Conclusions and future directions

The neuropeptide 26RFa/QRFP belongs to the most recently identified group of the RFamide peptide family and was first identified in the brain of the European green frog. Subsequently, the cDNAs encoding the 26RFa/QRFP precursors have been characterized in various animals, including goldfish, quail, chicken, zebra finch, mouse, rat, bovine, and humans, and these analyses have shown the existence of the 26RFa/QRFP-encoding gene in representative species of the vertebrate phylum. In mammals, 26RFa/QRFP has been found to be a high-affinity endogenous ligand for the previously identified orphan GPCR, GPR103 (QRFPR). In rodents and monkeys, 26RFa/QRFP exerts diverse biological actions, including regulation of food intake and energy homeostasis, hormone secretion, nociception, and bone formation. Recently, the mature sequences of 26RFa/QRFP have been identified by structural analysis in quail and zebra finch. In birds, as in mammals, 26RFa/QRFP-producing neurons are only located in the hypothalamus, while QRFPR is widely distributed throughout the brain. In birds, 26RFa/QRFP also exerts an orexigenic action, as it does in rodents, and a similar effect of 26RFa/QRFP has been suggested in fish, because of upregulation of 26RFa/qrfp mRNA by a negative energy state. Thus, the structure, distribution pattern, and biological actions of the 26RFa/QRFP–QRFPR system have been conserved across the vertebrate phylum, from fish to mammals. However, further studies are clearly required to fully elucidate the molecular evolution and functional significance of the 26RFa/QRFP–QRFPR pair in vertebrates. In particular, in vitro and in vivo studies on development, morphogenesis, and behavior in non-mammalian model organisms, such as Xenopus and zebrafish, should bring to light previously unknown physiological actions of the 26RFa/QRFP–QRFPR system. Recent studies have shown that a number of neuropeptide/GPCR pairs initially discovered in vertebrates/deuterostomes actually possess homologs in protostomes (Sherwood et al. 2006, Roch et al. 2011, Frooninckx et al. 2012, Grimmelikhuijzen & Hauser 2012, Mirabeau & Joly 2013). It would thus be interesting to look for the existence of 26RFa/QRFP and/or QRFPR orthologs in representative species of protostomes.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the review.

Funding

This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan (20770056 and 22687004 to K U and 18107002, 22132004, and 22227002 to K T), the Toray Science Foundation to K U, the Programme for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry to K U, and a France–Japan exchange program (INSERM-JSPS) to K T and H V.

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This paper is one of eight papers that form part of a thematic review section on the Molecular Evolution of GPCRs. The Guest Editor for this section was Hubert Vaudry, European Institute for Peptides Research, University of Rouen, France. He was not involved in the handling of this paper, on which he is listed as an author.

T Osugi is now at Suntory Foundation for Life Sciences, Bioorganic Research Institute, Osaka 618-8503, Japan

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    Phylogenetic tree of the RFamide peptide family in vertebrates. Studies over the past decade have demonstrated that the brain of vertebrates produces a variety of RFamide peptides. To date, five groups have been identified within this family: the neuropeptide FF (NPFF) group, the prolactin-releasing peptide (PrRP) group, the gonadotropin-inhibitory hormone (GnIH) group, the kisspeptin group, and the 26RFa/QRFP group.

  • View in gallery

    Alignments of the amino acid sequences of identified 26RFa/QRFP peptides (A) and their precursor proteins (B) deduced from mammalian (human, bovine, rat, and mouse), avian (chicken, quail, and zebra finch), amphibian (Xenopus), and fish (goldfish) cDNAs. The predicted signal peptide sequences are underlined with a dashed line. <E represents pyroglutamic acid. The positions of identified mature peptides in the precursor proteins are underlined with solid lines. The human and Xenopus 26RFa/QRFP precursors may also generate a nine-amino acid peptide, termed 9RFa (boxed). Fully conserved amino acids are highlighted with red boxes and highly conserved amino acids with gray boxes respectively. The Lys (K)-Arg (R) dibasic processing sites in birds and Xenopus, the single Arg (R) putative processing sites in mammals and fish, and the Gly (G) C-terminal amidation signals are shown in bold. Gaps marked by hyphens were inserted to optimize homology. The GenBank accession numbers of these sequences are as follows: human 26RFa/QRFP, NP_937823; bovine 26RFa/QRFP, NP_937865; rat 26RFa/QRFP, NP_937843; mouse 26RFa/QRFP, NP_906269; chicken 26RFa/QRFP, XP_001235089; quail 26RFa/QRFP, BAI81890; zebra finch 26RFa/QRFP, BAK32798; Xenopus tropicalis 26RFa/QRFP, XP_002936227; and goldfish 26RFa/Qrfp, ACI46681.

  • View in gallery

    Synteny analysis around 26RFa/QRFP gene loci. Orthologous or paralogous genes are linked by horizontal lines. The 26RFa/QRFP genes are shown white in black boxes. The nucleotide position of each gene on the chromosome is shown under each gene. The GenBank accession numbers of 26RFa/QRFP genes are as follows: human 26RFa/QRFP, AB109625.1; mouse 26RFa/Qrfp, AB109628.1; chicken 26RFa/QRFP, XM_001235088.2; Xenopus tropicalis 26RFa/qrfp, XM_004916673.1; zebrafish 26RFa/qrfp, XP_00133883; medaka 26RFa/qrfp, XP_004073955.1.

  • View in gallery

    Demonstrated biological actions of 26RFa/QRFP–QRFPR system in vertebrates. Both 26RFa/QRFP and QRFPR have been found to exert a wide array of biological activities.

  • View in gallery

    Alignment of the amino acid sequences of the G protein-coupled receptor for 26RFa/QRFP, QRFPR, in mammals (human and rat), birds (chicken and zebra finch), frog (Xenopus), and fish (zebrafish). Fully conserved amino acids are highlighted with green boxes and highly conserved amino acids with gray boxes. Putative transmembrane domains (TMD) are underlined. The disulfide bridge between the two Cys (C) residues located in the first and second extracellular loops is indicated by a line. The Asp (D) residue in TMD2 involved in G protein coupling, the conserved Glu (E)-Arg (R) residues in the second intracellular loop, and the conserved Phe (F), Pro (P), and Asn (N) residues in TMD6 and TMD7 are represented by colored letters. A hyphen has been inserted to obtain optimal homology. The GenBank accession numbers of these sequences are as follows: human QRFPR, NP_937822; rat QRFPR, NP_937842; chicken QRFPR, NP_001120642; zebra finch QRFPR, NP_001243137; Xenopus tropicalis QRFPR, NP_001072295; and zebrafish Qrfpr, XP_001920042.

  • View in gallery

    Synteny analysis around QRFPR gene loci. Orthologous or paralogous genes are linked by horizontal lines. The QRFPR genes are shown white in black boxes. The nucleotide position of each gene on the chromosome is shown under each gene. The GenBank accession numbers of QRFPR genes are as follows: human QRFPR, JF810892.1; mouse Qrfpr1, BC096610.1; chicken QRFPR, NM_001127170.1; Xenopus tropicalis qrfpr, NM_001078827.1; and medaka qrfpr, XP_004080459.1. Ensembl genome database accession numbers are as follows: mouse Qrfpr2, ENSMUSG00000029917; zebrafish qrfpr1, ENSDARG00000039349; zebrafish qrfpr2, ENSDARG00000068422; zebrafish qrfpr3, ENSDARG00000092652; coelacanth qrfpr, ENSLACG00000016226; and sea lamprey qrfpr, ENSPMAG00000005451. GENSCAN (http://genes.mit.edu/GENSCAN.html) was used to predict putative coelacanth Qrfpr3 precursor protein.

  • View in gallery

    Phylogenetic analysis of QRFPR precursor proteins. Drosophila melanogaster peptide GPCR was used as an outgroup. NPY receptors are included in the phylogenetic tree as a reference group of vertebrate GPCR. Scale bar refers to a phylogenetic distance of 0.1 nucleotide substitutions per site. Numbers on the branches indicate bootstrap percentage following 1000 replications in constructing the tree. The GenBank accession numbers of the NPY1R genes are as follows: human NPY1R, NM_000909; mouse Npy1r, NM_010934; chicken NPY1R, NM_001031535; anole lizard NPY1R, XM_003221700; Xenopus laevisnpy1r, NM_001085879; zebrafish npy1r, NM_001102391; and Drosophila melanogaster peptide GPCR, AY217746.1.

References

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