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Review 1
2
Molecular evolution and function of 26RFa/QRFP and its cognate receptor 3
4
Kazuyoshi Ukenaa, Tomohiro Osugi
b,d, Jérôme Leprince
c, Hubert Vaudry
c, 5
Kazuyoshi Tsutsuib,* 6
7
aSection of Behavioral Sciences, Graduate School of Integrated Arts and Sciences, 8
Hiroshima University, Higashi-Hiroshima, 739-8521, Japan 9
bLaboratory of Integrative Brain Sciences, Department of Biology, Waseda 10
University, Tokyo, 162-8480, Japan 11
cNormandy University, INSERM U982, Institute for Research and Innovation in 12
Biomedicine (IRIB), 76821 Mont-Saint-Aignan, France 13
Current address: dSuntory Foundation for Life Sciences, Bioorganic Research 14
Institute, Osaka, 618-8503, Japan 15
16
Keywords: 26RFa/QRFP, food intake, G protein-coupled receptor, hypothalamus, 17
neuropeptide 18
19
*Corresponding author: 20
Kazuyoshi Tsutsui, Ph. D., Professor 21
Laboratory of Integrative Brain Sciences, Department of Biology, Waseda 22
University, Center for Medical Life Science of Waseda University 23
2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan 24
Tel.: +81-3-5369-7311 25
Fax: +81-3-3355-0316 26
E-mail: [email protected]
Page 1 of 36 Accepted Preprint first posted on 14 February 2014 as Manuscript JME-13-0207
Copyright © 2014 by the Society for Endocrinology.
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ABSTRACT 28
Neuropeptides possessing the Arg-Phe-NH2 (RFamide) motif at their C-termini 29
(designated RFamide peptides) have been characterized in a variety of animals. 30
Among these, neuropeptide 26RFa (also termed QRFP) is the latest member of the 31
RFamide peptide family to be discovered in the hypothalamus of vertebrates. 32
26RFa/QRFP is a 26-amino acid residue peptide that was originally identified 33
from the frog brain. It has been shown to exert orexigenic activity in mammals and 34
to be a ligand of the previously identified orphan G protein-coupled receptor, 35
GPR103, viz., QRFPR. The cDNAs encoding 26RFa/QRFP and QRFPR have now 36
been characterized in representative species of mammals, birds, and fish. 37
Functional studies have shown that, in mammals, the 26RFa/QRFP-QRFPR 38
system may regulate various functions, including food intake, energy homeostasis, 39
bone formation, pituitary hormone secretion, steroidogenesis, nociceptive 40
transmission, and blood pressure. Several biological actions have also been 41
reported in birds and fish. This review summarizes the current state of 42
identification, localization, and understanding of the functions of 26RFa/QRFP 43
and its cognate receptor, QRFPR, in vertebrates. 44
45
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Introduction 46
Neuropeptides that possess the Arg-Phe-NH2 motif at their C-termini (i.e., 47
RFamide peptides) have been characterized both in invertebrates and vertebrates. 48
The first RFamide peptide to be identified was the cardioexcitatory peptide 49
Phe-Met-Arg-Phe-NH2 (FMRFamide), which was isolated from the ganglia of the 50
Venus clam Macrocallista nimbosa (Price & Greenberg 1977). Since then, a 51
number of RFamide peptides have been identified in invertebrates, where these 52
peptides seem to act as neurotransmitters and neuromodulators (for review, see 53
Walker et al. 2009). 54
A number of immunohistochemical studies that used antisera against 55
FMRFamide suggested that the nervous system of vertebrates also contained 56
neuropeptides immunologically related to FMRFamide (Raffa 1988, Vallarino et 57
al. 1991, 1994, 1995, Rastogi et al. 2001). In fact, several neuropeptides harboring 58
the RFamide sequence at their C-terminal end have been characterized in the brain 59
of various vertebrates. In the past, the existence of 5 groups within the RFamide 60
peptide family has been recognized in vertebrates, viz., the neuropeptide FF 61
(NPFF) group, the prolactin-releasing peptide (PrRP) group, the 62
gonadotropin-inhibitory hormone (GnIH) group, the kisspeptin group, and the 63
26RFa/QRFP group (for reviews, see Bruzzone et al. 2006, Chartrel et al. 2011, 64
Leprince et al. 2013, Osugi et al. 2006, Tsutsui 2009, Tsutsui & Ukena 2006, 65
Tsutsui et al. 2010a,b, Ukena & Tsutsui 2005) (Fig. 1). These RFamide peptides 66
have been shown to exert important neuroendocrine, behavioral, sensory, and 67
autonomic functions (for reviews, see Chartrel et al. 2002, 2006a, Tsutsui & 68
Ukena 2006, Ukena & Tsutsui 2005). Among these vertebrate RFamide peptides, 69
NPFF is well-documented as a morphine modulatory peptide (Panula et al. 1999). 70
Page 3 of 36
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In addition, GnIH and kisspeptin appear to play key roles in the regulation of the 71
reproductive axis (Tsutsui et al. 2010b). This review summarizes the current state 72
of knowledge on the molecular evolution and functions of 26RFa/QRFP, the latest 73
member of the RFamide peptide family to be discovered in vertebrates, and of its 74
cognate receptor, QRFPR. This review also indicates future directions in this 75
research field. 76
77
Unity and diversity of the structure of 26RFa/QRFP in vertebrates 78
The 26-amino acid residue RFamide peptide 26FRa/QRFP was identified for 79
the first time in the brain of an amphibian species (Chartrel et al. 2003). An 80
antibody against the RFamide motif was used to screen, by radioimmunoassay, 81
peptide fractions purified from a brain extract of the European green frog (Rana 82
esculenta). After high-performance liquid chromatography (HPLC) purification, 83
the sequence of the isolated substance was analyzed by mass spectrometry MS/MS 84
fragmentation; it turned out to be a 26-amino acid peptide possessing the RFamide 85
motif at its C-terminus, viz., VGTALGSLAEELNGYNRKKGGFSFRFamide. 86
This neuropeptide had not been reported in any animals previously and was 87
designated 26RFa (Fig. 2A) (Chartrel et al. 2003). 88
The amino acid sequence of frog 26RFa was employed to identify the cDNA 89
encoding the counterpart of 26RFa in rat and humans (Chartrel et al. 2003). 90
Concurrently, two other research groups independently identified 26RFa/QRFP 91
precursors using a bioinformatic approach in the rat, mouse, bovine, and human 92
genomes and paired 26RFa/QRFP with a previously identified orphan G 93
protein-coupled receptor, GPR103, also known as AQ27 or SP9155 (Fukusumi et 94
al. 2003, Jiang et al. 2003) (Fig. 2B). GPR103 has thus been re-named QRFPR by 95
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the HUGO Gene Nomenclature Committee (http://www.genenames.org/). The 96
mature 43-amino acid residue RFamide peptide was identified from the culture 97
medium of CHO cells that expressed the human peptide precursor (Fukusumi et al. 98
2003). Because the N-terminal amino acid was pyroglutamic acid, this RFamide 99
peptide was also named pyroglutamylated RFamide peptide (QRFP) (Fukusumi et 100
al. 2003). Subsequently, the cDNAs encoding the 26RFa/QRFP precursors have 101
been characterized in goldfish (Liu et al. 2009), quail (Ukena et al. 2010), chicken 102
(Ukena et al. 2010), and zebra finch (Tobari et al. 2011) (Fig. 2B). Although the 103
26RFa/QRFP cDNA has not been characterized in the European green frog, the 104
corresponding sequence in the African clawed frog (Xenopus tropicalis) is present 105
in the database (Fig. 2B). Furthermore, homologous sequences have been listed in 106
the genome database of reptilian (lizard) and fish (stickleback, medaka, fugu, and 107
zebrafish) species (Liu et al. 2009). These data have revealed the existence of the 108
26RFa/QRFP-encoding gene in representative species of the whole vertebrate 109
phyla, including fish, amphibians, reptilians, birds, and mammals (Chartrel et al. 110
2011, Ukena et al. 2011). 111
As there are several monobasic processing sites in the 26RFa/QRFP precursor 112
protein, alternative cleavage may yield various N-terminally elongated forms of 113
26RFa/QRFP (Chartrel et al. 2006b, 2011). HPLC analysis combined with 114
radioimmunoassays indicated the existence of both 26- and 43-amino acid residue 115
RFamide peptide-like immunoreactivities in the hypothalamus and spinal cord of 116
humans (Bruzzone et al. 2006). Indeed, an N-terminally extended peptide of 43 117
residues, called 43RFa or QRFP, has been characterized in rat brain extracts, as 118
well as in PC12 cells and the culture medium of CHO cells that express the human 119
precursor, as described above (Bruzzone et al. 2006, Fukusumi et al. 2003, 120
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Takayasu et al. 2006) (Fig. 2A). The human and Xenopus 26RFa/QRFP precursors 121
may also generate a 9-amino acid peptide, termed 9RFa, located upstream of 122
26RFa/QRFP (Fig. 2B). However, 9RFa has not been detected in tissue extracts to 123
date. Structure–activity relationship studies have revealed that the synthetic 124
C-terminal heptapeptide (26RFa20–26; GGFSFRFamide) is responsible for the 125
biological activity of 26RFa/QRFP (Le Marec et al. 2011, Neveu et al. 2012). A 126
reverse pharmacological study has demonstrated that 26RFa/QRFP is a natural 127
ligand for the previously identified orphan receptor, GPR103, viz., QRFPR, as 128
described below (Fukusumi et al. 2003, Jiang et al. 2003, Takayasu et al. 2006). 129
As reported above, the mature forms of 26RFa/QRFP have been identified in 130
the brains of amphibians and mammals (Bruzzone et al. 2006, Chartrel et al. 2003, 131
Takayasu et al. 2006) but, until recently, the existence of 26RFa/QRFP had not 132
been investigated in birds. Only the GnIH group among the RFamide peptide 133
family had been found in avian at the time we started this study (for reviews, see 134
Tsutsui 2009, Tsutsui & Ukena 2006, Tsutsui et al. 2010a,b, Ukena & Tsutsui 135
2005). We have therefore looked for 26RFa/QRFP in the avian brain and found the 136
presence of a gene encoding the 26RFa/QRFP precursor in chicken after searching 137
the genomic database. Subsequently, the cDNA of the 26RFa/QRFP precursor was 138
sequenced in the quail hypothalamus (Ukena et al. 2010). The quail precursor 139
protein demonstrates 88% overall similarity with the chicken sequence, 47% with 140
the corresponding human sequence, and 40% with the rat sequence (Ukena et al. 141
2011). A Lys-Arg dibasic cleavage site is present in the C-terminal region of the 142
quail and chicken precursor sequences, but not in that of mammalian sequences 143
(Fig. 2B). This indicates that the mature peptide consists of 27 amino acid residues 144
in quail and chicken, unlike the 26 residues in the amphibian 26RFa /QRFP 145
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sequence (Chartrel et al. 2003). In fact, MS analysis combined with 146
immunoaffinity purification has revealed that the 27-amino acid sequence 147
corresponds to the mature form of the peptide in the quail hypothalamus, 148
indicating that the peptide is actually produced from the precursor in the 149
hypothalamus (Ukena et al. 2010; Fig. 2A). More recently, a 26RFa/QRFP 150
orthologue, consisting of 25 amino acids, and the related cDNA have been 151
characterized in the brain of zebra finch (Tobari et al. 2011; Fig. 2). 152
Synteny analysis of the 26RFa/QRFP gene revealed that the chromosomal 153
region encompassing the 26RFa/QRFP gene is highly conserved from amphibians 154
to human. Indeed, all these regions contain paralogs of several other genes and 155
thus clearly constitute a paralogon (Fig. 3). However, this paralogon has not been 156
preserved in fish (Fig. 3), possibly because of the specific genome duplication and 157
rearrangements that have occurred during the evolution in the fish lineage. To 158
date, the existence of 26RFa/QRFP gene in coelacanth and lamprey is still unclear 159
(Fig. 3). 160
161
Comparative aspects of biological actions of 26RFa/QRFP in vertebrates 162
Mammals 163
The mRNAs encoding 26RFa/QRFP and its cognate receptor QRFPR are 164
highly expressed in the dorsolateral and mediobasal hypothalamic areas of rodents 165
(Bruzzone et al. 2007, Chartrel et al. 2003, Takayasu et al. 2006). These two areas 166
are known to be involved in the regulation of energy homeostasis. In a similar 167
way, in human, 26RFa/QRFP-producing cells are localized in the paraventricular 168
and ventromedial nuclei of the hypothalamus (Bruzzone et al. 2006) which are 169
also known to regulate food intake. Indeed, intracerebroventricular (i.c.v.) 170
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injection of 26RFa/QRFP has been demonstrated to stimulate food intake in 171
rodents (Chartrel et al. 2003, Do Régo et al. 2006, Lectez et al. 2009, Moriya et 172
al. 2006, Primeaux 2011, Primeaux et al. 2008, 2013, Takayasu et al. 2006). 173
In addition to its orexigenic effects, 26RFa/QRFP has been reported to exert a 174
wide range of biological actions (Fig. 4). In an earlier report, intravenous 175
administration of 26RFa/QRFP in rats was found to increase plasma aldosterone 176
levels in a dose-dependent manner (Fukusumi et al. 2003). Recently, it has been 177
reported that 26RFa/QRFP and QRFPR are present in the human and rat adrenal 178
gland and that 26RFa/QRFP stimulates corticosteroid secretion by human 179
adrenocortical cells (Ramanjaneya et al. 2013). In the rat pancreas, 180
glucose-evoked insulin secretion is reduced by perfusion of 26RFa/QRFP (Egido 181
et al. 2007). In the adipocyte cell line 3T3-L1, 26RFa/QRFP inhibits 182
isoproterenol-induced lipolysis (Malumba et al. 2010). Since 3T3-L1 cells express 183
the QRFPR-encoding gene, it appears that 26RFa/QRFP may act in an 184
autocrine/paracrine manner to regulate adipogenesis (Malumba et al. 2010). 185
26RFa/QRFP is also expressed in human prostate cancer and stimulates the 186
neuroendocrine differentiation and migration of cancer cells (Alonzeau et al. 187
2013). Administration of 26RFa/QRFP in the brain increases plasma luteinizing 188
hormone (LH) levels in both sexes in rat (Navarro et al. 2006, Patel et al. 2008) 189
and stimulates prolactin and growth hormone secretion in male rhesus monkeys 190
(Qaiser et al. 2012, Wahab et al. 2012). Intrathecally administered 26RFa/QRFP 191
induces analgesic effects in rat under formalin and carrageenan tests (Yamamoto et 192
al. 2008). Central injection of 26RFa/QRFP in mice causes a rise in blood pressure 193
and heart rate (Takayasu et al. 2006). Mice deficient in the receptor for 194
26RFa/QRFP (QRFPR) suffer from osteopenia (Baribault et al. 2006). This 195
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observation indicates that 26RFa/QRFP plays a major role in bone formation, via 196
QRFPR that is expressed in bone (Baribault et al. 2006). 197
198
Non-mammalian vertebrates 199
In goldfish, quantitative RT-PCR analysis demonstrated high expression of 200
26RFa/QRFP mRNA in the hypothalamus, optic tectum–thalamus, and testis. The 201
expression of 26RFa/QRFP mRNA in the hypothalamus is augmented at 4 days 202
after food deprivation (Liu et al. 2009). In addition, serum LH levels are 203
significantly increased at 1 h, but not at 3 and 6 h after intraperitoneal injection of 204
26RFa/QRFP (Liu et al. 2009). As 26RFa/QRFP has no effect on LH release from 205
pituitary cells in primary culture, it is thought that, in fish, the peptide may 206
stimulate the gonadotropic axis by acting exclusively at the hypothalamic level. 207
These results suggest that 26RFa/QRFP regulates energy homeostasis and the 208
hypothalamic–pituitary–gonadal (HPG) axis in fish, as also observed in mammals. 209
In birds, the expression of 26RFa/QRFP mRNA in the quail brain has been 210
investigated in different brain regions, i.e., the cerebrum, diencephalon, 211
mesencephalon, and cerebellum, by quantitative PCR analysis. A high level of 212
expression of 26RFa/QRFP mRNA is present in the diencephalon, including the 213
hypothalamus, while 26RFa/QRFP mRNA is almost undetectable in other brain 214
regions (Ukena et al. 2010). 26RFa/QRFP-immunoreactive cell bodies were found 215
only in the anterior hypothalamic nucleus in the diencephalon of 216
colchicine-treated birds (quail and chicken) (Ukena et al. 2010). Furthermore, in 217
situ hybridization has shown specific expression of 26RFa/QRFP mRNA in the 218
anterior hypothalamic nucleus in the chick brain, and the distribution of 219
26RFa/QRFP mRNA-containing perikarya clearly matches with that of 220
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26RFa/QRFP-immunoreactive neurons (Ukena et al. 2010). In the zebra finch, in 221
situ hybridization analysis has revealed that expression of 26RFa/QRFP mRNA is 222
localized to the anterior-medial hypothalamic area, the ventromedial nucleus of 223
the hypothalamus, and the lateral hypothalamic area (Tobari et al. 2011). These 224
neuroanatomical data suggest that, in birds, 26RFa/QRFP produced in the 225
hypothalamus participates in the control of feeding behavior, as previously shown 226
in rodents (Chartrel et al. 2006b, Do Régo et al. 2006, Moriya et al. 2006, 227
Primeaux et al. 2008). 228
To assess the above speculation, the effect of central injection of 26RFa/QRFP 229
has been surveyed in both broiler and layer chick lines. I.c.v. injection of 230
26RFa/QRFP stimulates feeding behavior in broiler chicks, but not in layer chicks 231
(Ukena et al. 2010). It is likely that the different effects in these two chick lines 232
can be explained by the following reports. It has been demonstrated that the effect 233
of 26RFa/QRFP on feeding behavior in rodents differs according to the energy 234
status and/or the species (Primeaux et al. 2013). Although 26RFa/QRFP hardly 235
effects food intake in normally fed rats (Fukusumi et al. 2003, Kampe et al. 2006, 236
Patel et al. 2008), at least under a low-fat diet (Primeaux et al. 2008), 237
26RFa/QRFP induces a marked orexigenic effect in mice and food-restricted rats 238
(Chartrel et al. 2003,2005, Do Régo et al. 2006, Lectez et al. 2009, Moriya et al. 239
2006, Takayasu et al. 2006). In addition, it has been demonstrated that 240
26RFa/QRFP selectively increases the intake of a high fat diet in rats (Primeaux 241
2011, Primeaux et al. 2008, 2013). On the other hand, to determine the 242
biologically active core of 26RFa/QRFP, the effect of a synthetic C-terminal 243
octapeptide (26RFa-8; KGGFAFRFamide) of 26RFa/QRFP has been tested on 244
chicken feeding behavior. This C-terminal sequence is highly conserved from fish 245
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to mammals (Fig. 2). 26RFa-8 stimulates food intake in broiler chicks, but not in 246
layer chicks, in much the same manner as the full length peptide (Ukena et al. 247
2010). Consistent with this observation, a synthetic C-terminal heptapeptide of 248
26RFa/QRFP (26RFa20–26; GGFSFRFamide) exerts an orexigenic effect in mice 249
(Do Régo et al. 2006). In addition, 26RFa20–26 evokes a significant increase in 250
serum LH levels in female rats (Navarro et al. 2006). Taken together, it appears 251
that the C-terminal region of 26RFa/QRFP is responsible for the biological 252
activity of the peptide. In addition to the chick data, it has been reported that 253
central injection of 26RFa/QRFP in free-feeding male zebra finches stimulates 254
food intake for 24 h, without a change in body mass (Tobari et al. 2011). These 255
results also indicate that 26RFa/QRFP exerts an orexigenic activity in various 256
avian species. 257
258
Comparative aspects of QRFPR in vertebrates 259
Mammals 260
In humans, 26RFa/QRFP has been found to be an endogenous ligand of the 261
orphan receptor GPR103 (now re-named QRFPR), which is a class A G 262
protein-coupled receptor (GPCR) (Fukusumi et al. 2003, Jiang et al. 2003). 263
QRFPR shares relatively high sequence similarity with other RFamide receptors, 264
notably those for NPFF, PrRP, kisspeptin, and GnIH, and to a lesser extent with 265
the other peptidergic receptors for neuropeptide Y (NPY), galanin, orexin, and 266
cholecystokinin (CCK) (Jiang et al. 2003, Lee et al. 2001). Surprisingly, 267
26RFa/QRFP displays a moderate affinity for NPFF-2 (the receptor for NPFF) and 268
a low affinity for NPFF-1 (the receptor for GnIH) (Gouardères et al. 2007). In 269
addition, QRFPR possesses several characteristic features of class A GPCRs, such 270
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as (i) a disulfide bridge between the two Cys (C) residues located in the first and 271
second extracellular loops (EL1 and EL2), (ii) the existence of an Asp (D) residue 272
within the second transmembrane domain (TM2) that seems to play a pivotal role 273
in G protein coupling, (iii) a conserved Glu (E)–Arg (R) doublet sequence at the 274
N-terminal end of the second intracellular loop (IL2), and (iv) three conserved 275
residues, i.e. Phe (F), Pro (P) and Asn (N), within TM6 and TM7, which are 276
crucial for receptor activation (Fig. 5). 26- and 43-amino acid residue QRFPs bind 277
to QRFPR with high affinity (EC50 = 3.2 and 0.52 nM, respectively) (Fukusumi et 278
al. 2003, Jiang et al. 2003). It has also been demonstrated that 26- and 43-amino 279
acid residue QRFPs inhibit cAMP formation with similar efficacy in 280
QRFPR-transfected CHO cells (Fukusumi et al. 2003). Furthermore, 26RFa/QRFP 281
markedly increases intracellular Ca2+ concentration ([Ca
2+]i) in a pertussis toxin 282
(PTX)-independent manner. These results suggest that QRFPR is coupled to a Gi/0 283
and/or to a Gq protein (Fukusumi et al. 2003). The affinity and potency of the 284
C-terminal heptapeptide 26RFa20–26 (GGFSFRFamide) have been investigated and 285
were found to be lower than those of 26RFa/QRFP. These data indicate that this 286
heptapeptide is a relatively weak ligand for QRFPR (Fukusumi et al. 2003, Le 287
Marec et al. 2011). Furthermore, it has been reported that 26RFa/QRFP enhances 288
corticosteroid secretion in human adrenocortical cells by regulating key 289
steroidgogenic enzymes involving MAPK/PKC and Ca2+ signaling pathways via 290
QRFPR (Ramanjaneya et al. 2013). 291
In contrast to humans, who only have a single QRFPR-encoding gene, two 292
isoforms of the receptor for 26RFa/QRFP have been characterized in rodents. 293
These 26RFa/QRFP receptor isoforms have been designated QRFPR1 and 294
QRFPR2 in rat and mouse (Kampe et al. 2006, Takayasu et al. 2006). 295
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26RFa/QRFP stimulates inositol triphosphate in rat QRFPR1 and QRFPR2 with 296
similar efficacy (Kampe et al. 2006) and binds mouse QRFPR1 and QRFPR2 with 297
similar affinity (Takayasu et al. 2006). 298
The distribution of QRFPR mRNA and its peptide binding sites have been 299
studied by in situ hybridization and autoradiography, respectively. In rat, QRFPR 300
mRNA-containing cells are notably expressed in the midbrain, the pons, and the 301
medulla oblongata, while 26RFa/QRFP binding sites are widely distributed 302
throughout the brain and spinal cord (Bruzzone et al. 2007). These results suggest 303
that 26RFa/QRFP can bind to a receptor(s) other than QRFPR. Indeed, it has been 304
found by competition experiments that 26RFa/QRFP interacts with NPFF-2, the 305
cognate receptor for NPFF (Bruzzone et al. 2007). The widespread distribution of 306
26RFa/QRFP binding sites suggests that 26RFa/QRFP exerts multiple functions in 307
the brain and spinal cord that are mediated by at least 2 distinct receptors, QRFPR 308
and NPFF-2 (Bruzzone et al. 2007). 309
310
Non-mammalian vertebrates 311
In birds, the cDNAs encoding QRFPR have been characterized in the brain of 312
chicken and zebra finch (Tobari et al. 2011, Ukena et al. 2010). The sequence of 313
chicken QRFPR is highly similar to those of human and rat QRFPR (Fig. 5). The 314
action of 26RFa/QRFP on chicken QRFPR has been studied by measuring [Ca2+]i 315
in HEK293T cells that had been transiently transfected with chicken QRFPR. In 316
these cells, 26RFa/QRFP increases [Ca2+]i in a dose-dependent manner, with an 317
EC50 value of around 40 nM (Ukena et al. 2010). The mRNA of QRFPR is widely 318
expressed in chicken and zebra finch brains and the highest concentration of 319
mRNA is observed in the diencephalon (Tobari et al. 2011, Ukena et al. 2010). As 320
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the mRNA of QRFPR is expressed in the brain outside the diencephalon in 321
chicken, as it is in rat (Bruzzone et al. 2007), 26RFa/QRFP may exert multiple 322
functions in addition to regulating food intake (Ukena et al. 2010). 323
Synteny analysis has revealed the existence of species specific paralogous 324
genes of QRFPR in mouse, zebrafish and coelacanth (Fig. 6). These paralogous 325
genes may have emerged along with the species specific gene or genome 326
duplications that occurred during the course of vertebrate evolution. Phylogenetic 327
analysis data are consistent with synteny analysis (Fig. 7). Although there are 328
homologous sequences to QRFPR in the genome database of Xenopus, zebrafish, 329
coelacanth and lamprey (Figs. 5 and 6), QRFPR has been studied only in 330
mammals and birds. Further characterization of QRFPR is thus needed to 331
determine the functional significance of the 26RFa/QRFP-QRFPR system in other 332
vertebrate phyla, such as reptilians, amphibians, and fish. 333
334
Conclusions and future directions 335
26RFa/QRFP belongs to the most recently identified group of the RFamide 336
peptide family. 26RFa/QRFP was firstly identified in the brain of the European 337
green frog. Subsequently, the cDNAs encoding the 26RFa/QRFP precursors have 338
been charaterized in various animals, including goldfish, quail, chicken, zebra 339
finch, mouse, rat, bovine, and humans and these analyses have shown the 340
existence of the 26RFa/QRFP-encoding gene in representative species of the 341
vertebrate phylum. 26RFa/QRFP has been found to be a high-affinity endogenous 342
ligand of the previously identified orphan G protein-coupled receptor GPR103 343
(QRFPR) in mammals. In rodents and monkeys, 26RFa/QRFP exerts diverse 344
biological actions, including regulation of food intake and energy homeostasis, 345
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hormone secretion, nociception, and bone formation. Recently, the mature 346
sequences of 26RFa/QRFP have been identified by structural analysis in quail and 347
zebra finch. In birds, as in mammals, 26RFa/QRFP-producing neurons are only 348
located in the hypothalamus while QRFPR is widely distributed throughout the 349
brain. 26RFa/QRFP also exerts an orexigenic action in birds, as it does in rodents, 350
and a similar effect of 26RFa/QRFP has been suggested in fish, because of 351
up-regulation of 26RFa/QRFP mRNA by a negative energy state. Thus, the 352
structure, distribution pattern, and biological actions of the 26RFa/QRFP-QRFPR 353
system have been conserved across the vertebrate phylum, from fish to mammals. 354
However, further studies are clearly required to fully elucidate the molecular 355
evolution and functional significance of the 26RFa/QRFP-QRFPR pair in 356
vertebrates. In particular, in vitro and in vivo studies on development, 357
morphogenesis, and behavior in non-mammalian model organisms, such as 358
Xenopus and zebrafish, should bring to light previously unknown physiological 359
actions of 26RFa/QRFP-QRFPR. Recent studies have shown that a number of 360
neuropeptide/GPCR pairs initially discovered in vertebrates/deuterostomes 361
actually possess homologs in protostomes (Sherwood et al. 2006, Roch et al. 362
2011, Frooninckx et al. 2012, Grimmelikhuijzen & Hauser 2012; Mirabeau & 363
Joly, 2013). It would thus be interesting to look for the existence of 26RFa/QRFP 364
and/or QRFPR orthologs in representative species of protostomes.365
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Acknowledgments 366
This work was supported by Grants-in-Aid for Scientific Research from the 367
Ministry of Education, Science, and Culture, Japan (20770056 and 22687004 to 368
K.U. and 18107002, 22132004, and 22227002 to K.T.), the Toray Science 369
Foundation to K.U., the Programme for Promotion of Basic and Applied 370
Researches for Innovations in Bio-oriented Industry to K.U., and a France-Japan 371
exchange program (INSERM-JSPS) to K.T. and H.V. 372
373
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Cellular Biology 26 709–717. 383
Bruzzone F, Lectez B, Tollemer H, Leprince J, Dujardin C, Rachidi W, Chatenet D, 384
Baroncini M, Beauvillain JC, Vallarino M et al. 2006 Anatomical 385
distribution and biochemical characterization of the novel RFamide peptide 386
26RFa in the human hypothalamus and spinal cord. Journal of 387
Neurochemistry 99 616–627. 388
Bruzzone F, Lectez B, Alexandre D, Jégou S, Mounien L, Tollemer H, Chatenet D, 389
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sites and GPR103 mRNA in the central nervous system of the rat. Journal of 391
Comparative Neurology 503 573–591. 392
Chartrel N, Bruzzone F, Dujardin C, Leprince J, Tollemer H, Anouar Y, Vallarino 393
M, Costentin J & Vaudry H 2005 Identification of 26RFa from frog brain: a 394
novel hypothalamic neuropeptide with orexigenic activity in mammals. 395
Annals of the New York Academy of Sciences 1040 80–83. 396
Chartrel N, Dujardin C, Leprince J, Desrues L, Tonon MC, Cellier E, Cosette P, 397
Jouenne T, Simonnet G & Vaudry H 2002 Isolation, characterization, and 398
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18
distribution of a novel neuropeptide, Rana RFamide (R-RFa), in the brain of 399
the European green frog Rana esculenta . Journal of Comparative 400
Neurology 448 111–127. 401
Chartrel N, Dujardin C, Anouar Y, Leprince J, Decker A, Clerens S, Do Régo JC, 402
Vandesande F, Llorens-Cortes C, Costentin J et al. 2003 Identification of 403
26RFa, a hypothalamic neuropeptide of the RFamide peptide family with 404
orexigenic activity. Proceedings of the National Academy of Sciences of the 405
USA 100 15247–15252. 406
Chartrel N, Tsutsui K, Costentin J & Vaudry H 2006a The RFamide-related 407
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Ségalas-Milazzo I, Guilhaudis L, Cosette P, Jouenne T et al. 2006b Structure 411
and functions of the novel hypothalamic RFamide neuropeptides R-RFa and 412
26RFa in vertebrates. Peptides 27 1110–1120. 413
Chartrel N, Alonzeau J, Alexandre D, Jeandel L, Alvear-Perez R, Leprince J, 414
Boutin J, Vaudry H, Anouar Y & Llorens-Cortes C 2011 The RFamide 415
neuropeptide 26RFa and its role in the control of neuroendocrine functions. 416
Frontiers in Neuroendocrinology 32 387–397. 417
Do Régo JC, Leprince J, Chartrel N, Vaudry H & Costentin J 2006 Behavioral 418
effects of 26RFamide and related peptides. Peptides 27 2715–2721. 419
Egido EM, Hernández R, Leprince J, Chartrel N, Vaudry H, Marco J & Silvestre 420
RA 2007 26RFa, a novel orexigenic neuropeptide, inhibits insulin secretion 421
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Zajac JM 2007 Functional differences between NPFF1 and NPFF2 receptor 431
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Kampe J, Wiedmer P, Pfluger PT, Castaneda TR, Burget L, Mondala H, Kerr J, 440
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relationships of a series of analogues of the RFamide-related peptide 26RFa. 446
Journal of Medicinal Chemistry 54 4806–4814. 447
Lectez B, Jeandel L, El-Yamani FZ, Arthaud S, Alexandre D, Mardargent A, Jégou 448
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2342–2350. 452
Lee DK, Nguyen T, Lynch KR, Cheng R, Vanti WB, Arkhitko O, Lewis T, Evans 453
JF, George SR & O'Dowd BF 2001 Discovery and mapping of ten novel G 454
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Tena-Sempere M, Tsutsui K & Vaudry H 2013 26RFa. In Handbook of 457
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cloning and functional characterization of the first non-mammalian 26RFa/ 460
QRFP orthologue in goldfish, Carassius auratus. Molecular and Cellular 461
Endocrinology 303 82–90. 462
Malumba M, Jossart C, Granata R, Gallo D, Escher E, Ghigo E, Servant MJ, 463
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in bilaterians. Proceedins of the National Academy of Sciences of the USA 467
110 E2028–E2037. 468
Moriya R, Sano H, Umeda T, Ito M, Takahashi Y, Matsuda M, Ishihara A, 469
Kanatani A & Iwaasa H 2006 RFamide peptide QRFP43 causes obesity with 470
hyperphagia and reduced thermogenesis in mice. Endocrinology 147 471
2916–2922. 472
Navarro VM, Fernández-Fernández R, Nogueiras R, Vigo E, Tovar S, Chartrel N, 473
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Le Marec O, Leprince J, Aguilar E, Pinilla L et al. 2006 Novel role of 26RFa, 474
a hypothalamic RFamide orexigenic peptide, as putative regulator of the 475
gonadotropic axis. Journal of Physiology 573 237–249. 476
Neveu C, Lefranc B, Tasseau O, Do Rego JC, Bourmaud A, Chan P, Bauchat P, Le 477
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molecular weight, stable, potent and long-lasting GPR103 479
aza-β3-pseudopeptide agonist. Journal of Medicinal Chemistry 55 480
7516–7524. 481
Osugi T, Ukena K, Sower SA, Kawauchi H & Tsutsui K 2006 Evolutionary origin 482
and divergence of PQRFamide peptides and LPXRFamide peptides in the 483
RFamide peptide family. Insights from novel lamprey RFamide peptides. 484
FEBS Journal 273 1731–1743. 485
Panula P, Kalso E, Nieminen M, Kontinen VK, Brandt A & Pertovaara A 1999 486
Neuropeptide FF and modulation of pain. Brain Research 848 191–196. 487
Patel SR, Murphy KG, Thompson EL, Patterson M, Curtis AE, Ghatei MA & 488
Bloom SR 2008 Pyroglutamylated RFamide peptide 43 stimulates the 489
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administration of the RFamide peptides, QRFP-26 and QRFP-43, increases 498
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high fat food intake in rats. Peptides 29 1994–2000. 499
Primeaux SD, Barnes MJ & Braymer HD 2013 Hypothalamic QRFP: regulation 500
of food intake and fat selection. Hormone and Metabolic Research 45 501
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& Shahab M 2012 Study of the role of novel RF-amide neuropeptides in 504
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aldosterone production via PKC and T-type calcium channel-mediated 511
pathways in human adrenocortical cells: evidence for a novel role of 512
GPR103. American Journal of Physiology - Endocrinology and Metabolism 513
305 E1049–E1058. 514
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Iela L 2001 FMRFamide in the amphibian brain: a comprehensive survey. 516
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insights from Ciona genomics. Comparative Biochemistry and Physiology A 521
144 254–271. 522
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H, Kawasawa YI, Ikeda Y, Sakakibara I et al. 2006 A neuropeptide ligand of 524
the G protein-coupled receptor GPR103 regulates feeding, behavioral 525
arousal, and blood pressure in mice. Proceedings of the National Academy of 526
Sciences of the USA 103 7438–7443. 527
Tobari Y, Iijima N, Tsunekawa K, Osugi T, Haraguchi S, Ubuka T, Ukena K, 528
Okanoya K, Tsutsui K & Ozawa H 2011 Identification, localisation and 529
functional implication of 26RFa orthologue peptide in the brain of zebra 530
finch (Taeniopygia guttata). Journal of Neuroendocrinology 23 791–803. 531
Tsutsui K 2009 A new key neurohormone controlling reproduction, gonadotropin- 532
inhibitory hormone (GnIH): biosynthesis, mode of action and functional 533
significance. Progress in Neurobiology 88 76–88. 534
Tsutsui K & Ukena K 2006 Hypothalamic LPXRF-amide peptides in vertebrates: 535
identification, localization and hypophysiotropic activity. Peptides 27 536
1121–1129. 537
Tsutsui K, Bentley GE, Bedecarrats G, Osugi T, Ubuka T & Kriegsfeld LJ 2010a 538
Gonadotropin-inhibitory hormone (GnIH) and its control of central and 539
peripheral reproductive function. Frontiers in Neuroendocrinology 31 540
284–295. 541
Tsutsui K, Bentley GE, Kriegsfeld LJ, Osugi T, Seong JY & Vaudry H 2010b 542
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and kisspeptin: new key neuropeptides controlling reproduction. Journal of 544
Neuroendocrinology 22 716–727. 545
Ukena K & Tsutsui K 2005 A new member of the hypothalamic RF-amide peptide 546
family, LPXRF-amide peptides: structure, localization, and function. Mass 547
Spectrometry Reviews 24 469–486. 548
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24
Ukena K, Tachibana T, Iwakoshi-Ukena E, Saito Y, Minakata H, Kawaguchi R, 549
Osugi T, Tobari Y, Leprince J, Vaudry H et al. 2010 Identification, 550
localization, and function of a novel avian hypothalamic neuropeptide, 551
26RFa, and its cognate receptor, Gprotein-coupled receptor-103. 552
Endocrinology 151 2255–2264. 553
Ukena K, Vaudry H, Leprince J & Tsutsui K 2011 Molecular evolution and 554
functional characterization of the orexigenic peptide 26RFa and its receptor 555
in vertebrates. Cell and Tissue Research 343 475–481. 556
Vallarino M, Salsotto-Cattaneo MT, Feuilloley M & Vaudry H 1991 Distribution 557
of FMRFamide-like immunoreactivity in the brain of the elasmobranch fish 558
Scyliorhinus canicula. Peptides 12 1321–1328. 559
Vallarino M, Feuilloley M, D’Aniello B, Rastogi RK & Vaudry H 1994 560
Distribution of FMRFamide-like immunoreactivity in the brain of the lizard 561
Podarcis sicula. Peptides 15 1057–1065. 562
Vallarino M, Feuilloley M, Thoumas JL, Demorgny R, Masini MA & Vaudry H 563
1995 Distribution of FMRFamide-like immunoreactivity in the brain of the 564
lungfish Protopterus annectens. Peptides 16 1187–1196. 565
Wahab F, Salahuddin H, Anees M, Leprince J, Vaudry H, Tena-Sempere M & 566
Shahab M 2012 Study of the effect of 26RFand 43RF-amides on 567
testosterone and prolactin secretion in the adult male rhesus monkey 568
(Macaca mulatta). Peptides 36 23–28. 569
Walker RJ, Papaioannou S & Holden-Dye L 2009 A review of FMRFamide- and 570
RFamide-like peptides in metazoa. Invertebrate Neuroscience 9 111–153. 571
Yamamoto T, Wada T & Miyazaki R 2008 Analgesic effects of intrathecally 572
administered 26RFa, an intrinsic agonist for GPR103, on formalin test and 573
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25
carrageenan test in rats. Neuroscience 157 214–222. 574
575
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26
Figure Legends 576
Fig. 1. Phylogenetic tree of the RFamide peptide family in vertebrates. Studies 577
over the past decade have demonstrated that the brain of vertebrates produces a 578
variety of RFamide peptides. To date, 5 groups have been identified within this 579
family: the neuropeptide FF (NPFF) group, the prolactin-releasing peptide (PrRP) 580
group, the gonadotropin-inhibitory hormone (GnIH) group, the kisspeptin group, 581
and the 26RFa/QRFP group. 582
583
Fig. 2. Alignments of the amino acid sequences of identified 26RFa/QRFP 584
peptides (A) and their precursor proteins (B) deduced from mammalian (human, 585
bovine, rat, and mouse), avian (chicken, quail, and zebra finch), amphibian 586
(Xenopus), and fish (goldfish) cDNAs. The predicted signal peptide sequences are 587
underlined with a dashed line. <E represents pyroglutamic acid. The positions of 588
identified mature peptides in the precursor proteins are underlined with solid lines. 589
The human and Xenopus 26RFa/QRFP precursors may also generate a 9-amino 590
acid peptide, termed 9RFa (boxed). Fully conserved amino acids are highlighted 591
with red boxes and highly conserved amino acids with gray boxes, respectively. 592
The Lys (K)–Arg (R) dibasic processing sites in birds and Xenopus, the single Arg 593
(R) putative processing sites in mammals and fish, and the Gly (G) C-terminal 594
amidation signals are shown in bold. Gaps marked by hyphens were inserted to 595
optimize homology. The GenBank accession numbers of these sequences are as 596
follows: human 26RFa/QRFP, NP_937823; bovine 26RFa/QRFP, NP_937865; rat 597
26RFa/QRFP, NP_937843; mouse 26RFa/QRFP, NP_906269; chicken 598
26RFa/QRFP, XP_001235089; quail 26RFa/QRFP, BAI81890; zebra finch 599
Page 26 of 36
27
26RFa/QRFP, BAK32798; Xenopus tropicalis 26RFa/QRFP, XP_002936227; and 600
goldfish 26RFa/QRFP, ACI46681. 601
602
Fig. 3. Synteny analysis around 26RFa/QRFP gene loci. Orthologous or 603
paralogous genes are linked by horizontal lines. The 26RFa/QRFP genes are 604
shown white in black boxes. The nucleotide position of each gene on the 605
chromosome is shown under each gene. Genbank accession numbers of 606
26RFa/QRFP genes are as follows: human 26RFa/QRFP, AB109625.1; mouse 607
26RFa/QRFP, AB109628.1; chicken 26RFa/QRFP, XM_001235088.2; Xenopus 608
tropicalis 26RFa/QRFP, XM_004916673.1; zebrafish 26RFa/QRFP, 609
XP_00133883; medaka 26RFa/QRFP, XP_004073955.1. 610
611
Fig. 4. Demonstrated biological actions of 26RFa/QRFP-QRFPR in vertebrates. 612
26RFa/QRFP and QRFPR have been found to exert a wide array of biological 613
activities. 614
615
Fig. 5. Alignment of the amino acid sequences of the G-protein coupled receptor 616
for 26RFa/QRFP, QRFPR, in mammals (human and rat), birds (chicken and zebra 617
finch), frog (Xenopus), and fish (zebrafish). Fully conserved amino acids are 618
highlighted with green boxes and highly conserved amino acids with gray boxes. 619
Putative transmembrane domains (TMD) are underlined. The disulfide bridge 620
between the 2 Cys (C) residues located in the first and second extracellular loops 621
is indicated by a line. The Asp (D) residue in TMD2 involved in G protein 622
coupling, the conserved Glu (E)–Arg (R) in the second intracellular loop, and the 623
conserved Phe (F), Pro (P), and Asn (N) residues in TMD6 and TMD7 are 624
Page 27 of 36
28
represented by colored letters. A hyphen has been inserted to obtain optimal 625
homology. The GenBank accession numbers of these sequences are as follows: 626
human QRFPR, NP_937822; rat QRFPR, NP_937842; chicken QRFPR, 627
NP_001120642; zebra finch QRFPR, NP_001243137; Xenopus tropicalis QRFPR, 628
NP_001072295; and zebrafish QRFPR, XP_001920042. 629
630
Fig. 6. Synteny analysis around QRFPR gene loci. Orthologous or paralogous 631
genes are linked by horizontal lines. The QRFPR genes are shown white in black 632
boxes. The nucleotide position of each gene on the chromosome is shown under 633
each gene. Genbank accession numbers of QRFPR genes are as follows: human 634
QRFPR, JF810892.1; mouse QRFRP1, BC096610.1; chicken QRFPR, 635
NM_001127170.1; Xenopus tropicalis QRFPR, NM_001078827.1; medaka 636
QRFPR, XP_004080459.1. Ensembl genome database accession numbers are as 637
follows: mouse QRFPR2, ENSMUSG00000029917; zebrafish QRFPR1, 638
ENSDARG00000039349; zebrafish QRFPR2, ENSDARG00000068422; 639
zebrafish QRFPR3, ENSDARG00000092652; coelacanth QRFPR, 640
ENSLACG00000016226; sea lamprey QRFPR, ENSPMAG00000005451. 641
Genscan (http://genes.mit.edu/GENSCAN.html) was used to predict putative 642
coelacanth QRFPR3 precursor protein. 643
644
Fig. 7. Phylogenetic analysis of QRFPR precursor proteins. Drosophila 645
melanogaster peptide GPCR was used as an out group. NPY receptors are 646
included in the phylogenetic tree as a reference group of vertebrate GPCR. Scale 647
bar refers to a phylogenetic distance of 0.1 nucleotide substitutions per site. 648
Numbers on the branches indicate bootstrap percentage following 1000 649
Page 28 of 36
29
replications in constructing the tree. Genbank accession numbers of the NPY1-R 650
genes are as follows: human NPY1-R, NM_000909; mouse NPY1-R, 651
NM_010934; chicken NPY1-R, NM_001031535; anole lizard NPY1-R, 652
XM_003221700; Xenopus laevis NPY1-R, NM_001085879; zebrafish NPY1-R, 653
NM_001102391; Drosophila melanogaster peptide GPCR, AY217746.1. 654
Page 29 of 36
0.1
LampreyGnIH
FuguNPFF
Zebrafish NPFF
Human NPFFRat NPFF
Mouse NPFF
Tilapia PrRP
Goldfish PrRP
Rat PrRP
Human PrRP
Ovine PrRP
Bovine PrRP
Hagfish NPFF
Lamprey NPFF
Newt LPXRFa
Bovine RFRP
Human RFRPMonkey RFRP
Rat RFRP
Mouse RFRP
Quail GnIHChicken GnIH
Starling GnIHSparrow GnIH
Zebra finch GnIH
Frog GRP
Zebrafish LPXRFa
Goldfish LPXRFa
X. tropicalis 26RFa/QRFP
Bovine 26RFa/QRFP
Ovine 26RFa/QRFP
Human 26RFa/QRFP
Rat 26RFa/QRFP
Mouse 26RFa/QRFP
Zebra finch26RFa/QRFP
Quail26RFa/QRFP
Chicken26RFa/QRFP
Medaka26RFa/QRFPGoldfish
26RFa/QRFP
Zebrafish26RFa/QRFP
Human Kiss1
Rat Kiss1
Grass puffer Kiss2
Zebrafish Kiss-2
Grass lizard Kiss-2
X. laevis Kiss-2
X. tropicalisKiss-2
26RFa/QRFP group
Kisspeptingroup
GnIH group
NPFF group
PrRP group
Fig. 1
Page 30 of 36
mature peptide (26RFa/QRFP)
signal peptide
Fig. 2
putative peptide (9RFa)
Human ------MVR-PYPLIYFLFLPLGACFPLLDRREPTDA----------MGGLGAGER 39Bovine -------MRSPYSLPYLLFLPLGACFPVLDTEEPVDA----------VGGTGREMS 39Rat -------MR-CLCSWLCLLLPLSACFPLLDRRGPTD-----------IGDIGARMS 37Mouse -------MR-GFRPLLSLLLPLSACFPLLDRRGPTD-----------IGDIGARMN 37Chicken -------MRAPYSLTCLILLSLGACCPPHEHHEPRRP------------EDVLEPC 37Quail -------MRASYSLSCLILLSLAACCPPHQHHQPRHP------------EDVLEPR 37Zebra finch -------MRAPYSLSCLFLLSLGACIPPGERWEPAEPGEGAALGGGWQRAAEGRGP 49Xenopus -------MS------VLVLLSLGYTFALQDSREQSDP----------WERIRLLRM 33Goldfish MKFQVIHLSSTLQTTILFLLVLLVQPPRGLMLPHHPMVYLPMLDNPEWEAALLQLQ 56
Human WADLAM-----------GPR-PHSVWGSSRWLRASQPQALLVIARGLQTSGREHAG 83Bovine WMDPAR-----------GRPFP---WGSPGWPRAPYPHALLVTAKELRASGKARAG 81Rat WVQLTE-----------GHT-PRSVQ-------SPRPQALLVVAKEQQASRREHTG 74Mouse WAQLAE-----------GHP-PNSVQ-------NPQPQALLVVAREQQASHREHTG 74Chicken WRG------------AVAEAVGPCGWAAPMRRRSEELGALLGIARVLRGYGQQHSV 81Quail WRG--------------AEAVGPCVWAAPMRRRSEELGTLLGIARGLRGYGQQHSV 78Zebra finch GWRAGARRRRSEELDELLSITRGPGWRAGARRRRSEELEALLSIARELRGYSAAGA 105Xenopus MADGEE-----------NSAGALWYPLAPRQRKSTDPASLFSVAKELQGFGKERAG 78Goldfish ASLGAAGGKAVVEIQPWALIQEPGPEELLERVKAELGWGQKGIVKGQKREELGWKP 112
Human CRFRF--GR-QDEGSEATGFLPAAGEKTSGPLGNLAEELNGYSRKKGGFSFRFGRR 136Bovine FQLRL--GR-QDDGSEATGLLLGEAEKVGGLLGTLAEELNGYSRKKGGFSFRFGRR 134Rat --FRL--GR-QDSGSEATGFLPTDSEKASGPLGTLAEELSSYSRRKGGFSFRFGR- 124Mouse --FRL--GR-QDGSSEAAGFLPADSEKASGPLGTLAEELSSYSRRKGGFSFRFGR- 124Chicken GPR----GR-PEG-SE--------KRGGGGTLGDLAEELNGYGRKKGGFAFRFGR- 122Quail GTR----GR-QEG-SE--------KRGGGGTLGDLAEELNGYSRKKGGFAFRFGR- 120Zebra finch -------GQRPGG-SGGPGALPVVGEKRSGTLGNLAEEINGYNRRKGGFTFRFGR- 152Xenopus FRFRF--GRQEEGNEFEDFEQQDEEKRGGTALGSLAEELNGYNRKKGGFSFRFGRR 132Goldfish MGTFPDNLIVDVPYPQGGEVEEEGGEKQNEALTSIAGGLQAFNRQKGGFGFRFGKK 168
Human <EDEGSEATGFLPAAGEKTSGPLGNLAEELNGYSRKKGGFSFRFamideRat <EDSGSEATGFLPTDSEKASGPLGTLAEELSSYSRRKGGFSFRFamideQuail GGGGTLGDLAEELNGYSRKKGGFAFRFamideZebra finch SGTLGNLAEEINGYNRRKGGFTFRFamideGreen frog VGTALGSLAEELNGYNRKKGGFSFRFamide
A
B
Page 31 of 36
Human
26RFA/QRFP133.77 MB
GPR107132.81 MB
FAM78A134.13 MB
Mouse
31.67 MB
31.00 MB
Ch. 17
6.65 MB
5.82 MB
Zebrafish
19.82 MB
34.08 MB
1.65 MB
1.51 MB
1.98 MB
1.12 MB
X. tropicalisScaffold
EXOSC2133.56 MB 6.56 MB31.52 MB
75.25 MB
6.76 MB31.90 MB
75.10 MB
Gpr107 GPR107 gpr107gpr107
exosc2EXOSC2Exosc2
exosc2
26Rfa/Qrfp 26RFA/QRFP 26rfa/qrfp 26rfa/qrfp
fam78aFAM78AFam78a
fam78a
Ch. 11
26rfa/qrfp1.98 MB
fam78a10.04 MB
Ch. 12
12.38 MB
exosc2
Ch. 5
CoelacanthScaffold GL476397
81.22 KB
gpr107
JH126633.1
1.92 MB
fam78aPOMT1134.37 MB
Pomt132.23 MB
POMT16.22 MB
pomt11.04 MB
pomt12.09 MB
pomt152.80 MB
Ch. 9
pomt128.21 MB
1.28 MB
exosc2
591.58 KB
fam78a
dnm1a441.68 KB
DNM1130.97 MB 5.02 MB
DNM132.31 MB
Dnm1 dnm1591.58 KB
dnm1a1.60 MB 2.01 MB
dnm1
5.86 MB 213.38 KB
ncs1NCS1132.93 MB
NCS131.25 MB
Ncs1 ncs11.93 MB
GL483090
2.91 KB
ncs1
133.05 MBHMCN2
31.31 MBHmcn2 hmcn2
242.57 KB
Ch. 19
Ch. 5
246.83 KB
rgs3b116.21 MB
RGS3
JH126677.1
JH128425.1
1.37 MB
RGS3
GL172827.1
GL476397
Medaka
GL476397
73.01 MB
rgs3
11.66 MB
dnm1a
Ch. 12
Ch. 9
Ch. 2
ChickenLampreyScaffold
Fig. 3
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Fig. 4
Page 33 of 36
TMD1
TMD2 TMD3
TMD4
TMD5
TMD6 TMD7
Fig. 5
Human MQALNITPEQFSRLLRDHNLTREQFIALYRLRPLVYTPELPGRAKLALVLTGVLIFALALFGNALVFYVV 70Rat MQALNITAEQFSRLLSAHNLTREQFIHRYGLRPLVYTPELPARAKVAFALAGALIFALALFGNSLVIYVV 70Chicken MRSLNITPEQFAQLLRDNNVTREQFIALYGLQPLVYVPELPGRTKVAFVLICVLIFALTLFGNCLVLYVV 70Zebra finch MRSLNITPEQFAQLLRDNNVTREQFIALYGLQPLVYIPELPRRTKVAFVLICVLIFALALFGNCLVLYVV 70Xenopus MQSLNITPEQFARLLQENNVTREQFIELYQLQPLVYIPELPFRTKIAFVTICVLIFVLALFGNSLVLYVV 70Zebrafish MGDKKITPEVLEQLLQFYNLTRQEFIETYQIEPLVYIPELPAGAKTTFVIVYTVIFLLALVGNSVVVYIV 70
Human TRSKAMRTVTNIFICSLALSDLLITFFCIPVTMLQNISDNWLGGAFICKMVPFVQSTAVVTEILTMTCIA 140Rat TRSKAMRTVTNIFICSLALSDLLIAFFCIPVTMLQNISDKWLGGAFICKMVPFVQSTAVVTEILTMTCIA 140Chicken TRSRAMRTVTNIFICSLALSDLLIAFFCVPFTMLQNISSEWLGGAFACKMVPFVQSTAIVTEILTMTCIA 140Zebra finch TRSKAMRTVTNIFICSLALSDLFIAFFCVPFTMLQNISSNWLGGAFACKMVPFVQSTAIVTEILTMTCIA 140Xenopus TRSKAMRTVTNIFICSLALSDLLIAFFCIPFTMLQNISSNWLGGAFACKMVPFVQSTAIVTEILTMTCIA 140Zebrafish LRKRGIQTATNIFICSLAVSDLLISFFCIPFTLLQNISSEWFGGVLVCKTVPFVQTTAVVTGILTMTCIA 140
Human VERHQGLVHPFKMKWQYTNRRAFTMLGVVWLVAVIVGSPMWHVQQLEIKYDFLYEKEHICCLEEWTSPVH 210Rat VERHQGLVHPFKMKWQYTTRRAFTILGVVWLAAIIVGSPMWHVQRLEIKYDFLYEKEHICCLEEWASPVH 210Chicken VERHQGIVHPLKMKWQYTNKRAFTMLGIVWLLAIIVGSPMWHVQRLEVKYDFLYEKVHICCLEEWASPTY 210Zebra finch VERHQGIVHPLKMKWQYTNKRAFTMLGIVWLLALIVGSPMWYVQRLEVKYDFLYEKVHVCCLEEWASPIY 210Xenopus VERHQGIVHPLKMKWQYTNRRAFTMLGIVWLIAAVVGIPMWHAQRLEVKYDFLYEKQYVCCLEAWNSQVH 210Zebrafish VERYQGIVHPLKIKRQCTPQRAYRMLGVVWIAAMMVGSPMLFVQQLEVKYDFLYDNHHVCCQERWRSSAH 210
Human QKIYTTFILVILFLLPLMVMLILYSKIGYELWIKKRVGDGSVLRTIHGKEMSKIARKKKRAVIMMVTVVA 280Rat QRIYSTFILVILFLLPLVVMLVLYSKIGYELWIKKRVGDSSALQTIHGKEMSKIARKKKRAVIMMVTVVA 280Chicken QKIYTTFILVILFLFPLILMLFLYTKIGYELWIKKRVGDASVLQTIHGSEMSKISRKKKRAIVMMVTVVF 280Zebra finch QKIYTTFILVILFLLPLMLMLFLYTKIGYELWIKKRVGDASVLQTIHGNEMSKISRKKKRAIVMMVTVVF 280Xenopus QKIYTTFILVILFLLPLTVMLLLYSKIGYELWIKKRVGDASVLQTIHGSEMSKIARKKKRAIIMMITVVV 280Zebrafish RKRYATFILVFLFLLPLAAMLILYTRIGIELWIRKQVGDSSVLNAMNQREVSKIARKKRRAIKMMVTIVV 280
Human LFAVCWAPFHVVHMMIEYSNFEKEYDDVTIKMIFAIVQIIGFSNSICNPIVYAFMNENFKKNVLSAVCYC 350Rat LFAACWAPFHVVHMMVEYSNFEKEYDDVTIKMVFAVAQTIGFFNSICNPFVYAFMNENFKKNFLSAVCYC 350Chicken LFAVCWAPFHIIHMMMEYSNFEKQYDDVTIKMIFAIVQIIGFFNSICNPIVYAFMNENFKKNFLSAICFC 350Zebra finch LFAVCWAPFHVIHMMMEYSNFEKEYDDVTIKMIFAIVQIIGFFNSICNPIVYAFMNENFKKNFLSALCFC 350Xenopus LFAVCWAPFHVVHMMIEYSNFENEYDDVTIKIIFAIVQIIGFFNSICNPIVYAFMNENFKKNFLSALCFC 350Zebrafish LFTVCWAPFHTVHILFEYSYLNKKYDDVTVNMIIAVAQAIGFSNSFNNPIIYAFMNENFQKNCMSTLSVC 350
Human IVNKTFSPAQRHGNSGITMMRKKAKFSLRENP-VEETKGEAFSDGNIEVKLCEQTEEKKKLKRHLALFRS 419Rat IVKESSSPARKPGNSGISMMQKRAKLSRPQRP-VEETKGDTFSDASIDVKLCEQPREKRQLKRQLAFFSS 419Chicken VVKENASPTRQLGNSGITMRRQKAGDSQRAPTDSDEARREAFSDGNIEVKFCDQPSSKRNLKRHLTLFSS 420Zebra finch IMKDSTSPGRQLGNSGITMRRQKPSASQRDLMHSDEGRREAFSDGNIEVKFCDQPASKRNLKRHLVLFSS 420Xenopus FLRDPSSPSRRPGNSGITLIQQKSSSSRRENT-CEDTRREAFSEGNIEVKFFDQPVSK---KRHLHLFTS 416Zebrafish IRRSNLT 357
Human ELAENSPLDSGH 431Rat ELSENSTFGSGHEL 433Chicken ELPAHSASAQ 430Zebra finch ELTVHSAVGNGQ 432Xenopus ELTVHS 422
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HumanCh. 4
122.25 MB
QRFPR
MouseCh. 3
ChickenCh. 4
36.18 MB
Qrfpr1 QRFPR
MouseCh. 6
53.49 MB65.38MB
Qrfpr2
X. tropicalisScaffold
GL173236.1
qrfpr41.85 KB
139.23 KB
tnip3
178.79 KB
ndnf
267.77 KB
prdm5
ZebrafishCh. 13
CoelacanthScaffold
JH127078.1
LampreyScaffold
122.05 MB
TNIP3
121.96 MB
NDNF
121.61 MB
PRDM5
65.59 MB
Tnip3
65.67 MB
Ndnf
65.78 MB
Prdm5
53.54 MB
TNIP3
53.61 MB
NDNF
PRDM553.64 MB
122.72 MBEXOSC9 Exosc9
36.55 MB 53.36 MBEXOSC9
891.70 KBanxa5
GL173104.1
53.40 MBANXA5
36.45 MBAnxa5ANXA5
122.59 MB
927.56 KBexosc9
ZebrafishCh. 12
ZebrafishCh. 21
qrfpr1 qrfpr2 qrfpr330.44 MB 49.12 MB 14.35 MB
164.25 MB
NPY1R
166.28 MB
CPE
1.01 MBqrfpr1
CoelacanthScaffold
JH126583.1
CoelacanthScaffold
JH126808.1
qrfpr2 qrfpr32.62 MB 1.49 MB
0.75 MB
ndnf
0.83 MB
tnip3
0.40 MBprdm5
1.20 MBexosc9
1.06 MBanxa5
2.71 MBanxa4
69.75 KBqrfpr
GL476500
ndnf200.08 KB
40.19 KB
prdm5
GL476494
GL478833
32.85 KBexosc9
ZebrafishCh. 23
2.08 MB
ndnf
1.95 MB
prdm5
Ch. 1
17.83 MBexosc9
cpe19.62 MB
npy1r20.03 MB
Medaka
9.76 MBqrfpr
30.56 MB
Ch. 19
Ch. 4
ndnf
30.59 MBprdm5
anxa523.42 MB
Ch. 1
4.81 MB
exosc9
23.66 MB
cpe
22.82 MB
NPY1R1.89 MB
GL172746.1
npy1r
23.34 MB
CPE2.47 MB
cpe
21.43 MB
anxa5
16.33 MB
anxa11a50.25 MB
anxa11b
Fig. 6
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QRFPR group
NPYR group
Drosophila GPCR
Coelacanth QRFPR3
Zebrafish NPY1-R
X. laevis NPY1-R
Anole lizard NPY1-R
Chicken NPY1-R
Human NPY1-R
Mouse NPY1-R
Lamprey QRFPR
Zebrafish QRFPR3
Coelacanth QRFPR2
Zebrafish QRFPR1
Zebrafish QRFPR2
Medaka QRFPR
Coelacanth QRFPR1
X. tropicalis QRFPR
Zebra finch QRFPR
Anser QRFPR
Chicken QRFPR
Mouse QRFPR2
Human QRFPR
Bovine QRFPR
Rat QRFPR2
Rat QRFPR1
Fig. 70.1
1000
636
931
1000
1000
1000
1000
998
547
1000
994
1000
1000
993
750
985
1000
937
990
1000
550
994
Mouse QRFPR1
Page 36 of 36
