The Arg–Phe‐amide peptide 26RFa/glutamine RF‐amide peptide and its receptor: IUPHAR Review 24

Abstract

The RFamide neuropeptide 26RFa was first isolated from the brain of the European green frog on the basis of cross‐reactivity with antibodies raised against bovine neuropeptide FF (NPFF). 26RFa and its N‐terminally extended form glutamine RF‐amide peptide (QRFP) have been identified as cognate ligands of the former orphan receptor GPR103, now renamed glutamine RF‐amide peptide receptor (QRFP receptor). The 26RFa/QRFP precursor has been characterized in various mammalian and non‐mammalian species. In the brain of mammals, including humans, 26RFa/QRFP mRNA is almost exclusively expressed in hypothalamic nuclei. The 26RFa/QRFP transcript is also present in various organs especially in endocrine glands. While humans express only one QRFP receptor, two isoforms are present in rodents. The QRFP receptor genes are widely expressed in the CNS and in peripheral tissues, notably in bone, heart, kidney, pancreas and testis. Structure–activity relationship studies have led to the identification of low MW peptidergic agonists and antagonists of QRFP receptor. Concurrently, several selective non‐peptidic antagonists have been designed from high‐throughput screening hit optimization. Consistent with the widespread distribution of QRFP receptor mRNA and 26RFa binding sites, 26RFa/QRFP exerts a large range of biological activities, notably in the control of energy homeostasis, bone formation and nociception that are mediated by QRFP receptor or NPFF2. The present report reviews the current knowledge concerning the 26RFa/QRFP‐QRFP receptor system and discusses the potential use of selective QRFP receptor ligands for therapeutic applications.

Abbreviations

[Ca²⁺]_i

intracellular calcium concentration

AD

Alzheimer's disease

ARC

arcuate nucleus

CD

circular dichroism

CRH

corticotropin‐releasing hormone

DPC

dodecylphosphocholine

EST

expressed sequence tag

FLP

FMRFamide‐like peptides

FSH

follicle‐stimulating hormone

GLP‐1

glucagon‐like peptide 1

GnIH

gonadotropin‐inhibitory hormone

GnRH

gonadotropin‐releasing hormone

GSIS

glucose‐stimulated insulin secretion

GTEx

genotype tissue expression

Gα_i

Gα inhibitory protein

Gα_s

Gα stimulatory protein

h26RFa

human 26RFa

IP‐1

inositol‐1‐phosphate

LH

luteinizing hormone

LHyp

lateral hypothalamus

NPAF

neuropeptide AF

NPFF

neuropeptide FF

NPFF1

NPFF receptor type‐1

NPY

neuropeptide Y

POMC

pro‐opiomelanocortin

PRL

prolactin

PrRP

prolactin‐releasing peptide

PVN

paraventricular nucleus

QRFP

glutamine RF‐amide peptide

RFRP

RFamide‐related peptide

SAR

structure–activity relationships

SNP

single‐nucleotide polymorphism

TM

transmembrane

VMH

ventro‐median nucleus

WT

wild‐type

Introduction

In 1977, Price and Greenberg published a paper, now a citation classic, in which they reported the purification and characterization of an α‐amidated tetrapeptide, H–Phe–Met–Arg–Phe–NH₂ (FMRFamide), from the ganglia of the Venus clam Macrocallista nimbosa on the basis of its cardioexcitatory activity (Price and Greenberg, 1977). Since then, hundreds of peptides that exhibit the –Arg–Phe–NH₂ motif at their C‐terminal extremity have been identified in invertebrates (Walker et al., 2009; Elphick and Mirabeau, 2014; Peymen et al., 2014). These peptides, collectively termed RFamide peptides, exert a wide range of biological activities (Marks et al., 1997; Nelson et al., 1998; Brownlee et al., 2000).

In vertebrates, the first RFamide peptide, H–Leu–Pro–Leu–Arg–Phe–NH₂, was isolated from chicken Gallus gallus (Dockray et al., 1983). Soon after, two RFamide peptides called neuropeptide FF (NPFF) and neuropeptide AF (NPAF) were characterized in mammals (Yang et al., 1985). To date, five groups of RFamide peptides have been identified, namely, the NPFF group (Yang et al., 1985), the prolactin‐releasing peptide (PrRP) group (Hinuma et al., 1998), the RFamide‐related peptide (RFRP)/gonadotropin‐inhibitory hormone (GnIH) group (Fukusumi et al., 2001; Yoshida et al., 2003; Tsutsui, 2009), the metastin/kisspeptins group (Kotani et al., 2001; Ohtaki et al., 2001) and the 26RFa/glutamine RF‐amide peptide (QRFP) group (Chartrel et al., 2003; Fukusumi et al., 2003; Jiang et al., 2003).

A 26‐amino acid peptide harboring the RFamide signature at its C‐terminus (26RFa) was initially isolated from a frog Pelophylax lessonae X Pelophylax ridibundus hybrid brain extract (Chartrel et al., 2003). Simultaneously, the search for endogenous ligands of the orphan GPCR GPR103/AQ27/SP9155 through bioinformatic analysis resulted in the identification of a novel RFamide peptide precursor that could potentially generate two ligands with 26 (Jiang et al., 2003) and 43 amino acid residues (Fukusumi et al., 2003). The latter peptide, designated QRFP, has been subsequently isolated from the rat brain (Takayasu et al., 2006). The 26RFa/QRFP and/or GPR103 cDNAs have now been characterized in a number of species of cephalochordates, fish, amphibians, birds and mammals (Sandvik et al., 2014; Ukena et al., 2014; Xu et al., 2015; Osugi et al., 2016) indicating that these neuropeptides and their receptors have been strongly conserved during evolution (Ukena et al., 2014).

The genes encoding the 26RFa/QRFP precursor and GPR103 are actively expressed in the hypothalamic nuclei implicated in the control of feeding behaviour including the arcuate nucleus (ARC) and the dorsal hypothalamic area (Chartrel et al., 2003; Bruzzone et al., 2007). Indeed, i.c.v. injection of 26RFa or QRFP stimulates food intake in mouse, rat and chicken (Chartrel et al., 2003; Takayasu et al., 2006; Ukena et al., 2010). GPR103 mRNA is also detected in a number of hypothalamic and extrahypothalamic regions (Takayasu et al., 2006; Bruzzone et al., 2007). Consistent with the widespread distribution of the receptor, 26RFa and QRFP have been found to regulate many physiological functions including energy homeostasis (Chartrel et al., 2016), bone formation (Baribault et al., 2006), hypothalamo‐pituitary‐gonadal activity (Navarro et al., 2006; Patel et al., 2008), insulin secretion (Egido et al., 2007; Granata et al., 2014; Prévost et al., 2015), locomotor activity (Do Rego et al., 2006) and analgesia (Yamamoto et al., 2008). The potential implication of these neuropeptides in various pathologies has prompted medicinal chemists to study the structure–activity relationships (SAR) of 26RFa in order to design selective agonists and antagonists (Le Marec et al., 2011; Neveu et al., 2012, 2014; Georgsson et al., 2014, 2015; Nordqvist et al., 2014).

26RFa/QRFP peptides

Discovery

Since the identification of FMRFamide in bivalve mollusc ganglia by Price and Greenberg (1977), a large number of FMRFamide‐like peptides (FLPs) ending with the RFamide sequence have been characterized in various classes of invertebrates including cnidarians (Grimmelikhuijzen et al., 2004), plathelminths (McVeigh et al., 2005; Mousley et al., 2005), nematodes (McVeigh et al., 2006; Husson et al., 2007; Peymen et al., 2014), annelids (Salzet, 2001), molluscs (López‐Vera et al., 2008; Bigot et al., 2014) and arthropods (Roller et al., 2008; Verleyen et al., 2009; Christie, 2015; Christie and Chi, 2015). Generally, each invertebrate FLP gene encodes a precursor protein that has the potential to generate several mature FLPs of variable length, from 4 to 45 amino acids (Walker et al., 2009; Orchard and Lange, 2013). In addition, each invertebrate species usually possesses numerous FLP genes. For instance, in Caenorhabditis elegans, no less than 33 genes encoding 70 distinct FLPs have been characterized (Li, 2005; Husson et al., 2007; Masler, 2013). Besides authentic FLPs which contain the RFamide signature at their C‐terminal end, a number of invertebrate neuropeptides terminate in –Arg–Tyr–NH₂ (RYa), –Arg–Trp–NH₂ (RWa) or –Xxx–Phe–NH₂ (XFa), X being a Gly, Ser, Cys, Ala, Met, Val, Leu, Ile, Thr or Tyr residue (Walker et al., 2009). These peptides exert a vast array of biological activities on various organs and tissues, notably the nervous system, heart, muscular plexus, digestive tract and reproductive system (Mercier et al., 2003; McVeigh et al., 2006; Orchard and Lange, 2013; Peymen et al., 2014).

The number of FLPs characterized in vertebrates is much lower than in invertebrates (Orchard and Lange, 2013). In mammals, five distinct genes, designated farp‐1 to farp‐5 (Dockray, 2004), encoding seven FLPs have been identified so far (Quillet et al., 2016).

The first two mammalian FLPs, NPFF and NPAF, were isolated from bovine brain (Yang et al., 1985) using a non‐selective antibody directed against FMRFamide (Dockray et al., 1983). Molecular cloning of the cDNA encoding NPFF revealed that NPFF and NPAF originate from the same gene termed farp‐1 (Perry et al., 1997; Vilim et al., 1999). NPFF and NPAF modulate the anti‐nociceptive action of morphine through activation of two GPCRs named NPFF receptor type‐1 (NPFF1) and NPFF receptor type‐2 (NPFF2) (Bonini et al., 2000; Elshourbagy et al., 2000). These two receptors share about 30–35% sequence identity with neuropeptide Y (NPY) receptors (Bonini et al., 2000; Elshourbagy et al., 2000).

The second FLP, called PrRP, has been characterized from a bovine brain extract, through a reverse pharmacology approach, as the natural ligand of the orphan receptor GPR10 (Hinuma et al., 1998; Kutzleb et al., 2005). The PrRP precursor, encoded by the farp‐2 gene, can generate two mature peptides of 20 and 31 amino acids, designated PrRP₂₀ and PrRP₃₁ (Hinuma et al., 1998). The latter peptide exhibits high affinity not only for GPR10 but also for NPFF2 (Engström et al., 2003). Although PrRP was initially reported to stimulate prolactin (PRL) release from pituitary cells (Hinuma et al., 1998), subsequent studies have cast doubt on its hypophysiotropic activity (Lawrence et al., 2000; Samson et al., 2003; Hinuma and Iijima, 2013).

A third FLP precursor has been identified by screening an expressed sequence tag (EST) library (Hinuma et al., 2000). This precursor, encoded by the farp‐3 gene, encompasses two authentic FLPs, named RFRP‐1 and RFRP‐3, plus a peptide terminating in –Arg–Ser–NH₂ (RFRP‐2). RFRP‐1, which inhibits gonadotropin secretion in birds and mammals, is also termed GnIH (Tsutsui, 2009; Tsutsui and Osugi, 2009). The RFRP‐1/GnIH and RFRP‐3 peptides have been isolated from human, bovine, rat and quail brain tissues (Tsutsui et al., 2000; Fukusumi et al., 2001; Ukena et al., 2002; Yoshida et al., 2003; Ubuka et al., 2009). They both activate GPR147/OT7T022, an orphan GPCR that exhibits about 35 and 32% sequence identity with orexin OX₁ and OX₂ receptors and NPY receptors respectively (Hinuma et al., 2000). RFRP‐1 and RFRP‐3 display high affinity for NPFF1, in addition to OT7T022 (Yoshida et al., 2003).

A fourth gene, farp‐4, encodes a polypeptide called metastin, initially isolated from the human placenta, that activates the orphan GPCR GPR54 (Ohtaki et al., 2001). In humans, loss of function of the GPR54 gene causes idiopathic hypogonadotropic hypogonadism (de Roux et al., 2003; Seminara et al., 2003) indicating that metastin and/or its processed fragments kisspeptins play a pivotal role in the control of the hypothalamo–pituitary–gonadal axis (Pinilla et al., 2012). Although metastin/kisspeptins exhibit an RFamide C‐terminal signature, senteny analysis reveals that the evolutionary origin of the metastin/kisspeptin precursor family is distinct from that of the other FLPs (Pasquier et al., 2012; Kim et al., 2014; Yun et al., 2014).

The fifth gene, farp‐5 encoding the 26RFa/QRFP precursor, has been identified concomitantly by three independent teams through different approaches. A peptidomic study was conducted on an extract of the whole brain of the European green frog Rana ridibunda, now renamed P. ridibundus (Conlon et al., 2009). Screening of the purified fractions by using a polyclonal antibody against bovine NPFF (Allard et al., 1991; Labrouche et al., 1993) led to the identification of two immunoreactive peptides displaying the RFamide motif, that is, a 12‐amino acid peptide, termed Rana RFamide, that is orthologous to RFRP‐1/GnIH (Chartrel et al., 2002), and a totally novel 26‐amino acid peptide, termed 26RFa, that does not belong to any known FLP family (Chartrel et al., 2003). Cloning of the human and rat 26RFa precursor cDNAs revealed that the sequence of the C‐terminal region, which contains 26RFa, has been strongly preserved during evolution of tetrapods (Chartrel et al., 2003; Fukusumi et al., 2003). Independently, a novel FLP precursor sequence has been identified from human and mouse genomic databases (Jiang et al., 2003). The predicted mature peptides encompass 26 amino acid residues and contain the RFamide signature at their C‐terminal extremity (Jiang et al., 2003). A similar bioinformatic study has been conducted on a human genome database and the mature forms of the derived peptides have been isolated by affinity column chromatography using a monoclonal antibody against rat RFRP‐1 (Fukusumi et al., 2001). Maldi‐TOF mass spectrometry analysis indicated that the major immunoreactive species is a 43‐amino acid peptide starting with an N‐terminal pyroglutamic acid (<Q) and ending with a C‐terminal –Arg–Phe–NH₂ moiety, which was termed QRFP (Fukusumi et al., 2003). The 26RFa/QRFP (farp‐5) gene has now been identified in a number of representative species of vertebrates from cephalochordates to mammals (see 2.4 section; Table 1). The predicted length of the 26RFa peptide ranges from 24 amino acids for the spotted green pufferfish (Tetraodon nigroviridis), the spotted gar (Lepisosteus oculatus) and the saker falcon (Falco cherrug) to 27 amino acids for the Chinese soft‐shelled turtle (Pelodiscus sinensis), the chicken (G. gallus), the Japanese quail (Coturnix japonica), the turkey (Meleagris gallopavo), the Sunda flying lemur (Galeopterus variegates) and the American pika (Ochotona princeps) mature peptides. As shown in Table 1 and Figure 1, the 26RFa sequence has been relatively well preserved whereas the sequence of the N‐terminal region of QRFP is far more variable (Ukena et al., 2011).

Table 1. Amino acid sequence alignment of QRFP peptide precursors

Speciesaa Inside a group, species are arranged alphabetically. It should be noted that taxonomic units do not have equal rank.		Sequencebb Dots mark N‐terminal residues. Dash represents gap for maximizing sequence similarity. The number into brackets corresponds to the number of residues after the last noted C‐terminal amino acid. Residues in green, red and blue represent the up‐stream putative processing site, the mature or putative 26RFa sequence and the C‐terminal amidation signal respectively. Residues in orange represent the putative processing site generating the N‐terminal extended form of 26RFa, QRFP. Residues in pink highlight the presence of a FLP just upstream QRFP. Residues identical to the human sequence of 26RFa or to the counterpart C‐teminal region of QRFP are shade‐boxed in tetrapods. The underlined Q corresponds to a pyroglutamic residue in the mature sequence. X, unidentified residue.	Residue number and origincc The number series corresponds to the number of residues in the precursor, in QRFP and in 26RFa respectively. C, cDNA cloning. D, genomic data.	Reference or accession number
Mammalia
Human	Homo sapiens	. . . RFGR‐QDE‐GSEATGFL‐‐‐‐‐PAAG‐EK‐‐TSGPLGNLAEELNGY‐‐SRKKGGFSFRFGRR	136|43|26|C	Chartrel et al., 2003; Fukusumi et al., 2003; Jiang et al., 2003
Chimpanzee	Pan troglodytes	. . . R‐QDE‐GSEATGFL‐‐‐‐‐PAAG‐EK‐‐TSGPLGNLAEELNGY‐‐SRKKGGFSFRFGRR	136|43|26|D	XP_009455785.1
	Pan paniscus	. . . RFGR‐QDE‐GTEATGFL‐‐‐‐‐PAAG‐EK‐‐TSGPLGNLAEELNGY‐‐SRKKGGFSFRFGRR	247|43|26|D	XP_008974188.2
Gorilla	Gorilla gorilla gorilla	. . . RFGR‐QDE‐GGEATGFL‐‐‐‐‐SAAR‐EK‐‐TSGPLGNLAEELNGY‐‐SRKKGGFSFRFGRR	136|43|26|D	XP_004048799.1
Orangutan	Pongo abelii	. . . RFGR‐QDE‐GSEATGFL‐‐‐‐‐PAAG‐EK‐‐ASGPLRNLAEELNGY‐‐SRKKGGFSFRFGRR	136|43|26|D	XP_002833229.1
Gibbon	Nomascus leucogenys	. . . RFGR‐QDE‐GSEATGFL‐‐‐‐R‐AAG‐EK‐‐ASGPLGNLAEELNGY‐‐SRKKGGFSFRFGRR	169|43|26|D	XP_003276794.2
Baboon	Papio anubis	. . . RFGR‐QDE‐GSEADDFL‐‐‐‐‐PAGG‐VK‐‐ASGPLGNLAEELNGY‐‐SRKKGGFSFRFGRR	136|43|26|D	XP_009186264.1
Colobus	Colobus angolensis palliatus	. . . RFGR‐QDE‐GSEASGF‐‐‐‐‐RPAGG‐VK‐‐ASGPLGNLAEELNGY‐‐SRKKGGSSFHFGRR	137|43|26|D	XP_011786583.1
Drill	Mandrillus leucophaeus	. . . RFGR‐QDE‐GSEADDFL‐‐‐‐‐PAGG‐VK‐‐ASSPLGNLAEELNGY‐‐SRKKGGFSFRFGRR	169|43|26|D	XP_011826536.1
Macaque	Macaca nemestrina	. . . RFGR‐QDE‐GSEADDFL‐‐‐‐‐PAGG‐VK‐‐ASGPLGNLAEELNGY‐‐SRKKGGFSFRFGRR	136|43|26|D	XP_011709965.1
	Macaca fascicularis	. . . RFGR‐QDE‐GSEADDFL‐‐‐‐‐PAGG‐VK‐‐ASGPLGNLAEELNGY‐‐SRKKGGFSFRFGRR	136|43|26|D	XP_005580679.1
Mangabey	Cercocebus atys	. . . RFGR‐QDE‐GSEADDFL‐‐‐‐‐PAGE‐VK‐‐ASSPLGNLAEELNGY‐‐SRKKGGFSFRFGRR	136|43|26|D	XP_011894201.1
Monkey	Rhinopithecus roxellana	. . . R‐QDE‐GSEANDFL‐‐‐‐‐PAGG‐VK‐‐ASGLLGNLAEELNGY‐‐SRKKGGFSFRFGRR	136|43|26|D	XP_010359256.1
	Chlorocebus sabaeus	. . . RFGR‐QDE‐GSEANDFL‐‐‐‐‐PAGG‐VK‐‐ASGPLGNLAEELNGY‐‐SRKKGGFSFRFGRR	214|43|26|D	XP_008004106.1
Marmoset	Callithrix jacchus	. . . RRQDQ‐GSEATSFL‐‐‐‐‐PAEG‐EK‐‐ASDPLGNLAEELNGY‐‐SRKKGGFSFRFGRR	136|43|26|D	XP_002743449.1
Ma's night monkey	Aotus nancymaae	. . . RRQDQ‐GSEAAEFL‐‐‐‐‐PAEG‐EK‐‐ASGPLGNLAEELNGY‐‐SRKKGGFSFRFGRR	136|43|26|D	XP_012313305.1
Squirrel monkey	Saimiri boliviensis boliviensis	. . . RRQDQ‐DSEATGFL‐‐‐‐‐PAEG‐DK‐‐ASGPLGNLAEELNGY‐‐SRKKGGFSFRFGRR	136|43|26|D	XP_010339691.1
Tarsier	Tarsius syrichta	. . . RFGR‐QEE‐GSEATGFL‐‐‐‐‐P‐DG‐EK‐‐ASGPLGNLAEELSGY‐‐SRKKGGFSFRFGRR[11]	514|42|26|D	XP_008054832.1
Sifaka	Propithecus coquereli	. . . RFGR‐QDE‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐AASVLLGNLAEELKGY‐‐SRKTGGFRFHFGR[1]	112|30|      |D	XP_012495100.1
Galago	Otolemur garnettii	. . . RRQDE‐GSEGTSFL‐‐‐‐‐PVDG‐EK‐‐ASGLLGNLAEELNGY‐‐SRKKGGFSFRFGRR	136|43|26|D	XP_003794941.1
Deer mouse	Peromyscus maniculatus bairdii	. . . RR‐DGSSEEAAGFL‐‐‐‐‐PSDS‐EK‐‐ASGPLGTLAEELSSF‐‐IRRKGGFSFRFGR	133|43|26|D	XP_006983508.1
Hamster	Cricetulus griseus	. . . RR‐DRSS‐QAAGFL‐‐‐‐‐PTDA‐EK‐‐ASRPLGTLAEELSSY‐‐SRRKGGFSFRFGR	175|42|26|D	XP_007618132.1
	Mesocricetus auratus	. . . R‐QD‐SSGQAAGFL‐‐‐‐‐PTDS‐EK‐‐ASSPLGTLAEELSSY‐‐SRRKGGFSFRFGR	132|43|26|D	XP_012980294.1
Vole	Microtus ochrogaster	. . . R‐QDG‐DSQAAGFL‐‐‐‐‐PTDS‐EK‐‐ASGPLGTLAEELSSY‐‐SRRKGGFSFRFGR	132|43|26|D	XP_005346152.1
Blind mole‐rat	Nannospalax galili	. . . R‐QD‐VSGEAAGFL‐‐‐‐‐PAD‐‐LK‐‐TSGPLGSLAEELNSY‐‐SRRKGGFSFRFGR	134|42|26|D	XP_008824628.1
Mouse	Mus musculus	. . . R‐QD‐GSSEAAGFL‐‐‐‐‐PAD‐SEK‐‐ASGPLGTLAEELSSY‐‐SRRKGGFSFRFGR	124|43|26|C	Fukusumi et al., 2003; Jiang et al., 2003.
Rat	Rattus norvegicus	. . . R‐QD‐SGSEATGFL‐‐‐‐‐PTDS‐EK‐‐ASGPLGTLAEELSSY‐‐SRRKGGFSFRFGR	124|43|26|C	Chartrel et al., 2003; Fukusumi et al., 2003
Vole	Microtus ochrogaster	. . . R‐QDG‐DSQAAGFL‐‐‐‐‐PTDS‐EK‐‐ASGPLGTLAEELSSY‐‐SRRKGGFSFRFGR	132|43|26|D	XP_005346152.1
Kangaroo‐rat	Dipodomys ordii	. . . RRQ‐‐SEAEAGGFP‐‐‐‐‐PEAG‐EK‐‐TSTLLGNLAQELNKL‐‐SRKKGGFNFRFGRR	130|42|26|D	XP_012876741.1
Jerboa	Jaculus jaculus	. . . R‐QE‐‐GGQATAFL‐‐‐‐‐PGGG‐EK‐‐ASGPLGSLAEELSSY‐‐SRKKGGFRFRFGR	127|42|26|D	XP_004671800.1
Ground‐squirrel	Ictidomys tridecemlineatus	. . . R‐QD‐‐GSEAAG‐‐‐‐‐‐‐‐‐‐‐‐EK‐‐AGGPLGSLAEELSGY‐‐SRKKGGFSFRFGRR[50]	254|36|26|D	XP_005321238.1
Chinchilla	Chinchilla lanigera	. . . R‐QE‐‐GSETAGFL‐‐‐‐‐PVDGSEK‐‐ATGPLGSLAEELNGY‐‐SRRKGGFSFRFGR[1]	134|43|26|D	XP_005408406.1
Degu	Octodon degus	. . . R‐QE‐‐GSKATGFL‐‐‐‐‐PADGNEK‐‐ATGPLGNLAEELNGY‐‐SRKKGGFSFRFGR[1]	135|43|26|D	XP_004640620.1
Guinea pig	Cavia porcellus	. . . R‐QE‐‐SSETSSFL‐‐‐‐‐PAEGSEK‐‐ATSPLGNLAEELNGY‐‐SRRKGGFSFRFGR	131|43|26|D	H0V2R1
Mole‐rat	Fukomys damarensis	. . . R‐QE‐‐GSEATSFL‐‐‐‐‐PADGSEK‐‐ATGPLGNLAEELTGY‐‐SRRKGGFNFRFGR[211]	552|43|26|D	XP_010625759.1
	Heterocephalus glaber	. . . R‐QE‐‐GTEATSFL‐‐‐‐‐SAHGSEK‐‐ATGPLGNLAEELTGY‐‐SRRKGGFSFRFGR[1]	126|43|26|D	XP_004849654.1
Pika	Ochotona princeps	. . . RRHED‐‐SE‐‐G‐‐‐‐‐‐‐D‐SGGEK‐AGSRSLGSLAEELGSY‐‐SRKKGGFSFRFGRR	129|39|27|D	XP_004593740.1
Rabbit	Oryctolagus cuniculus	. . . RFGR‐‐ED‐‐‐EGT‐‐‐‐‐‐‐‐DADGGDK‐‐AAGLLGSLAEELGGY‐‐SRKKGGFSFRFGRR	131|38|26|D	XP_008250432.1
Flying‐lemur	Galeopterus variegatus	. . . RFGR‐QD‐‐SSEVTSLL‐‐‐‐‐PDDS‐EK‐AGGGTLGNLAEELSGY‐‐SRKKGGFSFRFGRR	136|43|27|D	XP_008588732.1
Tree shrew	Tupaia chinensis	. . . R‐QADGGRKGTG‐‐‐‐‐‐‐‐‐‐‐‐‐KK‐SNELLDKLAQSFKAY‐‐TRQKGGFWFRYGRR	131|38|26|D	ELV12687.1
Bison	Bison bison bison	. . . R‐QDD‐GSEAT‐GLL‐‐‐‐‐GEA‐EK‐‐VGGLLGTLAEELNGY‐‐SRKKGGFSFRFGR[1]	133|42|26|D	XP_010855098.1
Buffalo	Bubalus bubalis	. . . R‐QDD‐GSEAAGGLL‐‐‐‐‐GEA‐EK‐‐VGGPLGTLAEELNGY‐‐SRKKGGFSFRFGR[1]	134|43|26|D	XP_006043366.1
Cattle	Bos mutus	. . . R‐QDD‐GSEATGLL‐‐‐‐‐‐GEA‐EK‐‐VGGLLGTLAEELNGY‐‐SRKKGGFSFRFGR[1]	133|42|26|D	XP_005902834.1
	Bos taurus	. . . R‐QDD‐GSEATGLLL‐‐‐‐‐GEA‐EK‐‐VGGLLGTLAEELNGY‐‐SRKKGGFSFRFGR[1]	134|43|26|C	Fukusumi et al., 2003
Chiru	Pantholops hodgsonii	. . . R‐QDD‐GSE‐‐‐VL‐‐‐‐‐‐EEA‐EK‐‐VGGPLGTLAEELNGY‐‐SRKKGGFSFRFGR[1]	130|39|26|D	XP_005982768.1
Goat	Capra hircus	. . . R‐QDD‐SSEATGLLL‐‐‐‐‐EEA‐EK‐‐VGGPLGTLAEELNGY‐‐SRKKGGFSFRFGR[1]	134|43|26|D	XP_005687329.1
Sheep	Ovis aries	. . . R‐QDD‐GSEATG‐LL‐‐‐‐LEEA‐EK‐‐VGGPLGTLAEELNGY‐‐SRKKGGFSFRFGR[1]	134|43|26|D	NP_001119841.1
Pig	Sus scrofa	. . . RFGR‐QD‐NGSEAAGFL‐‐‐‐‐PGDG‐EK‐‐ASGLLGTLAEELHGY‐‐SRKKGGFSFRFGR[1]	134|43|26|D	XP_003122292.1
Dolphin	Lipotes vexillifer	. . . RQQDD‐GSEATGFLL‐‐‐‐‐GDG‐EK‐‐AGGLLGTLAEELNGY‐‐SREKGGFSFRFGR[1]	135|44|26|D	XP_007472218.1
Killer‐whale	Orcinus orca	. . . RQQDD‐GSEATGFLL‐‐‐‐‐GDG‐EK‐‐AGGLLGTRAEELDGY‐‐SREKGGFSFRFGR[1]	519|44|26|D	XP_012388228.1
Sperm whale	Physeter catodon	. . . RQQDD‐GSEATGFLLG‐‐‐‐DG‐EK‐‐AGGLLGTLAEELNGY‐‐SREKGGFSFCFGQG	136|47|29|D	XP_007131067.1
Whale	Balaenoptera acutorostrata	. . . RQQDD‐GSEATGFLLG‐‐‐‐‐DG‐EE‐‐AGGLLGTLAEELNSY‐‐SRKEGGFSFRFGQG	135|47|      |D	XP_007194552.1
Alpaca	Vicugna pacos	. . . RFGR‐QDN‐GSEAAGFL‐‐‐‐‐PADG‐EK‐‐TSGPLGTLAEELSGY‐‐SRKKGGFSFRFGRR	129|43|26|D	XP_006216676.1
Camel	Camelus dromedarius	. . . RFGR‐QDN‐GSEAAGFL‐‐‐‐‐PAGE‐‐‐‐‐‐‐‐‐‐‐‐LAEELSGY‐‐SRKKGGFSFRFGRR	126|34|      |D	XP_010982517.1
	Camelus bactrianus	. . . RFGR‐QDD‐GSEPAGFL‐‐‐‐‐PADG‐EK‐‐TSGPLGTLAEELSGY‐‐SRKKGGFSFRFGRR	135|43|26|D	XP_010954179.1
	Camelus ferus	. . . RFGR‐QDD‐GSEAAGFL‐‐‐‐‐PADG‐EK‐‐TSGPLGTLAEELSGY‐‐SRKKGGFSFRFGRR	135|43|26|D	XP_006181990.1
Bear	Ursus maritimus	. . . R‐QDD‐GSEAPSFL‐‐‐‐‐PADG‐EK‐‐ASGPLGTLAEELSTY‐‐SRKKGGFSFRFGRR	136|43|26|D	XP_008696907.1
Panda	Ailuropoda melanoleuca	. . . R‐QDD‐GSEAPSFF‐‐‐‐‐PADG‐EK‐‐ASGPLGTLAEELSTY‐‐SRKKGGFSFRFGRR	136|43|26|D	XP_002915255.1
Walrus	Odobenus rosmarus divergens	. . . RFGR‐QDD‐GSEATGFL‐‐‐‐‐PAGG‐EK‐‐ASGPLGTLAEELSSY‐‐SRKKGGFSFRFGRR	138|43|26|D	XP_004392513.1
Seal	Leptonychotes weddellii	. . . RFGR‐QDD‐GSEATGFL‐‐‐‐‐PADG‐EK‐‐ASGPLGTLAEELSSY‐‐SRKKGGFSFRFGRR	136|43|26|D	XP_006746182.1
Ferret	Mustela putorius furo	. . . RFGR‐QDD‐GSKATGFL‐‐‐‐‐PADG‐EK‐‐ASGPLGTLAEELSSY‐‐SRKKGGFSFRFGRR	136|43|26|D	XP_012914087.1
Dog	Canis lupus familiaris	. . . RFGR‐QDD‐GSEATGFL‐‐‐‐‐PADG‐EK‐‐AHGPLGTLAEELSSY‐‐SRKKGGFSFRFGRR	136|43|26|D	XP_850751.2
Cat	Felis catus	. . . RFGR‐HDD‐GSEATGFLL‐‐‐‐‐ADG‐DK‐‐ASGPLGTLAEELSGY‐‐SRKKGGFSFRFGRR	131|43|26|D	XP_006939618.1
Tiger	Panthera tigris altaica	. . . RFGR‐QDD‐GSEATGFLL‐‐‐‐‐ADG‐DK‐‐ASGPLGTLAEELSGY‐‐SRKKGGFSFRFGRR	178|43|26|D	XP_007078896.1
Bat	Eptesicus fuscus	. . . RFGR‐QDE‐GRGAAGFLL‐‐‐‐‐ADG‐EK‐‐AGSPLGTLAGELSGY‐‐SRKKGGFSFRFGRR	136|43|26|D	XP_008150734.1
	Myotis lucifugus	. . . RRQDD‐GLGEAGSLL‐‐‐‐‐ADG‐‐KK‐AGSQLGTLADEISGH‐‐NKNKGGFHFRFGRR	172|43|26|D	XP_006105734.2
	Myotis brandtii	. . . RRQDD‐GRGEAGSLL‐‐‐‐‐ADG‐EK‐‐ASSPLGTLADEISGH‐‐DKKKGGFHFRFGRR	173|43|26|D	XP_005884863.1
Flying‐fox	Pteropus vampyrus	. . . XFGR‐QDD‐GNEAASF‐‐‐‐‐‐PLADGE‐‐‐ASDPLGTLAEELNGY‐‐SRKKSGFSFRFGRR	115|42|      |D	XP_011368360.1
	Pteropus alecto	. . . RFGR‐QDD‐GNEAASF‐‐‐‐‐‐PPADGE‐‐‐ASGPLGTLAEELNGY‐‐SRKKGGFSFRFGRR	83|42|      |D	ELK07292.1
Myotis	Myotis davidii	. . . RRQDD‐GREVAGFLL‐‐‐‐‐ADG‐E‐‐TASSPLGTLADEFSGY‐‐GKKKGGFSFRFGRR	136|43|      |D	XP_006754269.1
Shrew	Sorex araneus	. . . RR‐ED‐DQEAPDDP‐‐‐‐‐PADG‐‐RK‐APGQLGTLVEGLQGY‐‐GRKKGGFSFRFGR[1]	165|42|26|D	XP_004613507.1
Hedgehog	Erinaceus europaeus	. . . RFGR‐QDD‐SSEAAGFL‐‐‐‐‐QADGGEK‐‐ASGPLGTLAEELNGY‐‐SRKKGGFSFRFGRR	135|44|26|D	XP_007530369.1
Star‐nosed mole	Condylura cristata	. . . R‐QDPGGSEAAGFL‐‐‐‐‐PAEG‐EK‐‐AGGPLGTLAEELTSY‐‐SRKKGGFSFRFGRR	129|43|26|D	XP_004677935.1
Horse	Equus caballus	. . . RFGR‐QDD‐GSEATGFLL‐‐‐‐‐ADG‐EK‐‐ESGPLETLAEELNGY‐‐SRKKGGFSFRFGR[3]	138|43|26|D	XP_001499477.1
	Equus przewalskii	. . . RFGR‐QDD‐GSEATGFLL‐‐‐‐‐ADG‐EK‐‐ESGPLETLAEELNGY‐‐SRKKGGFSFRFGR[3]	155|43|26|D	XP_008536129.1
Rhinoceros	Ceratotherium simum simum	. . . RFGR‐QD‐AGSEATGFLL‐‐‐‐‐ADG‐EK‐‐ASSPLGTLAEELNGY‐‐SRKKGGFSFRFGRR	136|43|26|D	XP_004423643.1
Aardvark	Orycteropus afer afer	. . . RFGR‐QDD‐GGEATSFL‐‐‐‐‐PADG‐EK‐‐ASGPLGNLAEELNGY‐‐SRKKGGFSFRFGRR	171|43|26|D	XP_007942537.1
Elephant	Loxodonta africana	. . . RFGR‐QDD‐GDEATRFL‐‐‐‐‐PADG‐EK‐‐TSGPLGSLAEELTGY‐‐SRKKGGFSFRFGRR	136|43|26|D	XP_003407599.1
Elephant‐shrew	Elephantulus edwardii	. . . RFGR‐QDD‐SGEATSFL‐‐‐‐‐PADG‐EK‐‐ASGPLGNLAEELSGY‐‐SRKKGGFSFRFGRR	137|43|26|D	XP_006892510.1
Manatee	Trichechus manatus latirostris	. . . RFGR‐QDD‐GGEATSFL‐‐‐‐‐SADG‐EK‐‐ASGPLGNLAEELNGY‐‐SRKKGGFSFRFGRR	134|43|26|D	XP_004375898.2
Mole	Chrysochloris asiatica	. . . RFGR‐QDD‐GSEATSFL‐‐‐‐‐PADG‐EK‐‐ATGPLGNLAEELNGY‐‐‐RKKGGFSFRFGRR	178|42|25|D	XP_006835052.1
Armadillo	Dasypus novemcinctus	. . . RFGR‐QDD‐GSEATSF‐F‐‐‐‐TADS‐EK‐‐ASGPLGMLAEELSGY‐‐SRKKGGFSFRFGRR	136|43|26|D	XP_004463118.1
Opossum	Monodelphis domestica	. . . RFGR‐QEE‐DSKATNIY‐‐‐‐‐PAS‐EEK‐‐QNSLLGSLAEELSGY‐‐IRKKGGFSFRFGR	141|43|26|D	XP_007475223.1
Platypus	Ornithorhynchus anatinus	. . . RFGR‐QE‐EES‐‐PGAHFV‐‐‐PASG‐EKR‐‐SGLLGNLAEELNGY‐‐RRKKGGFSFRFGRR	198|43|25|D	XP_007660564.1
Archosauria/Aves
Canary	Serinus canaria	. . . RR‐‐‐GGPE‐‐‐‐LL‐‐‐‐PAGG‐EKR‐‐SGTLGNLAEELNGY‐‐NRKKGGFTFRFGR	129|38|25|D	XP_009091665.1
Crow	Corvus brachyrhynchos	. . . RR‐‐‐GDPE‐‐‐‐LL‐‐‐‐PVGG‐EKR‐‐SGTLGNLAEEINGY‐‐NRKKGGFTFRFGR	128|38|25|D	XP_008640427.1
Flycatcher	Ficedula albicollis	. . . RR‐‐‐GDTE‐‐‐‐LL‐‐‐‐PSAG‐EKR‐‐SGTLGNLAEELNGY‐‐NRRKGGFTFRFGR	137|38|25|D	XP_005063171.1
Ground‐tit	Pseudopodoces humilis	. . . RR‐‐‐GDPE‐‐‐‐LL‐‐‐‐TMAG‐EKR‐‐SGALGNLAEELNGY‐‐NRKKGGFTFRFGR	123|38|25|D	XP_005527338.1
Sparrow	Zonotrichia albicollis	. . . R‐‐‐SGGPEA‐‐‐L‐‐‐‐‐PAGG‐EKR‐‐SGALGNLAEELTGY‐‐NRRKGGFTFRFGR	120|39|25|D	XP_005495284.1
Zebra finch	Taeniopygia guttata	. . . R‐‐‐PGGSGGPGAL‐‐‐‐‐PVVG‐EKR‐‐SGTLGNLAEEINGY‐‐NRRKGGFTFRFGR	152|42|25|C	Tobari et al., 2011
Eagle	Haliaeetus leucocephalus	. . . R‐Q‐‐GGSE‐‐‐‐LL‐‐‐‐PAGG‐EKR‐‐SGTLGNLAEELNGY‐‐NRKKGGFTFRFGR	128|39|25|D	XP_010569415.1
	Aquila chrysaetos canadensis	. . . R‐Q‐‐GGSE‐‐‐‐LL‐‐‐‐PAGG‐LKR‐‐SGTLGNLAEELNGY‐‐NRKKGGFTFRFGR	128|39|25|D	XP_011578360.1
Falcon	Falco peregrinus	. . . R‐Q‐‐GGSE‐‐‐‐LL‐‐‐‐PVGG‐EKR‐‐SGTLGNLAEELNGY‐‐NRKKGGFTFRFGR	129|39|25|D	XP_005231361.1
	Falco cherrug	. . . R‐Q‐‐GGSE‐‐‐‐LC‐‐‐RWEGG‐‐R‐‐‐‐GTLGNLAEELNGY‐‐NRKKGGFTFRFGR	127|38|24|D	XP_005447490.1
Egret	Egretta garzetta	. . . RR‐‐‐GGSE‐‐‐‐L‐‐‐‐RPVGG‐EKR‐‐SGTLGNLAEELNGY‐‐NRKKGGFTFRFGR	128|38|25|D	XP_009641085.1
Ibis	Nipponia nippon	. . . RR‐‐‐GGSE‐‐‐‐LL‐‐‐‐PVGG‐EKR‐‐SGTLGNLAEELNGY‐‐NRKKGGFTFRFGR	129|38|25|D	XP_009475349.1
Pelican	Pelecanus crispus	. . . RR‐‐‐GGSM‐‐‐‐LL‐‐‐‐PAGG‐EKR‐‐SGTLGNLAEELNGY‐‐NRKKGGFTFRFGR	128|38|25|D	XP_009487762.1
Chicken	Gallus gallus	. . . R‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐PEGS‐EKRGGGGTLGDLAEELNGY‐‐GRKKGGFAFRFGR	122|34|27|D	Ukena et al., 2010
Quail	Coturnix japonica	. . . R‐QE‐‐GSE‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐KRGGGGTLGDLAEELNGY‐‐SRKKGGFAFRFGR	120|34|27|C	Ukena et al., 2010
Turkey	Meleagris gallopavo	. . . R‐QE‐‐GSE‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐KRGGGGTLGDLAEELNGY‐‐SRKKGGFAFRFGR	119|34|27|D	XP_010719204.1
Seriema	Cariama cristata	. . . KRRR‐‐‐SEELSTLLGITRELRSYGK‐‐EGTALGNLAEELNGY‐‐NRRKGGFTFRFGR	108|44|26|D	XP_009693562.1
Roatelo	Mesitornis unicolor	. . . R‐QG‐‐GSE‐‐‐‐LL‐‐‐‐PAGGEER‐‐‐SGTLGSLAEGVSGY‐‐NRKKGGFTFRFGR	124|39|25|D	XP_010182464.1
Penguin	Aptenodytes forsteri	. . . R‐Q‐‐GGSK‐‐‐‐LL‐‐‐‐PVGG‐EKR‐‐SGTLGNLAEELNSY‐‐NRKKGGFTFRFGR	128|39|25|D	XP_009285293.1
Pigeon	Columba livia	. . . R‐Q‐‐GGL‐‐P‐‐LL‐‐‐‐PAGG‐EKR‐‐SGTLGDLAEELTGY‐‐NRKKGGFTFRFGR	126|39|25|D	XP_005512965.1
Loon	Gavia stellata	. . . R‐‐‐EGTPLKPS‐‐‐‐‐‐‐WQAAAAR‐‐‐SGTLGNLAEELNGY‐‐NRKKGGFTFRFGR	73|40|25|D	XP_009807973.1
Hornbill	Buceros rhinoceros silvestris	. . . R‐QG‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐EKR‐‐SGTLGNLAEELNGY‐‐NRKKGGFTFRFGR	129|30|25|D	XP_010133912.1
Chuck‐will's widow	Caprimulgus carolinensis	. . . PAAGPXXXXXXXXXX‐‐‐XXXXX‐EKR‐‐SGTLGNLAEELNGY‐‐NRRKGGFTFRFGR	142|      |25|D	XP_010172496.1
Cuckoo	Cuculus canorus	. . . R‐Q‐‐RGSE‐‐‐‐LL‐‐‐‐PVGG‐EKR‐‐SGTLGNLAEELNGY‐‐NRKKGGFTFRFGR	130|39|25|D	XP_009567895.1
Kiwi	Apteryx australis mantelli	. . . R‐‐‐‐‐‐SH‐‐‐FL‐‐‐‐‐PAGG‐EKR‐‐SGTLGDLAEELNSY‐‐DRKKGGFTFRFGR	89|36|25|D	XP_013800043.1
Archosauria/Crocodylia
Alligator	Alligator mississippiensis	. . . R‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐DDG‐EKR‐‐SGVLGSLAKELNGY‐‐NRKKGGFSFRFGRR	126|31|25|D	XP_006260077.1
	Alligator sinensis	. . . R‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐DDG‐EKR‐‐SGVLGSLAEKLNGY‐‐NRKKGGFSFRFGRR	126|31|25|D	XP_006024464.1
Reptilia/Testudinata
Turtle	Chelonia mydas	. . . R‐‐EEE‐GEGVGFL‐‐‐‐R‐GDG‐EKR‐‐SSTLGNLAEELNGY‐‐NRKKGGFSFRFGRR	140|42|25|D	XP_007061178.1
	Chrysemys picta bellii	. . . R‐‐EEE‐GEGVGFL‐‐‐‐R‐GDG‐EKR‐‐SGTLGNLAEELNGY‐‐NRKKGGFSFRFGRR	135|42|25|D	XP_005293537.1
	Pelodiscus sinensis	. . . R‐‐E‐‐‐GEEVGFL‐‐‐‐‐WGDG‐EKR‐‐SGTLGNLAEELNGYSYNRKKGGFSFRFGRR	137|42|27|D	XP_006128457.1
Reptilia/Squamata
Anole	Anolis carolinensis	. . . RREKADDNEAAS‐‐‐‐‐‐‐‐ENG‐EKR‐‐AGGLEGLAEELHGY‐‐SRRKGGFSFRFGRR[18]	134|41|25|D	XP_008123041.1
Cobra	Ophiophagus hannah	. . . RFGRQDADEKPKAIMDLL‐‐‐‐EEGD‐EK‐‐GAGSLETLTEEQNGY‐YKRKKTGISIWFGRR[12]	146|47|27|D	ETE68258.1
Garter snake	Thamnophis sirtalis	. . . R‐‐‐‐‐‐‐‐‐‐‐DL‐‐‐‐‐PEGDGEK‐‐GA‐‐‐‐‐LAEQRDGH‐PNRKKPRFSTWFGRK[22]	172|31|22|D	XP_013928945.1
Python	Python bivittatus	. . . RFGRRDMDENPNAVKDLL‐‐‐‐EEGG‐EK‐‐GASSLEALAEELNGY‐YNGKKAGFSIWFGR[18]	149|46|27|D	XP_007439020.1
Amphibia
Clawed frog	Xenopus tropicalis	. . . RFGR‐QEE‐GNEFEDF‐‐‐‐‐‐EQQDEEKR‐GGTALGSLAEELNGY‐‐NRKKGGFSFRFGRR	138|44|26|D	XP_004916730.2
Teleostei
Grouper	Epinephelus coioides	. . . GEDGG‐KR‐‐NEDLTSIIGGLQAV‐‐SREKGGFGFRFGRK[9]	165|      |25|C	Shu et al., 2014
Stickleback	Gasterosteus aculeatus	. . . EDGG‐‐KR‐‐NEALTSIAGGLQAV‐‐GREKGGIAFRFGKR[7]	155|      |25|D	926 659 430
Yellow croaker	Larimichthys crocea	. . . EDGG‐‐KR‐‐NEALTSIAGGLQAV‐‐SREKGGFGFRFGRK[19]	204|      |25|D	A0A0F8AIW9
Fugu	Takifugu rubripes	. . . RR‐QG‐‐‐‐‐‐‐‐‐‐‐‐‐‐ENGG‐‐KK‐‐NEALTSIAGELQAV‐‐SREKGGFGFRFGRK[13]	155|33|25|C	Mirabeau and Joly, 2013
Pufferfish	Tetraodon nigroviridis	. . . RRRRQ‐G‐‐‐‐‐‐‐‐‐‐‐‐DGGG‐‐RKK‐‐EPLSSMAGGLQAV‐‐SREKGGFGFRFGRR[39]	169|33|24|Ddd Partial sequence.	ALD51297.1
Medaka	Oryzias latipes	. . . GEDG‐WKR‐‐NDALTSIAGGLQAV‐‐SREKGGFGFRFGRK[16]	183|      |25|D	Liu et al., 2009
Tilapia	Oreochromis niloticus	. . . GEDG‐WKR‐‐NDALTSIAGGLQAV‐‐SREKGGFGFRFGRK	163|      |25|D	I3JBY6
Goldfish	Carassius auratus	. . . EEGG‐EK‐‐QNEALTSIAGGLQAF‐‐NRQKGGFGFRFGKK	168|      |26|C	Liu et al., 2009
Zebrafish	Danio rerio	. . . EVGG‐EKR‐‐NEALTSIAGGLQAF‐‐NRQKGGFGFRFGRK	168|      |25|D	Liu et al., 2009
Holostei
Spotted gar	Lepisosteus oculatus	. . . R‐‐DG‐‐PLLLQLVL‐‐‐KPAH‐‐‐KR‐‐ADDLEMLAEELNGY‐‐‐RRKGGFSFRFGRK	144|40|24|D	XP_006640805.1
Branchiostomidae
Amphioxus	Branchiostoma floridae	. . . PAD‐‐‐KR‐‐SAMLQQLA‐QQLKNRP‐REKGGFTFRFGKR[9]	133|      |25|C	Xu et al., 2015
		. . . DRE‐‐‐KK‐‐ALDLSSLA‐QSLRTMG‐SRKAGIILRFGKR[14]	156|      |25|C	Mirabeau and Joly, 2013
		. . . ERE‐‐‐KK‐‐TTRLLSWA‐AEMLRMM‐STKGGFQFRFGKR[8]	143|      |25|C	Mirabeau and Joly, 2013

• a Inside a group, species are arranged alphabetically. It should be noted that taxonomic units do not have equal rank.
• b Dots mark N‐terminal residues. Dash represents gap for maximizing sequence similarity. The number into brackets corresponds to the number of residues after the last noted C‐terminal amino acid. Residues in green, red and blue represent the up‐stream putative processing site, the mature or putative 26RFa sequence and the C‐terminal amidation signal respectively. Residues in orange represent the putative processing site generating the N‐terminal extended form of 26RFa, QRFP. Residues in pink highlight the presence of a FLP just upstream QRFP. Residues identical to the human sequence of 26RFa or to the counterpart C‐teminal region of QRFP are shade‐boxed in tetrapods. The underlined Q corresponds to a pyroglutamic residue in the mature sequence. X, unidentified residue.
• c The number series corresponds to the number of residues in the precursor, in QRFP and in 26RFa respectively. C, cDNA cloning. D, genomic data.
• d Partial sequence.

Figure 1

Alignment of mammalian 26RFa sequences. The data obtained with Weblogo 2.8.2 (weblogo.berkeley.edu) consist of 73 26RFa sequences. A few sequences with rare absence of cleavage site upstream and/or downstream 26RFa (sifaka, sperm whale, whale, Arabian camel, flying‐foxes and myotis) were removed for alignment clarity. The overall height of the stack indicates the sequence conservation at each position, while the height of each symbol within a stack indicates the relative frequency of a given residue at each position.

Primary structure of the 26RFa/QRFP precursor and post‐translational processing

The cDNAs encoding the 26RFa/QRFP precursors have been characterized in various species belonging to diverse vertebrate phyla (Ukena et al., 2014). To date, the 26RFa/QRFP precursor cDNAs have been cloned in goldfish (Carassius auratus), orange‐spotted grouper (Epinephelus coioides), zebra finch (Taeniopygia guttata), Japanese quail (C. japonica), chicken (G. gallus), mouse (Mus musculus), rat (Rattus norvegicus), bovine (Bos taurus) and human (Homo sapiens) (Ukena et al., 2014). Furthermore, homologous sequences have been listed in the genome database of the whole vertebrate phylum, from fish to mammals (Table 1). The size of the predicted precursor is quite variable, from 73 amino acids in the red‐throated loon (Gavia stellata) to 552 amino acids in the Damara mole‐rat (Fukomys damarensis) (Table 1). In all species investigated so far, the 26RFa/QRFP precursors exhibit a similar organization with a predicted 15‐ to 39‐amino acid signal peptide (Petersen et al., 2011) and a 8‐ to more than 450‐amino acid N‐terminal flanking peptide; the 26RFa/QRFP bioactive sequence being generally located at the C‐terminal extremity of the precursor (Table 1). Also the 26RFa/QRFP sequence found in the Florida lancelet ‘amphioxus’ (Branchiostoma floridae), the only invertebrate in which the peptide has been identified so far, displays these features (Mirabeau and Joly, 2013; Xu et al., 2015). The 26RFa/QRFP sequence is followed by a Gly amidation signal and single Arg or dibasic amino acid motifs (Arg–Arg, Arg–Lys, or Lys–Lys) at the C terminus (Table 1). In addition, in a number of species, the 26RFa/QRFP sequence is flanked by one or several amino acids on its C‐terminal side. For instance, in the amphioxus (B. floridae), the spotted green pufferfish (T. nigroviridis) or the green anole (Anolis carolinensis), the bioactive sequence is extended by a 9‐, 13‐ or 18‐amino acid peptide after the amidation signal respectively (Xu et al., 2015; Mirabeau and Joly, 2013; Table 1). These cryptic peptides are as short as one residue, that is, in the goat (Capra hircus) and the dolphin (Lipotes vexillifer) precursors and can reach 211 residues for the Damara mole‐rat (F. damarensis) (Table 1). All 26RFa/QRFP precursors display several mono‐ or dibasic amino acids that constitute potential cleavage sites by prohormone convertases (Artenstein and Opal, 2011; Seidah et al., 2013), but these cleavage motifs have been poorly conserved. For instance, a canonic Lys–Arg/Lys dibasic site is present upstream of 26RFa in amphioxus (B. floridae) (Xu et al., 2015), chicken (G. gallus), Japanese quail (C. japonica) and zebra finch (T. guttata) (Ukena et al., 2011), while a single Lys residue flanks the 26RFa sequence in goldfish (C. auratus), red‐legged seriema (Cariama cristata) and most mammalian species (Leprince et al., 2013; Table 1), and a single Arg residue is present in the saker falcon (F. cherrug) and the brown roatelo (Mesitornis unicolor) precursors. The fact that 26RFa has been purified and sequenced in the European green frog (P. ridibundus) (Chartrel et al., 2003), the Japanese quail (C. japonica) (Ukena et al., 2010), the zebra finch (T. guttata) (Tobari et al., 2011) and in human brain tissues (Bruzzone et al., 2006) indicates that these mono‐ or dibasic cleavage sites are actually recognized by prohomone convertases. In contrast, the precursors of the Arabian camel (Camelus dromaderius), the flying foxes (Pteropus vampyrus and P. alecto), the David's myotis (Myotis davidii), the Coquerel's sifaka (Propithecus coquereli) and the Minke whale (Balaenoptera acutorostrata) are devoid of canonical cleavage sites upstream of the 26RFa sequence suggesting that QRFP is the only mature bioactive peptide in these species (Table 1). Interestingly, in the two latter species, the C‐terminal sequences of QRFP exhibit HFamide and RFGQamide motifs respectively.

In mammals, the QRFP sequence is generally flanked at its N‐terminus by a single Arg residue (Chartrel et al., 2003; Fukusumi et al., 2003; Jiang et al., 2003) that is efficiently cleaved to generate the 43‐amino acid form, at least in rat (Fukusumi et al., 2003; Takayasu et al., 2006) and human (Bruzzone et al., 2006). Indeed, the mature 43‐amino acid residue RFamide peptides were identified from the rat hypothalamus (Takayasu et al., 2006) and from the culture medium of CHO cells which express the human peptide precursor (Fukusumi et al., 2003). In birds, a similar single Arg residue could potentially generate a 34‐amino acid QRFP in chicken (G. gallus) and Japanese quail (C. japonica) (Ukena et al., 2010) and a 42‐amino acid QRFP in zebra finch (T. guttata) (Tobari et al., 2011). However, to date, none of these peptides has been biochemically characterized in birds. It should also be noted that this single Arg residue upstream of QRFP is not present in the amphioxus (B. floridae) (Xu et al., 2015), the goldfish (C. auratus), the Japanese medaka (Oryzias latipes), the zebrafish (Danio rerio) (Liu et al., 2009), the three‐spined stickleback (Gasterosteus aculeatus), the Nile tilapia (Oreochromis niloticus) and the yellow croaker (Larimichthys crocea) 26RFa/QRFP precursors.

In humans, a 9‐amino acid FLP is present upstream of QRFP (Chartrel et al., 2003; Jiang et al., 2003). This peptide, which is delimited by single Arg residues at both its N‐ and C‐terminal extremities, shares the –Phe–Arg–NH₂ sequence with 26RFa/QRFP. A similar 7‐ to 16‐residue long RFamide peptide is found in the 26RFa/QRFP precursors of several species including horse (Equus caballus), pygmy chimpanzee (Pan paniscus) and clawed frog (Xenopus tropicalis) but not in other species (Liu et al., 2009; Ukena et al., 2014; Tables 1 and 2). It should be mentioned however that this peptide, termed 9RFa (Chartrel et al., 2003) or P51 (Jiang et al., 2003), has not been characterized in human (Bruzzone et al., 2006) or frog brain extracts (Chartrel et al., 2003).

Table 2. Sequence alignment of residues upstream the N‐terminal cleavage site of QRFP

Speciesaa Inside a group, species are arranged alphabetically. It should be noted that taxonomic units do not have equal rank.		Sequencebb Dots mark N‐terminal residues. Dash represents gap for maximizing sequence similarity. Residues in green, red and blue represent monobasic putative processing site, dibasic putative processing site and the C‐terminal amidation signal respectively. Residues identical to the human sequence are shade‐boxed in tetrapods.	Reference or accession number
Mammalia
Human	Homo sapiens	. . . R‐PHSVWGSSRWLRAS‐QPQALLVIARGLQTSGR‐EHAGCRFRFGR	Chartrel et al., 2003; Fukusumi et al., 2003; Jiang et al., 2003
Chimpanzee	Pan paniscus	. . . R‐PHSVWGSSRWLRAS‐QPQALLVIARGLQTSGR‐EHAGCRFRFGR	XP_008974188.2
Gorilla	Gorilla gorilla gorilla	. . . R‐PHSVWGSSRWLRAS‐QPQALLVIARGLHTSGR‐EHAGCRFRFGR	XP_004048799.1
Orangutan	Pongo abelii	. . . R‐PHSVWGSSRWLRAS‐QPQALLVIARGLQTSGR‐EHAGCRFRFGR	XP_002833229.1
Gibbon	Nomascus leucogenys	. . . R‐PHSVWGSSRWLRAS‐QPQALLVITRGLQTSGR‐EHAGCRFRFGR	XP_003276794.2
Baboon	Papio anubis	. . . R‐PHSVWGSSRWLRAS‐QPQALLVIARGLQTSGR‐EHAGCRFRFGR	XP_009186264.1
Colobus	Colobus angolensis palliatus	. . . R‐SHSVWGSSRWLRAS‐QPQTLLVTAWGLQTSGR‐EHAGCRFRFGR	XP_011786583.1
Drill	Mandrillus leucophaeus	. . . R‐PHSVWGSSRWLRAS‐QPQALLVIARGLQTSGR‐EHAGCRFRFGR	XP_011826536.1
Macaque	Macaca nemestrina	. . . R‐PHSVWGSSRWPRAS‐QPQALLVITRGLQTSGR‐EHAGCRFRFGR	XP_011709965.1
	Macaca fascicularis	. . . R‐PHSVWGSSRWPRAS‐QPQALLVITRGLQTSGR‐EHAGCRFRFGR	XP_005580679.1
Mangabey	Cercocebus atys	. . . R‐PHSVWGSSRWLRAS‐QPQALLVIARGLQTSGR‐EHAGCRFRFGR	XP_011894201.1
Monkey	Clorocebus sabaeus	. . . R‐PHSVWGSSRWLRAS‐QPQALLVIARGLQTSGR‐EHAGCRFRFGR	XP_008004106.1
Tarsier	Tarsius syrichta	. . . R‐SHIMWGSSRWPRAP‐RPQALLVIAKELRTLGR‐EHAGFRFRFGR	XP_008054832.1
Sifaka	Propithecus coquereli	. . . H‐PHSMWGSSRWSKDP‐RPQALLVEAKGLWTMGR‐EHAGFRFRFGR	XP_012495100.1
Rabbit	Oryctolagus cuniculus	. . . ‐‐PHLARGTSRGLRAP‐AHGPLLVLARQLQTWGQ‐EHTGFHFRFGR	XP_008250432.1
Flying‐lemur	Galeopterus variegatus	. . . ‐‐PHFVW‐SSEWPRAP‐QPQALLFMAKELQTSGR‐EHAGFRFRFGR	XP_008588732.1
Pig	Sus scrofa	. . . R‐‐HFLWGSPGWQRAP‐HPGAWLIMAKELQAVGR‐ARASFGLRFGR	XP_003122292.1
Alpaca	Vicugna pacos	. . . R‐‐‐FPWGFPGWPRVQ‐HPLA‐‐‐‐‐EELQTTGR‐GRAGFKLRFGR	XP_006216676.1
Camel	Camelus dromedarius	. . . RR‐‐FPWGSPGWPRVQ‐HPLALLVMAEELQTTGRGGRAGFKLRFGR	XP_010982517.1
	Camelus bactrianus	. . . RR‐‐FPWGSPGWPRVQ‐HPLALLVMAEEPQTTGRGGRAGFKVRFGR	XP_010954179.1
	Camelus ferus	. . . RR‐‐FPWGSPGWPRVQ‐HPLALLVMAEELQTTGRGGRAGFKLRFGR	XP_006181990.1
Walrus	Odobenus rosmarus divergens	. . . RVP‐LPRGSSPWPRAP‐QPHGLLVMAKELQTSGR‐QRAGFRFRFGR	XP_004392513.1
Seal	Leptonychotes weddellii	. . . RVP‐LPRGSSRWPRAP‐QPHGLLVMAKELQTSGQ‐QHAGFRFRFGR	XP_006746182.1
Ferret	Mustela putorius furo	. . . R‐MLPPRGSPGWPGAP‐QPHGLLVMAKELQVSGQ‐RRPGFTFRFGR	XP_012914087.1
Dog	Canis lupus familiaris	. . . RAP‐LPRGSSRWLRAP‐HPHGLLVTAKELQMSGR‐QRAGFRFRFGR	XP_850751.2
Cat	Felis catus	. . . RVS‐LPRGSPRRPRAP‐HPLGPPVTAKELQMSGR‐QRAGFRVRFGR	XP_006939618.1
Tiger	Panthera tigris altaica	. . . RVS‐LPRGSPRRPRAP‐HPLGPLVTAKELQTSGR‐QRAGFRVRFGR	XP_007078896.1
Bat	Eptesicus fuscus	. . . RAVHLPW‐PPPWPRAP‐RPHALLVTAKELQVSGR‐ARAGFGLRFGR	XP_008150734.1
Flying‐fox	Pteropus alecto	. . . RPARH‐‐‐SRGAADV‐RQRACRCQVRFGR	ELK07292.1
Hedgehog	Erinaceus europaeus	. . . H‐‐‐FSWGPSRWRRAP‐APPTLLMVAKELQTSGR‐ERAGFRFRFGR	XP_007530369.1
Horse	Equus caballus	. . . RV‐HSLWGSSPWPRAP‐QSPALLVTAKELQAAGR‐ERAGFRFRFGR	XP_001499477.1
	Equus przewalskii	. . . RV‐HSLWGSSPWPRAP‐QSPALLVTAKELQAAGR‐ERAGFRFRFGR	XP_008536129.1
Rhinoceros	Ceratotherium simum simum	. . . RV‐HSRWGSSPWPRAP‐HSPALLVTAEELQTSGS‐QRAGFRFRFGR	XP_004423643.1
Aardvark	Orycteropus afer afer	. . . R‐PNFVWGSSRQPRAP‐QPLALLVIAKELPS‐‐‐‐‐‐ASFRFRFGR	XP_007942537.1
Elephant	Loxodonta africana	. . . R‐PNFRWGSSWQPRAP‐QPLALLVVAKELQTSGK‐ERAGFRFRFGR	XP_003407599.1
Elephant‐shrew	Elephantulus edwardii	. . . R‐PDLRWGSSRQPRAP‐QPLALLVIAKELQTSNK‐EHAGFRFRFGR	XP_006892510.1
Manatee	Trichechus manatus latirostris	. . . R‐PNFVWGSSRQPRAP‐QPLALLVIAKELQTSGK‐ERAGFRFRFGR	XP_004375898.2
Mole	Chrysochloris asiatica	. . . R‐PNFAWGSSQQPRAPPESLALLIIAKELPTSDQ‐ESASFRFRFGR	XP_006835052.1
Armadillo	Dasypus novemcinctus	. . . HQPNFLWGSSQWLTAP‐LPHSLLMPAKELQTSGK‐EPAAFRLRFGR	XP_004463118.1
Opossum	Monodelphis domestica	. . . HWPGFLKGTPKWKRFQPDS‐SLFVMAKKLQGLGK‐ERAGFRFRFGR	XP_007475223.1
Platypus	Ornithorhynchus anatinus	. . . SPPGWPWGASTQKK‐‐‐DPNPLFGLAKEPQGFGK‐ERASFGFRFGR	XP_007660564.1
Reptilia/Squamata
Cobra	Ophiophagus hannah	. . . EPGFGEAGRTEWRRSSVGLNPLFSIAKELQSFGK‐EKAGIQFRFGR	ETE68258.1
Python	Python bivittatus	. . . DPGLWETPSLKWRRSPMDLSSLFSIAKELQSFGK‐EKAGIQFRFGR	XP_007439020.1
Amphibia
Clawed frog	Xenopus tropicalis	. . . WYP‐LA‐‐‐PRQRKST‐DPASLFSVAKELQGFGK‐ERAGFRFRFGR	XP_004916730.2

• a Inside a group, species are arranged alphabetically. It should be noted that taxonomic units do not have equal rank.
• b Dots mark N‐terminal residues. Dash represents gap for maximizing sequence similarity. Residues in green, red and blue represent monobasic putative processing site, dibasic putative processing site and the C‐terminal amidation signal respectively. Residues identical to the human sequence are shade‐boxed in tetrapods.

26RFa/QRFP gene

The 26RFa sequence has been used for the cloning of the human and rat 26RFa/QRFP precursor cDNAs (Chartrel et al., 2003). Concurrently, a bioinformatic search in human genome databases has led to the identification of the 26RFa/QRFP gene (Fukusumi et al., 2003). The 26RFa/QRFP gene (farp‐5) is the most recently discovered FLP gene (farp‐1 to −5) in mammals (see 2.1 section) and, as predicted (Fukusumi et al., 2006), no new mammalian FLP gene has been reported since the identification of farp‐5.

Comparison of the different human FLP gene structures reveals that the DNA sequence encoding the 26RFa/QRFP preproprotein is not interspersed by introns, while those of other genes (farp‐1 to −4) display 1 or 2 introns (Figure 2). It should be noted, however, that, in the amphioxus (B. floridae), the 26RFa/QRFP gene exhibits an intron in the DNA sequence encoding the preprotein (Xu et al., 2015), suggesting that intron gain and loss have occurred in farp‐5 during species diversification, but the driving mechanisms behind intron gain and loss in the vertebrate genomes are unclear. Finally, the farp‐1‐5 genes are located on different chromosomal loci (Figure 2). Altogether, these observations support the notion that the 26RFa/QRFP gene diverged relatively early during evolution (see 2.4 section).

Figure 2

Comparison of the structures of the five human RFamide peptide genes and their chromosome localization. NCBI reference sequences of farp‐1 to ‐5 are NM_003717.3 (NPFF, NPAF), NM_015893.1 (PrRP), NM_022150.3 (RFRP‐1/GnIH, RFRP‐3), NM_002256.3 (metastin/kisspeptins) and NM_198180.2 (26RFa/QRFP) respectively. Gene loci are indicated on the right.

A single exon of the human 26RFa/QRFP gene encodes a preproprotein with 136 amino acid residues (Figure 3). This preproprotein consists of an N‐terminal signal peptide with 18 hydrophobic amino acid residues, potential cleavage sites with arginine or lysine residues, and a C‐terminal RFGRR motif which is the typical progenitor of RFamide peptides. Several mature peptides have been isolated and characterized, including human QRFP with 43 amino acid residues (Fukusumi et al., 2003), frog 26RFa with 26 amino acid residues (Chartrel et al., 2003) and the avian 26RFa ortholog with 25 amino acid residues (Tobari et al., 2011).

Figure 3

Alignment of the amino acid sequences of the human QRFP precursor protein (deduced from the corresponding cDNA), of purified 26RFa from avian (zebra finch), and of purified 26RFa from amphibian (European green frog). The putative signal peptide sequence is designated by the upper line, and the sequence of 26RFa is underlined. Potential cleavage sites are marked by stars. The N‐terminal residue of human QRFP is arrowed. The amino acids of human precursor are numbered on the right. Fully conserved amino acids are highlighted with black box and frequently conserved amino acids with grey boxes respectively.

Molecular evolution of the 26RFa/QRFP gene family

Because a 26RFa/QRFP gene has been identified in amphioxus (B. floridae) (Mirabeau and Joly, 2013; Xu et al., 2015), it is obvious that the gene existed before even the first of the two tetraploidizations (genome doublings) that gave rise to the vertebrate lineage (Nakatani et al., 2007). Nevertheless, all vertebrates so far investigated seem to display a single QRFP gene, implying that the duplicates must have been lost. Likewise, no duplicate seems to have survived the third tetraploidization in the teleost ancestor. It remains to be investigated in detail whether lineages or species that have undergone additional independent tetraploidizations have retained any duplicates (Xenopus laevis, salmonids, cyprinids, sturgeons, paddlefish, etc.). Thus, 26RFa/QRFP appears to be a single‐member ‘family’ in the vertebrates, possibly with the reservation for some recent duplicates in some lineages. The lack of duplicates appears somewhat suprising in consideration of the receptor situation with (at least) four receptor subtypes in the vertebrate ancestor (see below).

In the light of the absence of QRFP duplicates in vertebrates, it appears almost ironic that no less than three QRFP‐like peptides have been identified in amphioxus (Mirabeau and Joly, 2013; Table 1). However, apart from the peptide used in the functional receptor study (Xu et al., 2015), the other two QRFP‐like peptides raise serious questions of structure and function, because their signal peptides surprisingly are almost identical although the propeptides differ considerably outside the mature QRFP regions, as noted by Xu et al. (2015). Clearly, further studies are necessary to ascertain that these genes actually give rise to functional QRFP‐like mature peptides.

Distribution of 26RFa/QRFP in the CNS

26RFa was initially isolated and characterized from extracts of whole brain from frogs (Chartrel et al., 2003) indicating that the 26RFa/QRFP gene is expressed in the CNS. The tissue distribution of 26RFa/QRFP mRNA has been investigated in rat, mouse and human by RT‐PCR. In all three species, the highest concentrations of 26RFa/QRFP transcript are found in the brain, notably in the diencephalon (Fukusumi et al., 2003; Jiang et al., 2003; Kampe et al., 2006; Takayasu et al., 2006). In situ hybridization studies have shown that the 26RFa/QRFP gene is primarily expressed in the hypothalamus, specifically in the ARC, the ventro‐median nucleus (VMH), the lateral hypothalamus (LHyp) and the retrochiasmatic areas (Chartrel et al., 2003; Fukusumi et al., 2003; Kampe et al., 2006; Takayasu et al., 2006; Figure 4A). Immunohistochemical labelling has revealed that, in the human hypothalamus, 26RFa/QRFP‐immunoreactive neurons are present in the dorsal and medial aspects of the paraventricular nucleus (PVN) along the third ventricle (Bruzzone et al., 2006). Similarly, in the ventromedial hypothalamic nucleus, 26RFa/QRFP‐positive neurons are gathered along the edge of the third ventricle (Bruzzone et al., 2006). The occurrence of 26RFa/QRFP mRNA has also been detected in the human spinal cord (Bruzzone et al., 2006). Immunoreactive neurons are mainly located in the dorsal horn and, to a lesser extent, in the lateral horn of the spinal cord (Bruzzone et al., 2006; Figure 4B).

Figure 4

Distribution of 26RFa/QRFP, QRFP receptor 1 and QRFP receptor 2 in the central nervous system. (A) Parasagittal section of the rat brain and pituitary depicting the localization of 26RFa/QRFP (), QRFP receptor 1 () and QRFP receptor 2 () mRNAs. Acb, accumbens nucleus; Ambg, nucleus ambiguus; Amy, amygdala; BST, bed nucleus of the stria terminalis; C, cerebellum; CC, corpus callosum; CPut, caudate putamen; Cx, cerebral cortex; DBB, horizontal limb of the diagonal band of Broca; DG, dentate gyrus; DMH, dorsomedian hypothalamus; DR, dorsal raphe nucleus; GP, globus palidus; Hi, hippocampus; Hpt, hypothalamus; Hyp, pituitary; LC, locus cœruleus; LRN, lateral reticular nucleus; ME, median eminence; MPA, median preoptic area; NST, nucleus of the solitary tract; OB, olfactory bulb; PAG, periaqueducal grey; PBN, parabrachial nucleus; PVN, paraventricular nucleus; Rch, retrochiasmatic area; S, septum; Sch, suprachiasmatic nucleus; SN, substantia nigra; SON, supraoptic nucleus; Th, thalamus; VP, ventral globus pallidus; VR, ventral raphe nucleus. (B) Coronal section of the human spinal cord depicting the localization of 26RFa/QRFP‐like immunoreactivity and/or 26RFa/QRFP mRNA (), QRFP receptor mRNA and/or 26RFa‐binding sites (). Cc, central canal; DH, dorsal horn; LHo, lateral horn; VH, ventral horn; WM, white matter. (Chartrel et al., 2003; Fukusumi et al., 2003; Bruzzone et al., 2006, 2007; Kampe et al., 2006; Takayasu et al., 2006).

In the brain of birds, the 26RFa/QRFP gene is exclusively expressed in the diencephalon (Ukena et al., 2010). Thus, in quail (C. japonica) and chick (G. gallus), 26RFa/QRFP mRNA‐containing cells are found only in the anterior hypothalamic nucleus and the distribution of 26RFa‐immunoreactive neurons totally matches with that of the 26RFa/QRFP mRNA‐expressing perikaria (Ukena et al., 2010).

In the goldfish (C. auratus) brain, RT‐PCR analysis indicates that 26RFa/QRFP mRNA is abundant in the hypothalamus and optic tectum (Liu et al., 2009). However, the precise distribution of 26RFa/QRFP‐expressing neurons has not yet been determined in fish.

Distribution of 26RFa/QRFP in peripheral organs

In rodents, the 26RFa/QRFP gene is highly expressed in the eye, trachea, mammary gland and testis (Fukusumi et al., 2003; Jiang et al., 2003; Takayasu et al., 2006). Moderate expression also occurs in the thymus, salivary gland, duodenum and uterus (Fukusumi et al., 2003). In humans, the 26RFa/QRFP gene is expressed in endocrine glands including the pituitary, thyroid and parathyroid glands, and testis, as well as in the coronary artery, large intestine, bladder and prostate (Jiang et al., 2003), indicating the existence of species differences in the distribution of 26RFa/QRFP in peripheral tissues.

In the human pancreas, preproQRFP mRNA and 26RFa‐like immunoreactivity are found in endocrine islets (Prévost et al., 2015). PreproQRFP mRNAs are also expressed in rat INS‐1E beta cells (Granata et al., 2014) and in mouse insulinoma MIN6 cells (Prévost et al., 2015).

QRFP mRNA is expressed in the human adrenal gland (Fukusumi et al., 2003; Ramanjaneya et al., 2013). The presence of QRFP‐like immunoreactivity has been confirmed in all zones of the cortex in both fetal and adult human and rat adrenal glands (Ramanjaneya et al., 2013). Notably, in the human fetal adrenal, the fetal zone is intensively immunostained, whereas in the definitive zone, staining intensity is lower than in fetal zone cells (Ramanjaneya et al., 2013). In the human adult and rat adrenal cortex, immunostaining is particularly intense in the zona fasciculata and reticularis (Ramanjaneya et al., 2013).

In the human prostate, intense expression of QRFP mRNA is observed, the expression level being higher in the prostate than in the hypothalamus (Jiang et al., 2003).

26RFa/QRFP in tumour cells

26RFa/QRFP‐immunoreactive material is present in carcinomatous foci of human benign prostate hyperplasia and prostate tumours at different stages (Alonzeau et al., 2013). Interestingly, the proportion of 26RFa/QRFP‐like immunoreactive cancer cells is significantly correlated to the severity of prostate tumour (Alonzeau et al., 2013). The QRFP/26RFa gene is expressed in several human prostatic cell lines including the androgen‐responsive cell line LNCaP and the androgen‐nonresponsive cell lines DU145 and PC3 (Alonzeau et al., 2013). 26RFa/QRFP mRNA is also expressed in the human adrenal corticocarcinoma cell line H295R (Ramanjaneya et al., 2013), suggesting the potential value of 26RFa/QRFP as prognostic markers for various types of cancers.

26RFa/QRFP knockout

QRFP (farp‐5) gene deficient mice (QRFP^−/−) have been generated by homologous recombination in ES cells of the 129SvJ strain and implanted in C57 blastocyts (Okamoto et al., 2016). QRFP^−/− mice are hypophagic and lean and display increased anxiety‐like behaviour, decreased wakefulness time during the dark period and reduced locomotor activity (Okamoto et al., 2016; see 4.1 section).

Secondary structure of QRFP peptides

The secondary structure of human 26RFa (h26RFa) has been predicted using the Agadir program, an algorithm based on the helix/coil transition theory (Muñoz and Serrano, 1997). The sequence spanning from residues Leu⁸ to Gly²⁰ of h26RFa displays a helical tendency (Thuau et al., 2005). Circular dichroism (CD) spectra reveal that, in water, h26RFa has a disordered structure. In trifluoroethanol/H₂O or MeOH/H₂O solvents, h26RFa exhibits patterns characteristic of a mixture of α‐helical and random conformations with a positive band at 195 nm and negative bands around 210 and 220 nm (Thuau et al., 2005). Conformational analysis of h26RFa in water and methanol has been performed by two‐dimensional NMR spectroscopy and restrained molecular dynamics (Thuau et al., 2005). Consistent with the CD spectra, h26RFa encompasses mainly a random coil conformation in water although a nascent helix occurs between residues 6 and 15. In methanol, h26RFa adopts a well‐defined conformation consisting of an amphipathic α‐helical structure (Pro⁴–Arg¹⁷) flanked by two N‐ and C‐terminal disordered regions (Thuau et al., 2005). Of note, NMR studies have shown that the structures of NPAF and PrRP also present an α‐helix and a flexible region (d'Ursi et al., 2002; Miskolzie and Kotovych, 2003). The Agadir program predicts the same organization for RFRP‐1 and RFRP‐3 (Thuau et al., 2005). In particular, RFRP‐3 shares with h26RFa, NPAF and PrRP a central helix flanked by two flexible regions, suggesting a similar ligand–receptor recognition process.

Consistent with the existence of a C‐terminal disordered region in h26RFa, the CD spectrum of 26RFa_(20–26) in water displays a negative band around 200 nm and a moderate positive maximum at 218 nm, suggesting a random coil structure (Neveu et al., 2012). Similarly, NMR structure of the heptapeptide in water (<10% DMSO) exhibits a high degree of conformational flexibility, that is, the peptide backbone mainly adopts an extended conformation (β‐strand‐like) with the hydrophobic side chains (Phe²², Phe²⁴ and Phe²⁶) grouped together on one side of the molecule, the hydrophilic residues (Ser²³ and Arg²⁵) being located on the opposite site (Georgsson et al., 2014). However, in dodecylphosphocholine (DPC) micelles, CD spectrum of 26RFa_(20–26) shows the presence of a turn motif. Further investigations in eukariotic membrane mimetic media are required to precisely characterize the type of turn motif occurring in 26RFa_(20–26) and possibly in the C‐terminal part of 26RFa, in order to rationally design novel GPR103 agonists.

26RFa/QRFP receptor(s)

Cloning and characterization of the QRFP receptor and isoforms

The QRFP receptor belongs to the class‐A or rhodopsin‐like receptor family that represents the largest group of GPCRs (Alexander et al., 2015a). GPR103 (AF411117) was first identified as an orphan GPCR in the human hypothalamus by searching EST and high throughput genome sequencing databases with GPCR‐encoding motifs (Lee et al., 2001). The sequence originally isolated does not code for a typical seven transmembrane GPCR since it has an incorrect assembly at the 5′ end with an insertion of 154 bp which has high homology with mitochondrial DNA, and a 113‐bp deletion at the exon4/intron4 junction. Later, a new GPCR called AQ27 or SP9155 was isolated from a human hypothalamus cDNA library and turned out to be similar to GPR103 and closely related to various peptide receptors including NPFF1 (49% amino acid identity), NPFF2 (48%), OX₁ (48%) and OX₂ (47%) receptors (Jiang et al., 2003). GPR103, or AQ27/SP9155, was subsequently deorphanized independently by two groups using bioinformatic identification of novel RF‐amide peptides and found to be activated by QRFP, 26RFa and shorter segments (Fukusumi et al., 2003; Jiang et al., 2003). Based on this finding, GPR103 was renamed QRFP receptor by the Human Genome Organization Gene Nomenclature Committee (Bonner et al., 2016). Human QRFP receptor exhibits several typical motifs of class‐A GPCRs: LxxxD^2.50 in transmembrane (TM) domain 2, ER^3.50H in TM3 instead of the classical E/DR^3.50Y, CWxP^6.50 in TM6, NP^7.50xxY in TM7 and a putative cystine linkage between TM3 and extracellular loop 2. The Trp residue of the canonical toggle switch CWxP^6.50 and the F^5.47 on TM5 are thought to establish an aromatic locking interaction which holds the Trp moiety in a position that allows movement of the TM helices to promote receptor activation (Schwartz et al., 2006). While humans possess only one receptor for 26RFa/QRFP, two isoforms have been described in rodents and named QRFP receptor 1 and QRFP receptor 2 (Kampe et al., 2006; Takayasu et al., 2006). QRFP receptor 1, the isoform with the highest similarity to human QRFP receptor, was isolated by RT‐PCR from mouse (NP_937835) and rat (NP_937842) brain poly(A)+ RNA (Fukusumi et al., 2003; Jiang et al., 2003). Rat QRFP receptor 1 shares 96 and 84% amino acid identity with the mouse and human homologues respectively. Interestingly, QRFP receptor 1 is localized on chromosome 3 in mouse and chromosome 2 (2q25) in rat, while the human orthologue is on chromosome 4 (4q27). Another QRFP receptor was identified in rat (NP_001102709 or EDL91598, Kampe et al., 2006) and mouse (AX665940, Takayasu et al., 2006) and named QRFP receptor 2. Rat QRFP receptor 2, isolated from a rat thalamus cDNA library, shares 82 and 78% amino acid identity with human QRFP receptor and rat QRFP receptor 1, respectively, and is localized on chromosome 4 (4q31). Mouse QRFP receptor 2, that was isolated from mouse brain RNA, shares 79 and 75% amino acid identity with human QRFP receptor and mouse QRFP receptor 1, respectively, and is localized on chromosome 6. Both QRFP receptor 1 and QRFP receptor 2 have been also identified in other rodents such as the Chinese hamster (Cricetulus griseus, XP_007614055.1, ERE66087.1), the golden hamster (Mesocricetus auratus, XP_005069758.1, XP_005076165.1), the prairie vole (Microtus ochrogaster, XP_005360686.1) and the deer mouse (Peromyscus maniculatus bairdii, XP_006971161.1, XP_006991698.1) (Ukena et al., 2014). An evolutionary analysis of the QRFP receptor family indicated that this duplication happened early in the rodent lineage (Larhammar et al., 2014). In vertebrates, two additional receptor family members have been reported (Ukena et al., 2014), and parallel analyses showed that no less than four receptor subtypes exist in vertebrates, representing four ancestral vertebrate receptor lineages, see below (Larhammar et al., 2014).

Structural model of QRFP receptor and peptide docking

Human QRFP receptor exhibits 26% sequence homology with the β₂‐adrenoceptor. Thus, a 3D molecular model of the human QRFP receptor has been built from the X‐ray structure of the β₂‐adrenoceptor as a class‐A receptor scaffold (Neveu et al., 2014). In addition to the seven helical TM domains, the human QRFP receptor model, like the β₂‐adrenoceptor template, displays one extracellular (Ile¹⁸⁸–Lys¹⁹⁶) and one intracellular (Glu³³⁷–Val³⁴⁷) helices (Figure 5) which are generally involved in the first step of the ligand recognition process (Dror et al., 2011; González et al., 2011; Kruse et al., 2012) and in G‐protein binding (Katragadda et al., 2004) respectively. The TM helix bundle defines a narrow and deep orthosteric binding pocket on the extracellular side of the human QRFP receptor for binding endogenous and synthetic ligands (Neveu et al., 2014). The bioactive C‐terminal octapeptide 26RFa_(19–26), KGGFSFRF‐NH₂, has been docked in the human QRFP receptor model, and the ligand–receptor complex has been submitted to energy minimization. In the most stable complex, the –Phe–Arg–Phe–NH₂ motif is found to be oriented inside the receptor cavity whereas the N‐terminal Lys¹⁹ points outside, suggesting that the amphipathic α‐helix of 26RFa (see 2.9 section) remains at the cell surface (Neveu et al., 2014). Among the repertoire of interactions between 26RFa_(19–26) and the human QRFP receptor, the strongest intermolecular contact is predicted between the Arg²⁵ residue of the peptide and the Gln¹²⁵ residue located in the TM3 helix of the receptor (Neveu et al., 2014), which is known to play a key role in the structure and function of class‐A GPCR (Venkatakrishnan et al., 2013). Similar ligand–receptor binding interactions have been reported between two insect RFamide peptides, sulfakinin and dromyosuppressin, and their respective receptors (Bass et al., 2014; Rasmussen et al., 2015).

Figure 5

Structure of the final model of the human QRFP receptor monomer after energy minimization (PDB entries: Q96P65 (QRFP receptor) and 2RH1 (β₂‐adrenoceptor), resolution: 2.4 Å, sequence identity: 26%) (Neveu et al., 2014). TM and nonTM (upper: extracellular loop, down: intracellular loop) helices are shown as ribbons in red and orange respectively. Structure was examined using MacPyMOL (v1.74).

Molecular evolution of the QRFP receptor family

When the analysis of QRFP receptor was extended beyond mammals, it became clear that additional ancient, that is, early vertebrate, QRFP receptor lineages could be identified. One study used sequence‐based phylogenetic analyses to suggest that three distinct receptors existed in early vertebrates (Ukena et al., 2014). An independent analysis combined sequence‐based phylogenies with comparison of chromosomal locations in multiple vertebrate species and concluded that as many as four receptor subtypes existed at the origin of vertebrates (Larhammar et al., 2014). The phylogenetic analysis separated these four subtypes into two major clades, one consisting of QRFP receptor 1–3 and the other consisting of the gene named QRFP receptor 4 (Figure 6). Before the two vertebrate tetraploidizations (Nakatani et al., 2007), the vertebrate predecessor had two QRFP receptor genes, the ancestors of QRFP receptor 1–3 and QRFP receptor 4, respectively, whereupon the genome doublings led to triplication of one of these (QRFP receptor 1–3), whereas the other ancestral receptor (QRFP receptor 4) must have lost all duplicates and remained single (Larhammar et al., 2014).

Figure 6

Gene duplication events for the QRFP receptor family in early vertebrate evolution including local duplications and the two basal vertebrate tetraploidizations. NPYR, NPY receptors. Crosses mark gene losses. The human Y6 gene is a pseudogene. Numbers under gene boxes show map positions along the chromosomes in megabase pairs. Modified from Larhammar et al. (2014).

Interestingly, no single species has yet been found that has retained all four of these ancestral vertebrate QRFP receptor genes. Not even the coelacanth (Latimeria chalumne) and the basal non‐teleost rayfinned fish, the spotted gar (L. oculatus), which often have kept more of the ancestral vertebrate tetraploidization duplicates, were found to have kept more than three genes: the coelacanth has suptypes 1, 2 and 4, and the spotted gar has subtypes 2, 3 and 4 (Larhammar et al., 2014; Figure 6).

The teleost fish tetraploidization is not known to have duplicated the QRFP peptide gene, which makes it less surprising that very few receptor duplicates have survived. Of the species studied, only QRFP receptor 3 in zebrafish (D. rerio) is present in duplicate as QRFP receptor 3a and QRFP receptor 3b (Larhammar et al., 2014). Whether these have undergone any sub‐ and/or neo‐functionalization remains to be explored.

Nevertheless, it is indeed striking that a single known peptide ligand had as many as four receptor subtypes at an early point in vertebrate evolution. This imbalance between peptide and receptor number may explain why receptor gene losses continued to take place differentially in the vertebrate lineages (Larhammar et al., 2014). Of these, human and birds seem to have been most severely affected, having retained only QRFP receptor 1 (Figure 6), as there are two receptors, QRFP receptor 1 and QRFP receptor 2, in rodent, reptilian and amphibian species (Larhammar et al., 2014). The roles of the other three subtypes in those lineages which still have them will be interesting to investigate, as this may indicate if some QRFP functions have been lost in human and birds or taken over by the QRFP receptor 1 subtype (or other peptide‐receptor systems). Of note, in the Tianfu meat goose (Anser cygnoides), five QRFP receptor variants including the full‐length form and four alternatively spliced variants have been identified, and these variants exhibit differential tissue expression patterns (Xiao et al., 2014).

Beyond the QRFP receptor family, the closest relatives are receptors for other RFamide or RYamide peptides. Several of these receptor genes are located in the same chromosomal regions as the QRFP receptors, such as the NPY receptor family (Larhammar et al., 2014) and the NPFF and PRL receptor families (Yun et al., 2015). These observations suggest that the ancestors of the different receptor families arose by local duplications before the first vertebrate tetraploidization. Then, the two tetraploidizations multiplied these ancestral receptor genes, whereupon several were lost. It thus appears that these RFamide peptide‐receptor systems may have already been established before the origin of the vertebrates.

Signalling mechanisms

As all seven TM‐spanning receptors, the QRFP receptor acts via heterotrimeric guanine nucleotide regulatory proteins (G proteins). Initial studies conducted in native cells indicated that 26RFa provokes a dose‐dependent increase in cAMP production in cultured rat anterior pituitary cells preincubated with forskolin, suggesting that the QRFP receptor is primarily coupled to adenylyl cyclase (AC) through a stimulatory Gα subunit (Gα_s) (Chartrel et al., 2003; Figure 7). The involvement of AC in QRFP receptor signalling has been confirmed in INS‐1E beta cells and in human islet cells (Granata et al., 2014). Interestingly, in the two latter cell types, 26RFa exerts an insulinostatic effect, whereas QRFP displays an insulinotropic effect through inhibitory Gα subunit (Gα_i/o) and Gα_s signalling, respectively (Granata et al., 2014; Figure 7), suggesting the possibility of a Gα_i/o‐Gα_s switch of for the QRFP receptor, as previously reported for the V₂ vasopressin receptor (Ren et al., 2005) and the gonadotropin‐releasing hormone (GnRH) receptor (Krsmanovic et al., 2003). In the perfused rat pancreas, 26RFa reduces glucose‐stimulated insulin secretion (GSIS) via a Pertussis toxin‐sensitive Gα_i protein negatively coupled to AC (Egido et al., 2007; Figure 7). Similarly, in human H295R adrenocortical cells, activation of the QRFP receptor by QRFP leads to a decrease in forskolin‐evoked cAMP production (Ramanjaneya et al., 2013).

Figure 7

Signalling pathways associated with QRFP receptors after 26RFa/QRFP activation in different cells. Akt, protein kinase B; CREB, cAMP response element‐binding protein; HSL, hormone‐sensitive lipase; OXR, orexin receptor; P, phosphorylation; PDE3B, phosphodiesterase 3B; SRC, sarcoma kinase.

The QRFP receptor is also coupled to Gα_q/11, leading to activation of the MAPK pathways. In particular, in H295R cells, QRFP causes calcium influx from the extracellular space via mibeframil‐sensitive T‐type voltage‐operated Ca²⁺ channels, leading to PKC activation and, subsequently, to phosphorylation of ERKs 1/2 (Ramanjaneya et al., 2013). In INS‐1E beta cells, QRFP also stimulates phosphorylation of ERK 1/2 (Granata et al., 2014; Figure 7). Similarly, in the QRFP receptor‐OX receptor functional heterodimer, QRFP, like orexin‐A or orexin‐B, induces ERK 1/2 phosphorylation (Davies et al., 2015).

In HEK‐293 cells transfected by the chicken QRFP receptor (Ukena et al., 2010) or the human QRFP receptor (Takayasu et al., 2006), 26RFa increases intracellular calcium concentration ([Ca²⁺]_i).

It thus appears that QRFP receptors, like most GPCRs, displays multiple signalling pathways resulting from multiple G protein couplings (for review, see Perez and Karnik, 2005) that might account for the versatile activities of 26RFa/QRFP. Further studies should help to elucidate which particular Gα subunits the QRFP receptor couples to in native cells and whether signal transduction mechanisms are altered in certain pathologies.

Structure–activity relationships

The in vitro activity of QRFP, 26RFa and 26RFa_(20–26) has been initially assessed by three distinct approaches, that is, displacement of [¹²⁵I–Tyr³²]QRFP binding, measurement of forskoline‐induced cAMP production and monitoring of [Ca²⁺]_i in human QRFP receptor‐transfected HEK293 or CHO cells (Fukusumi et al., 2003; Jiang et al., 2003). Subsequently, the pharmacological characterization of 26RFa analogues was conducted using these three assays (Le Marec et al., 2011; Neveu et al., 2012, 2014). Consistent with its slightly better affinity for the human QRFP receptor, QRFP is more potent than 26RFa in inhibiting cAMP formation and stimulating [Ca²⁺]_i mobilization (Fukusumi et al., 2003). The C‐terminal fragment 26RFa_(20–26) does not displace radio‐iodinated QRFP (Fukusumi et al., 2003) but competes with [¹²⁵I–Tyr¹⁵]26RFa with a micromolar IC₅₀ (Neveu et al., 2012). Consistently, the in vitro activity of 26RFa_(20–26) is considerably lower than that of QRFP and 26RFa in cAMP and calcium‐mobilizing assays (Fukusumi et al., 2003; Jiang et al., 2003; Le Marec et al., 2011; Neveu et al., 2012). As reported, the EC₅₀ ratio between 26RFa_(20–26) and 26RFa/QRFP ranges from 35 (Jiang et al., 2003; Le Marec et al., 2011) to >500 (Fukusumi et al., 2003). Indeed, serial sequence shortening from the N‐terminal region of 26RFa is accompanied by a gradual loss of affinity and functionality for the human QRFP receptor (Fukusumi et al., 2003; Le Marec et al., 2011). Conversely, C‐terminal truncations yield downsized analogues, such as 26RFa_(1–18) and 26RFa_(1–16) that are devoid of agonistic activity for human GPR103 (Le Marec et al., 2011). These data clearly indicate that the biologically active domain of 26RFa is located within its C‐terminal region and that 26RFa_(20–26), whose sequence is very well conserved from fish to mammals (see 2.2 section; Table 1), is the shortest analogue that retains substantial in vitro activity (Fukusumi et al., 2003; Le Marec et al., 2011). As a matter of fact, 26RFa_(20–26) mimics the orexigenic and hypophysiotropic effects of 26RFa (Do Rego et al., 2006; Navarro et al., 2006). The gradual decline in the biological activity is likely to be accounted for by the destabilization of the central α‐helix (see 2.9 section) and the loss of intermolecular interactions between truncated peptides and the receptor (Fukusumi et al., 2003; Le Marec et al., 2011). It is important to note that non‐amidated 26RFa is almost totally devoid of affinity and activity (Fukusumi et al., 2003; Le Marec et al., 2011) as usually observed for biologically active amidated peptides (Merkler, 1994). The fact that mono‐ and disubstituted carboxamide 26RFa analogues are less potent than the parent peptide suggests that the amide function is involved in hydrogen bond(s) within the binding pocket of QRFP receptor or that the cavity is too narrow for harboring modification at this point (Le Marec et al., 2011).

As 26RFa_(20–26) is the shortest peptide sequence (Figure 8A) that retains substantial affinity and activity (Le Marec et al., 2011), this peptide has served as a scaffold for designing low MW agonists and antagonists of QRFP receptor. Alanine and D‐amino acid scanning studies (Eustache et al., 2016) converge to demonstrate that the chemical feature and the correct orientation of the side chain of each residue of 26RFa_(20–26) play critical roles in the activity of the peptide (Le Marec et al., 2011). For instance, alanine‐ and D‐residue substitutions in the N‐terminal triad –Phe²⁴–Arg²⁵–Phe²⁶–NH₂ generate analogues without agonistic and antagonistic properties (Le Marec et al., 2011), suggesting that this motif is directly involved in activation of QRFP receptors, as already reported for NPFF and PrRP (Mazarguil et al., 2001; Boyle et al., 2005). It should be noticed that docking experiments confirm the involvement of the –Phe²⁴–Arg²⁵–Phe²⁶–NH₂ sequence in the activation of QRFP receptors (see 2.2 section). In contrast, the Ser²³ residue, which is not fully conserved across vertebrate species (Table 1), is the less Ala‐sensitive amino acid, suggesting that its side chain could be optimized by differently branched aliphatic moieties. Consistent with this notion, [Nva²³]26RFa_(20–26) (Nva, norvaline) appears to be threefold more potent than 26RFa_(20–26) to increase [Ca²⁺]_i and thus represents the most potent downsized analogue with an EC₅₀ of 233 ± 72 nM (Le Marec et al., 2011). The propensity of aliphatic residues such as Ile, norleucine (Nle), 2‐amino‐butyric acid (Abu), Val and probably Nva (Arfmann et al., 1977; Padmanabhan et al., 1990; Chin et al., 2005) to induce a β sheet or a turn formation may explain this gain of potency and the switch from a disordered structure in water (Thuau et al., 2005) to a nascent structure more favourable to receptor activation. N‐acylation of 26RFa_(20–26) does not significantly modify the biological activity although it may protect the heptapeptide from aminopeptidase activity. In contrast, N‐benzoylation of 26RFa_(17–26) reduces by approximately 100‐fold the calcium‐mobilizing effect of the peptide (Rouméas et al., 2015). Finally, mono‐ and disubstitutions of the carboxamide function impair the agonist activity of 26RFa_(20–26) (Le Marec et al., 2011).

Figure 8

Chemical structures of three pepditic or pseudopeptidic human QRFP receptor ligands. (A) 26RFa_(20–26). (B) LV‐2172 [cmpi²¹, aza‐β³‐Hht²³]26RFa_(19–26) (Neveu et al., 2012). (C) LV‐2185 [ADMA²⁵]26RFa_(20–26) (Neveu et al., 2014).

Turns are privileged target‐recognition patterns of bioactive peptides stabilized by intramolecular hydrogen bond(s) (Chou, 2000). As stated above (see also 2.9 section), in DPC micelles, 26RFa_(20–26) exhibits the features of a turn still uncharacterized. In order to favour the formation of turn motifs within 26RFa_(20–26), a series of aza‐β³ pseudopeptides has been prepared (Neveu et al., 2012). Sequential aza‐β³‐amino acid substitution of glycine at positions 20 and 21 enhances by twofold and fivefold the potency of 26RFa_(20–26) on [Ca²⁺]_i, while the aza‐β³‐Phe²² analogue is three times less potent than 26RFa_(20–26). Replacement of the native Ser²³ residue by the aza‐β³ surrogate of (γ‐OH)homoThr (aza‐β³‐Hht) generates an analogue that is twice as potent. In contrast, the [aza–β³–Phe²⁴]–, [aza–β³–Arg²⁵]– and [aza–β³–Phe²⁶]26RFa_(20–26) pseudopeptides are totally devoid of effect on calcium mobilization indicating that incorporation of intramolecular H‐bonds at these positions impairs the biological activity, whereas the N‐terminal part of 26RFa_(20–26) is likely to establish a structure close to a hydrazine turn (Neveu et al., 2012). Surprisingly, the triple aza‐β³ substitution, leading to the combined pseudopeptide [aza–β³–(Gly²⁰,Gly²¹,Hht²³)]26RFa_(20–26), does not enhance the activity of 26RFa_(20–26). On the other hand, the pseudopeptide [Cmpi²¹,aza‐β³‐Hht²³]26RFa_(21–26) (LV‐2172; Figure 8B), in which the –Gly–Gly– moiety is replaced by a 4‐(carboxymethyl)piperazine unit, is eight times more potent than 26RFa_(20–26) (Neveu et al., 2012). Congruent with an increase in their agonistic property, the [aza‐β³‐Gly²¹]26RFa_(20–26) and LV‐2172 pseudopeptides are four and five times more potent in displacing [¹²⁵I–Tyr¹⁵]26RFa than 26RFa_(20–26) respectively. In fact, the half‐life time of the latter compound in human serum is three times higher than that of 26RFa_(20–26). Consistent with these findings, i.c.v. injection of LV‐2172 in food‐restricted mice induces a dose‐dependent increase in food intake that lasts for 120 min, instead of 60 min for 26RFa_(20–26). In conclusion, the pseudopeptide [Cmpi²¹,aza‐β³‐Hht²³]26RFa_(21–26) exhibits a stronger potency, a higher affinity, a better stability and exerts a longer in vivo effect than 26RFa_(20–26) (Neveu et al., 2012). Some fluoro‐olefin pseudopeptides such as LV‐2094 and LV‐2098 have been designed for improving the serum stability of 26RFa analogues (Pierry et al., 2013). These data constitute the first step towards the development of new GPR103 ligands that should prove useful for the treatment of feeding disorders and/or osteoporosis.

Elucidating the structures of GPCRs and characterizing the mechanisms controlling ligand–receptor binding are required for rational drug design. Docking studies predict a strong intermolecular interaction between the Arg²⁵ residue of 26RFa_(19–26) and the Gln¹²⁵ residue located in the TM3 helix of the human QRFP receptor. In order to confirm this interaction, the ability of Arg‐modified 26RFa analogues to activate QRFP receptors (see 3.2 section) has been assessed. Replacement of the Arg²⁵ residue by a lysine, an ornithine or a citrulline moiety leads to analogues that are totally devoid of agonistic and antagonistic activities in the calcium mobilization assay (Neveu et al., 2014). Similarly, substitution of the Arg²⁵ residue by a symmetric dimethyl arginine generates an analogue, [SDMA²⁵]26RFa_(20–26), that does not exhibit agonistic or antagonistic activities. Moreover, asymmetric dimethylation of the side chain of arginine leads to a 26RFa analogue, [ADMA²⁵]26RFa_(20–26) LV‐2185 (Figure 8C), which, at concentrations ranging from 10⁻¹⁰ to 3 × 10⁻⁵ M, is unable to activate the QRFP receptor but antagonizes by 67.5% 26RFa‐evoked [Ca²⁺]_i increase at high concentration (Neveu et al., 2014). Altogether, these data provide strong evidence for a functional interaction between the Arg²⁵ residue of 26RFa and the Gln¹²⁵ residue of the QRFP receptor upon ligand–receptor activation, which can be exploited for the rational design of potent agonists and antagonists of this receptor.

While 26RFa/QRFP and its fragment peptides specifically activate the QRFP receptor, these peptides also exhibit significant affinity for other related receptors. In particular, the IC₅₀ of human QRFP, h26RFa and 26RFa_(20–26) for human NPFF2 are 53.0, 10.1 and 76.3 nM respectively. Their affinity for human NPFF1 is 2.5 to 6.2 times lower (Gouardères et al., 2007). However, h26RFa stimulates [³⁵S]GTPγS binding with an EC₅₀ of 5.3 nM on NPFF2 and 5.4 nM on NPFF1 (Gouardères et al., 2007). Conversely, 26RFa does not show any affinity for GPR10 or GPR54 (Elhabazi et al., 2013). Thus, the design of selective ligands for the QRFP receptor should take into account possible cross‐specificity with related receptors, notably NPFF2 and NPFF1.

Site‐directed mutagenesis in QRFP receptor

So far, there are only few mutagenesis data available. Based on the interaction between the positively charged C‐terminal arginine of NPY and the Asp^6.59 residue of TM6 in all Y‐receptors (Merten et al., 2007), Findeisen et al. (2011a) have hypothesized the same interaction between the arginine of the –Arg–Phe–NH₂ motif of RFRP‐1 and ‐3, NPFF, NPAF, PrRP and 26RFa and the acidic residue on the top of TM6 in their cognate receptors. Ala‐substituted Asp^6.59 mutants of human NPFF1, NPFF2 and GPR10 (Findeisen et al., 2011b) or the Glu^5.59 mutant of QRFP receptor (Findeisen et al., 2011a) display significant loss in ligand affinity and receptor activity. As the acidic moiety in position 6.59, which interacts with the RFamide motif, is close to the extracellular face rather than deeply embedded in the TM bundle, we can suppose that either 26RFa does not enter into the binding pocket but remains at the cell surface or only its N‐terminal region dives into the cavity for interacting and activating the QRFP receptor. These views are not consonant with initial SAR studies showing that N‐terminal 26RFa fragments are unable to activate human QRFP receptors (Le Marec et al., 2011) and the docking studies indicating that the Arg²⁵ and Phe²⁶ residues of 26RFa_(19–26) interact with mid‐TM Gln^3.32, Glu^3.39 and Val^3.36, Val^3.37, Ile^5.42 and Leu^5.46 moieties, respectively (Neveu et al., 2014). Indeed, docking studies strongly suggest that the positively charged side chain of the Lys¹⁹ residue rather than those of the Arg²⁵ moiety interacts with Glu^6.59 of QRFP receptors. Accordingly, in this pose, Cα of lysine‐19 is 8 Å far from that of Glu^6.59, whereas the Cα‐Cα distance between Arg²⁵ and Glu^6.59 is >14 Å (Neveu et al., 2014).

As mentioned above, the strongest intermolecular interaction is thought to be a H‐bond between Arg²⁵ residue of 26RFa_(20–26) and the Gln^3.32 of the QRFP receptor (Neveu et al., 2014). In order to confirm this contact, the ability of 26RFa and Arg‐modified 26RFa analogues to activate the wild‐type (WT) and the Q125A‐mutated QRFP receptor transiently expressed in CHO cells has been investigated by assessing their calcium‐mobilizing response. 26RFa enhanced [Ca²⁺]_i in WT QRFP receptor‐ but totally failed to increase [Ca²⁺]_i in Q125A‐mutated receptor‐expressing cells, confirming a functional interaction between the Arg²⁵ and Gln^3.32 residues (Neveu et al., 2014).

QRFP receptor dimerization

It has been recently reported that QRFP receptors and OX receptors form constitutive and induced functional hetero‐dimers in nerve cells (Davies et al., 2015). Treatment of the neuroblastoma SH‐SY5Y cell line with the β‐amyloid peptide Aβ42 reduces the expression of both QRFP receptors and OX receptors. In accordance, mRNAs for these receptors are down‐regulated in the anterior hippocampus of Alzheimer's disease (AD) patients. These results suggest that a reduction in QRFP receptor‐OX receptor heterodimers in AD patients may promote cellular damages resulting in memory deficit (Davies et al., 2015).

Surprisingy, in orexin knockout mice, the effect of QRFP on food intake is similar to that observed in WT animals, suggesting that QRFP‐induced feeding behaviour does not depend on the orexin signalling pathway (Takayasu et al., 2006). However, this finding does not exclude transactivation of OX receptors by 26RFa/QRFP.

Design of non‐peptidic agonists and antagonists

The QRFP receptor is a class‐A GPCR first described in 2001 (Lee et al., 2001). The observation that endogenous QRFP receptor peptide agonists stimulate food intake and increase body weight (Chartrel et al., 2003; Do Rego et al., 2006; Takayasu et al., 2006; Neveu et al., 2012) has prompted research for the development of peptidic as well as non‐peptidic QRFP receptor ligands (Carlebur et al., 2015). Indeed, QRFP receptor antagonists are expected to be useful for the prevention or the treatment of a variety of metabolic disorders such as bulimia, angiospasm, obesity, diabetes mellitus, endocrinopathies, hypercholesterolemia, hyperlipidaemia, gout, and fatty liver. Antagonists of the QRFP receptor may also be of therapeutic value for cardiovascular diseases including arterial sclerosis or heart insufficiency, and for renal diseases. Since 2009, several non‐peptidic QRFP receptor antagonists have been designed (see below), but no non‐peptidic agonists have been reported so far.

Indole derivatives

In 2010, Banyu Pharmaceutical has issued two patents describing two structurally close families of compounds, which act both as human QRFP receptor antagonists: (i) aryl indole derivatives (Kishino et al., 2010) and (ii) indole‐2‐carboxamide derivatives (Haga et al., 2010a) (Figure 9). In these series, compounds 1–5 were evaluated using a binding assay with [¹²⁵I–Tyr³²]QRFP as a radioligand and were found to inhibit [¹²⁵I–Tyr³²]QRFP binding to QRFP receptors. For the aryl indole derivatives 1 and 2, the measured IC₅₀ values are 33 and 50 nM, respectively, and for the indole‐2‐carboxamide derivatives 3, 4 and 5, they are even better, that is, 49.0, 5.5 and 6.1 nM respectively. The patent on indole‐2‐carboxamide derivatives highlights the importance of the dimethylamine substituent to the affinity (replacement by a methylamine substituent impairs affinity). Although the addition of a chlorine atom on the indole ring did not improve the affinity (see 4 vs. 5), this halogen was retained in the next generation of QRFP receptor ligands (see below).

Figure 9

Chemical structures of various non‐peptidic QRFP receptor antagonists from Banyu Pharmaceutical based on 3‐aryl and heteroaryl‐substituted indole scafold. Compounds 1 and 2 are arylindole derivatives; compounds 3–5 are indole‐2‐carboxamide derivatives.

In 2010, a new series of indole derivatives, including 3‐aryl and heteroaryl‐substituted indoles, was patented in the US by the same company (Fujimura et al., 2010). Indole derivatives which are substituted by a heteroaryl (specially a furan group) at the 3‐position displayed improved affinities (6 vs. 7 with IC₅₀ = 22.56 vs. 9.82 nM; Figure 10). Further, the incorporation of an alkylaminocarbonyl substituent (a more classical chemical group compared to previous substitutions) at the 2‐position of the indolic system yields compound 8 (Figure 10), which exhibits a better affinity (IC₅₀ = 2.26 nM) than 7. Finally, derivative 9, with another furan group close to the amine function in this case, reaches an IC₅₀ of 0.58 nM (Figure 10).

Figure 10

Chemical structures of four non‐peptidic QRFP receptor antagonists from Banyu Pharmaceutical based on 3‐aryl and heteroaryl‐substituted indole scafolds.

A series of pyrrolo[2,3‐c]pyridines as low MW antagonists of QRFP receptors, based on the first compounds patented by Banyu Pharmaceutical has been developed (Georgsson et al., 2014, 2015). First, the authors found that compound 5 designed by Banyu Pharmaceutical (Figure 9) displays disadvantages in terms of metabolic stability, solubility and cytochrome P450 inhibition, in addition to narrow margins on cardiac targets. Therefore, they focused their study on the design and synthesis of a series of compounds with metabolism, pharmacokinetics and safety properties suitable for drug development. Thus, they identified the indole motif as a driver for lipophilicity, and its replacement with pyrrolo[2,3‐c]pyridine (10 and 11; Figure 11) led to compounds with increased metabolic stability and solubility as well as improved margins in terms of cytochrome P450 inhibition and cardiac targets. However, the IC_50s of the latter compounds in radiobinding assays remain in the same range, that is, IC₅₀ = 40 nM for compound 10 and IC₅₀ = 280 nM for compound 11 compared with IC₅₀ = 70 nM for compound 5. Of note, the IC₅₀ for compound 5 measured by Georgsson et al. (2014) is 10 times higher than the IC₅₀ for this compound reported in the original patent (IC₅₀ = 6.1 nM) (Haga et al., 2010a). Preclinical tests revealed for the first time an effect of a QRFP receptor antagonist: in a 3 day automated food intake measurement study, compound 10 (Figure 11) provoked a significant and dose‐dependent reduction on food intake compared to vehicle‐treated animals (Georgsson et al., 2014).

Figure 11

Chemical structures of two non‐peptidic QRFP receptor antagonists from AstraZeneca based on a pyrrolo[2,3‐c]pyridine scafold.

The same authors then investigated the free solution structure of the C‐terminal motif of the endogenous QRFP receptor ligand, 26RFa_(20–26), and compared it to that of compound 11 (Georgsson et al., 2014; see 2.9 section). From these NMR data, they have selected eight dominant conformational families and compared them with low‐energy conformations of compound 11. An overlay of key pharmacophore features of the terminal –Arg–Phe–NH₂ and 11 is observed for one of the most populated family (Figure 12). The conformation of 11 used in this superimposition is only 0.8 kcal·mol⁻¹ higher than the lowest energy conformation of 11 identified after optimization by quantum mechanics. These observations suggest that the antagonists designed by Georgsson et al. (2014) mimic the C‐terminal Arg²⁵–Phe²⁶ residues of 26RFa/QRFP.

Figure 12

Structural superposition between 26RFa_(20–26) (grey) and compound 11 (pink). Reprinted from Georgsson et al. (2014). Used with permission.

2‐Aryl‐imidazoline derivatives

In 2010, Banyu Pharmaceutical issued a patent describing another family of potential antagonists of QRFP receptors, that is, 2‐aryl‐imidazoline derivatives (Haga et al., 2010b). The inventors reported that compounds having a diphenylmethyl substituent at position 1 of imidazoline and an aryl substituent at position 2 act as antagonists of the human QRFP receptor. Among the aryl substituents, the phenyl ring substituted in para by little bulky groups such as a methoxy radical generates the higher affinity compounds of the series. For instance, compound 12 (Figure 13) exhibits an IC₅₀ of 11 nM.

Figure 13

Chemical structure of a QRFP receptor antagonist from Banyu Pharmaceutical based on a 2‐aryl‐imidazoline scafold.

Carboximidamide derivatives

To identify potential QRFP receptor antagonists, Nordqvist et al. (2014) have performed a high throughput screening on the 900 000 in‐house compound library. The authors first tested the effect of a single concentration of each compound on the production of inositol‐1‐phosphate (IP‐1) followed up in a concentration–response IP‐1 assay that led to the identification of about 35 000 active molecules. Subsequently, they carried out an orthogonal cell based screening test using a ten‐point concentration–response assay and about 18 000 compounds were confirmed active. Finally, among these 18 000 compounds, 100 molecules were selected as the most attractive and evaluated in a [¹²⁵I–Tyr³²]QRFP radioligand binding assay with a hit rate of 3% (three active compounds). Thus, only a small fraction of the compounds selected in the IP‐1 assay were confirmed in the radioligand binding assay. The authors propose that this low confirmation rate could be ascribed to the presence of artefacts in the IP‐1 assay, such as binding to allosteric sites of QRFP receptors, or IP‐1 production through off‐target interactions. They have thus decided to revisit all compounds flagged as active on IP‐1 in the primary high throughput screening and in the IP‐1 concentration–response assay. In this way, 963 compounds were selected for a second screening in the [¹²⁵I–Tyr³²]QRFP radiobinding assay at a single concentration. The compounds considered active, 123, were subsequently followed up in radiobinding concentration–response tests. This approach allowed the authors to enlarge the set of confirmed QRFP receptor antagonists and enabled them to identify a total of 17 new chemical clusters. However, in their publication, the authors have selected representative compounds from only three clusters (compounds 13–15; Table 3) (Nordqvist et al., 2014). Two of the identified clusters have previously been reported in the context of ligands for GPCRs, that is, compound 14 as a 5‐HT₆ receptor ligand (Nordvall et al., 2006) and compound 15 as an adenosine receptor antagonist (Webb et al., 2003). So the authors carried out a SAR study on compound 13.

Table 3. Biological data for compounds 13–15

Compound		[^125I]‐QRFP radiobinding assay, IC50 (nM)	Measurement of production on IP‐1, IC50 (nM)
	13	160 ± 5	50 ± 1
	14	80 ± 1	80 ± 1
	15	5000 ± 300	2510 ± 9

The pharmacomodulation of compound 13 led to the most potent compound 16 (Figure 14) with an IC₅₀ of 12 nM and with an improved ligand lipophilic efficiency. Among the different chemical groups ‐ amidine aryl ring, amidine moiety, methoxy radical and cyclopentylsulfanylmethyl substituent ‐ substitution of the last group is the only modification that improves the QRFP receptor antagonist activity of the compounds.

Figure 14

Chemical structure of N‐[3‐(cyclopentylsulfanyl‐methyl)‐4‐methoxy‐phenyl]thiophene‐2‐carboximidamide, a QRFP receptor antagonist from AstraZeneca.

Distribution of QRFP receptors in the CNS

The localization of the mRNA for QRFP receptors has been determined in the CNS by Northern blot, RT‐PCR and in situ hybridization histochemistry (Lee et al., 2001; Chartrel et al., 2003; Fukusumi et al., 2003; Jiang et al., 2003; Kampe et al., 2006; Takayasu et al., 2006; Bruzzone et al., 2007). In rat, the highest concentrations of QRFP receptor 1 mRNA are found in the piriform cortex, the amygdalohippocampal area, the lateral septum, the reuniens thalamic nucleus, the ARC, the VMH, the zona incerta, the intergeniculate leaf, the raphe nucleus, the locus coeruleus, the medial parabrachial nucleus, the nucleus of the solitary tract and in the dorsal horn of the spinal cord (Fukusumi et al., 2003; Bruzzone et al., 2007). Intense expression of the QRFP receptor 1 gene is also observed in other brain regions including the hippocampus, the hypothalamus, the medial geniculate nucleus, the amygdala, the interpeduncular nucleus, the superior and inferior colliculus and the vestibular nucleus (Fukusumi et al., 2003; Bruzzone et al., 2007). The distribution of QRFP receptor 2 mRNA markedly differs from that of QRFP receptor 1 mRNA. Thus, the highest density of QRFP receptor 2 in the rat brain is observed in the medial part of the medial preoptic nucleus, the anterior hypothalamic area, the reuniens and parafascicular thalamic nuclei, the lateral paragigantocellular nucleus, the facial and the hypoglossal nuclei (Kampe et al., 2006; Figure 4A). Similarly, in the mouse brain, QRFP receptor 1 and QRFP receptor 2 mRNAs are differentially expressed (Takayasu et al., 2006). In particular, the mitral cell layer of the olfactory bulb, the island of Calleja and the solitary tract that contain high concentrations of QRFP receptor 1 mRNA are devoid of QRFP receptor 2 mRNA. Reciprocally, several brain regions that exhibit intense expression of QRFP receptor 2 mRNA – including the caudate putamen, the olfactory tubercle, the triangular septal nucleus, the suprachiasmatic nucleus, the magnocellular nucleus of the hypothalamus, the medial supramammilary nucleus and the facial nucleus – do not express the QRFP receptor 1 gene (Takayasu et al., 2006). The distribution of [¹²⁵I–Tyr¹⁵]26RFa binding sites in the rat brain matches relatively well with the areas of expression of QRFP receptor 1 and QRFP receptor 2 mRNAs as well as NPFF2 mRNA (Bruzzone et al., 2007). In particular, a high density of binding sites is observed in the piriform cortex, the hippocampal formation, the amygdaloid complex, the lateral septum, the medial preoptic area, the reuniens and parafascicular thalamic nuclei, the anterior hypothalamic area, the ARC, the VMH, the zona incerta, the locus coeruleus, the raphe nucleus and the dorsal horn of the spinal cord that are all enriched with QRFP receptor 1 and/or QRFP receptor 2 mRNAs (Kampe et al., 2006; Bruzzone et al., 2007).

In the human brain, the QRFP receptor is primarily expressed in the cerebral cortex, the hypothalamus, the thalamus, the vestibular nucleus and the trigeminal ganglion (Lee et al., 2001; Jiang et al., 2003). Moderate expression also occurs in the amygdala, the caudate nucleus, the hippocampus and the ventral tegmental area (Jiang et al., 2003).

In the chicken brain, GPR103 mRNA is widely expressed, the highest concentrations being found in the diencephalon and mesencephalon (Ukena et al., 2010).

Distribution of QRFP receptors in peripheral organs

In mouse, a high concentration of QRFP receptor 1 mRNA occurs in the eyes (Baribault et al., 2006; Takayasu et al., 2006). Moderate expression is detected in the spine, the thymus, the adrenal gland and the testis (Jiang et al., 2003; Baribault et al., 2006; Takayasu et al., 2006). The QRFP receptor 2 gene is primarily expressed in the mouse eye and testis (Takayasu et al., 2006). In rat, the QRFP receptor 1 gene is intensely expressed in the adrenal gland and, to a lesser extent, in the eye, kidney and testis (Fukusumi et al., 2003).

In human, the highest level of QRFP receptor mRNA is found in fetal bone (Genotype Tissue Expression (GTEx) Consortium, n.d.; Baribault et al., 2006). The QRFP receptor gene is also expressed in the heart, thyroid and parathyroid glands, kidney and testis (Jiang et al., 2003; Baribault et al., 2006; GTEx Consortium, n.d.) as well as in the pituitary (Lee et al., 2001; GTEx Consortium, n.d.). In human osteoblasts in primary culture, dexamethasone induces a concentration‐dependent decrease of the expression levels of QRFP receptors (Baribault et al., 2006).

QRFP receptor mRNAs were not initially detected in rat and mouse pancreas (Fukusumi et al., 2003; Jiang et al., 2003). However, RT‐PCR analyses show that QRFP receptor mRNA is expressed in cultured rat INS‐1E beta cells (Granata et al., 2014) and mouse insulinoma MIN6 cells (Prévost et al., 2015) as well as in cultured human pancreatic islets (Granata et al., 2014). Immunohistochemical studies confirm the presence of QRFP receptors in these cells and in the human pancreas (Granata et al., 2014; Prévost et al., 2015).

In the human adrenal gland, the QRFP receptor is exclusively expressed during embryogenesis in the fetal zone but not in the zona glomerulosa or adrenal medulla, whereas in the adult human adrenal gland, QRFP receptor mRNA is present in all three zones of the cortex. In the rat adrenal gland, the medulla is devoid of QRFP receptor mRNA (Ramanjaneya et al., 2013).

QRFP receptor knockout

So far, only QRFP receptor 1 knockout animals have been reported in the literature. QRFP receptor 1 knockout mice on a C57Bl6/J background suffer from kyphosis and display osteopenia (Baribault et al., 2006). This phenotype is more frequent in females than males (80 vs. 10% respectively) and is enhanced by ovariectomy. Detailed analysis using microcomputed tomography reveals a decrease in trabecular bone density in the spine in both sexes. The reduction in bone density is more pronounced in female than male knockout mice with a thinning of the osteochondral growth plate. In contrast, no evidence for calcium, phosphate, vitamin D or parathyroid hormone changes can be detected in the knockout mice. Despite this phenotype, knockout mice appear to grow normally and no difference in body weight was reported (Baribault et al., 2006).

Interestingly, SAMP6 osteopenic mice display a single‐nucleotide polymorphism (SNP) in the promoter region and three SNPs, including one silent SNP, in the coding region of the QRFP prepropeptide, upstream the sequence of QRFP (Zhang et al., 2007). In these mice, QRFP expression is reduced in the lumbar spine and in the hypothalamus, suggesting that the SNP in the promoter region of the gene results in generation of a putative repressive transcription factor binding site (Zhang et al., 2007).

In human, three SNPs within the QRFP receptor gene show significant association with Hashimoto's thyroiditis in the Japanese (Ban et al., 2016) and Caucasian (Tomer et al., 2015) populations.

QRFP receptors in tumour cells

QRFP receptor transcript is detected in the androgen‐unresponsive prostate cancer cell line DU145 but not in the androgen‐unresponsive PC3 cell line and in the androgen‐responsive LNCaP cell line (Alonzeau et al., 2013). The human adrenal corticocarcinoma cell line H295R also expresses QRFP receptor mRNA (Ramanjaneya et al., 2013).

Biological and pharmacological effects of QFRP peptides

Effects of QRFP peptides on the CNS

Central regulation of feeding behaviour by QRFP peptides

The observation that the QRFP precursor is primarily expressed in hypothalamic nuclei involved in the control of energy homeostasis, such as the ARC, PVN, VMH and LHyp (Chartrel et al., 2003; Fukusumi et al., 2003; Bruzzone et al., 2006; Kampe et al., 2006; Takayasu et al., 2006), has led researchers to investigate the possible effect of 26RFa/QRFP on food intake. The earliest report on the action of 26RFa/QRFP on feeding behaviour revealed that i.c.v. administration of 26RFa in food‐restricted mice causes a dose‐dependent increase in chow consumption (Chartrel et al., 2003). Numerous studies have supported this initial observation and expanded the original findings by investigating the effects of 26RFa/QRFP on food intake in fully satiated mice and following chronic administration of 26RFa/QRFP (Do Rego et al., 2006; Moriya et al., 2006; Takayasu et al., 2006). Notably, chronic administration of QRFP produced a transient increase in food intake, weight gain and adiposity in mice consuming a standard chow diet and increased body weight and adiposity in mice fed a moderately fat diet (Moriya et al., 2006). The recent observation that QRFP‐deficient mice are hypophagic and lean (Okamoto et al., 2016) strongly supports the notion that 26RFa/QRFP is a potent orexigenic peptide.

Original studies of 26RFa/QRFP effects on feeding behaviour in mice have been expanded to include rat, chick and zebra finch (Kampe et al., 2006; Patel et al., 2008; Primeaux et al., 2008; Lectez et al., 2009; Ukena et al., 2010; Primeaux, 2011; Tobari et al., 2011; Primeaux et al., 2013; Zagorácz et al., 2015). In rats, the effects of centrally administered QRFP on food intake have been inconsistent and appear to depend on the macronutrient profile of the diet (Primeaux et al., 2013). The first report on the effects of 26RFa/QRFP in rats suggests a subtle orexignic response to 26RFa when the peptide is i.c.v. injected prior to the beginning of the light phase without any significant effect on energy expenditure (Kampe et al., 2006). Central administration of QRFP (the elongated form of 26RFa) also fails to significantly influence food intake in ad libitum‐fed rats (Patel et al., 2008). In contrast, in food‐restricted rats, centrally administered 26RFa increases chow intake (Lectez et al., 2009). In addition, site‐specific micro‐injection of 26RFa into the medial hypothalamus of adult male rats increases « liquid food » (i.e. milk) intake (Zagorácz et al., 2015). The macronutrient profile of the diet is thought to be an important predictor of QRFP‐induced hyperphagia in rats (Primeaux et al., 2013). Central administration of 26RFa and QRFP in male rats selectively increases intake of high‐fat diet (55–60% kcal from fat) but fails to increase the intake of low‐fat diet (10% kcal from fat) (Primeaux et al., 2008; Primeaux et al., 2013). Similar effects have been reported in female rats (Primeaux, 2011; Primeaux et al., 2013). The lack of effect of QRFP administration on low‐fat food intake is consistent with previous data obtained with standard chow intake, likely to be due, at least in part, to the fat content of the diets. Recent evidence suggests that QRFP also exerts an orexigenic effect in birds (e.g. broiler chick and zebra finch) (Ukena et al., 2010; Tobari et al., 2011). Interestingly, 26RFa stimulates food intake in broiler chicks (that have been genetically selected for meat production and consume considerably more food than other strains) but not in layer chicks (Ukena et al., 2010), suggesting that 26RFa/QRFP may contribute to the higher feeding activity of broiler chicks.

Regulation of hypothalamic 26RFa/QRFP expression by nutritional status

Understanding the role of 26RFa/QRFP on feeding behaviour requires consideration of the effects of the nutritional status and circulating factors on hypothalamic 26RFa/QRFP expression. Studies conducted in mice indicate that a 48 h fast significantly increases hypothalamic preproQRFP mRNA levels (Takayasu et al., 2006), while food deprivation in the orange‐spotted grouper (E. coioides) does not alter 26RFa/QRFP expression, though refeeding leads to an increase in 26RFa/QRFP expression (Shu et al., 2014). The macronutrient profile of the diet also affects hypothalamic preproQRFP mRNA levels in rats (Primeaux et al., 2008; Beck and Richy, 2009; Schreiber et al., 2016). Consumption of a high‐fat diet (55 and 60% kcal from fat) significantly increases the expression of preproQRFP mRNA in the VMH/ARC region of the hypothalamus (Primeaux et al., 2008; Schreiber et al., 2016). Reciprocally, consumption of a moderately fat diet (40% kcal from fat) decreases expression of 26RFa/QRFP in the VMH (Beck and Richy, 2009). Altogether, these observations support the hypothesis that the nutritional status regulates the expression of the preproQRFP gene in regions associated with feeding behaviour.

Peripheral signals, such as leptin, that control feeding behaviour also regulate hypothalamic 26RFa/QRFP expression. Genetically altered mouse models, which have a deficiency in leptin regulation (i.e. ob/ob and db/db), have increased hypothalamic expression of preproQRFP mRNA, compared to WT controls (Takayasu et al., 2006). As leptin is an adipose tissue hormone that acts as a satiety signal and leads to a decrease in food intake (Schwartz et al., 2000; Barsh and Schwartz, 2002), it appears that a decrease in circulating satiety signals produces a compensatory increase in 26RFa/QRFP expression, leading to restoration of food intake. Circulating oestradiol levels, which are known to affect food intake and body weight, also regulate hypothalamic 26RFa/QRFP expression (Eckel et al., 2000; Messina et al., 2006; Santollo and Eckel, 2008; Schreiber et al., 2016). In female rats, preproQRFP mRNA levels in the VMH/ARC are elevated during diestrus, a phase associated with low oestradiol levels, and decrease during estrus, a phase associated with high oestradiol levels. Inversely, ovariectomy increases preproQRFP mRNA expression and oestrogen replacement attenuates this increase in the VMH/ARC (Schreiber et al., 2016).

Role of QRFP peptides in hypothalamic feeding circuitry

Expression of QRFP in hypothalamic regions that control food intake suggests that there is an interaction between QRFP and other neuropeptide systems that are known to regulate feeding behaviour. Several studies have reported that pretreatment with an NPY Y₁ receptor antagonist attenuates the effects of 26RFa/QRFP on food intake in rats and mice (Takayasu et al., 2006; Lectez et al., 2009; Primeaux et al., 2013; Zagorácz et al., 2015). Administration of a Y₅ receptor antagonist also reduces the effect of 26RFa on food intake (Lectez et al., 2009). Double in situ hybridization labelling reveals that QRFP receptor is expressed in NPY neurons of the ARC (Lectez et al., 2009). Further analyses indicate that central administration of 26RFa increases preproNPY mRNA expression in the VMH and leads to a 37‐fold increase in NPY release from rat hypothalamic explants (Lectez et al., 2009). The effect of 26RFa on NPY expression is reduced by co‐administration of Y₁ and Y₅ receptor antagonists (Lectez et al., 2009). Central administration of 26RFa also leads to a 38% decrease in hypothalamic pro‐opiomelanocortin (POMC) mRNA expression, and a 55% decrease in α‐melanocyte‐stimulating hormone (α‐MSH) release, although QRFP receptors are not present in POMC neurons (Lectez et al., 2009). Pretreatment with an MC₃/MC₄ receptor agonist attenuates the effect of QRFP on high‐fat food intake (Primeaux et al., 2013).

Overall, these studies suggest that the nutritional status plays a pivotal role on the effect of 26RFa/QRFP on food intake and hypothalamic 26RFa/QRFP expression and that QRFP expression is regulated by peripheral and central signals which influence food intake.

Effects of QRFP peptides on locomotor activity

It is known that i.c.v. injections of FMRFamide and NPFF in mice decrease locomotor activity (Kavaliers and Hirst, 1986; Quelven et al., 2004) and reverse morphine‐induced locomotor hyperactivity (Raffa, 1988; Marco et al., 1995; Cador et al., 2002). Conversely, i.c.v. injection of 26RFa and QRFP dose‐dependently increases locomotor activity in mice (Do Rego et al., 2006; Takayasu et al., 2006). The observation that qrfp^−/− mice are hypoactive (Okamoto et al., 2016) supports a physiological role of the 26RFa/QRFP‐QRFP receptor system in the control of locomotor activity. Interestingly, the effect of 26RFa on locomotion is mimicked by the N‐terminal peptide 26RFa_(1–16), whereas the central segment 26RFa_(8–16) and the C‐terminal segment 26RFa_(20–26) are devoid of effect on horizontal and vertical locomotor activities. Chronic i.c.v. infusion of QRFP in mice under regular diet condition does not alter the cumulative motor activity during either the light or dark cycle (Moriya et al., 2006). Pretreatment with naloxone does not inhibit the hyperlocomotor action of 26RFa (Do Rego et al., 2006), indicating that, in contrast to NPFF, the locomotor effect of 26RFa is not mediated through modulation of opioid neurotransmission. In contrast to what is observed in mice, acute i.c.v. administration of 26RFa in rat has no effect on locomotion (Kampe et al., 2006). These divergent responses may be ascribed to the occurrence of two isoforms of QRFP receptors in rodents (Kampe et al., 2006; Takayasu et al., 2006) and/or to the substantial affinity of 26RFa for NPFF2 (Gouardères et al., 2007). Alternatively, 26RFa and its derivatives may behave as biased ligands inducing subtle conformational changes in a particular isoform of the QRFP receptor, which differently trigger downstream responses.

Overexpression of the QRFP gene in zebrafish larvae attenuates their daytime locomotor activity without inducing sleep (Chen et al., 2016). Reciprocally, in qrfp^−/− zebrafish larvae, the locomotor activity while awake is enhanced, and the number of sleep bouts are reduced during the day but not at night, suggesting that 26RFa/QRFP‐ QRFP receptor signalling is necessary to maintain normal locomotor activity and daytime sleep levels in zebrafish (Chen et al., 2016).

Implication of QRFP peptides in anxious behaviour

QRFP receptor 1 and QRFP receptor 2 mRNAs are differentially expressed in mouse brain regions involved in anxiety and stress such as the bed nucleus of the stria terminalis, the lateral septum and the periaqueductal gray (Takayasu et al., 2006). Intracerebroventricular injection of 26RFa in mice reduces anxious behaviour in elevated plus maze test, and this effect is mediated via GABAergic and β‐adrenergic transmission (Palotai and Telegdy, 2016). Consistent with an anxiolytic effect of 26RFa, QRFP‐deficient mice exhibit exacerbated anxiety‐like behaviour (Okamoto et al., 2016). Conversely, i.c.v. administration of QRFP does not affect anxiety‐like behaviour in mice (Takayasu et al., 2006). The divergent effects of the two peptides are attributable to the better affinity of 26RFa than QRFP for NPFF2 (Gouardères et al., 2007) whose activation causes the release of corticotropin‐releasing hormone (CRH). Nevertheless, QRFP stimulates grooming bouts and the time spent grooming which are marks of elevated stress levels. As a matter of fact, QRFP stimulates CRH mRNA expression in 4B hypothalamic cells (Ishigame et al., 2016), suggesting that both 26RFa and QRFP may contribute to stress responses.

Involvement of QRFP peptides in nociception

The parafascicular thalamic nucleus, the locus coeruleus, the dorsal raphe nucleus and the parabrachial nucleus, which are involved in pain transmission, are all enriched with QRFP receptor mRNA and/or 26RFa binding sites (Bruzzone et al., 2006, 2007). In rat, i.t. or i.c.v. injection of 26RFa reverses the agitation behaviour induced by formalin injection in the paw (Yamamoto et al., 2008, 2009), while i.t. administration of 26RFa attenuates the mechanical allodynia induced by carrageenan injection in the paw (Yamamoto et al., 2008). Conversely, i.c.v. injection of 26RFa in mice induces NPFF1/NPFF2‐mediated hyperalgesia (Elhabazi et al., 2013). In a partial sciatic nerve ligation rat model, i.t. or i.c.v. injection of 26RFa attenuates the level of mechanical allodynia by increasing the mechanical nociceptive threshold independently of the activation of Y₁ and NPFF1 receptors (Yamamoto et al., 2011). Immunoneutralization of endogenous QRFP by antibodies does not affect the level of allodynia induced by partial sciatic nerve injury (Yamamoto et al., 2011). Taken together, these data suggest that the 26RFa/QRFP‐QRFP receptor system is involved in nociceptive transmission from the spinal cord to the brain during inflammation and/or neuropathic pain.

Involvement of QRFP peptides in the central control of cardiovascular activity

In conscious mice, i.c.v. administration of QRFP leads to a long‐lasting rise in mean arterial blood pressure and heart rate within 20–30 min after injection, which can be likely ascribed to stimulation of the sympathetic tone (Takayasu et al., 2006). As preproQRFP mRNA is up‐regulated in obese animals, 26RFa/QRFP may be involved in hypertension of patients suffering from metabolic syndrom (Takayasu et al., 2006).

Involvement of QRFP peptides in the peripheral control of cardiovascular activity

Intravenous injection of 26RFa in anaesthetized rats produces a biphasic change in mean arterial blood pressure consisting in a rapid (~30 s after injection) hypotension followed by a hypertensive response and a concomitant increase in heart rate (Fang et al., 2009). Interestingly, i.v. injections of 26RFa_(8–26) and 26RFa_(19–26) produce only hypertensive and tachycardiac responses at lower doses than those needed for a response of 26RFa, with a different sensitivity towards bilateral vagotomy and pretreatment with the β‐adrenoreceptor antagonist propranolol (Fang et al., 2009). Thus, the hypo‐ and/or hypertensive effects of 26RFa and its C‐terminal fragments may be mediated via different pathways involving another receptor besides QRFP receptors (Fang et al., 2009).

In isolated adult rat cardiac myocytes paced at 0.2 Hz, 26RFa_(8–26) and 26RFa_(19–26) do not modify the sarcomere length, the peak shortening, the departure velocity and the return velocity, whereas the same dose of RFRP‐1 decreases shortening amplitude and the shortening and re‐lengthening rates without changes in resting sarcomere length (Nichols et al., 2010). These findings exclude the RFRP‐1 receptor, GPR147/OT7T022, and favour the involvement of NPFF2 in the versatile effects of 26RFa on arterial pressure.

Effects of QRFP peptides on skeletal muscle cells

The effects of 26RFa and QRFP on insulin‐stimulated glucose uptake have been investigated on rat L6 myotubes employed as an in vitro model of skeletal muscle (Allerton and Primeaux, 2015). 26RFa, but not QRFP, potentiates the effects of insulin on glycogen synthesis and on 2‐deoxy‐D‐glucose uptake in L6 myotubes, indicating that 26RFa enhances insulin‐sensitivity in skeletal muscle (Allerton and Primeaux, 2015).

Effects of QRFP peptides on the pituitary–gonadal axis

Most FLPs including GnIH/RFRP‐1, PrRP and kisspeptins (see 2.1 section) are known to be involved in the control of reproduction (Seal et al., 2000; Tsutsui et al., 2010; Pinilla et al., 2012). Several studies indicate that 26RFa/QRFP also play a role in the control of reproduction (Fukusumi et al., 2003; Navarro et al., 2006; Patel et al., 2008; Liu et al., 2009; Primeaux, 2011; Parhar et al., 2012; Schreiber et al., 2016).

In rodents, QRFP receptors are expressed in the preoptic area, the anterior hypothalamus and in other hypothalamic nuclei involved in the regulation of the pituitary–gonadal axis (Kampe et al., 2006; Takayasu et al., 2006; Bruzzone et al., 2007; see ‘Distribution of QRFP receptors in the CNS’ section). Intracerebroventricular injection of 26RFa, 26RFa_(20–26) or QRFP to cyclic and ovariectomized rats significantly elevates plasma luteinizing hormone (LH) level (Navarro et al., 2006). Central administration of QRFP also increases LH and follicle‐stimulating hormone (FSH) in male rats (Patel et al., 2008). In vitro, QRFP triggers GnRH release from male rat hypothalamic fragments (Patel et al., 2008). Both 26RFa/QRFP precursor and QRFP receptor genes are also expressed in the pituitary gland of male and female rats (Navarro et al., 2006). Consistent with this observation, 26RFa stimulates LH and FSH release from the anterior pituitary of male and female rats and enhances GnRH‐induced secretion of gonadotropins in male rats (Navarro et al., 2006). In contrast, 26RFa does not affect PRL release, indicating that the peptide acts selectively on gonadotrope cells (Navarro et al., 2006). The stimulatory effect of 26RFa on LH is mimicked by 26RFa_(20–26) and by QRFP, but not by 26RFa_(1–16) (Navarro et al., 2006). Taken together, these data indicate that, in rodents, 26RFa/QRFP may control the pituitary–gonadal axis by acting both at the hypothalamic level on GnRH neurons and at the pituitary level on gonadotrope cells.

In the rhesus monkey Macaca mullata, i.v. administration of 26RFa or QRFP increases plasma PRL but does not affect testosterone levels (Wahab et al., 2012b). Of note, in the same species, i.v. injection of 26RFa stimulates, while QRFP inhibits, growth hormone secretion (Qaiser et al., 2012).

In the chicken G. domesticus, i.v. administration of 26RFa reduces plasma testosterone concentration, and this effect is mimicked by kisspeptin (Wahab et al., 2012a).

Finally, in the goldfish C. auratus, intraperitoneal injection of a high dose of 26RFa increases serum LH level, while incubation of goldfish pituitary cells with 26RFa has no effect on LH release (Liu et al., 2009).

Effects of QRFP peptides on the adrenal gland

There is a high level of expression of QRFP receptors in the rat and human adrenal gland (Fukusumi et al., 2003; Ramanjaneya et al., 2013) and 26RFa/QRFP precursor mRNA is present in the adrenal cortex and medulla (Ramanjaneya et al., 2013). The effect of 26RFa/QRFP on corticosteroid secretions has thus been studied in various experimental settings. Intravenous administration of QRFP to male rats causes a dose‐dependent increase in plasma aldosterone concentration but does not affect plasma levels of corticosterone and testosterone, two other steroid hormones secreted by the adrenal cortex (Fukusumi et al., 2003). In the human adrenocortical cell line H295R, QRFP stimulates aldosterone and to some extent cortisol secretion (Ramanjaneya et al., 2013). The steroidogenic action of QRFP can likely be accounted for by the increased expression of the steroidogenic acute regulatory protein and the cytochrome P450 steroidogenic enzymes CYP11B1 and CYP11B2 (Ramanjaneya et al., 2013). The stimulatory effect of QRFP on H295 cells is mediated via the MAPK/PKC signalling pathway and involves T‐type calcium channel activation (Ramanjaneya et al., 2013; Figure 7). Interestingly, QRFP receptor knockdown with siRNA partially blocks QRFP‐induced corticosteroid secretion (Ramanjaneya et al., 2013), suggesting the involvement of another receptor in addition to QRFP receptor (Ramanjaneya et al., 2013). As a matter of fact, 26RFa and QRFP both exhibit substantial affinity for the human NPFF2 receptor (Gouardères et al., 2007), which is expressed in the rat adrenal cortex (Bonini et al., 2000). Altogether, these data support the notion that 26RFa/QRFP produced within the adrenal cortex and/or medulla may act locally to regulate corticosteroid secretion through a paracrine/autocrine mode of communication.

Effects of QRFP peptides on the pancreas

The preservation of pancreatic beta cell mass is essential for maintaining normal glucose metabolism. Both type 1 and type 2 diabetes are characterized by decreased beta cell function and survival and reduced capacity of the endocrine pancreas to maintain an adequate insulin secretion (Muoio and Newgard, 2008). Therefore, one of the major goals in diabetes research is to identify strategies to prevent beta cell loss and increase beta cell function (Vetere et al., 2014).

QRFP receptors show sequence similarity with NPY and galanin receptors (Lee et al., 2001). Moreover, like NPY and galanin (Schwartz et al., 2000), in addition to stimulating food consumption (Chartrel et al., 2003; Do Rego et al., 2006), 26RFa regulates insulin secretion (Egido et al., 2007). Indeed, 26RFa infusion in the perfused rat pancreas inhibits insulin release in response to glucose, arginine and exendin‐4, without affecting basal insulin secretion. Furthermore, 26RFa does not affect basal glucagon output or glucagon release in response to arginine (Egido et al., 2007). 26RFa‐induced inhibition of insulin secretion involves a Pertussis toxin‐sensitive Gα_i protein coupled to the AC pathway. Noteworthy, FMRF‐NH₂, a peptide sharing with 26RFa the C‐terminal RFamide sequence, was previously found to inhibit insulin secretion without modifying glucagon output (Sorenson et al., 1984). An inhibitory effect on insulin secretion has also been described for two other FMRFamide‐related peptides, A‐18‐Famide or NPAF and F‐8‐Famide or NPFF (Fehmann et al., 1990). Interestingly, in their study, Egido et al. (2007) were unable to identify the receptor mediating the inhibitory effect of 26RFa in the rat pancreas, and this was ascribed to the fact that other investigators failed to demonstrate expression of QRFP receptor in the pancreas (Fukusumi et al., 2003; Jiang et al., 2003). It should be noted that expression of either QRFP receptor or 26RFa/QRFP gene and protein has been recently shown in both rat INS‐1E beta cells, mouse MIN6 beta cells and human pancreatic islets (Granata et al., 2014; Prévost et al., 2015). Therefore, the involvement of QRFP receptors in the inhibitory effects of 26RFa described by Egido et al. cannot be excluded.

In agreement with the initial findings of Egido et al. (2007), 26RFa was subsequently found to inhibit both basal and GSIS in INS‐1E beta cells and human pancreatic islets through Gα_i‐mediated mechanisms and attenuation of intracellular cAMP levels (Granata et al., 2014). However, the insulinostatic effect of 26RFa in beta cells is not blunted by knocking down QRFP receptor expression, suggesting engagement of a different receptor. Interestingly, QRFP, differently from 26RFa, increases both basal and GSIS in beta cells and human islets by activating a QRFP receptor‐dependent Gα_s/cAMP pathway and also promotes glucose uptake (Granata et al., 2014). Furthermore, QRFP enhances, whereas 26RFa attenuates the insulinotropic effect of the GLP‐1 receptor agonist exendin‐4 in both beta cells and human islets (Granata et al., 2014). Therefore, both peptides exert opposite effects on beta cell function by activating distinct signalling pathways.

In addition to beta cell function, the role of 26RFa and QRFP has been recently studied on survival and apoptosis of pancreatic beta cells and human pancreatic islets (Granata et al., 2014). At variance with the opposite effects observed for insulin secretion, both 26RFa and QRFP promote survival, increase proliferation and reduce apoptosis of INS‐1E beta cells cultured in either serum‐deprived conditions or exposed to diabetogenic stimuli, such as inflammatory cytokine synergism or a combination of high glucose and palmitate, also known as glucolipotoxicity (Donath et al., 2008; Poitout and Robertson, 2008). Interestingly, the protective effect of the peptides are similar to those elicited by exendin‐4, whose GLP‐1 receptor‐mediated survival action in beta cells is firmly established (Campbell and Drucker, 2013). Similar protective effects also occur in human pancreatic islets, where 26RFa and QRFP equally increased islet cell survival and inhibited apoptosis induced by the same detrimental stimuli (Granata et al., 2014). QRFP‐induced protection in both beta cells and human islets involves activation of the survival and proliferative pathways PI3K/Akt and ERK1/2, whereas the survival effect of 26RFa requires ERK1/2 but not PI3K/Akt phosphorylation. Overall, these data indicate a novel common protective role for 26RFa and QRFP in beta cells and human pancreatic islets, but opposite effects on insulin secretion, probably through activation of distinct receptors and mechanisms.

At variance with the studies of Egido et al. (2007) and Granata et al. (2014), a recent study indicates that 26RFa may act as an incretin hormone (Prévost et al., 2015). In fact, 26RFa is abundantly produced in the gastrointestinal tract in mice and humans, as well as in the human endocrine pancreas. In addition, 26RFa was reported to promote insulin secretion, at low, but not at high concentrations of glucose in mouse MIN6 beta cells, via QRFP receptor‐mediated mechanisms (Prévost et al., 2015). Exendin‐4 similarly increases insulin secretion at low glucose concentrations, but this effect is also observed with high glucose (Prévost et al., 2015). In mice, 26RFa attenuates hyperglycaemia induced by a glucose load, potentiates insulin sensitivity and increases plasma insulin concentrations. Moreover, glucose load induces a massive secretion of 26RFa by the small intestine in mice, both in vivo and in vitro. Furthermore, Prévost et al. (2015) show a moderate positive correlation between plasma 26RFa level, plasma insulin level and the plasma insulin resistance marker HOMA‐R in patients with diabetes, suggesting a possible link between the 26RFa system and glucose homeostasis. Accordingly, plasma 26RFa levels also increase in response to an oral glucose tolerance test, suggesting that an oral glucose load influences circulating 26RFa concentrations (Prévost et al., 2015).

In conclusion, expression of either 26RFa, QRFP or the QRFP receptor has been demonstrated in the human endocrine pancreas, the human and rodent gastrointestinal tract and in pancreatic beta cell lines. Although the role of 26RFa on beta cell function is still controversial, most likely due to different experimental conditions, it is clear that both peptides exert survival and antiapoptotic effects and influence insulin secretion, suggesting that they may represent potential therapeutic targets for insulin resistance and diabetes.

Effects of QRFP peptides on adipose tissue

26RFa/QRFP and its receptor have been shown to play a role in energy balance and glucose metabolism, the majority of studies indicating that central administration of 26RFa increases energy intake (Chartrel et al., 2003; Do Rego et al., 2006; Moriya et al., 2006; Takayasu et al., 2006; Primeaux et al., 2008; Lectez et al., 2009; Mulumba et al., 2010; Primeaux, 2011; Primeaux et al., 2013; Chartrel et al., 2016; Schreiber et al., 2016). In mice and rats, i.c.v. injection of 26RFa and QRFP increases intake of high‐fat diet, and chronic administration of QRFP causes hyperphagia, increases body weight and fat mass in mice consuming a moderately fat (32% kcal from fat) diet (Moriya et al., 2006; Primeaux et al., 2008; Primeaux, 2011; Primeaux et al., 2013). In mice, chronic central administration of QRFP also yields an increase in circulating leptin levels (Moriya et al., 2006). Leptin is an adipose hormone that is positively correlated with fat mass and acts as a peripheral adipose signal, which interacts with the brain to alter feeding behaviour (Elmquist et al., 1999; Barsh and Schwartz, 2002). Dysregulation of the leptin system, as seen in genetic models of leptin deficiency (ob/ob and db/db mice), leads to an increase in hypothalamic preproQRFP mRNA expression (Takayasu et al., 2006). Further investigation of the interaction between centrally administered 26RFa/QRFP and leptin indicates that 26RFa and leptin have opposite effects on the expression and release of hypothalamic neuropeptides that regulate feeding behaviour (i.e. NPY, POMC and αMSH) (Lectez et al., 2009). Central administration of 26RFa attenuates the effects of leptin on POMC mRNA, while leptin administration attenuates the effects of 26RFa on NPY mRNA expression and NPY release (Lectez et al., 2009).

In addition to their central effect on adiposity and adipose hormones, 26RFa/QRFP also acts peripherally on adipose tissue. QRFP and its receptor, QRFP receptor 2, are expressed in fat depots of mice and in 3T3‐L1 adipocyte cells. Consistent with this observation, administration of 26RFa or QRFP increases triglyceride accumulation and fatty acid uptake in these cells (Mulumba et al., 2010; Jossart et al., 2014). The inhibitory effect of QRFP on isoproterenol‐induced lipolysis is mediated via the PI3K/Akt pathway leading to the activation of phosphodiesterase 3B (Figure 7) which inhibits the phosphorylation of the hormone‐sensitive lipase. Concomitantly, activation of QRFP receptor 2 by QRFP attenuates Src kinases/PKC signalling (Mulumba et al., 2015). Diet‐induced obesity in mice provokes a significant decrease in preproQRFP mRNA expression in epididymal fat depots and a 16‐fold increase in QRFP receptor 2 mRNA expression (Mulumba et al., 2010). Clinical studies reveal that circulating 26RFa levels are elevated in anorexia nervosa patients who present low levels of body adiposity (Galusca et al., 2012). 26RFa/QRFP overexpression could thus represent a mechanism to promote energy intake and increase fat stores.

Intracerebroventricular chronic infusion of QRFP reduces rectal temperature in ad libitum‐ and pair‐fed mice (Moriya et al., 2006). In brown adipose tissue of both groups, QRFP decreases mRNA expression of uncoupling protein 1 (also called thermogenin), which plays an important role in thermogenesis regulation (Moriya et al., 2006).

Effects of QRFP peptides on tumour cells

Although there are few studies investigating the role of QRFP and its receptors in tumour regulation, QRFP and QRFP receptors are expressed in a number of cancer cell lines and tumours, most notably, colorectal, testicular, pancreatic and liver cancers and also in breast, ovarian and prostate cancer (Human Protein Atlas www.proteinatlas.org). Because neuropeptides produced by neuroendocrine cells influence the aggressiveness of prostate cancer by affecting growth, invasiveness, metastatic processes and/or angiogenesis (Hansson and Abrahamsson, 2001), it is conceivable that 26RFa/QRFP may play a role in tumour regulation. Thus, the role of 26RFa and QRFP receptors in prostate cancer, notably in hormone refractory prostate cancer which is often associated with advanced prostate cancer, has been investigated (Alonzeau et al., 2013). 26RFa/QRFP and the QRFP receptor are present in human prostate tumours, as shown by immunohistochemistry, and the number of 26RFa/QRFP‐ and QRFP receptor‐stained cells increases with the grade or severity of the tumour. To further examine the role of 26RFa/QRFP and QRFP receptors in prostate cancer, the androgeno‐independent cancer cell line, DU145, was used to examine the effects of 26RFa on migration, proliferation and neuroendocrine cell differentiation. 26RFa promotes migration of the cells, but not proliferation, and stimulates neuroendocrine cell differentiation (Alonzeau et al., 2013). These data support a role for 26RFa in prostate tumour development, specifically in hormone‐independent tumours. Further studies are required to elucidate the possible role of 26RFa/QRFP and QRFP receptor on tumour growth and differentiation.

The 26RFa/QRFP gene (farp‐5) has been identified as a key candidate gene during the transformation of normal buccal mucosa to precancerous lesions in the Syrian golden hamster (M. auratus) by the chemical carcinogen 7,12‐dimethylbenz(a)anthracene (Chen et al., 2011). Down‐regulation of the 26RFa/QFRP gene in precancerous lesions of buccal mucosa suggests that stimulation of farp‐5 or QRFP receptor signalling may improve treatment methods and chemoprophylaxis of precancerous lesions (Chen et al., 2011).

Conclusions and perspectives

Since the discovery of 26RFa/QRFP and the QRFP receptor (Chartrel et al., 2003; Fukusumi et al., 2003; Jiang et al., 2003), a number of studies have been conducted to elucidate the functional significance of this novel peptide‐receptor system. The structure of the 26RFa/QRFP cDNA and the C‐terminal sequence of the 26RFa/QRFP peptide have been strongly preserved in the vertebrate lineage (Ukena et al., 2014; Xu et al., 2015), suggesting that 26RFa/QRFP and its receptor exert vital biological functions.

Consistent with the expression of 26RFa/QRFP and its receptor in hypothalamic nuclei involved in the control of feeding behaviour, several studies have shown that the peptide exerts a potent orexigenic activity in rodents and birds (Chartrel et al., 2003; Do Rego et al., 2006; Moriya et al., 2006; Takayasu et al., 2006; Ukena et al., 2010; Tobari et al., 2011; Primeaux et al., 2013; Zagorácz et al., 2015). 26RFa/QRFP also influences insulin secretion from pancreatic beta cells (Egido et al., 2007; Granata et al., 2014; Prévost et al., 2015) and induces lipid accumulation in adipocytes (Mulumba et al., 2015). Molecular design of peptidic or non‐peptidic, selective and high‐affinity antagonists may thus contribute to the development of new compounds with therapeutic value for the treatment of metabolic disorders and obesity.

The phenotype of QRFP‐deficient mice (Okamoto et al., 2016) has confirmed pharmacological data showing that 26RFa/QRFP displays orexigenic and anxiogenic properties (Chartrel et al., 2003; Do Rego et al., 2006; Moriya et al., 2006; Takayasu et al., 2006; Primeaux et al., 2013) and stimulates locomotor activity (Do Rego et al., 2006; Takayasu et al., 2006). Search for association between SNPs within the human QRFP gene with eating and mood disorders will be required to determine whether 26RFa/QRFP exerts similar activities in humans.

QRFP receptor 1 knockout female mice exhibit severe kyphosis and osteopenia (Baribault et al., 2006), indicating that 26RFa/QRFP is likely involved in osteochondral bone formation. Thus, rational design of stable, selective and high‐affinity peptidic agonists may lead to the development of innovative therapeutic agents for the treatment of osteoporosis. Concurrently, generation of QRFP receptor 2‐deficient mice would help to elucidate other physiological roles of 26RFa/QRFP. In addition, creation of mice with tissue‐specific disruption of the QRFP receptor 1/2 genes may reveal novel functions exerted by the peptide.

There is strong evidence that 26RFa/QRFP and the QRFP receptor are involved in the regulation of the hypothalamo–pituitary–gonadal axis (Kampe et al., 2006; Navarro et al., 2006; Patel et al., 2008). Since several other peptides harboring the RF‐amide or the RY‐amide motifs at their C‐terminus (i.e. GnIH and kisspeptin) are also involved in the control of reproduction (Pinilla et al., 2012; Tsutsui and Ubuka, 2016), cross‐activities of the different peptides with other FLP receptors should be carefully examined.

The C‐terminal hexapeptide of 26RFa/QRFP, that is, 26RFa_(20–26), is the biologically active determinant of the peptide that mimics most of its behavioural and metabolic effects (Do Rego et al., 2006; Navarro et al., 2006). Surprisingly, however, the N‐terminal region of 26RFa, that is, 26RFa_(1–16), appears to be responsible for its hyperlocomotor activity (Do Rego et al., 2006). Whether the effect of 26RFa/QRFP on locomotion is mediated through a receptor distinct from QRFP receptor deserves further investigation.

Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Southan et al., 2016), and are permanently archived in the Consise Guide to PHARMACOLOGY 2015/16 (Alexander et al., 2015a, 2015b, 2015c).

Acknowledgements

The authors thank Dr Eliot Ohlstein (Drexel University College of Medicine, Philadelphia, PA, USA) for his valuable cooperation. This work was supported by INSERM, the University of Normandy Rouen, the LARC‐Neuroscience Network, the European Regional Development Fund (ERDF, PeReNE), the Institute for Research and Innovation in Biomedicine (IRIB) and the Region Normandy.

Conflict of interest

The authors declare no conflicts of interest.