Handbook of Hormones: Comparative Endocrinology for Basic and Clinical Research

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Part I

Peptides and Proteins in Vertebrates

Outline

Section I.1 Neuropeptides

Section I.2 Adenohypophysial Hormones

Section I.3 Gastrointestinal Hormones

Section I.4 Parathyroid Gland, Ultimobranchial Gland, and Stannius Corpuscle Hormones

Section I.5 Other Peripheral Hormones

Section I.1

Neuropeptides

Outline

Chapter 1 RFamide Peptide Family

Subchapter 1A Gonadotropin-Inhibitory Hormone

Subchapter 1B Kisspeptin

Subchapter 1C PQRFamide Peptide

Subchapter 1D Pyroglutamylated RFamide Peptide

Subchapter 1E Prolactin-Releasing Peptide

Chapter 2 Corticotropin-Releasing Hormone Family

Subchapter 2A Corticotropin-Releasing Hormone

Subchapter 2B Urotensin-I

Subchapter 2C Urocortins

Subchapter 2D Sauvagine

Chapter 3 Gonadotropin-Releasing Hormone

Chapter 4 Thyrotropin-Releasing Hormone

Chapter 5 Somatostatin

Chapter 6 Neurohypophysial Hormone Family

Subchapter 6A Vasopressin

Subchapter 6B Vasotocin

Subchapter 6C Oxytocin

Subchapter 6D Non-Mammalian Oxytocin Family Peptides

Chapter 7 Opioid Peptide Family

Subchapter 7A Enkephalin

Subchapter 7B Dynorphin/α-Neo-endorphin

Subchapter 7C Nociceptin/Orphanin FQ

Subchapter 7D Endomorphin

Subchapter 7E Dermorphin

Chapter 8 Agouti Family

Subchapter 8A Agouti-Signaling Protein

Subchapter 8B Agouti-Related Protein

Chapter 9 Tachykinin Family

Subchapter 9A Substance P/Neurokinin A

Subchapter 9B Neurokinin B

Chapter 10 Appetite-Regulating Peptides

Subchapter 10A Melanin-Concentrating Hormone

Subchapter 10B Orexin

Subchapter 10C Cocaine- and Amphetamine-Regulated Transcript

Chapter 11 Urotensin II

Chapter 12 Neurotensin

Chapter 13 Neuromedin U/S

Chapter 1

RFamide Peptide Family

Kazuyoshi Tsutsui and Takayoshi Ubuka

Abstract

Neuropeptides that possess the Arg–Phe–NH2 motif at their C-termini that form an RFamide peptide family have been characterized in both invertebrates and vertebrates. The first RFamide peptide was the cardioexcitatory peptide Phe–Met–Arg–Phe–NH2 (FMRFamide), which was isolated from the ganglia of the Venus clam Macrocallista nimbosa. In addition to invertebrates, RFamide peptides have been characterized in vertebrates. In the past, the existence of five groups within the RFamide peptide family has been recognized in vertebrates: the gonadotropin-inhibitory hormone (GnIH) group, the kisspeptin group, the PQRFamide peptide (NPFF) group, the pyroglutamylated RFamide peptide (QRFP)/26RFamide group, and the prolactin-releasing peptide (PrRP) group. These RFamide peptides have been shown to exert important neuroendocrine, behavioral, sensory, and autonomic functions. This chapter summarizes the structural features and functions of RFamide peptides and their cognate receptors.

Keywords

Arg–Phe–NH2 motif; FMRFamide peptides; gonadotropin-inhibitory hormone (GnIH) group; kisspeptin group; PQRFamide peptide (NPFF) group; pyroglutamylated RFamide peptide (QRFP)/26RFamide group; prolactin-releasing peptide (PrRP) group

History

Neuropeptides that possess the Arg–Phe–NH2 motif at their C-termini (i.e., RFamide peptides) have been characterized both in invertebrates and vertebrates. The first RFamide peptide to be identified was the cardioexcitatory peptide Phe–Met–Arg–Phe–NH2 (FMRFamide), which was isolated from the ganglia of the Venus clam Macrocallista nimbosa [1]. Since then, a number of RFamide peptides have been identified in invertebrates, where these peptides seem to act as neurotransmitters and neuromodulators [2]. Subsequently, several RFamide peptides have been characterized in the brain of various vertebrates. In the past, the existence of five groups within the RFamide peptide family has been recognized in vertebrates: the gonadotropin-inhibitory hormone (GnIH) group, the kisspeptin group, the PQRFamide peptide (NPFF) group, the pyroglutamylated RFamide peptide (QRFP)/26RF amide group, and the prolactin-releasing peptide (PrRP) group (Table 1.1) [3–7]. These RFamide peptides have been shown to exert important neuroendocrine, behavioral, sensory, and autonomic functions [3–8].

Table 1.1

Comparison of Amino Acid Sequences of the RFamide Peptide Family in Humans*

*RFamide sequences are shown in bold.

Structure

Structural Features

A common structural feature of the family members is the Arg–Phe–NH2 motif at their C-termini (Table 1.1) [3–9]. Human GnIH precursor mRNA encodes a polypeptide that produces two mature peptides, GnIH1 (RFRP-1) and GnIH2 (RFRP-3) [9]. Human kisspeptin precursor has several potential N-terminal cleavage sites and one C-terminal cleavage and amidation site, which generate several lengths of biologically active kisspeptins (kisspeptin-54, kisspeptin-14, kisspeptin-13, and kisspeptin-10) [10]. Human PQRFamide peptide (NPFF) precursor mRNA encodes a polypeptide that produces two mature peptides (NPFF and NPAF) [8]. Human QRFP precursor has several potential N-terminal cleavage sites and one C-terminal cleavage and amidation site, which generate several lengths of biologically active QRFPs (43RFa and 26RFa) [8]. Human PrRP precursor has several potential N-terminal cleavage sites and one C-terminal cleavage and amidation site, which generate several lengths of biologically active PrRPs (PrRP-31 and PrRP-20) [8].

Molecular Evolution of Family Members

E-Figure 1.1 shows a phylogenetic tree of the RFamide peptide family in vertebrates. The scale bar refers to a phylogenetic distance of 0.1 amino acid substitutions per site among the precursors. The phylogenetic tree of the RFamide peptide family shows that the precursors of RFamide peptides are clustered into five groups – the GnIH group, the kisspeptin group, the PQRFamide peptide (NPFF) group, the QRFP/26RFamide group, and the PrRP group – due to their structural similarities. The kisspeptin group includes two closely related peptides, Kiss1 and Kiss2, which are produced from different precursors encoded in paralogous genes of each animal [7,8,10].

Receptors

Structure and Subtypes

The cognate receptors for GnIH, NPFF, kisspeptin, QRFP/26RFamide, and PrRP are seven-transmembrane G protein-coupled receptors (GPCRs), GPR147, GPR74, GPR54, GPR103, and GPR10, respectively (Table 1.2) [7,8,10]. GPR147, GPR74, GPR103, and GPR10 belong to the family of rhodopsin β-type GPCRs. GPR54 belongs to rhodopsin γ-type GPCR family. GPR147 and GPR74 are structurally similar, and they both bind GnIH and NPFF, although GnIH and NPFF have higher affinity for GPR147 and GPR74, respectively. GPR74 also binds kisspeptin, QRFP, and PrRP with lower affinity than GPR54, GPR103, and GPR10, respectively.

Table 1.2

Intracellular Effects of RFamide-Peptide Receptor Activation

Signal Transduction Pathway

GPR147, GPR74, GPR54, GPR103, and GPR10 are GPCRs, which interact with G proteins in the cell [7,8,10]. Agonist-bound GPCR activates various G proteins, such as Gαi, Gαq, and Gβγ subunits, and regulates the activity of AC and PLC (Table 1.2). AC is a membrane associated enzyme that catalyzes synthesis of the second messenger cAMP. PLC is a membrane associated enzyme that catalyzes the synthesis of IP3. cAMP and IP3 can modify intracellular Ca²+ concentration ([Ca²+]i) (Table 1.2). It has been reported that GPR147, GPR74, GPR103, and GPR10 can inhibit the activity of AC. GPR54 stimulates PLC and increases [Ca²+]i. GPR103 and GPR10 can also increase [Ca²+]i.

Biological Functions

Target Cells/Tissues and Functions

GnIH neurons project to the median eminence and gonadotropin-releasing hormone (GnRH) neurons [3,5]. GnIH receptor GPR147 is expressed in the gonadotropes and GnRH neurons [3,5]. Thus, GnIH may inhibit gonadotropin secretion by decreasing the activity of GnRH neurons as well as directly inhibiting gonadotropes. NPFF binds GPR74 expressed in the dorsal horn in the spinal cord and modulates pain transmission [7,8]. NPFF and NPAF attenuate the analgesic effect of morphine [7,8]. NPFF also modifies food intake [7,8]. Kisspeptin neurons project to GnRH neurons, and GnRH neurons express GPR54 [6,8]. Kisspeptin is thought to be important for puberty onset-to-adult regulation of gonadotropin secretion and fertility. Kisspeptin also has potent anti-metastasis activity in several tumors [8]. QRFP/26RFamide and its receptor GPR103 mRNAs are expressed in the hypothalamus [7,8]. QRFP/26RFamide increases food intake, stimulates the gonadotropic axis, inhibits insulin secretion, inhibits locomotor activity, and induces analgesic effects [7,8]. PrRP receptor GPR10 mRNA is widely expressed throughout the brain, but highest in the thalamus, hypothalamus, and pituitary [7,8]. PrRP stimulates prolactin secretion, and has a variety of regulatory functions on food intake, stress, blood pressure, and pain [8].

Supplemental Information Available on Companion Website

• Phylogenetic tree of the RFamide peptide family members/E-Figure 1.1

• Phylogenetic tree of the RFamide peptide receptors/E-Figure 1.2

References

1. Price DA, Greenberg MJ. Structure of a molluscan cardioexcitatory neuropeptide. Science. 1977;197:670–671.

2. Walker RJ, Papaioannou S, Holden-Dye L. A review of FMRFamide- and RFamide-like peptides in metazoa. Invert Neurosci. 2009;9:111–153.

3. Tsutsui K. A new key neurohormone controlling reproduction, gonadotropin-inhibitory hormone (GnIH): Biosynthesis, mode of action and functional significance. Prog Neurobiol. 2009;88:76–88.

4. Tsutsui K, Ukena K. Hypothalamic LPXRF-amide peptides in vertebrates: identification, localization and hypophysiotropic activity. Peptides. 2006;27:1121–1129.

5. Tsutsui K, Bentley GE, Bedecarrats G, et al. Gonadotropin-inhibitory hormone (GnIH) and its control of central and peripheral reproductive function. Front Neuroendocrinol. 2010;31:284–295.

6. Tsutsui K, Bentley GE, Kriegsfeld LJ, et al. Discovery and evolutionary history of gonadotrophin-inhibitory hormone and kisspeptin: new key neuropeptides controlling reproduction. J Neuroendocrinol. 2010;22:716–727.

7. Ukena K, Tsutsui K. A new member of the hypothalamic RF-amide peptide family, LPXRF-amide peptides: structure, localization, and function. Mass Spectrom Rev. 2005;24:469–486.

8. Chartrel N, Tsutsui K, Costentin, et al. Gonadotropin-inhibitory hormone. In: Kastin AJ, ed. Handbook of biologically active peptides, Section on brain peptides. Amsterdam: Elsevier; 2006;79–86.

9. Ubuka T, Morgan K, Pawson AJ, et al. Identification of human GnIH homologs, RFRP-1 and RFRP-3, and the cognate receptor, GPR147 in the human hypothalamic pituitary axis. PLoS One. 2009;4:e8400.

10. Pinilla L, Aguilar E, Dieguez C, et al. Kisspeptins and reproduction: physiological roles and regulatory mechanisms. Physiol Rev. 2012;92:1235–1316.

Supplemental Information

E-Figure 1.1 The unrooted phylogenetic tree of the vertebrate RFamide peptide family members.

Vertebrate brains produce a variety of RFamide peptides. To date, five groups in the RFamide peptide family have been documented: the gonadotropin-inhibitory hormone (GnIH) group, kisspeptin group, PQRFamide peptide (NPFF) group, pyroglutamylated RFamide peptide (QRFP)/26RFamide group, and prolactin-releasing peptide (PrRP) group. The scale bar refers to a phylogenetic distance of 0.1 amino acid substitutions per site among the precursors.

E-Figure 1.2 The unrooted phylogenetic tree of the vertebrate RFamide peptide receptors.

The cognate receptors for GnIH, NPFF, kisspeptin, QRFP/26RFamide, and PrRP are seven-transmembrane G protein-coupled receptors (GPCRs), GPR147, GPR74, GPR54, GPR103, and GPR10, respectively. Scale bar refers to a phylogenetic distance of 0.1 amino acid substitutions per site among the precursors. The accession numbers of the receptors are as follows: Human NPFFR1 (NP_071429.1), Mouse NPFFR1 (NP_001170982.1), Rat NPFFR1 (NP_071627.2), Xenopus tropicalis NPFFR1 (XP_004915843.1), Chicken NPFFR1 (NP_989693.1), Quail GnIHR (BAD86818.1), Zebrafish NPFFR1-like2 (NP_001165168.1), Zebrafish NPFFR1 (NP_001082858.1), Zebrafish QRFPR-like (XP_002665976.2), Zebrafish QRFPR (XP_001920042.3), Xenopus tropicalis QRFPR-like (XP_002935352.1), Human QRFPR (NP_937822.2), Mouse QRFPR (NP_937835.1), Rat QRFPR (NP_937842.1), Chicken QRFPR (NP_001120642.1), Xenopus tropicalis QRFPR (NP_001072295.1), Human PrRPR (AAC50504.1), Rat PrRPR (NP_631932.3), Mouse PrRPR (NP_963909.2), Chicken PrRPR (NP_001012295.1), Xenopus tropicalis PrRPR (XP_002940396.1), Xenopus tropicalis PrRPR-like1 (XP_002933014.1), Zebrafish PrRPR (NP_001034615.1), Zebrafish PrHR2b (NP_001108618.1), Zebrafish PrRPR-like (NP_001108620.1), Xenopus tropicalis PrRPR-like2 (XP_002940541.2), Zebrafish PrH2R (NP_001108619.1), Human GPR54 (AAK83235.1), Mouse GPR54 (AAK83236.1), Rat GPR54 (AAD19664.1), Zebrafish Kiss-1Rb (NP_001104001.1), Xenopus tropicalis Kiss-1R (NP_001163985.1), Xenopus tropicalis GPR54 isoform b (NP_001165295.1), Xenopus tropicalis GPR54 (NP_001165296.1), Zebrafish Kiss-1Ra (NP_001099149.1), Zebrafish LOC559950 (NP_001165167.1), Zebrafish NPFFR2.1 (NP_001098579.1), Zebrafish NPFFR2 (XP_690069.5), Chicken NPFFR2 (NP_001029997.2), Xenopus tropicalis NPFFR2 (XP_002940397.1), Human NPFFR2 (NP_444264.1), Mouse NPFFR2 (NP_573455.2), Rat NPFFR2 (NP_076470.1).

Subchapter 1A

Gonadotropin-Inhibitory Hormone

Kazuyoshi Tsutsui and Takayoshi Ubuka

Abstract

Gonadotropin-inhibitory hormone (GnIH) was originally identified in birds, and subsequently in mammals and other vertebrates. GnIH acts on the pituitary and on gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus via GPR147, a G protein-coupled receptor. GnIH decreases gonadotropin synthesis and release, inhibiting gonadal development and maintenance. Such a downregulation of the hypothalamo-pituitary–gonadal (HPG) axis may be conserved across vertebrates. GnIH also inhibits reproductive behaviors, such as sexual and aggressive behaviors. GnIH may operate at the level of the gonads as an autocrine/paracrine regulator of steroidogenesis and gametogenesis. GnIH acts in the HPG axis to appropriately regulate reproductive activity across the seasons and during times of stress.

Keywords

gonadotropin-inhibitory hormone (GnIH); GPR147; gonadotropin; hypothalamo-pituitary–gonadal axis; reproduction; reproductive behavior; melatonin; stress

Additional names/abbreviations: LPXRFamide peptide, RFamide-related peptide, Neuropeptide VF/GnIH, LPXRFa, RFRP, NPVF

A hypothalamic neuropeptide inhibiting gonadotropin secretion, GnIH inhibits sexual and aggressive behavior. It has an LPXRFamide (X=L or Q) sequence at its C-terminal.

Discovery

First identified in the hypothalamus of the Japanese quail in 2000 [1], GnIH’s structure has been determined in the hypothalamus of various vertebrates, including humans [2].

Structure

Structural Features

Human GnIH precursor polypeptide consists of 196 amino acid residues that produce two mature GnIH peptides, human GnIH1 (RFRP-1) and GnIH2 (RFRP-3) [2]. The human GnIH1 (RFRP-1) aa sequence in the precursor polypeptide follows a basic aa lysine that is a proteolytic cleavage site preceding its N-terminal. Subsequent MPHSFANLPLRF sequence is followed by glycine as an amidation signal, and arginine as an endoproteolytic basic amino acid (Table 1A.1). The human GnIH2 (RFRP-3) aa sequence in the precursor polypeptide follows two arginine preceding its N-terminal. The subsequent VPNLPQRF sequence is followed by glycine as an amidation signal and arginine as an endoproteolytic basic aa (Table 1A.1) [2]. Most mammalian GnIH precursor polypeptides may produce two LPXRFamide (X=L or Q) peptides, whereas non-mammalian GnIH precursor polypeptides may produce three or four LPXRFamide peptides [3].

Table 1A.1

Primary Structure of GnIH Peptides

Primary Structure

Most GnIH peptides have the LPXRFamide (X=L or Q) motif at their C-termini (Table 1A.1) [3]. Although the N-terminal sequences are less conserved across vertebrate species, many identified endogenous GnIH peptides start from serine at the N-terminal (macaque RFRP-3, bovine RFRP-1, hamster RFRP-1, quail GnIH, quail GnIH-RP-2, starling GnIH, zebra finch GnIH, frog GRP, frog GRP-RP-1, newt LPXRFa-1, newt LPXRFa-3, goldfish LPXRFa-3, lamprey LPXRFa-1a, and lamprey LPXRFa-2). Other GnIH peptides start from alanine (bovine RFRP-3, rat RFRP-3, frog GRP-RP-3, newt LPXRFa-4, and lamprey LPXRFa-1b), methionine (human RFRP-1, newt LPXRFa-2), valine (human RFRP-3), threonine (hamster RFRP-3), and tyrosine (frog GRP-RP-2) at their N-termini.

Properties

Human GnIH1 (RFRP-1): Mr 1,429, pI 10.55. Freely soluble in water. Human GnIH1 (RFRP-1) dissolved in water at 10−3 M is stable for more than a year at −20°C.

Human GnIH2 (RFRP-3): Mr 969, pI 10.55. Freely soluble in water. Human GnIH2 (RFRP-3) dissolved in water at 10−3 M is stable for more than a year at −20°C.

Synthesis and Release

Gene, mRNA, and Precursor

The human GnIH precursor gene (NPVF), located on chromosome 7 (p21–p15), consists of three exons. Human GnIH precursor mRNA has 1,190 bp. An orthologous GnIH precursor gene was found in sea lamprey, one of the most basal vertebrate species [4]. The gene structure and mRNA sizes are well conserved among vertebrates [3].

Distribution of mRNA

In the brain, GnIH precursor mRNA is exclusively expressed in the hypothalamus [1–5]. Transcripts are also found in the eye and the gonads [3,5].

Tissue and Plasma Concentrations

In the brain, abundant GnIH immunoreactive neuronal fibers are found in the hypothalamus followed by the midbrain [1–4]. The GnIH concentration in quail diencephalon is approximately 100 pmol/g tissue [1,6]. In the gonad, GnIH immunoreactive materials were found in ovarian thecal and granulosa cells, testicular interstitial cells, and germ cells [3]. The plasma concentration of GnIH is below its detection limit, except in the hypophysial portal system.

Regulation of Synthesis and Release

GnIH expression in the brain is regulated by nocturnal secretion of melatonin in birds [6] and mammals [3]. Melatonin may also stimulate GnIH release [3]. GnIH expression in the brain is also regulated by stress via the action of glucocorticoids [3]. Although glucocorticoid response elements (GREs) are found at −1,665 and −1,530 bp upstream of the GnIH precursor coding region in rats, the −1,530 GRE was identified to be critical for corticosterone responsiveness and recruitment of the glucocorticoid receptor (GR). GnIH expression in the testis is also regulated by melatonin and corticosterone in birds [3].

Receptors

Structure and Subtype

Quail GnIH binds the GnIH receptor (GnIHR) with high affinity (Kd=0.752 nM) [7]. GnIHR is a seven-transmembrane rhodopsin β-type G protein-coupled receptor (GPCR). GnIHR is also named GPR147 or NPFFR1 [2,5]. Mammalian GnIH (RFRP) also binds GPR74 (NPFFR2) with significantly lower affinity [3]. GPR147 (NPFFR1) and GPR74 (NPFFR2) genes are thought to be paralogous, positioned within the MetaHOX paralogons in human chromosomes 10 and 4, respectively. Human GnIHR (GPR147) has 430 aa residues, Mr 47,819. Vertebrate GnIHR has highly conserved seven-transmembrane domains as well as disulfide bridge sites between the first and second extracellular loops. Several glycosylation sites are predicted in the extracellular amino terminus and the extracellular loops.

Signal Transduction Pathway

Mammalian GnIH (RFRP) suppresses cAMP production in Chinese hamster ovarian cells transfected with GnIHR (GPR147) cDNA, suggesting that GPR147 couples to Gαi protein [5]. The cell signaling pathway by GnIH and GnIHR (GPR147) and its possible interaction with gonadotropin-releasing hormone (GnRH) signaling was investigated using a mouse gonadotrope cell line, LβT2 [8]. The results indicated that mouse GnIH peptides (mRFRPs) inhibit GnRH-induced gonadotropin subunit gene transcriptions by inhibiting AC/cAMP/PKA-dependent ERK activation in LβT2 cells [8].

Agonist

GnIHR (GPR147) binds with high affinity to GnIH peptides, which have a LPXRFamide (X=L or Q) motif at their C-termini [7]. GnIHR (GPR147) also binds neuropeptide FF (NPFF), a pain modulatory neuropeptide that has a PQRFamide motif at its C-terminus, with significantly lower affinity [3].

Antagonist

RF9 was reported as a selective and potent antagonist of GPR147 (NPFFR1) and GPR74 (NPFFR2) [9]. Central administration of RF9 evokes dose-dependent increases of LH and FSH levels in adult male and female rats, possibly by antagonizing GPR147 (NPFFR1) [3]. RF9 can block the increase in blood pressure and heart rate evoked by NPFF in rats, possibly by antagonizing GPR74 (NPFFR2) [3]. When chronically co-injected with heroin, RF9 blocks the delayed and long-lasting opioid-induced hyperalgesia and prevents the development of associated tolerance in rats [9].

Biological Functions

Target Cells/Tissues and Functions

GnIH neurons are located in the paraventricular nucleus (PVN) in birds [1], in the dorsomedial hypothalamic area (DMH) in mammals [2], and in the nucleus posterioris periventricularis in fish [3]. In many vertebrate species, GnIH neurons project to the median eminence or close to the pituitary to control anterior pituitary function [1,4]. GPR147 is expressed in the gonadotropes [2,3], and GnIH suppresses synthesis and release of gonadotropins in many vertebrate species (Table 1A.2) [2,3]. GnIH neurons also project to GnRH neurons in the preoptic area [2,3], and GnRH neurons express GPR147. Accordingly, GnIH may inhibit gonadotropin synthesis and release by decreasing the activity of GnRH neurons as well as directly acting on the gonadotropes [3]. However, GnIH can stimulate gonadotropin synthesis or release in fish and hamsters, depending on their reproductive states (Table 1A.2) [3]. GnIH also stimulates prolactin release in rats (Table 1A.2) [5]. GnIH can inhibit reproductive behavior by possibly acting within the brain (Table 1A.2) [3]. GnIH inhibits aggressive behavior of male quail by stimulating the activity of aromatase and increasing neuroestrogen concentration in the brain (Table 1A.2) [10]. GnIH and GnIHR expressed in the gonads may regulate gametogenesis and sex steroid secretion [3].

Table 1A.2

Biological Actions of GnIH

Phenotype in Gene-Modified Animals

Ablation of GPR147 elevated LH and FSH levels, and lowered the suppressive effect of stress on LH secretion in mice. There are reports showing the effect of RNA interference (RNAi) of GnIH precursor mRNA on the behavior of white-crowned sparrows and Japanese quail [10]. GnIH RNAi reduced the resting time of male and female birds, and spontaneous production of complex vocalizations, and stimulated brief agonistic vocalizations in male white-crowned sparrows. GnIH RNAi further stimulated aggressive song production of short duration in male birds when they were challenged by playbacks of novel male songs. These behaviors resembled the behavior of breeding birds during territorial defense. GnIH RNAi increased locomotor activity and aggressive behavior in the male quail [10]. These results suggest that GnIH RNAi induces arousal and aggressiveness in birds.

Pathophysiological Implications

Clinical Implications

Two endogenous human GnIH peptides, human GnIH1 (RFRP-1) and GnIH2 (RFRP-3), were identified in the human hypothalamus [2]. It was demonstrated that human RFRP-3 has a potent inhibitory action on gonadotropin secretion in ovines that has the same RFRP-3 aa sequence in its GnIH precursor polypeptide [3]. Many factors, such as stress, anorexia, diabetes, obesity, and photoperiod, inhibit gonadotropin secretion [3]. The possible role of GnIH in mediating these effects has been implicated in animal models [3,6]. Thus, GnIH has the potential of an alternative or adjunct therapeutic agent to inhibit gonadotropins and steroid hormones.

Use for Diagnosis and Treatment

The endogenous inhibitor of gonadotropin secretion, GnIH, has therapeutic potential in the treatment of gonadotropins or steroid hormone-dependent diseases, such as precocious puberty, endometriosis, uterine fibroids, benign prostatic hyperplasia, and prostatic and breast cancers. Human GnIH may also have potential as a novel contraceptive.

Supplemental Information Available on Companion Website

• Gene, mRNA, and precursor structures of human GnIH/E-Figure 1A.1

• Precursor and mature hormone sequences of various animals/E-Figure 1A.2

• Gene, mRNA, and precursor structures of human GnIH receptor/E-Figure 1A.3 [11–13]

• Primary structure of GnIH receptor of various animals/E-Figure 1A.4

• Accession numbers of genes and cDNAs for GnIH precursor and GnIH receptor/E-Tables 1A.1, 1A.2

References

1. Tsutsui K, Saigoh E, Ukena K, et al. A novel avian hypothalamic peptide inhibiting gonadotropin release. Biochem Biophys Res Commun. 2000;275:661–667.

2. Ubuka T, Morgan K, Pawson AJ, et al. Identification of human GnIH homologs, RFRP-1 and RFRP-3, and the cognate receptor, GPR147 in the human hypothalamic pituitary axis. PLoS One. 2009;4:e8400.

3. Tsutsui K, Bentley GE, Bedecarrats G, et al. Gonadotropin-inhibitory hormone (GnIH) and its control of central and peripheral reproductive function. Front Neuroendocrinol. 2010;31:284–295 (Review).

4. Osugi T, Daukss D, Gazda K, et al. Evolutionary origin of the structure and function of gonadotropin-inhibitory hormone: insights from lampreys. Endocrinology. 2012;153:2362–2374.

5. Hinuma S, Shintani Y, Fukusumi S, et al. New neuropeptides containing carboxy-terminal RFamide and their receptor in mammals. Nat Cell Biol. 2000;2:703–708.

6. Ubuka T, Bentley GE, Ukena K, et al. Melatonin induces the expression of gonadotropin-inhibitory hormone in the avian brain. Proc Natl Acad Sci USA. 2005;102:3052–3057.

7. Yin H, Ukena K, Ubuka T, et al. A novel G protein-coupled receptor for gonadotropin-inhibitory hormone in the Japanese quail (Coturnix japonica): identification, expression and binding activity. J Endocrinol. 2005;184:257–266.

8. Son YL, Ubuka T, Millar RP, et al. Gonadotropin-inhibitory hormone inhibits GnRH-induced gonadotropin subunit gene transcriptions by inhibiting AC/cAMP/PKA-dependent ERK pathway in LβT2 cells. Endocrinology. 2012;153:2332–2343.

9. Simonin F, Schmitt M, Laulin JP, et al. RF9, a potent and selective neuropeptide FF receptor antagonist, prevents opioid-induced tolerance associated with hyperalgesia. Proc Natl Acad Sci USA. 2006;103:466–471.

10. Ubuka T, Haraguchi S, Tobari Y, et al. Hypothalamic inhibition of socio-sexual behaviour by increasing neuroestrogen synthesis. Nat Commun. 2014;5:3061.

11. Hirokawa T, Boon-Chieng S, Mitaku S. SOSUI: classification and secondary structure prediction system for membrane proteins. Bioinformatics. 1998;14:378–379.

12. Horn F, Weare J, Beukers MW, et al. GPCRDB: an information system for G protein-coupled receptors. Nucleic Acids Res. 1998;26:277–281.

13. Gupta R, Jung E, Brunak S. Prediction of N-glycosylation sites in human proteins. Available at: <www.cbs.dtu.dk/services/NetNGlyc/>; 2004.

Supplemental Information

E-Figure 1A.1 Gene, mRNA, and precursor structures of human GnIH.

Human neuropeptide VF precursor: NPVF, location 7p21–p15. Rectangle in the mRNA shows the reading frame of human GnIH precursor. SP, signal peptide.

E-Figure 1A.2 Precursor and mature hormone sequences of various animals: the alignment of amino acid sequences of GnIH precursor polypeptides in vertebrates.

Conserved amino acid residues are shaded. Identified or predicted LPXRFamide (X=Q or L) peptide sequences are underlined with solid lines. Predicted LPXRFamide-like peptide sequences are underlined with broken lines. Glycine as an amidation donor and possible proteolytic basic amino acids are italicized. Accession number: human (Homo sapiens), NP_071433.3; quail (Coturnix japonica), BAB15932.1; turtle (Chrysemys picta bellii), XP_005290937.1; frog (Rana catesbeiana), BAC11854.1; goldfish (Carassius auratus), BAC06473.1.

E-Figure 1A.3 Gene, mRNA, and precursor structures of human GnIH receptor.

Human neuropeptide FF receptor 1: location 10q21–q22. Rectangle in the mRNA shows the reading frame of human GnIH receptor. N-Term, N-terminal; TM, transmembrane domain; IL, intracellular loop; EL, extracellular loop.

E-Figure 1A.4 Primary structure of GnIH receptor of various animals: the alignment of amino acid sequences of GnIH receptor in vertebrates.

Predicted transmembrane domains are shaded. Transmembrane domains predicted by SOSUI [11] are shaded. Cysteine (C) residues that are predicted by GPCRDB [12] are shown in red. Possible glycosylation sites predicted by NetNGlyc 1.0 Server [13] are italicized. Asterisk (*) indicates positions which have a single, fully conserved residue. Colon (:) indicates conservation between groups of strongly similar properties – scoring >0.5 in the Gonnet PAM 250 matrix. Period (.) indicates conservation between groups of weakly similar properties – scoring ≤0.5 in the Gonnet PAM 250 matrix. Accession number: human (Homo sapiens), NP_071429.1; quail (Coturnix japonica), BAD86818.1; turtle (Chrysemys picta bellii), XP_005281913.1; goldfish (Carassius auratus), AFY63167.1.

E-Table 1A.1

Accession Numbers of Genes and cDNAs for GnIH Precursor

E-Table 1A.2

Accession Numbers of Genes and cDNAs for GnIH Receptor

Subchapter 1B

Kisspeptin

Shinji Kanda and Yoshitaka Oka

Abstract

Kisspeptin is a neuropeptide that has been found to be essential for mammalian reproduction; this has been proven by the fact that, in rodents and humans, lack of Kiss1/KISS1 or its receptor Gpr54/GPR54 gene leads to hypogonadotropic hypogonadism. This peptidergic neuron expresses estrogen receptor α, which is also essential for mouse reproduction, and the peptide not only facilitates significant LH release in serum after injection but also directly or indirectly depolarizes GnRH neurons. In spite of its wide distribution in the vertebrate phylogeny, its regulatory functions on reproduction in non-mammalian vertebrates are still controversial. On the other hand, it has been generally agreed that kisspeptin neurons show prominent expression of sex steroid receptors and act as a potent sensor of peripheral sex steroid conditions, possibly modulating reproductive physiology of vertebrates in general.

Keywords

HPG axis; sex steroid; GnRH; hypothalamus; pituitary

Additional names/abbreviation: metastin

Kisspeptin is a neuropeptide that is encoded by the kiss1/kiss2 gene in vertebrates. Kisspeptin neurons are mainly localized in the hypothalamus, and there is a growing body of evidence to support their essentiality and importance for reproduction in mammals.

Discovery

The KISS1 gene and its product KISS1 were identified to be a natural ligand for an orphan receptor, GPR54, in humans in 2001 [1]. In addition to Kiss1, the existence of a highly homologous peptide was reported when the first kiss1 gene in a non-mammalian species was found [2]. Subsequent synteny analysis as well as reporter assays strongly suggested that: (1) both peptides are kisspeptins in terms of being ligands of GPR54; (2) kiss1 and kiss2 genes [3,4] are suggested to have been duplicated in the early vertebrate lineage; and (3) both are conserved in the majority of vertebrates, although placental mammals and marsupials lack Kiss2.

Structure

Structural Features

Both of the paralogous kiss1 and kiss2 products (Kiss1 and Kiss2 peptides) include widely conserved important C-terminal 10 aa residues.

Primary Structure

Although longer forms have been isolated, the 10 aa residues with amidated C-terminals have been shown to activate the kisspeptin receptor Gpr54 signaling pathways. For instance, human preprokisspeptin consists of 145 aa residues and is cleaved to a shorter C-terminus that varies from 10 to 54 residues (Figure 1B.1). The C-terminal RF/RY-NH2 is the most conserved motif within the RF-amide peptide family which kisspeptins belong to [5]. Native Kiss1/2 peptides have been isolated from tissues in a very limited number of species.

Figure 1B.1 Maturation process of the Kiss1/Kiss2 peptide and conserved peptide sequence of kisspeptins in vertebrates.

(A) Translated preprokisspeptin is cleaved in its dibasic site, the C-terminus of which is amidated. After this cleavage, degradation and/or modification may occur at the N-terminus. (B) Core sequence of Kiss1 and Kiss2 peptide sequence. Adapted with permission from Kanda and Oka, 2013 [9], © Springer.

Synthesis and Release

Gene, mRNA, and Precursor

Ancestral kisspeptin genes have been suggested to be duplicated prior to the emergence of agnathans, due to the fact that lampreys have both kiss1 and kiss2 genes. Moreover, because synteny relationship is conserved between these two paralogs, it has been also suggested that these genes were duplicated in either 1R or 2R whole genome duplication. On the other hand, there has been no report for the existence of two kiss1 or two kiss2 genes in teleosts, and it is therefore likely that one of the duplicated copies was lost immediately after the 3R whole genome duplication. Although the mechanisms have not been elucidated, some of the species or evolutionary branches lack either kiss1 or kiss2; kiss2 has been lost in placental mammals and marsupials, for instance. Interestingly, the avian species are supposed to have lost both kiss1 and kiss2 (Figure 1B.2). The length of kisspeptin precursors, which contain signal peptides in their N-termini, varies among species, and the precursors are cleaved into shorter mature peptides with their conserved 10 aa residues.

Figure 1B.2 The ancestral gene of kisspeptin has been suggested to be duplicated into kiss1 and kiss2 in the early vertebrate lineage, and either kiss1 or kiss2, or both, are lacking in some branches. WGD, whole genome duplication.

Distribution of mRNA

In all animals reported thus far, kisspeptin mRNA is expressed in the brain. In addition, high expression of KISS1 in human placenta has been reported. Although kisspeptin is expressed in some other peripheral tissues [6], most research has been focused on kisspeptin expressed in the brain as a neuropeptide.

Regulation of Synthesis and Release

In both teleosts and mammals, it has been demonstrated that some kisspeptin neurons show high steroid sensitivity in that they alter their expression in accordance with serum sex steroid concentration. This suggests that the cis-regulatory elements of the kisspeptin gene include estrogen responsive elements, and the presence of some transcription factors in a certain kisspeptin neuronal population determines whether positive or negative expressional regulation of kisspeptin genes occurs. Thus, kisspeptin neurons are sensitive to gonadal/breeding states, and their steroid-sensitivity of expression/release is widely conserved in vertebrates, at least in osteichthyes [7]. Recently, it was suggested that, in mouse, Kiss1 neurons in the arcuate nucleus co-express neurokinin B and dynorphin to enhance/suppress their firing activities, respectively, in autocrine/paracrine manners. Some physiological evidence further suggests a hypothesis that their interactions may underlie the pulsatile release of GnRH/LH in mammals [8].

Receptors

Structure and Subtype

Kisspeptin receptor Gpr54 is a G protein-coupled receptor. As is true with the ligands, Gpr54 also has paralogs, Gpr54-1 and -2, in vertebrates. Reporter assay studies have shown that both Kiss1 and Kiss2 activate both the Gpr54-1 and -2 signaling pathways. However, dose dependence analysis shows that Kiss1 prefers Gpr54-1, and Kiss2 prefers Gpr54-2 in many species [9].

Biological Functions

Target Cells/Tissues and Functions

In mammals, accumulating evidence strongly suggests that Kiss1, which is produced in hypothalamic Kiss1 neurons, directly and indirectly activates GnRH1 neuron firing activity and, eventually, the HPG axis. Given that mutations of the kisspeptin ligand or receptors lead to infertility, the kisspeptin system is believed to be essential for the regulation of reproduction in placental mammals. On the other hand, there is no clear evidence for kisspeptin regulation on reproduction in non-mammalian vertebrates. In addition to regulation of reproductive functions, kisspeptin is suggested to be involved in various other functions. Some of the candidate action sites outside of the HPG axis include neurons producing oxytocin, prolactin (mammals), vasotocin, isotocin [10], and somatostatin (teleosts).

Phenotype in Gene-Modified Animals

As described above, Kiss1 knockout mice and rats, as well as Gpr54 knockout mice, experience drastic gonadal regression and infertility. On the other hand, in spite of intensive previous research, no one has ever identified kiss1 or kiss2 in avian species, although there are some genome databases available for birds. Thus, the fact that the avian species completely lack the kisspeptin gene clearly shows that kisspeptin is not essential for reproduction, at least in this group.

Pathophysiological Implications

Clinical Implications

Mutation of the KISS1 gene or neurokinin B (TAC3) gene (an autocrine/paracrine neurotransmitter of arcuate kisspeptin neuron activities) causes idiopathic hypogonadotropic hypogonadism. GnRH application to patients with this disorder resulted in the partial recovery of their fertility, similar to cases in rodent and primate studies.

Use for Diagnosis and Treatment

Kisspeptin has not yet been used for diagnosis and treatment.

Supplemental Information Available on Companion Website

• Precursor and mature hormone sequences of various animals/E-Figure 1B.1

• Accession numbers or location of genes and cDNAs for kisspeptins and their receptor genes/E-Tables 1B.1, 1B.2

References

1. Ohtaki T, Shintani Y, Honda S, et al. Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor. Nature. 2001;411(6837):613–617.

2. Kanda S, Akazome Y, Matsunaga T, et al. Identification of KiSS-1 product kisspeptin and steroid-sensitive sexually dimorphic kisspeptin neurons in medaka (Oryzias latipes). Endocrinology. 2008;149(5):2467–2476.

3. Kitahashi T, Ogawa S, Parhar IS. Cloning and expression of kiss2 in the zebrafish and medaka. Endocrinology. 2009;150(2):821–831.

4. Lee YR, Tsunekawa K, Moon MJ, et al. Molecular evolution of multiple forms of kisspeptins and GPR54 receptors in vertebrates. Endocrinology. 2009;150(6):2837–2846.

5. Oakley AE, Clifton DK, Steiner RA. Kisspeptin signaling in the brain. Endocr Rev. 2009;30(6):713–743.

6. Akazome Y, Kanda S, Okubo K, Oka Y. Functional and evolutionary insights into vertebrate kisspeptin systems from studies of fish brain. J Fish Biol. 2010;76(1):161–182.

7. Kanda S, Oka Y. Evolutionary insights into the steroid sensitive kiss1 and kiss2 neurons in the vertebrate brain. Front Endocrinol. 2012;3 http://dx.doi.org/10.3389/fendo.2012.00028 2012.

8. Okamura H, Tsukamura H, Ohkura S, et al. Kisspeptin and GnRH Pulse Generation. Adv Exp Med Biol. 2013;784:297–323.

9. Kanda S, Oka Y. Structure, synthesis, and phylogeny of kisspeptin and its receptor. Adv Exp Med Biol. 2013;784:9–26.

10. Kanda S, Akazome Y, Mitani Y, et al. Neuroanatomical evidence that kisspeptin directly regulates isotocin and vasotocin neurons. PLoS One. 2013;8(4):e62776.

Supplemental Information

E-Figure 1B.1 Kisspeptin: precursor and mature hormone sequences of various animals.

The alignment of amino acid sequences of kisspeptin precursor polypeptides in vertebrates. Perfectly conserved residues are shaded in black, and highly conserved (>80%) are shaded in gray. Adapted with permission from Akazome et al., 2010 [6], © The Fisheries Society of the British Isles.

E-Table 1B.1

Accession Numbers or Location of Genes and cDNAs for Kisspeptins

E-Table 1B.2

Accession Numbers or Location of Genes and cDNAs for Kisspeptin Receptor Genes (gpr54/GPR54)

Subchapter 1C

PQRFamide Peptide

Tomohiro Osugi, Takayoshi Ubuka and Kazuyoshi Tsutsui

Abstract

PQRFamide peptides were originally identified in bovine medulla oblongata as pain modulatory neuropeptides. PQRFamide peptides possess the conserved C-terminal PQRFamide sequence. PQRFamide peptides are present in mammals, teleosts, and agnathans, and putative genes are found in the genome database of reptiles, amphibians, and cartilaginous fish. PQRFamide peptides are involved in the regulation of pain transmission, food intake, water balance, and the cardiovascular system in mammals. In teleosts, PQRFamide peptides modulate gonadotropin-releasing hormone (GnRH) neurons involved in the regulation of behavior. In agnathans, PQRFamide peptides are involved in the regulation of reproductive function. The antagonists of PQRFamide peptide receptors have the potential to be used as therapeutic agents improving the efficacy of opioids in the treatments for chronic pain in humans.

Keywords

PQRF amide peptide; pain modulation; food intake; water balance; reproduction; neuroendocrine regulation

Abbreviation: PQRFa peptide/PQRFa

Additional names: Neuropeptide FF (NPFF)

A pain modulatory neuropeptide isolated from the medulla oblongata, PQRFamide peptide is also involved in the regulation of food intake, water balance, blood pressure, behavior, and reproduction.

Discovery

Neuropeptide FF (NPFF), a representative PQRFamide (PQRFa) peptide, was first discovered as a morphine modulating neuropeptide in 1985 [1]. NPFF was originally isolated from bovine medulla oblongata [1].

Structure

Structural Features

The PQRFa peptide precursors encode two PQRFa peptides in mammals and teleosts, and three PQRFa peptides in agnathans (Figure 1C.1) [2,3]. After cleavage of the signal peptide, biologically active PQRFa peptides, such as SQA-NPFF, SPA-NPFF, NPAF, and QFW-NPSF, are cleaved at the Arg–(Xaa)2n–Lys/Arg sequence (n=0, 1, or 2 in many cases) or a single basic amino acid at their N-termini (Figures 1C.1, 1C.2). NPFF and NPSF are exceptionally cleaved after alanine (Figures 1C.1, 1C.2). Hagfish possess two types of precursors that encode hagfish PQRFa, PQRFa-RP-1 and PQRFa-RP-2, or hagfish LPQRFa, PQRFa-RP-1 and PQRFa-RP-2 (Figures 1C.1, 1C.2) [3].

Figure 1C.1 Comparison of PQRFa peptide precursor amino acid sequences in representative species of vertebrates.

The peptide coding regions are boxed by broken lines.

Figure 1C.2 Comparison of PQRFa peptides in representative species of vertebrates. The identical amino acids are shaded. * indicates putative peptides.

Primary Structure

PQRFa peptides possess a conserved C-terminal PQRFa sequence. Their N-terminal sequences are variable across different classes.

Properties

Mr 729–2,325. Freely soluble in water.

Synthesis and Release

Gene, mRNA and Precursor

Human PQRFa peptide gene (NPFF), located on chromosome 12 (q13.13), consists of three exons (E-Figure 1C.1) and has PIT1, AP2, and other elements in the promoter region [4]. Human PQRFa peptide precursor cDNA consists of 591 nt and encodes a 113-aa precursor protein [4]. PQRFa peptide genes are present in mammals, teleosts, and agnathans. In the genome database, putative PQRFa peptide genes are present in reptiles, amphibians, and cartilaginous fish. The presence of the PQRFa peptide gene in birds is not clarified.

Distribution of mRNA

PQRFa peptide mRNA is highly expressed in the dorsal spinal cord and the nucleus of the solitary tract in the rat [4]. PQRFa peptide mRNA is expressed at some level in rat hypothalamus [4]. In teleosts, PQRFa peptide mRNA is selectively expressed in the terminal nerve gonadotropin-releasing hormone (GnRH) neurons in the embryo and larva of zebrafish, and in the hypothalamus in grass puffer [5]. In agnathans, PQRFa peptide mRNA is strongly expressed around the third ventricle in the hypothalamus and is moderately expressed in the medulla oblongata [2,3].

Tissue and Plasma Concentrations

NPFF concentration is the highest (1,008 pmol/g protein) in the pituitary in the rat. In the rat central nervous system, NPFF is highly concentrated in the spinal cord (368 pmol/g protein), hypothalamus (202 pmol/g protein), and pons-medulla (136 pmol/g protein). In the plasma, NPFF concentration is 2.2±0.5 pg/ml in humans.

Receptors

Structure and Subtype

In mammals, the two genes of NPFF receptors, NPFF receptor 1 (NPFFR1) and NPFF receptor 2 (NPFFR2), have been identified [4]. NPFFR1 and NPFFR2 are G protein-coupled receptors, and are also referred to as OT7TO22/GPR147/NPFF1/GnIH-R and HLWAR77/GPR74/NPFF2, respectively. NPFFR2 is considered to be the receptor for PQRFa peptides in mammals, because PQRFa peptides have higher binding affinities for NPFFR2. Human NPFFR2 is located on chromosome 4 (q13.3) and consists of four exons (E-Figure 1C.2). In teleosts, two subtypes of the NPFFR2 gene (npffr2-1 and npffr2-2) have been reported in zebrafish and Takifugu (E-Figure 1C.3) [6]. The fish NPFF receptors may have been duplicated by fish-specific genome duplication.

Signal Transduction Pathway

NPFFR2 is coupled to Gαi/o, and the activation of NPFFR2 inhibits forskolin stimulated adenylyl cyclase activity, intracellular cAMP levels, and phosphorylation of extracellular signal-regulated kinase 2 (ERK2) [4]. In addition, NPFFR2 and μ-opioid receptors are able to heteromerize [7]. The activation of NPFFR2 induces phosphorylation of μ-opioid receptors mediated by GPCR kinase 2 (GRK2) and results in the inactivation of μ-opioid receptors [7].

Agonist

[D-Tyr¹, (NMe)Phe³]NPFF (1DMe), which is an N-terminally modified NPFF, is broadly used as a NPFF agonist. 1DMe binds to both NPFFR1 and NPFFR2, with higher affinity for NPFFR2.

Antagonist

RF9 (2-adamantanecarbonyl–Arg–Phe–NH2) was found by screening of small peptides that antagonize NPFFR1 and NPFFR2. RF9 displays an equally high affinity for both NPFFR1 and NPFFR2. RF9 blocks opioid-induced hyperalgesia, and prevents the development of associated opioid tolerance in the rat. RF9 also blocks the increase in blood pressure and heart rate evoked by NPFF.

Biological Functions

Target Cells/Tissues and Functions

PQRFa peptides bind to NPFFR2, which is expressed in the superficial layers of the dorsal horn in the spinal cord and modulates pain transmission at the spinal level. NPFF and NPAF attenuate the analgesic effect of morphine when administered intracerebroventricularly in rodents [4]. This anti-opioid effect of NPFF is considered to be due to the interaction between NPFFR2 and μ-opioid receptor [7]. On the other hand, NPFF also exerts an antinociceptive effect when administered intrathecally by modulating μ- and δ-opioid receptors in the rat [4]. Intrathecally injected NPFF potentiates the antinociceptive effect of morphine by modulating δ-opioid receptors [4]. NPFF and NPSF also exert pronociceptive actions by potentiating the acid sensing ion channel [4]. Moreover, NPFF inhibits food intake at low doses and stimulates food intake at high doses via opioid receptors expressed in the parabrachial nucleus in the rat [4]. Central administration of NPFF evokes an elevation in arterial blood pressure and heart rate, and stimulates water intake in the rat [4,8]. Central administration of NPFF also activates hypothalamic oxytocin neurons that are involved in the ascending visceral autonomic pathways in the rat [4,8]. In teleosts, PQRFa peptides modulate pacemaker activity of terminal nerve GnRH neurons in dwarf gourami [9]. In agnathans, one of the PQRFa peptides increases GnRH concentration in lampreys [10] and stimulates gonadotropin-β mRNA expression in hagfish [3].

Pathophysiological Implications

Clinical Implications

PQRFa peptides comprise one of the anti-opioid factors that may solve clinical problems of opiate tolerance and dependence. Thus, the antagonists of PQRFa peptide receptors, such as RF9, have the potential to be used as therapeutic agents that improve the efficacy of opioids in treatment for chronic pain in humans. It was reported that the density of NPFF fibers in the dorsal motor nucleus of vagus and the ambiguus nucleus in the medulla was significantly lower in hypertensive patients than in controls [8].

Supplemental Information Available on Companion Website

• Gene, mRNA, and precursor structure of human PQRFamide peptide /E-Figure 1C.1

• Gene, mRNA and precursor structures of human PQRFamide peptide receptor (NPFFR2)/E-Figure 1C.2

• Primary structure of PQRFamide peptide receptor (NPFFR2) of various animals /E-Figure 1C.3 [11,12]

• Accession numbers of genes and cDNAs for PQRFamide peptide precursor and PQRFamide peptide receptor/E-Tables 1C.1, 1C.2

References

1. Yang HY, Fratta W, Majane EA, et al. Isolation, sequencing, synthesis, and pharmacological characterization of two brain neuropeptides that modulate the action of morphine. Proc Natl Acad Sci USA. 1985;82:7757–7761.

2. Osugi T, Ukena K, Sower SA, et al. Evolutionary origin and divergence of PQRFamide peptides and LPXRFamide peptides in the RFamide peptide family Insights from novel lamprey RFamide peptides. FEBS J. 2006;273:1731–1743.

3. Osugi T, Uchida K, Nozaki M, et al. Characterization of novel RFamide peptides in the central nervous system of the brown hagfish: isolation, localization, and functional analysis. Endocrinology. 2011;152:4252–4264.

4. Yang HY, Tao T, Iadarola MJ. Modulatory role of neuropeptide FF system in nociception and opiate analgesia. Neuropeptides. 2008;42:1–18 (Review).

5. Ando H, Shahjahan M, Hattori A. Molecular neuroendocrine basis of lunar-related spawning in grass puffer. Gen Comp Endocrinol. 2013;181:211–214.

6. Zhang Y, Li S, Liu Y, et al. Structural diversity of the GnIH/GnIH receptor system in teleost: its involvement in early development and the negative control of LH release. Peptides. 2010;31:1034–1043.

7. Moulédous L, Froment C, Dauvillier S, et al. GRK2 protein-mediated transphosphorylation contributes to loss of function of μ-opioid receptors induced by neuropeptide FF (NPFF2) receptors. J Biol Chem. 2012;287:12736–12749.

8. Jhamandas JH, Goncharuk V. Role of neuropeptide FF in central cardiovascular and neuroendocrine regulation. Front Endocrinol (Lausanne). 2013;4:8.

9. Saito TH, Nakane R, Akazome Y, et al. Electrophysiological analysis of the inhibitory effects of FMRFamide-like peptides on the pacemaker activity of gonadotropin-releasing hormone neurons. J Neurophysiol. 2010;104:3518–3529.

10. Daukss D, Gazda K, Kosugi T, et al. Effects of lamprey PQRFamide peptides on brain gonadotropin-releasing hormone concentrations and pituitary gonadotropin-β mRNA expression. Gen Comp Endocrinol. 2012;177:215–219.

11. Hirokawa T, Chieng SB, Mitaku S. SOSUI: classification and secondary structure prediction system for membrane proteins. Bioinformatics. 1998;14:378–379.

Supplemental Information

E-Figure 1C.1 Gene, mRNA, and precursor structure of human PQRFamide peptide.

Human neuropeptide FF precursor: NPFF, location 12q13.13. Rectangle in the mRNA shows the reading frame of human NPFF precursor. SP, signal peptide.

E-Figure 1C.2 Gene, mRNA, and precursor structures of human PQRFamide peptide receptor (NPFFR2).

Human neuropeptide FF receptor 2: location 4q13.2–q13.3. Rectangle in the mRNA shows the reading frame of human NPFF receptor. TM, transmembrane domain; IL, intracellular loop; EL, extracellular loop.

E-Figure 1C.3 Primary structure of PQRFamide peptide receptor (NPFFR2) of various animals.

The alignment of amino acid sequences of PQRFamide peptide receptor (NPFFR2) in vertebrates. Conserved amino acid residues are shadowed. Transmembrane domains predicted by SOSUI [11] are boxed. The seventh transmembrane domains of Xenopus tropicalis NPFFR2 and zebrafish NPFFR2-2 could not be predicted by SOSUI. Possible glycosylation sites predicted by NetNGlyc 1.0 Server are shaded in red. TM: transmembrane domain. Genbank accession numbers: human (Homo sapiens), AF268899; chimpanzee (Pan troglodytes), XM_001158705.1; mouse (Mus musculus), NM_133192; rat (Rattus norvegicus), NM_023980; ovine (Ovis aries), KC119399; chicken (Gallus gallus), NM_001034825; mallard (Anas platyrhynchos), XM_005012732; anole lizard (Anolis carolinensis), XM_003221835; green sea turtle (Chelonia mydas), XM_007065513.1; Xenopus tropicalis, XM_002940351; zebrafish (Danio rerio) NPFFR2-1, NM_001105109; zebrafish (Danio rerio) NPFFR2-2, XP_690069; takifugu (Takifugu rubripes) NPFFR2-1, AB193137.1; takifugu (Takifugu rubripes) NPFFR2-2, AB193138.1.

E-Table 1C.1

Accession Numbers of Genes and cDNAs for PQRFamide Peptide Precursor

E-Table 1C.2

Accession Numbers of Genes and cDNAs for PQRFamide Peptide Receptor

Subchapter 1D

Pyroglutamylated RFamide Peptide

Kazuyoshi Ukena and Kazuyoshi Tsutsui

Abstract

QRFP was first identified in the brain of the European green frog. Subsequently, the cDNAs encoding the QRFP precursors were characterized in various animals, including goldfish, quail, chicken, zebra finch, mouse, rat, bovines, and human. QRFP was found to be a high-affinity endogenous ligand of the previously identified orphan G protein-coupled receptor GPR103 (QRFPR) in mammals. In mammals, QRFP exerts diverse biological actions, including regulation of food intake, energy homeostasis, pituitary hormone secretion, nociception, and bone formation. QRFP also exerts an orexigenic action in birds, as it does in rodents, and a similar effect of QRFP is suggested in fish, because of upregulation of QRFP mRNA by a negative energy state. Thus, the structure, distribution pattern, and biological actions of the QRFP–QRFPR system are conserved across vertebrates, from fish to mammals.

Keywords

pyroglutamylated RFamide peptide (QRFP); QRFPR; GPR103; orexigenic peptide; hypothalamus; energy homeostasis; bone formation; pituitary hormone secretion

Abbreviation: QRFP

Additional names: 26RFamide, 26RFa, P518, QRFP-43, QRFP-26, 43RFa

A 26-aa residue peptide originally isolated from the frog brain, QRFP is the latest member of the RFamide peptide family discovered in the hypothalamus of vertebrates.

Discovery

QRFP was independently discovered by three research groups in 2003. One group identified the 26-aa residue RFamide peptide in the brain of the European green frog (Rana esculenta) and designated it 26RFa [1]. Concurrently, two other research groups found QRFP precursors using a bioinformatic approach in rat, mouse, bovine, and human genomes, and paired QRFP with a previously identified orphan GPCR, GPR103, now termed QRFPR [2,3].

Structure

Structural Features

Human QRFP precursor consists of 136 aa residues, encoding one RFamide peptide with 43 aa residues. This RFamide peptide is flanked at the N-terminus by a monobasic amino acid cleavage site and at the C-terminus by a glycine amidation signal. The mature 43-aa residue RFamide peptide was identified from the culture medium of CHO cells that expressed the human peptide precursor [2]. Because the N-terminal amino acid was pyroglutamic acid, this RFamide peptide was named pyroglutamylated RFamide peptide (QRFP).

Primary Structure

The amino acid lengths of mature peptides depend on species – for example, 43 aa residues in human and rat, 27 aa residues in quail, 25 aa residues in zebra finch, and 26 aa residues in frog (Figure 1D.1) [4,5]. As there are several monobasic processing sites in the QRFP precursor protein, alternative cleavage may yield various N-terminally elongated forms of QRFP (E-Figure 1D.1). Indeed, the existence of both 26- and 43-aa residue RFamide peptide-like immunoreactivities was detected in the hypothalamus and spinal cord of humans [6]. The human and Xenopus QRFP precursors may also generate a 9-aa residue peptide, termed 9RFa, located upstream of QRFP (E-Figure 1D.1). However, 9RFa has not been detected in tissue extracts to date. The sequence of C-terminal octapeptide of QRFP, KGGFXFRF-NH2 (X=S, A, T, or G), is highly conserved in vertebrates.

Figure 1D.1 Primary structure of identified QRFP.

Properties

Human QRFP: Mr 4,504, pI 4.95. Moderately soluble in water. Human QRFP dissolved in water at 10−3 M is stable for more than a year at −20°C.

Synthesis and Release

Gene, mRNA, and Precursor

Human QRFP precursor gene (QRFP) locates on chromosome 9 (at 9q34.12). Because only the open reading frame of the precursor is deposited in the database, the exon–intron structure is obscure. Human QRFP precursor mRNA has 411 bp. The cDNAs encoding QRFP have been are identified in human, rat, mouse, quail, chicken, zebra finch, and goldfish. Furthermore, homologous sequences are listed in the genome database of reptilian (lizard), amphibian (Xenopus), and fish (stickleback, medaka, fugu, and zebrafish) species. These data reveal the existence of the QRFP-encoding gene in representative species of vertebrates, including fish, amphibians, reptilians, birds, and mammals.

Distribution of mRNA

The mRNA encoding QRFP is highly expressed in paraventricular and ventromedial nuclei of the hypothalamus in humans, and the dorsolateral and mediobasal hypothalamic areas in rodents. In birds, QRFP mRNA is expressed in the anterior hypothalamic nucleus in the chick brain, and in the anterior-medial hypothalamic area, the ventromedial nucleus of the hypothalamus, and the lateral hypothalamic area in the zebra finch brain. In goldfish, QRFP mRNA is expressed in the hypothalamus, optic tectum–thalamus, and testis.

Regulation of Synthesis and Release

The expression of QRFP mRNA in the hypothalamus is upregulated in fasted and genetically obese ob/ob and db/db mice [7]. In goldfish, mRNA expression is also augmented at 4 days after food deprivation.

Receptors

Structure and Subtype

In humans, QRFP is found to be an endogenous ligand of the orphan receptor GPR103, also known as AQ27 or SP9155 (now re-named QRFPR), which is a class A G protein-coupled receptor (GPCR) (E-Figure 1D.2) [2,3]. QRFPR shares relatively high sequence similarity with other RFamide receptors, notably those for neuropeptide FF (NPFF), prolactin-releasing peptide (PrRP), kisspeptin, and gonadotropin-inhibitory hormone (GnIH). Two isoforms of the receptor for QRFP are characterized in rodents. These QRFP receptor isoforms are designated QRFPR1 and QRFPR2 in rat and mouse. In birds, the cDNAs encoding QRFPR are characterized in the brain of chicken and zebra finch. The sequence of chicken QRFPR is highly similar to those of human and rat QRFPR. QRFP increases [Ca²+]i in a dose-dependent manner, with an EC50 value of around 40 nM [8]. The mRNA of QRFPR is widely expressed in chicken and zebra finch brains, and the highest concentration of mRNA is observed in the diencephalon. Although there are homologous sequences to QRFPR in the genome database of Xenopus, zebrafish, coelacanth, and lamprey, QRFPR has been studied only in mammals and birds to date (E-Figure 1D.3).

Signal Transduction Pathway

The 26- or 43-aa residue QRFP binds to QRFPR with high affinity (EC50=3.2 or 0.52 nM, respectively). QRFP inhibits cAMP formation with similar efficacy in QRFPR-transfected CHO cells. Furthermore, QRFP markedly increases intracellular Ca²+ concentration ([Ca²+]i) in a pertussis toxin (PTX)-independent manner. These results suggest that QRFPR couples to Gi/0 and/or Gq protein.

Agonist

A synthetic C-terminal heptapeptide of QRFP (26RFa20–26; GGFSFRF-NH2) is responsible for the biological activity of the peptide via QRFPR.

Biological Functions

QRFP is thought to be one of the orexigenic peptides in vertebrates. Although QRFP hardly affects food intake in normally fed rats (at least under a low-fat diet), QRFP induces a marked orexigenic effect in mice, food-restricted rats, and rats fed with a high-fat diet. In addition to its orexigenic effects, in mammals QRFP regulates various functions, including energy homeostasis, bone formation, pituitary hormone secretion, steroidogenesis, nociceptive transmission, and blood pressure. In an earlier report, intravenous administration of QRFP in rats was found to increase plasma aldosterone levels [2]. In birds, intracerebroventricular injection of QRFP stimulates feeding behavior in broiler chicks, but not in layer chicks [8]. Central injection of QRFP in free-feeding male zebra finches stimulates food intake for 24 hours, without a change in body mass. These results also indicate that QRFP exerts an orexigenic activity in various avian species. In goldfish, serum LH levels are significantly increased at 1 hour after intraperitoneal injection of QRFP. As QRFP has no effect on LH release from pituitary cells in primary culture, it is thought that, in fish, the peptide may stimulate the gonadotropic axis by acting exclusively at the hypothalamic level.

Phenotype in Gene-Modified Animals

Mice deficient in the receptor for QRFP (QRFPR1) suffer from osteopenia [9]. This observation indicates that QRFP plays a major role in bone formation, via QRFPR that is expressed in bone.

Pathophysiological Implications

Clinical Implications

Chronic intracerebroventricular injection of QRFP increases body weight and fat mass with a hyperphagic behavior in mice [10]. These data indicate that QRFP is able to cause obesity by affecting food intake and reducing energy expenditure. QRFPR1 knockout mice display osteopenia. Four single-nucleotide polymorphisms (SNPs) are identified in the promoter region of QRFP of osteoporosis-prone strain SAMP6 mice. The lower expression of QRFP mRNA in the spine and the hypothalamus of SAMP6 mice may associate with osteoporosis.

Use for Diagnosis and Treatment

QRFP/26RFa has not yet been used for diagnosis and treatment.

Supplemental Information Available on Companion Website

Precursor sequences of QRFP of various animals/E-Figure 1D.1

Gene, mRNA and precursor structures of human QRFPR/E-Figure 1D.2

Primary structure of QRFPR of various animals/E-Figure 1D.3

References

1. Chartrel N, Dujardin C, Anouar Y, et al. Identification of 26RFa, a hypothalamic neuropeptide of the RFamide peptide family with orexigenic activity. Proc Natl Acad Sci USA. 2003;100:15247–15252.

2. Fukusumi S, Yoshida H, Fujii R, et al. A new peptidic ligand and its receptor regulating adrenal function in rats. J Biol Chem. 2003;278:46387–46395.

3. Jiang Y, Luo L, Gustafson EL, et al. Identification and characterization of a novel RF-amide peptide ligand for orphan G-protein-coupled receptor SP9155. J Biol Chem. 2003;278:27652–27657.

4. Chartrel N, Alonzeau J, Alexandre D, et al. The RFamide neuropeptide 26RFa and its role in the control of neuroendocrine functions. Front Neuroendocrinol. 2011;32:387–397 (Review).

5. Ukena K, Vaudry H, Leprince J, et al. Molecular evolution and functional characterization of the orexigenic peptide 26RFa and its receptor in vertebrates. Cell Tissue Res. 2011;343:475–481 (Review).

6. Bruzzone F, Lectez B, Tollemer H, et al. Anatomical distribution and biochemical characterization of the novel RFamide peptide 26RFa in the human hypothalamus and spinal cord. J Neurochem. 2006;99:616–627.

7. Takayasu S, Sakurai T, Iwasaki S, et al. A neuropeptide ligand of the G protein-coupled receptor GPR103 regulates feeding, behavioral arousal, and blood pressure in mice. Proc Natl Acad Sci USA. 2006;103:7438–7443.

8. Ukena K, Tachibana T, Iwakoshi-Ukena E, et al. Identification, localization, and function of a novel avian hypothalamic neuropeptide, 26RFa, and its cognate receptor, G protein-coupled receptor-103. Endocrinology. 2010;151:2255–2264.

9. Baribault H, Danao J, Gupte J, et al. The G- protein-coupled receptor GPR103 regulates bone formation. Mol Cell Biol. 2006;26:709–717.

10. Moriya R, Sano H, Umeda T, et al. RFamide peptide QRFP43 causes obesity with hyperphagia. Endocrinology. 2006;147:2916–2922.

Supplemental Information

E-Figure 1D.1 Precursor sequences of QRFP of various animals.

The alignment of the amino acid sequences of QRFP