Prolactin-releasing Peptide Receptor Research Articles

Identification and Functional Analysis of G Protein-Coupled Receptors in 20-Hydroxyecdysone Signaling From the Helicoverpa armigera Genome.

G protein-coupled receptors (GPCRs) are the largest family of membrane receptors in animals and humans, which transmit various signals from the extracellular environment into cells. Studies have reported that several GPCRs transmit the same signal; however, the mechanism is unclear. In the present study, we identified all 122 classical GPCRs from the genome of Helicoverpa armigera, a lepidopteran pest species. Twenty-four GPCRs were identified as upregulated at the metamorphic stage by comparing the transcriptomes of the midgut at the metamorphic and feeding stages. Nine of them were confirmed to be upregulated at the metamorphic stage. RNA interference in larvae revealed the prolactin-releasing peptide receptor (PRRPR), smoothened (SMO), adipokinetic hormone receptor (AKHR), and 5-hydroxytryptamine receptor (HTR) are involved in steroid hormone 20-hydroxyecdysone (20E)-promoted pupation. Frizzled 7 (FZD7) is involved in growth, while tachykinin-like peptides receptor 86C (TKR86C) had no effect on growth and pupation. Via these GPCRs, 20E regulated the expression of different genes, respectively, including Pten (encoding phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase), FoxO (encoding forkhead box O), BrZ7 (encoding broad isoform Z7), Kr-h1 (encoding Krüppel homolog 1), Wnt (encoding Wingless/Integrated) and cMyc, with hormone receptor 3 (HHR3) as their common regulating target. PRRPR was identified as a new 20E cell membrane receptor using a binding assay. These data suggested that 20E, via different GPCRs, regulates different gene expression to integrate growth and development.

Prolactin-releasing peptide receptor in GtoPdb v.2021.3

The precursor (PRLH, P81277) for PrRP generates 31 and 20-amino-acid versions. QRFP43 (43RFa) (named after a pyroglutamylated arginine-phenylalanine-amide peptide) is a 43 amino acid peptide derived from QRFP (P83859) and is also known as P518 or 26RFa. RFRP is an RF amide-related peptide [31] derived from a FMRFamide-related peptide precursor (NPVF, Q9HCQ7), which is cleaved to generate neuropeptide SF, neuropeptide RFRP-1, neuropeptide RFRP-2 and neuropeptide RFRP-3 (neuropeptide NPVF).

Impact of novel palmitoylated prolactin-releasing peptide analogs on metabolic changes in mice with diet-induced obesity.

Analogs of anorexigenic neuropeptides, such as prolactin-releasing peptide (PrRP), have a potential as new anti-obesity drugs. In our previous study, palmitic acid attached to the N-terminus of PrRP enabled its central anorexigenic effects after peripheral administration. In this study, two linkers, γ-glutamic acid at Lys11 and a short, modified polyethylene glycol at the N-terminal Ser and/or Lys11, were applied for the palmitoylation of PrRP31 to improve its bioavailability. These analogs had a high affinity and activation ability to the PrRP receptor GPR10 and the neuropeptide FF2 receptor, as well as short-term anorexigenic effect similar to PrRP palmitoylated at the N-terminus. Two-week treatment with analogs that were palmitoylated through linkers to Lys11 (analogs 1 and 2), but not with analog modified both at the N-terminus and Lys11 (analog 3) decreased body and liver weights, insulin, leptin, triglyceride, cholesterol and free fatty acid plasma levels in a mouse model of diet-induced obesity. Moreover, the expression of uncoupling protein-1 was increased in brown fat suggesting an increase in energy expenditure. In addition, treatment with analogs 1 and 2 but not analog 3 significantly decreased urinary concentrations of 1-methylnicotinamide and its oxidation products N-methyl-2-pyridone-5-carboxamide and N-methyl-4-pyridone-3-carboxamide, as shown by NMR-based metabolomics. This observation confirmed the previously reported increase in nicotinamide derivatives in obesity and type 2 diabetes mellitus and the effectiveness of analogs 1 and 2 in the treatment of these disorders.

Transcriptome profiling of the hypothalamus and pituitary at gonadal maturation and regression phases in marbled flounder (Pseudopleuronectes yokohamae)

The Pseudopleuronectes yokohamae (marbled flounder) features one of the most commercially important marine fishes in P.R. China. Previous studies were mainly focused on the biology and genetic breeding of the fish, little have been reported about the molecular mechanism of its reproduction. In this study, we used second generation Illumina sequencing to investigate the genes of Pseudopleuronectes yokohamae that regulate gonadal development and obtained 40,882 unigenes from the hypothalamus and pituitary samples of the fish at maturation and regression phases. Homogonous sequences of 29,129 unigenes (71.25 %) were identified and 308 unigenes among which were associated with the regulation of reproductive endocrine. These genes are a very useful resource for future studies of P.yokohamae. All unigenes with eggNog annotations were divided into 26 categories that are mostly involved in signal transduction, translation mechanism, physiological and biochemical processes, etc. Based on KEGG analysis, all unigenes expressed at the maturation phase were involved in 107 metabolic pathways, with 184 up-regulated sequences, while the unigenes expressed at the regression phase were related to 210 metabolic pathways, with 858 up-regulated sequences. Additionally, the quantitative real-time PCR results indicated that follicle stimulating hormone and prolactin-releasing peptide receptor are highly expression at the regression phase, while growth hormone releasing hormone, kisspeptin, gonadotropin and insulin growth-like factors expressed higher at the maturation phase. This study provides valuable data for better understanding the transcriptome profiles of marbled flounder and may help to improve artificial reproduction technologies of the fish.

Prolactin-releasing peptide: a new tool for obesity treatment.

Obesity is an escalating epidemic, but an effective noninvasive therapy is still scarce. For obesity treatment, anorexigenic neuropeptides are promising tools, but their delivery from the periphery to the brain is complicated because peptides have a low stability and limited ability to cross the blood-brain barrier. In this review, we summarize results of several studies with our newly designed lipidized analogs of prolactin-releasing peptide (PrRP). PrRP is involved in feeding and energy balance regulation as demonstrated by obesity phenotypes of both PrRP- and PrRP-receptor-knockout mice. Lipidized PrRP analogs showed binding affinity and signaling in PrRP receptor-expressing cells similar to natural PrRP. Moreover, these analogs showed high binding affinity also to anorexigenic neuropeptide FF (NPFF)-2 receptor. Acute peripheral administration of myristoylated and palmitoylated PrRP analogs to mice and rats induced strong and long-lasting anorexigenic effects and neuronal activation in the brain areas involved in food intake regulation. Two-week-long subcutaneous administration of palmitoylated PrRP31 and myristoylated PrRP20 lowered food intake, body weight, improved metabolic parameters and attenuated lipogenesis in mice with diet-induced obesity. A strong anorexigenic, body weight-reducing and glucose tolerance-improving effect of palmitoylated-PrRP31 was shown also in diet-induced obese rats after its repeated 2-week-long peripheral administration. Thus, the strong anorexigenic and body weight-reducing effects of palmitoylated PrRP31 and myristoylated PrRP20 make these analogs attractive candidates for antiobesity treatment. Moreover, PrRP receptor might be a new target for obesity therapy.

Analysis of transcriptome profiling from the brain at maturation and regression phases in starry flounder (Platichthys stellatus)

This study was to investigate the regulatory mechanisms of reproduction and endocrinology in starry flounder (Platichthys stellatus). Total RNA from the mixed sample of pituitary gland and hypothalamus of brain at the maturation and regression phases was isolated. The RNA sequencing (RNA-Seq) library was prepared and sequenced on Illumina Hiseq 2000 platform. A total of 53,567,296 and 75,630,680 short fragments were obtained from the samples of maturation and regression phases, respectively. After splicing, 30,640 unigene sequences were obtained based on the blasting results from National Center for Biotechnology Information (NCBI) database. Homogonous sequences of 24,128 unigenes (78.8%) were found and analyzed with eggNOG and KEGG (Kyoto Encyclopedia of Genes and Genomes). All 24,128 unigenes with eggNOG annotations were divided into 26 categories that are mostly associated with physiological processes, including signaling transduction, translation mechanism, etc. Based on KEGG analysis, all unigenes at the maturation phase were involved in 98 metabolic pathways, with 135 up-regulated sequences, while the unigenes at the regression phase were related to 192 metabolic pathways, with 648 up-regulated sequences. Moreover, 334 differentially expressed genes (DEGs) were up-regulated at the maturation phase, while 987 up-regulated DEGs were found at the regression phase. Total 408 unigenes were associated with the regulation of reproduction and endocrinology. The mRNA expression of some reproduction-associated DEGs including growth hormone releasing hormone (GnRH), neurokinin B (NKB), gonadotropin (GtH), follicle stimulating hormone (FSH), kisspeptin (Kiss) and prolactin-releasing peptide receptor (PrRPR) in the hypothalamus and pituitary gland at two phases was verified by qRT-PCR. Except FSH, all other 5 DEGs showed higher expression at the regression phase. This study provides valuable data for understanding the transcriptome profiles of starry flounder brain and improving the technologies of artificial breeding.

Involvement of Mammalian RF-Amide Peptides and Their Receptors in the Modulation of Nociception in Rodents.

Mammalian RF-amide peptides, which all share a conserved carboxyl-terminal Arg–Phe–NH2 sequence, constitute a family of five groups of neuropeptides that are encoded by five different genes. They act through five G-protein-coupled receptors and each group of peptide binds to and activates mostly one receptor: RF-amide related peptide group binds to NPFFR1, neuropeptide FF group to NPFFR2, pyroglutamylated RF-amide peptide group to QRFPR, prolactin-releasing peptide group to prolactin-releasing peptide receptor, and kisspeptin group to Kiss1R. These peptides and their receptors have been involved in the modulation of several functions including reproduction, feeding, and cardiovascular regulation. Data from the literature now provide emerging evidence that all RF-amide peptides and their receptors are also involved in the modulation of nociception. This review will present the current knowledge on the involvement in rodents of the different mammalian RF-amide peptides and their receptors in the modulation of nociception in basal and chronic pain conditions as well as their modulatory effects on the analgesic effects of opiates.

In vitro modification of substituted cysteines as tool to study receptor functionality and structure–activity relationships

Mutagenic investigations of expressed membrane proteins are routine, but the variety of modifications is limited by the twenty canonical amino acids. We describe an easy and effective cysteine substitution mutagenesis method to modify and investigate distinct amino acids in vitro. The approach combines the substituted cysteine accessibility method (SCAM) with a functional signal transduction readout system using different thiol-specific reagents. We applied this approach to the prolactin-releasing peptide receptor (PrRPR) to facilitate biochemical structure–activity relationship studies of eight crucial positions. Especially for D6.59C, the treatment with the positively charged methanethiosulfonate (MTS) ethylammonium led to an induced basal activity, whereas the coupling of the negatively charged MTS ethylsulfonate nearly reconstituted full activity, obviously by mimicking the wild-type charged side chain. At E5.26C, W5.28C, Y5.38C, and Q7.35C, accessibility was observed but hindered transfer into the active receptor conformation. Accordingly, the combination of SCAM and signaling assay is feasible and can be adapted to other G-protein-coupled receptors (GPCRs). This method circumvents the laborious way of inserting non-proteinogenic amino acids to investigate activity and ligand binding, with rising numbers of MTS reagents allowing selective side chain modification. This method pinpoints to residues being accessible but also presents potential molecular positions to investigate the global conformation.

The activity of prolactin releasing peptide correlates with its helicity

The prolactin releasing peptide (PrRP) is involved in regulating food intake and body weight homeostasis, but molecular details on the activation of the PrRP receptor remain unclear. C-terminal segments of PrRP with 20 (PrRP20) and 13 (PrRP8-20) amino acids, respectively, have been suggested to be fully active. The data presented herein indicate this is true for the wildtype receptor only; a 5-10-fold loss of activity was found for PrRP8-20 compared to PrRP20 at two extracellular loop mutants of the receptor. To gain insight into the secondary structure of PrRP, we used CD spectroscopy performed in TFE and SDS. Additionally, previously reported NMR data, combined with ROSETTANMR, were employed to determine the structure of amidated PrRP20. The structural ensemble agrees with the spectroscopic data for the full-length peptide, which exists in an equilibrium between α- and 3(10)-helix. We demonstrate that PrRP8-20's reduced propensity to form an α-helix correlates with its reduced biological activity on mutant receptors. Further, distinct amino acid replacements in PrRP significantly decrease affinity and activity but have no influence on the secondary structure of the peptide. We conclude that formation of a primarily α-helical C-terminal region of PrRP is critical for receptor activation.

Ligand-mimicking Receptor Variant Discloses Binding and Activation Mode of Prolactin-releasing Peptide

The prolactin-releasing peptide receptor and its bioactive RF-amide peptide (PrRP20) have been investigated to explore the ligand binding mode of peptide G-protein-coupled receptors (GPCRs). By receptor mutagenesis, we identified the conserved aspartate in the upper transmembrane helix 6 (Asp(6.59)) of the receptor as the first position that directly interacts with arginine 19 of the ligand (Arg(19)). Replacement of Asp(6.59) with Arg(19) of PrRP20 led to D6.59R, which turned out to be a constitutively active receptor mutant (CAM). This suggests that the mutated residue at the top of transmembrane helix 6 mimics Arg(19) by interacting with additional binding partners in the receptor. Next, we generated an initial comparative model of this CAM because no ligand docking was required, and we selected the next set of receptor mutants to find the engaged partners of the binding pocket. In an iterative process, we identified two acidic residues and two hydrophobic residues that form the peptide ligand binding pocket. As all residues are localized on top or in the upper part of the transmembrane domains, we clearly can show that the extracellular surface of the receptor is sufficient for full signal transduction for prolactin-releasing peptide, rather than a deep, membrane-embedded binding pocket. This contributes to the knowledge of the binding of peptide ligands to GPCRs and might facilitate the development of GPCR ligands, but it also provides new targeting of CAMs involved in hereditary diseases.

Protein Interacting with C Kinase 1 (PICK1) Reduces Reinsertion Rates of Interaction Partners Sorted to Rab11-dependent Slow Recycling Pathway

The scaffolding protein PICK1 (protein interacting with C kinase 1) contains an N-terminal PSD-95/Discs large/ZO-1 (PDZ) domain and a central lipid-binding Bin/amphiphysin/Rvs (BAR) domain. PICK1 is thought to regulate trafficking of its PDZ binding partners but different and even opposing functions have been suggested. Here, we apply ELISA-based assays and confocal microscopy in HEK293 cells with inducible PICK1 expression to assess in an isolated system the ability of PICK1 to regulate trafficking of natural and engineered PDZ binding partners. The dopamine transporter (DAT), which primarily sorts to degradation upon internalization, did not form perinuclear clusters with PICK1, and PICK1 did not affect DAT internalization/recycling. However, transfer of the PICK1-binding DAT C terminus to the β(2)-adrenergic receptor, which sorts to recycling upon internalization, led to formation of PICK1 co-clusters in Rab11-positive compartments. Furthermore, PICK1 inhibited Rab11-mediated recycling of the receptor in a BAR and PDZ domain-dependent manner. In contrast, transfer of the DAT C terminus to the δ-opioid receptor, which sorts to degradation, did not result in PICK1 co-clusters or any change in internalization/recycling. Further support for a role of PICK1 determined by its PDZ cargo was obtained for the PICK1 interaction partner prolactin-releasing peptide receptor (GPR10). GPR10 co-localized with Rab11 and clustered with PICK1 upon constitutive internalization but co-localized with the late endosomal marker Rab7 and did not cluster with PICK1 upon agonist-induced internalization. Our data suggest a selective role of PICK1 in clustering and reducing the recycling rates of PDZ domain binding partners sorted to the Rab11-dependent recycling pathway.

Identification of the Receptors for Prolactin-Releasing Peptide (PrRP) and Carassius RFamide Peptide (C-RFa) in Chickens

Prolactin-releasing peptide (PrRP) and its structurally related peptide, Carassius Arg-Phe-amide peptide (C-RFa), have been reported to play similar roles in regulating food intake and pituitary functions in vertebrates. However, the identity, functionality, and expression of the receptor(s) for PrRP and C-RFa remain largely unknown in nonmammalian vertebrates, including birds. In this study, three receptors homologous to mammalian PrRP receptor (PrRPR), named cPrRPR1, cPrRPR2, and cC-RFaR, respectively, were cloned from chicken brain by RT-PCR. Using a pGL3-NFAT-RE-luciferase reporter system, we demonstrated that cPrRPR1 and cPrRPR2 expressed in Chinese hamster ovarian cells could be activated by cPrRP₂₀ and cC-RFa₂₀ potently, whereas cC-RFaR could only be activated effectively by cC-RFa₂₀ (EC₅₀, 0.11 nM), indicating that cPrRPR1 and cPrRPR2 can function as common receptors for PrRP and C-RFa, whereas cC-RFaR is a receptor specific to C-RFa. Using a pGL3-CRE-luciferase reporter system, cPrRPR1, cPrRPR2, and cC-RFaR expressed in Chinese hamster ovarian cells were also shown to activate intracellular protein kinase A signaling pathway upon cC-RFa₂₀ treatment (100 nM). Moreover, RT-PCR assay revealed that cPrRPR1, cPrRPR2, and cC-RFaR were widely expressed in most adult chicken tissues examined, including various regions of brain. These findings, together with evidence of PrRP and C-RFa encoded by separate genes in chicken, Xenopus, and zebrafish, and the differential expression of PrRP and C-RFa genes in chicken tissues, strongly suggest that PrRP and C-RFa may play similar yet distinctive roles in nonmammalian vertebrates, including chicken, and their actions are mediated by common receptor(s) or a specific C-RFa receptor.

Biological properties of prolactin-releasing peptide analogs with a modified aromatic ring of a C-terminal phenylalanine amide

Prolactin-releasing peptide (PrRP)-induced secretion of prolactin is not currently considered a primary function of PrRP, but the development of late-onset obesity in both PrRP and PrRP receptor knock-out mice indicates the unique anorexigenic properties of PrRP. In our recent study, we showed comparable potencies of peptides PrRP31 and PrRP20 in binding, intracellular signaling and prolactin release in pituitary RC-4B/C cells, and anorexigenic effect after central administration in fasted mice. In the present study, eight analogs of PrRP20 with C-terminal Phe amide modified with a bulky side chain or a halogenated aromatic ring revealed high binding potency, activation of mitogen-activated protein kinase/extracellular-regulated kinase (MAPK/ERK1/2) and cAMP response element-binding protein (CREB) and prolactin release in RC-4B/C cells. In particular, [PheNO231]PrRP20, [1-Nal31]PrRP20, [2-Nal31]PrRP20 and [Tyr31]PrRP20 showed not only in vitro effects comparable or higher than those of PrRP20, but also a very significant and long-lasting anorexigenic effect after central administration in fasted mice. The design of potent and long-lasting PrRP analogs with selective anorexigenic properties promises to contribute to the study of food intake disorders.

Characterization of prolactin-releasing peptide: Binding, signaling and hormone secretion in rodent pituitary cell lines endogenously expressing its receptor

The recently discovered prolactin-releasing peptide (PrRP) binds to the PrRP receptor and is involved in endocrine regulation and energy metabolism. However, its main physiological role is currently unknown. Two biologically active isoforms of PrRP exist: the 31 (PrRP31) and the 20 (PrRP20) amino acid forms, which both contain a C-terminal Phe amide sequence. In the present study, the PrRP receptor was immunodetected in three rodent tumor pituitary cell lines: GH3, AtT20 and RC-4B/C cells. The saturation binding of radioiodinated PrRP31 to intact cells demonstrated a K d in the 10 −9 M range and a B max in the range of tens of thousands binding sites per cell. For binding to RC-4B/C cells, both PrRP31 and PrRP20 competed with 125I-PrRP31 with a similar K i. The C-terminal analog PrRP13 showed lower binding potency compared to PrRP31 and PrRP20. All PrRP analogs increased the phosphorylation of MAPK/ERK1/2 (mitogen-activated phosphorylase/extracellular-regulated kinase) and CREB (cAMP response element-binding protein) in RC-4B/C cells. Additionally, prolactin release was induced by the PrRP analogs in a dose-dependent manner in RC-4B/C cells. Finally, food intake after intracerebroventricular administration of PrRP analogs in fasted mice was followed. Both PrRP31 and PrRP20 decreased food intake, but PrRP13 did not show significant effect. Studies on pituitary cell lines expressing the PrRP receptor are more physiologically relevant than those on cells transfected with the receptor. This cell type can be used as a model system for pharmacological studies searching for PrRP antagonists and stable effective PrRP agonists, as these drugs may have potential as anti-obesity agents.

Prolactin-releasing peptide receptor expressed in the pituitary in Mozambique tilapia Oreochromis mossambicus: An aspect of prolactin regulatory mechanisms

Prolactin (PRL)-releasing peptide (PrRP) has been reported as a strong candidate for a stimulating factor of both PRL secretion and expression in teleost species; however, there is no information available on its receptor. Here we report cDNA cloning and characterization of PrRP receptor expressed in the pituitary of Mozambique tilapia Oreochromis mossambicus. The deduced amino acid sequence of cDNA for tilapia PrRP receptor shared 50–83% homology with other vertebrate homologs. Intracellular calcium mobilization assay revealed that PrRP receptor responded to as low as 1 nM order of tilapia PrRP, indicating its high affinity to PrRP. The expression of PrRP receptor was detected in the brain, pituitary, heart, spleen, kidney and rectum of freshwater (FW)- and seawater (SW)-adapted fish. There was no significant difference between FW and SW fish in transcription levels of PrRP receptor in the rostral pars distalis (RPD) of the pituitary. Similarly, the PrRP expression level in the whole brain was not changed by environmental salinity. Immunohistochemistry with a specific antibody showed that PrRP receptor was mainly localized in the cells of the RPD and neurohypophysis in the pituitary of both FW and SW tilapia. We also examined the effects of PrRP on PRL expression in primary-incubated PRL cells of FW tilapia; PrRP failed to stimulate PRL expression in PRL cells in vitro. These results suggest that in vivo stimulatory effects of PrRP on PRL gene expression reported in teleosts are presumably mediated by an unknown regulator secreted from the neurohypophysis expressing PrRP receptor.

Increase of the trophoblast giant cells with prolactin‐releasing peptide (PrRP) receptor expression in p53‐null mice

Trophoblast giant cells in the mouse placentas are polyploid cells that form as a result of endoreduplication. The giant cells form the outermost layer of the extraembryonic compartment and produce a number of pregnancy-specific hormones, including prolactin family members. Here we demonstrate that trophoblast giant cells are increased, and display upregulation of prolactin releasing peptide (PrRP) receptor in the p53-null (p53(-/-)) embryonic placentas. At day 13.5 of gestation, the weight of p53(-/-) placentas was less than that of both wild-type and p53(+/-) placentas. In p53(-/-) placentas, the spongiotrophoblast layer was significantly decreased in thickness, and the trophoblast giant cells were observed not only in the outer layer of placentas but in both the spongiotrophoblast layer and the labyrinthine layer. The giant cells spread over the spongiotrophoblast and labyrinthine layer in p53(-/-) placentas displayed more intensive expression of immunoreactive PrRP receptor than in wild-type placentas. Previous studies indicated that the association between PrRP and PrRP receptor physiologically involves in the expression and secretion of the peptide hormones, including prolactin and growth hormones. These results suggest that p53 may regulate the differentiation of trophoblast giant cells, and may control the physiological PrRP stimuli in mouse placentas.

PDZ Domain Protein-Protein Interactions: A Case Study with PICK1

Using PICK1 as an example this review highlights PDZ domains support a repertoire of protein-protein interactions that regulate the subcellular localisation and function of receptors, ion channels and enzymes. PICK1 is a 416 amino acid protein that contains a PDZ domain, a coiled-coil motif/arfaptin homology domain and an acidic c-terminal. Nearly all proteins thus far reported to interact with PICK1 do so via its single PDZ domain. PICK1 self-associates via its coiled-coil motif and together with its PDZ domain has potential to act as a scaffolding protein. This molecule was first identified as a protein interacting with Calpha-kinase (PICK1) and interacts with several members of the glutamate receptor family and receptor tyrosine kinases. The PDZ domain of PICK1 has since been shown to interact with a plethora of proteins including dopamine transporter, prolactin-releasing peptide receptor, ion channels BNaC1/ASIC and many more. The single PDZ domain of PICK1 interacts with a network of proteins that is pivotal in processes such as synaptic plasticity, development and neural guidance as well as many diseased states. The proteins that interact with PICK1 and the functional roles of its PDZ domain are discussed and illustrated are ways to regulate PDZ protein-protein interactions.

Prolactin-Releasing Peptide Mediates Cholecystokinin-Induced Satiety in Mice

We have shown previously that prolactin-releasing peptide (PrRP) plays a role in the regulation of feeding and energy expenditure in rats. We hypothesize that PrRP may have a physiological action through its putative receptor, GPR10, to mediate the central anorexigenic effects of peripheral satiety factors. Here we examine the effects of PrRP and cholecystokinin (CCK) on feeding in mice, including PrRP receptor gene knockout animals (GPR10(-/-)). Intracerebroventricular administration of PrRP (1-4 nmol) inhibited feeding in C57B6/J mice under both fast-induced and nocturnal feeding conditions. In contrast to the observations made in wild-type mice, neither PrRP nor CCK reduced food intake in GRP10(-/-) mice. The reduction in feeding and the release of corticosterone induced by systemic injection of the stressor lipopolysaccharide was similar in both GPR10(+/+) and GPR10(-/-) mice. These findings suggest that PrRP, acting through GPR10, is involved in regulating food intake and may be a key intermediary in the central satiating actions of CCK.

Characterization of a naturally-occurring polymorphism in the UHR-1 gene encoding the putative rat prolactin-releasing peptide receptor

The rat orphan receptor UHR-1 and its human orthologue, GPR10, were first isolated in 1995. The ligand for this receptor, prolactin-releasing peptide (PrRP), was identified in 1998 by reverse pharmacology and has subsequently been implicated in a number of physiological processes. As supported by its localization and regulation in the hypothalamus and brainstem, we have shown previously that PrRP is involved in energy homeostasis. Here we describe a naturally occurring polymorphism in the UHR-1 gene that results in an ATG to ATA change at the putative translational initiation site. The presence of the polymorphism abolished the binding of 125I PrRP in rat brain slices but did not affect the ability of PrRP to reduce fast-induced food intake. Together this data suggest that PrRP may be exerting its feeding effects through a receptor other than UHR-1.

Prolactin‐releasing peptide affects gastric motor function in rat by modulating synaptic transmission in the dorsal vagal complex

Prolactin-releasing peptide (PrRP) is a recently discovered neuropeptide implicated in the central control of feeding behaviour and autonomic homeostasis. PrRP-containing neurones and PrRP receptor mRNA are found in abundance in the caudal portion of the nucleus tractus solitarius (NTS), an area which together with the dorsal motor nucleus of the vagus (DMV) comprises an integrated structure, the dorsal vagal complex (DVC) that processes visceral afferent signals from and provides parasympathetic motor innervation to the gastrointestinal tract. In this study, microinjection experiments were conducted in vivo in combination with whole-cell recording from neurones in rat medullary slices to test the hypothesis that PrRP plays a role in the central control of gastric motor function, acting within the DVC to modulate the activity of preganglionic vagal motor neurones that supply the stomach. Microinjection of PrRP (0.2 pmol (20 nl)(-1)) into the DMV at the level of the area postrema (+0.2 to +0.6 mm from the calamus scriptorius, CS) markedly stimulated gastric contractions and increased intragastric pressure (IGP). Conversely, administration of peptide into the DMV at sites caudal to the obex (0.0 to -0.3 mm from the CS) decreased IGP and reduced phasic contractions. These effects occurred without change in mean arterial pressure and were abolished by ipsilateral vagotomy, indicating mediation via a vagal-dependent mechanism(s). The pattern of gastric motor responses evoked by PrRP mimicked that produced by administration of L-glutamate at the same sites, and both the effects of L-glutamate and PrRP were abolished following local administration of NMDA and non-NMDA-type glutamate receptor antagonists. On the other hand, microinjection of PrRP into the medial or comissural nucleus of the solitary tract (mNTS and comNTS, respectively) resulted in less robust changes in IGP in a smaller percentage of animals, accompanied by marked alterations in arterial pressure. Superfusion of brain slices with PrRP (100-300 nm) produced a small depolarization and increased spontaneous firing in 10 of 30 retrogradely labelled gastric-projecting DMV neurones. The excitatory effects were blocked by administration of TTX (2 mum) or specific glutamate receptor antagonists, indicating that they resulted from interactions of PrRP at a presynaptic site. Congruent with this, PrRP increased the amplitude of excitatory postsynaptic currents (EPSCs, 154 +/- 33%, 12 of 25 neurones) evoked by electrical stimulation in mNTS or comNTS. In addition, administration of PrRP decreased the paired-pulse ratio of EPSCs evoked by two identical stimuli delivered 100 ms apart (from 0.95 +/- 0.08 to 0.71 +/- 0.11, P < 0.05), whereas it did not affect the amplitude of inward currents evoked by exogenous application of L-glutamate to the slice. The frequency, but not amplitude of spontaneous EPSCs and action potential-independent miniature EPSCs was also increased by administration of PrRP, suggesting that the peptide was acting at least in part at receptors on presynaptic nerve terminals to enhance glutamatergic transmission. In recordings obtained from a separate group of slices, we did not observe any direct effects of PrRP on spontaneous discharge or postsynaptic excitability in either mNTS or comNTS neurones (n = 31). These data indicate that PrRP may act within the DVC to regulate gastric motor function by modulating the efficacy of conventional excitatory synaptic inputs from the NTS onto gastric-projecting vagal motor neurones.