Identification of a novel neuromedin U receptor subtype expressed in the central nervous system

Neuromedin U is a neuropeptide prominently expressed in the upper gastrointestinal tract and central nervous system. Recently, GPR66/FM-3 (NmU-R1) was identified as a specific receptor for neuromedin U. A BLAST search of the GenBank(TM) genomic database using the NmU-R1 cDNA sequence revealed a human genomic fragment encoding a G protein-coupled receptor that we designated NmU-R2 based on its homology to NmU-R1. The full-length NmU-R2 cDNA was subsequently cloned, stably expressed in 293 cells, and shown to mobilize intracellular calcium in response to neuromedin U. This response was dose-dependent (EC(50) = 5 nm) and specific in that other neuromedins did not induce a calcium flux in receptor-transfected cells. Expression analysis of human NmU-R2 demonstrated its mRNA to be most highly expressed in central nervous system tissues. Based on these data, we conclude that NmU-R2 is a novel neuromedin U receptor subtype that is likely to mediate central nervous system-specific neuromedin U effects.     

Identification and functional analysis of a novel ligand for G protein-coupled receptor, Neuromedin S

We identified a novel 36-amino acid neuropeptide in rat brain as an endogenous ligand for the G protein-coupled receptors FM-3/GPR66 and FM-4/TGR-1, which were identified to date as the neuromedin U (NMU) receptors, and designated this peptide neuromedin S (NMS) because it was specifically expressed in the suprachiasmatic nucleus (SCN) of the hypothalamus. NMS shared a C-terminal core structure with NMU. NMS mRNA was highly expressed in the central nervous system, spleen and testis. In rat brain, NMS expression was restricted to the ventrolateral portion of the SCN and has a diurnal peak under light/dark cycling, but remains stable under constant darkness. Intracerebroventricular (ICV) administration of NMS in rats induced nonphotic type phase shifts in the circadian rhythm of locomotor activity. ICV injection of NMS also decreased 12-h food intake during the dark period in rats. This anorexigenic effect was more potent than that observed with the same dose of NMU. ICV administration of NMS increased proopiomelanocortin (POMC) mRNA expression in the arcuate nucleus (Arc) and corticotropin-releasing hormone mRNA in the paraventricular nucleus, and induced c-Fos expression in the POMC neurons in the Arc. These findings suggest that NMS is implicated in the regulation of circadian rhythm and feeding behavior.     

Identification of neuromedin S and its possible role in the mammalian circadian oscillator system

The discovery of neuropeptides has resulted in an increased understanding of novel regulatory mechanisms of certain physiological phenomena. Here we identify a novel neuropeptide of 36 amino-acid residues in rat brain as an endogenous ligand for the orphan G protein-coupled receptor FM-4/TGR-1, which was identified to date as the neuromedin U (NMU) receptor, and designate this peptide 'neuromedin S (NMS)' because it is specifically expressed in the suprachiasmatic nuclei (SCN) of the hypothalamus. NMS shares a C-terminal core structure with NMU. The NMS precursor contains another novel peptide. NMS mRNA is highly expressed in the central nervous system, spleen and testis. In rat brain, NMS expression is restricted to the core of the SCN and has a diurnal peak under light/dark cycling, but remains stable under constant darkness. Intracerebroventricular administration of NMS in rats activates SCN neurons and induces nonphotic type phase shifts in the circadian rhythm of locomotor activity. These findings suggest that NMS in the SCN is implicated in the regulation of circadian rhythms through autocrine and/or paracrine actions.     

Neuromedin S exerts an antidiuretic action in rats

We recently identified neuromedin S (NMS) as an endogenous ligand for the FM-4/TGR-1 receptor. Here, we examined the possible involvement of central NMS in regulation of urinary output and vasopressin (AVP) release in rats. Intracerebroventricular (icv) injection of NMS induced a dose-dependent increase in the plasma level of AVP, followed by a decrease of nocturnal urinary output. Expression of cFos after icv injection of NMS was observed in the supachiasmatic nucleus (SCN), arcuate nucleus, paraventricular nucleus (PVN), and supraoptic nucleus (SON). The cFos expressing cells in PVN and SON, but not SCN, were then double-stained using antibodies against the vasopressin. On the other hand, icv injection of neuromedin U, which also binds to the FM-4/TGR-1 receptor, required a concentration ten times higher than that of NMS in order to exert the same antidiuretic potency. These results suggest that central NMS may exert a physiological antidiuretic action via vasopressin release.     

Involvement of neuromedin S in the oxytocin release response to suckling stimulus

We recently identified neuromedin S (NMS) from the rat hypothalamus as an endogenous ligand for the FM-4/TGR-1 receptor distinct from neuromedin U. In the present study, we examined the role of NMS in the oxytocin release response to suckling stimulation by rat pups. Intracerebroventricular (icv) injection of NMS induced cFos expression in the paraventricular nucleus and supraoptic nucleus. Double immunohistochemical analysis revealed induction of cFos expression in a proportion of oxytocinergic neurons in both nuclei. In addition, icv injection of NMS stimulated oxytocin release dose-dependently in intact rats, and increased milk secretion in lactating rats. On the other hand, icv injection of anti-NMS antiserum into lactating rats significantly suppressed suckling-induced milk ejection. These results suggest that, in the rat, endogenous NMS plays an important role in the oxytocin release response to the suckling stimulus.     

The expression of urotensin II receptor (U2R) is up-regulated by interferon-gamma

Urotensin-II (U-II) was identified as the natural ligand of the G protein-coupled receptor GPR14, which has been correspondingly renamed Urotensin-II receptor (U2R). The tissue distribution of U2R and the pharmacological effects of U-II suggest a novel neurohormonal system with potent cardiovascular effects. We here report the human rhabdomyosarcoma cell line TE-671 as the first natural and endogenous source of functional U2R in an immortalized cell line. In TE-671 cells, U-II stimulated extracellular signal regulated kinase phosphorylation and increased c-fos mRNA expression. Furthermore, we demonstrate that the expression of U2R mRNA and functional U-II high affinity binding sites are serum-responsive and that they are specifically up-regulated by interferon gamma (IFNgamma). We propose that IFNgamma contributes to the previously observed increase of U2R density in the heart tissue of congestive heart failure (CHF) patients and we suggest that U2R up-regulation, as a consequence of an inflammatory response, could lead to a clinical worsening of this disease.     

Cloning and expression of a rat neuromedin K receptor cDNA

Functional cDNA clones for rat neuromedin K receptor were isolated from a rat brain cDNA library by cross-hybridization with the bovine substance K receptor cDNA. Injection of the mRNA synthesized in vitro from the cloned cDNA into Xenopus oocytes elicited electrophysiological responses to tachykinins, with the most potent sensitivity being to neuromedin K. Ligand-binding displacement in membranes of mammalian COS cells transfected with the cDNA indicated the rank order of affinity of the receptor to tachykinins: neuromedin K greater than substance K greater than substance P. The hybridization analysis showed that the neuromedin K receptor mRNA is expressed in both the brain and the peripheral tissues at different levels. The rat neuromedin K receptor consists of 452 amino acid residues and belongs to the family of G protein-coupled receptors, which are though to have seven transmembrane domains. The sequence comparison of the rat neuromedin K, substance P, and substance K receptors revealed that these receptors are highly conserved in the seven transmembrane domains and the cytoplasmic sides of the receptors. They also show some structural characteristics, including the common presence of histidine residues in transmembrane segments V and VI and the difference in the numbers and distributions of serine and threonine residues as possible phosphorylation sites in the cytoplasmic regions. This paper thus presents the first comprehensive analysis of the molecular nature of the multiple peptide receptors that exhibit similar but pharmacologically distinguishable activities.     

Distinguishing bombesin receptor subtypes using the oocyte assay.

Physiological responses to mammalian bombesin-like peptides were studied in Xenopus oocytes injected with mRNA isolated from Swiss 3T3 cells and rat esophagus in order to identify and characterize bombesin receptor subtypes. Both groups respond similarly to either gastrin releasing peptide or neuromedin B, but only the response to neuromedin B in oocytes expressing the esophagus mRNA is not blocked by a specific gastrin releasing peptide receptor antagonist, des-Met-[D-Phe6]Bn(6-13) ethyl ester. Complete desensitization of gastrin releasing peptide-evoked responses in oocytes expressing esophagus mRNA does not abolish neuromedin B-evoked responses. A single application of neuromedin B abolishes responses to subsequently applied gastrin releasing peptide in oocytes expressing esophagus, but not Swiss 3T3, mRNA. RNA blot hybridization studies using a Swiss 3T3 gastrin releasing peptide receptor cDNA probe show no detectable hybridization in esophagus mRNA samples. These data suggest that a gastrin releasing peptide receptor is expressed in the esophagus and that it is distinct from that expressed in Swiss 3T3 cells and may represent a third subtype of mammalian bombesin receptor.     

Molecular cloning and expression of a cDNA encoding the secretin receptor.

Secretin is a 27 amino acid peptide which stimulates the secretion of bicarbonate, enzymes and potassium ion from the pancreas. A complementary DNA encoding the rat secretin receptor was isolated from a CDM8 expression library of NG108-15 cell line. The secretin receptor expressed in COS cells could specifically bind the iodinated secretin with high and low affinities. Co-expression of the secretin receptor with the alpha-subunit of rat Gs protein increased the concentration of the high affinity receptor in the membrane fraction of the transfected COS cells. Secretin could stimulate accumulation of cAMP in COS cells expressing the cloned secretin receptor. The nucleotide sequence analysis of the cDNA has revealed that the secretin receptor consists of 449 amino acids with a calculated Mr of 48,696. The secretin receptor contains seven putative transmembrane segments, and belongs to a family of the G protein-coupled receptor. However, the amino acid sequence of the secretin receptor has no significant similarity with that of other G protein-coupled receptors. A 2.5 kb mRNA coding for the secretin receptor could be detected in NG108-15 cells, and rat heart, stomach and pancreatic tissue.     

Inflammatory stress increases receptor for lysophosphatidylcholine in human microvascular endothelial cells

The atherogenic serum lysophosphatidylcholine (LPC) is known to mediate vascular endothelial responses ranging from upregulation of adhesion molecules and growth factors to secretion of chemokines and superoxide anion. We investigated whether endothelial cells express receptors for LPC, which may account for their actions. Human brain microvascular (HBMEC) and dermal microvascular endothelial cells (HMEC) were prepared for RT-PCR analysis for possible expression of the G protein-coupled receptors, GPR4 and G2A, which are believed to be specific LPC receptors. Results indicated that HBMEC expressed low basal GPR4 mRNA, but stimulation with tumor necrosis factor-alpha (TNF-alpha) (100 U/ml) or H2O2 (50 micromol/l) for 2 h or overnight upregulated expression severalfold. In contrast, HMEC expressed high basal GPR4 mRNA, which was not further increased by either TNF-alpha or H2O2 stimulation. Another LPC receptor, G2A, was not detected in either endothelial cell type. Competition binding studies were made to evaluate specific binding of [3H]LPC to the intact endothelial cell monolayer. Basal specific [3H]LPC binding in HBMEC was approximately eight times lower than in HMEC; however, TNF-alpha or H2O2 stimulation increased [3H]LPC binding on HMBEC but not HMEC. The results indicated that GPR4 expression was consistent with specific [3H]LPC binding. Overall, we report that endothelial cells selectively expressed GPR4, a specific LPC receptor. Furthermore, GPR4 expression by HBMEC, but not HMEC, was increased by inflammatory stresses. We conclude that endogenous GPR4 in endothelial cells may be a potential G protein-coupled receptor by which LPC signals proinflammatory activities.     

Heterologous expression and comparative characterization of the human neuromedin U subtype II receptor using the methylotrophic yeast Pichia pastoris and mammalian cells

Neuromedin U (a neuropeptide) plays regulatory roles in feeding, anxiety, smooth muscle contraction, blood flow and pain. The physiological actions of NmU are mediated via two recently identified G protein-coupled receptors namely the neuromedin U type 1 receptor (NmU(1)R) and the neuromedin U type 2 receptor (NmU(2)R). Despite their crucial roles in cell physiology, structural information on these receptors is limited, mainly due to their low expression levels in native tissues. Here, we report the overexpression of the human NmU(2)R in the methylotrophic yeast Pichia pastoris and baby hamster kidney (BHK) cells using the Semliki Forest virus (SFV) system. The recombinant receptor was expressed as a fusion protein with three different affinity tags namely, the Flag tag, the histidine 10 tag and the biotinylation domain of Propionobacterium shermanii. Expression level of the recombinant receptor was 6-9pmol/mg under optimized conditions, which is significantly higher than the expression level in the native tissues. The recombinant receptor binds to its endogenous ligand neuromedin U with high affinity (Kd=0.8-1.0nM) and the binding constant for the recombinant receptor is similar to that of the wild type NmU(2)R. Enzymatic deglycosylation suggested that the recombinant NmU(2)R was glycosylated in P. pastoris, but not in BHK cells. Confocal laser scanning microscopy and immunogold labelling experiment revealed that the recombinant receptor was predominantly localized in the intracellular membranes. To our knowledge, this is the first report of heterologous overexpression of an affinity tagged recombinant NmU(2)R and it should facilitate further characterization of this receptor.     

The dopamine D(2) receptor is expressed and sensitizes adenylyl cyclase activity in airway smooth muscle

Dopamine receptors are G protein-coupled receptors that are divided into two subgroups, "D(1)-like" receptors (D(1) and D(5)) that couple to the G(s) protein and "D(2)-like" receptors (D(2), D(3), and D(4)) that couple to G(i). Although inhaled dopamine has been reported to induce bronchodilation in patients with asthma, functional expression of dopamine receptor subtypes has never been described on airway smooth muscle (ASM) cells. Acute activation of G(i)-coupled receptors inhibits adenylyl cyclase activity and cAMP synthesis, which classically impairs ASM relaxation. In contrast, chronic activation of G(i)-coupled receptors produces a paradoxical enhancement of adenylyl cyclase activity referred to as heterologous sensitization. We questioned whether the dopamine D(2)-like receptor is expressed on ASM, whether it exhibits classical G(i)-coupling, and whether it modulates ASM function. We detected the mRNA encoding the dopamine D(2) receptor in total RNA isolated from native human ASM and from cultured human airway smooth muscle (HASM) cells. Immunoblots identified the dopamine D(2) receptor protein in both native human and guinea pig ASM and cultured HASM cells. The dopamine D(2) receptor protein was immunohistochemically localized to both human and guinea pig ASM. Acute activation of the dopamine D(2) receptor by quinpirole inhibited forskolin-stimulated adenylyl cyclase activity in HASM cells, which was blocked by the dopamine D(2) receptor antagonist L-741626. In contrast, the chronic pretreatment (1 h) with quinpirole potentiated forskolin-stimulated adenylyl cyclase activity, which was inhibited by L-741626, the phospholipase C inhibitor U73122, or the protein kinase C inhibitor GF109203X. Quinpirole also stimulated inositol phosphate synthesis, which was inhibited by L-741626 or U73122. Chronic pretreatment (1 h) of the guinea pig tracheal rings with quinpirole significantly potentiated forskolin-induced airway relaxation, which was inhibited by L-741626. These results demonstrate that functional dopamine D(2) receptors are expressed on ASM and could be a novel therapeutic target for the relaxation of ASM.     

The Expression of Urotensin II Receptor (U2R) is Up‐regulated by Interferon‐γ

Abstract

Urotensin‐II (U‐II) was identified as the natural ligand of the G protein‐coupled receptor GPR14, which has been correspondingly renamed Urotensin‐II receptor (U2R). The tissue distribution of U2R and the pharmacological effects of U‐II suggest a novel neurohormonal system with potent cardiovascular effects. We here report the human rhabdomyosarcoma cell line TE‐671 as the first natural and endogenous source of functional U2R in an immortalized cell line. In TE‐671 cells, U‐II stimulated extracellular signal regulated kinase phosphorylation and increased c‐fos mRNA expression. Furthermore, we demonstrate that the expression of U2R mRNA and functional U‐II high affinity binding sites are serum‐responsive and that they are specifically up‐regulated by interferon γ (IFNγ). We propose that IFNγ contributes to the previously observed increase of U2R density in the heart tissue of congestive heart failure (CHF) patients and we suggest that U2R up‐regulation, as a consequence of an inflammatory response, could lead to a clinical worsening of this disease.     

Molecular cloning and expression of a pituitary-specific receptor for growth hormone-releasing hormone.

A novel cDNA was isolated from rat pituitary mRNA using the polymerase chain reaction to amplify sequences encoding G protein-coupled receptors. The human homolog of this cDNA was isolated and expressed in human kidney 293 cells, and membrane fractions from these cells were found to bind human GH-releasing hormone (GHRH) with high affinity and specificity. GHRH also stimulates intracellular cAMP production in these transfected cells. The encoded receptor protein contains seven potential membrane-spanning domains, a hallmark of G protein-coupled receptors, and is homologous to previously identified receptors for secretin and vasoactive intestinal peptide, ligands that are related to GHRH. The rat GHRH receptor mRNA is expressed predominantly, if not exclusively, in the anterior pituitary gland, the major target for GHRH action. These results define a mechanism for cellular signaling by GHRH and provide the opportunity to examine the role of the GHRH receptor in growth abnormalities that involve the GH axis.     

Cloning, functional expression, and mRNA tissue distribution of the rat 5-hydroxytryptamine1A receptor gene

G protein-coupled receptors comprise a family of genes that share significant sequence similarity. We have screened a rat genomic library under low stringency hybridization conditions with the coding portion of the hamster beta 2-adrenergic receptor gene to isolate new members of this gene family. We show that one of these clones, clone D, codes for a 5-hydroxytryptamine1A (5-HT1A) binding site since: 1) it possesses an intronless open reading frame encoding a protein with seven putative transmembrane domains and 89% amino acid identity with the human 5-HT1A receptor (G21); 2) when transfected into Ltk- cells, it expresses a ligand-binding site with the pharmacology of the 5-HT1A receptor subtype, including 5-HT- and spiroxatrine-displaceable binding of 8-hydroxy-(2-(N,N-di[2,3-3H]propylamino)-1,2,3,4-tetrahydronaphthalene (KH = 0.8 nM). We further show that clone D encodes a functional receptor because its binding site interacts with G proteins and because it mediates agonist-induced inhibition of basal and stimulated cAMP accumulation in transfected GH4C1 pituitary cells. Finally, we have analyzed the tissue distribution of 5-HT1A receptor mRNA in rat brain and have found that 5-HT1A mRNA is present with the expected distribution of the 5-HT1A receptor (highest in septum and hippocampus) but is present as three RNA species (3.9, 3.6, and 3.3 kilobases). These studies represent the first characterization of receptor function and brain distribution of the cloned rat 5-HT1A receptor.