Characterization of complementary DNA encoding the rat neuromedin U precursor.

162

151

Neuromedins are a family of peptides best known for their contractile activity on smooth muscle preparations. The biological mechanism of action of neuromedin U remains unknown, despite the fact that the peptide was first isolated in 1985. Here we show that neuromedin U potently activates the orphan G proteincoupled receptor FM3, with subnanomolar potency, when FM3 is transiently expressed in human HEK-293 cells. Neuromedins B, C, K, and N are all inactive at this receptor. Quantitative reverse transcriptase-polymerase chain reaction analysis of neuromedin U expression in a range of human tissues showed that the peptide is highly expressed in the intestine, pituitary, and bone marrow, with lower levels of expression seen in stomach, adipose tissue, lymphocytes, spleen, and the cortex. Similar analysis of FM3 expression showed that the receptor is widely expressed in human tissue with highest levels seen in adipose tissue, intestine, spleen, and lymphocytes, suggesting that neuromedin U may have a wide range of presently undetermined physiological effects. The discovery that neuromedin U is an endogenous agonist for FM3 will significantly aid the study of the full physiological role of this peptide. G protein-coupled receptors (GPCRs)1 represent one of the largest gene superfamilies identified to date, with more than 1000 members cloned from a wide range of species. The current explosion in the availability of human genomic sequence data is allowing many more members of this family to be identified in man. Most if not all of these newly identified GPCRs fall into the category of orphan receptors, for which the endogenous ligand(s) remain to be identified. Typically these orphan receptors show only low levels of similarity (less than 35% identity) with known GPCRs, too low to classify them with any confidence into a specific receptor subfamily, although one can often predict the likely class of ligand for these receptors, e.g. peptide, nucleotide, lipid, etc., by using phylogenetic analysis.Recently, naturally occurring ligands have been identified for a number of these orphan GPCRs using a "reverse-pharmacological" approach (1), that is using the recombinant orphan receptor as a specific sensor component of a bioassay. Tissue extracts have often been the source of these natural ligands (2, 3), although more recently the ligands for several orphans have been identified as a result of screening large libraries of known or putative GPCR ligands (4 -6). Here, we describe how this latter approach has been used to identify neuromedin U (NmU) as a naturally occurring ligand for the orphan receptor FM3.Neuromedin U was first isolated over 15 years ago from extracts of porcine spinal cord, using a uterine smooth muscle contraction bioassay to monitor purification (7). Two molecular forms were isolated; neuromedin U-8 (NmU-8) and neuromedin U-25 (NmU-25). NmU-like immunoreactivity has since been detected in neurones in the mammalian brain and gastrointestinal tracts of various species (8 -10) and in the thyroid and endocri...

Section: Neuromedin U Is a Potent Agonist At The Orphan Gpcr Fm3supporting

confidence: 90%

Neuromedin U Is a Potent Agonist at the Orphan G Protein-coupled Receptor FM3

Szekeres¹,

Muir²,

Spinage³

et al. 2000

162

151

“…The amino acid sequence for rat neuromedin U can be accessed in the Swiss-Prot Database under accession no. P12760 (42). The amino acid sequence for chicken neuromedin U can be accessed in the Swiss-Prot Database under accession no.…”

Section: Resultsmentioning

confidence: 99%

“…Virtually nothing is known of the physiological roles that skin peptides play in amphibian survival; however, they may be involved in antipredation, modes of reproduction, and mating behavior or involved in control in osmoregulation (42). Establishment of these functions is difficult, due of the large variety of endogenous skin compounds, which could interact together to generate complex effects not produced by a single compound alone.…”

Section: Discussionmentioning

confidence: 99%

“…Previous studies have demonstrated that the secretion of the African clawed frog, Xenopus laevis, promotes yawning and other orofacial movements in several predatory snakes, which slowed ingestion rate and facilitated escape of the frog (43,44). Cerulein, as well as the myoactive and neuromodulatory peptide, xenopsin, were reported to be responsible for some of these antipredator actions (42). In the arboreal L. caerulea, the high content of cerulein (13), in conjunction with NmU, may elicit peripheral or neurocentral effects to the predator that protect this species from being consumed.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Isolation, Structural Characterization, and Bioactivity of a Novel Neuromedin U Analog from the Defensive Skin Secretion of the Australasian Tree Frog, Litoria caerulea

Salmon¹,

Johnsen²,

Bienert³

et al. 2000

We report the isolation of a novel bioactive peptide, neuromedin U-23 (NmU-23), from the defensive skin secretion of the Australasian tree frog, Litoria caerulea. The primary structure of the peptide was established by a combination of microsequencing, mass spectroscopy and site-directed antiserum immunoreactivity as SDEEVQVPGGVISNGYFLFRPRN-amide (M r 2580.6). A synthetic replicate of frog NmU-23 displaced monoradioiodinated rat NmU-23 from uterine membranes in a dose-dependent fashion indistinguishable from nonisotopically labeled rat NmU-23. In a rat uterine smooth muscle strip preparation, synthetic frog NmU-23 produced dose-dependent contractions identical to porcine NmU-25. However, in a preparation of human urinary bladder muscle strip, the synthetic frog peptide was more potent than porcine NmU-25 in eliciting contraction and produced desensitization of the preparation to the latter peptide. This report demonstrates that the defensive skin secretion of a frog contains a novel peptide exhibiting a high degree of primary structural similarity to the endogenous vertebrate peptide, NmU, and that this frog skin analog displays biological activity in mammalian tissues.

“…The cDNAs for rat and human NmU have been cloned, and analysis of the nucleic acid sequence suggests that NmU is produced as a 174-amino acid precursor (10,11). The precursor contains a signal peptide and several dibasic cleavage sites that give rise to a number of possible secreted peptides, including NmU, which is present near the carboxyl terminus.…”

mentioning

confidence: 99%

Identification of a Novel Neuromedin U Receptor Subtype Expressed in the Central Nervous System

Shan¹,

Qiao²,

Crona³

et al. 2000

104

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.