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

Abstract

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...

Effects of neuromedin-U on immature rat adrenocortical cells: In vitro and in vivo studies

“…Our present RT-PCR and ICC studies clearly demonstrate the expression of NMUR1 as mRNA and protein in the cortex of immature rat adrenals, thereby confirming earlier findings obtained in adult animals (9,(27)(28)(29)33,34). Moreover, the detection of NMUR1 mRNA in dispersed zona glomerulosa and zona fasciculata-reticularis cells rules out the possibility that the expression was due to the presence in the specimens assayed of the non-parenchymal components of the gland (connective tissue and capillaries).…”

“…NPY, cholecystokinin, galanin, leptin, orexins, beacon and NPW) (11)(12)(13)(14)(15)(16)(17), also NMU appears to regulate the hypothalamic-pituitary-adrenal (HPA) axis. The expression of NMU and NMUR 2 was found in the hypothalamus and anterior pituitary (10,(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33), while in the adrenal glands only the expression of NMUR1 was detected (9,(27)(28)(29)33,34). NMU has been shown to enhance c-fos expression in the hypothalamic paraventricular nucleus (1,2,30,35), to increase CRH and vasopressin output from rat hypothalamic explants, and to stimulate ACTH and corticosterone secretion in the rat (2,4,5,(36)(37)(38).…”

Oral cancer in vivo gene expression profiling assisted by laser capture microdissection and microarray analysis

“…Of particular importance are the di erentially expressed genes that are not yet functionally characterized or genes that have not been studied by classic methods in head and neck/oral carcinogenesis. One such example is neuromedin U (Nmu), which is downregulated in 5/5 tumors (Szekeres et al, 2000). Nmu is a poorly understood protein that manifests potent contractile activities on smooth muscle cells.…”

Molecular Cloning and Functional Characterization of MCH2, a Novel Human MCH Receptor

“…Calcium Mobilization Assays-Intracellular calcium assays were carried out essentially as described previously (17). The maximum change in fluorescence above baseline, measured on a fluorometric imaging plate reader (FLIPR, Molecular Devices), was used to determine the agonist response.…”

“…The maximum change in fluorescence above baseline, measured on a fluorometric imaging plate reader (FLIPR, Molecular Devices), was used to determine the agonist response. For cross-screening studies, HEK293 cells were screened against a large library of over 1500 known and putative GPCR agonists including all known mammalian neuropeptides as described previously (17). Peptides in this library were tested at a final concentration of Ͼ100 nM, and other potential agonists were tested at a final concentration of Ͼ1 M. For antagonist studies, test substances were added 30 min before the addition of an EC 50 concentration of agonist.…”