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...
Melanin-concentrating hormone (MCH) is involved in the regulation of feeding and energy homeostasis. Recently, a 353-amino acid splice variant form of the human orphan receptor SLC-1 (1) (hereafter referred to as MCH 1 ) was identified as an MCH receptor. This report describes the cloning and functional characterization of a novel second human MCH receptor, which we designate MCH 2 , initially identified in a genomic survey sequence as being homologous to MCH 1 receptors. Using this sequence, a full-length cDNA was generated with an open reading frame of 1023 base pairs, encoding a polypeptide of 340 amino acids, with 38% identity to MCH 1 and with many of the structural features conserved in G protein-coupled receptors. This newly discovered receptor belongs to class 1 (rhodopsin-like) of the G protein-coupled receptor superfamily. HEK293 cells transfected with MCH 2 receptors responded to nanomolar concentrations of MCH with an increase in intracellular Ca 2؉ levels and increased cellular extrusion of protons. In addition, fluorescently labeled MCH bound with nanomolar affinity to these cells. The tissue localization of MCH 2 receptor mRNA, as determined by quantitative reverse transcription-polymerase chain reaction, was similar to that of MCH 1 in that both receptors are expressed predominantly in the brain. The discovery of a novel MCH receptor represents a new potential drug target and will allow the further elucidation of MCH-mediated responses. Melanin-concentrating hormone (MCH)1 is a cyclic neuropeptide that was first discovered in teleost fish, in which it acts as a skin color-regulating hormone (2). In rodents its tissue distribution in the perikarya of the lateral hypothalamus and the zona incerta suggests that MCH may be involved in a variety of behavioral responses (3). Similar tissue distributions have been reported in both bird (4) and monkey (5). Reports implicating MCH in the regulation of feeding behavior show that increased food intake occurs after direct administration of MCH into the brain (6) and that MCH is up-regulated after fasting and in obese leptin-deficient mice (7). There are also reports that suggest MCH may be involved in aggressive behavior, anxiety, and reproductive function (8, 9).Recently, several groups independently identified a 353-amino acid splice variant of the orphan G protein-coupled receptor (GPCR) SLC-1 (1) as an MCH receptor (10 -14). In view of the findings of the current study, we propose that this form of SLC-1 be hereafter referred to as MCH 1 . Southern blot and related studies have indicated the absence of additional MCH receptor subtypes that closely resemble MCH 1 at the DNA level (3, 10, 15). However, because degeneracy in receptor-ligand pairings throughout the GPCR superfamily is common, we reasoned that other MCH receptors with low homology to the MCH 1 receptor may exist. This suggestion is supported by reports of pharmacological differences between the MCH 1 receptor and MCH binding sites in various cell lines and tissues (10).Sequencing of the human...
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