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...
Endothelins are a family of peptide hormones having profound cardiovascular, mitogenic, and potential neuroregulatory functions. In mammals, the ET 1 peptide family is composed of three members, ET-1, ET-2, and ET-3, that are encoded by three separate genes, which are differentially expressed in the tissues of the periphery and central nervous system (for reviews, see Refs. 1 and 2). Mammalian ETs share high sequence homology and structural similarity with a family of 21 amino acid peptide toxins from the snake Atractaspis engaddensis, the sarafotoxins (3).Two major subtypes of ET receptors (ET A and ET B ) (4) have been identified based on the rank order potency of ET-1, ET-2, and S6c (5,6). ET A receptor is defined by high and equal affinity for ET-1 and ET-2, approximately 70 -100-fold lower affinity for ET-3, and a 1000-fold lower affinity for S6c. In contrast, the ET B receptor subtype displays equal high affinities for all ET-related peptides. Two additional receptors have been cloned and characterized from Xenopus melanophores (ET C ) and heart (ET AX ) (7,8). While ET C receptors display high affinity for ET-3 compared to ET-1, ET AX receptors displayed extremely weak affinity for BQ123 as well as S6c (ET A -and ET B -selective ligands, respectively). Receptors for ET are differentially expressed in a wide variety of tissues and cell types (9, 10). ETs and sarafotoxins bind to a common receptor and initiate a common signal transduction pathway, principally a G-protein-mediated activation of phospholipase C and subsequent inositol triphosphate-mediated increase in Ca 2ϩ levels (1, 11).ET mediates a number of physiological effects including vasoconstriction mitogenesis, and induction of c-fos transcription (12-23). These diverse and complex physiological effects mediated by ET in conjunction with the molecular heterogeneity and differential tissue distribution of the ET-related peptides and their receptors underscores the importance of utilizing molecular biological approaches to dissect the components of ET physiology. Several laboratories have postulated the presence of additional ET receptors to account for the diverse biochemical and physiological activities of various ETs (24). This hypothesis has been supported by binding as well as functional studies. We have previously reported the cloning, functional characterization, and regulation of the human ET A and ET B receptor subtypes (25). In this report, we describe the cloning and functional characterization of a novel ET B receptor splice variant from human placenta. EXPERIMENTAL PROCEDURESConstruction and Screening of the cDNA Libraries-The porcine cerebellum cDNA library (26) in pcDNA vector was screened by hybridization to nitrocellulose replicates using 32 P-labeled porcine ET B -R cDNA coding sequence as a probe in 20% formamide, 5 ϫ SSC (SSC is 150 mM NaCl, 15 mM sodium citrate), 5 ϫ Denhardt's, 0.1% SDS, and 0.2 mg/ml Escherichia coli tRNA at 42°C (27). Filters were washed with 2 ϫ SSC, 0.1% SDS at 42°C. Several positive recombinant clones wer...
The transcription program of bacteriophage 17 in vivo was analyzed by hybridizing T7 mRNAs, labeled at intervals after infection, to Hpa I restriction fragments of 17 DNA. Transcription of the late genes is temporally regulated: class II genes are transcribed between 4 and 16 min after infection; most class III genes are transcribed from 8 min after infection until lysis. Genes 8-10 are transcribed as both class II and class III genes. The rate of T7 RNA synthesis decreases sharply at 10 min after infection. The rapid decrease in the rate of 17 RNA synthesis and the shutoff of class II RNA synthesis were not observed in cells infected with phage defective in gene 3.5 (lysozyme). Although the decrease in the rate of T7 RNA synthesis is independent of DNA replication, the failure to shut off class II RNA synthesis normally in 3.5--infected cells may reflect the role of 17 lysozyme in DNA replication. In vitro, the regions of 17 DNA transcribed by the phage RNA polymerase were found to be dependent upon ionic conditions.The bacteriophage T7 proteins are synthesized in three classes during infection: class I proteins from 4 until 8 min after infection, class II proteins from 6 until 15 min after infection, and class III proteins from 6-8 min after infection until lysis (1, 2). Although the regulation of expression of the class I (early) genes has been studied in some detail (2-4), the mechanism(s) by which expression of the class II and class III genes are, regulated is poorly understood. Hopper et al. (5) have demonstrated that the time-dependent appearance of the T7 late proteins in vivo can be duplicated in a cell-free system programmed with mRNA isolated from T7-infected cells at intervals after infection. Thus, the control of synthesis of these proteins is dependent upon the availability of functional class II and class HII mRNAs and does not appear to involve modification of the ribosomes or other translational factors.Transcription of the class II and class III genes requires the synthesis of a phage-specified RNA polymerase, the product of gene 1 (a class I gene) (6, 7); no other phage genes are required for the expression of the class II or class III genes (2). In vitro, the purified phage RNA polymerase transcribes both the class II and class III regions of T7 DNA but synthesizes much more class III RNA than class II RNA (8-10). It had been suggested that a preferential synthesis of class III mRNA in vivo might account for the shutoff of class II protein synthesis midway during infection: as the more abundantly produced class III mRNAs accumulate in the infected cell, they would successfully compete for the available translational capacity of the cell (9). Preliminary evidence, however, indicated that the class II RNAs are synthesized to a much greater extent in vivo than in vitro (10).In this study we characterized the transcription program of bacteriophage T7 in vivo by hybridizing T7 mRNA, labeled at intervals after infection, to restriction fragments of T7 DNA.We found that transcription of t...
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