Urotensin-II (U-II) is a vasoactive 'somatostatin-like' cyclic peptide which was originally isolated from fish spinal cords, and which has recently been cloned from man. Here we describe the identification of an orphan human G-protein-coupled receptor homologous to rat GPR14 and expressed predominantly in cardiovascular tissue, which functions as a U-II receptor. Goby and human U-II bind to recombinant human GPR14 with high affinity, and the binding is functionally coupled to calcium mobilization. Human U-II is found within both vascular and cardiac tissue (including coronary atheroma) and effectively constricts isolated arteries from non-human primates. The potency of vasoconstriction of U-II is an order of magnitude greater than that of endothelin-1, making human U-II the most potent mammalian vasoconstrictor identified so far. In vivo, human U-II markedly increases total peripheral resistance in anaesthetized non-human primates, a response associated with profound cardiac contractile dysfunction. Furthermore, as U-II immunoreactivity is also found within central nervous system and endocrine tissues, it may have additional activities.

The cysteinyl leukotrienes (CysLTs) have been implicated in the pathophysiology of inflammatory disorders, in particular asthma, for which the CysLT receptor antagonists pranlukast, zafirlukast, and montelukast, have been introduced recently as novel therapeutics. Here we report on the molecular cloning, expression, localization, and pharmacological characterization of a CysLT receptor (CysLTR), which was identified by ligand fishing of orphan seven-transmembrane-spanning, G protein-coupled receptors. This receptor, expressed in human embryonic kidney (HEK)-293 cells responded selectively to the individual CysLTs, LTC(4), LTD(4), or LTE(4), with a calcium mobilization response; the rank order potency was LTD(4) (EC(50) = 2.5 nM) > LTC(4) (EC(50) = 24 nM) > LTE(4) (EC(50) = 240 nM). Evidence was provided that LTE(4) is a partial agonist at this receptor. [(3)H]LTD(4) binding and LTD(4)-induced calcium mobilization in HEK-293 cells expressing the CysLT receptor were potently inhibited by the structurally distinct CysLTR antagonists pranlukast, montelukast, zafirlukast, and pobilukast; the rank order potency was pranlukast = zafirlukast > montelukast > pobilukast. LTD(4)-induced calcium mobilization in HEK-293 cells expressing the CysLT receptor was not affected by pertussis toxin, and the signal appears to be the result of the release from intracellular stores. Localization studies indicate the expression of this receptor in several tissues, including human lung, human bronchus, and human peripheral blood leukocytes. The discovery of this receptor, which has characteristics of the purported CysLT(1) receptor subtype, should assist in the elucidation of the pathophysiological roles of the CysLTs and in the identification of additional receptor subtypes.

Uridine 5-diphosphoglucose (UDP-glucose) has a well established biochemical role as a glycosyl donor in the enzymatic biosynthesis of carbohydrates. It is less well known that UDP-glucose may possess pharmacological activity, suggesting that a receptor for this molecule may exist. Here, we show that UDP-glucose, and some closely related molecules, potently activate the orphan G protein-coupled receptor KIAA0001 heterologously expressed in yeast or mammalian cells. Nucleotides known to activate P2Y receptors were inactive, indicating the distinctly novel pharmacology of this receptor. The receptor is expressed in a wide variety of human tissues, including many regions of the brain. These data suggest that some sugar-nucleotides may serve important physiological roles as extracellular signaling molecules in addition to their familiar role in intermediary metabolism.

The underlying causes of obesity are poorly understood but probably involve complex interactions between many neurotransmitter and neuropeptide systems involved in the regulation of food intake and energy balance. Three pieces of evidence indicate that the neuropeptide melanin-concentrating hormone (MCH) is an important component of this system. First, MCH stimulates feeding when injected directly into rat brains; second, the messenger RNA for the MCH precursor is upregulated in the hypothalamus of genetically obese mice and in fasted animals; and third, mice lacking MCH eat less and are lean. MCH antagonists might, therefore, provide a treatment for obesity. However, the development of such molecules has been hampered because the identity of the MCH receptor has been unknown until now. Here we show that the 353-amino-acid human orphan G-protein-coupled receptor SLC-1 expressed in HEK293 cells binds MCH with sub-nanomolar affinity, and is stimulated by MCH to mobilize intracellular Ca2+ and reduce forskolin-elevated cyclic AMP levels. We also show that SLC-1 messenger RNA and protein is expressed in the ventromedial and dorsomedial nuclei of the hypothalamus, consistent with a role for SLC-1 in mediating the effects of MCH on feeding.

dehydrogenase by phosphorylation involves no long-range conformational change in the free enzyme.

1 Urotensin-II (U-II) and its G-protein-coupled receptor, GPR14, are expressed within mammalian cardiac and peripheral vascular tissue and, as such, may regulate mammalian cardiovascular function. The present study details the vasoconstrictor pro®le of this cyclic undecapeptide in di erent vascular tissues isolated from a diverse range of mammalian species (rats, mice, dogs, pigs, marmosets and cynomolgus monkeys). 2 The vasoconstrictor activity of human U-II was dependent upon the anatomical origin of the vessel studied and the species from which it was isolated. In the rat, constrictor responses were most pronounced in thoracic aortae and carotid arteries: 7log[EC 50 ]s 9.09+0.19 and 8.84+0.21, R max s 143+21 and 67+26% 60 mM KCl, respectively (compared, for example, to 7log[EC 50 ] 7.90+0.11 and R max 142+12% 60 mM KCl for endothelin-1 [ET-1] in thoracic aortae). Responses were, however, absent in mice aortae (7log [EC 50 ] 56.50). These ®ndings were further contrasted by the observation that U-II was a`coronary-selective' spasmogen in the dog (7log [EC 50 ] 9.46+0.11, R max 109+23% 60 mM KCl in LCX coronary artery), yet exhibited a broad spectrum of vasoconstrictor activity in arterial tissue from Old World monkeys (7log [EC 50 ]s range from 8.96+0.15 to 9.92+0.13, R max s from 43+16 to 527+135% 60 mM KCl). Interestingly, signi®cant di erences in reproducibility and vasoconstrictor e cacy were seen in tissue from pigs and New World primates (vessels which responded to noradrenaline, phenylephrine, KCl or ET-1 consistently). 3 Thus, human U-II is a potent, e cacious vasoconstrictor of a variety of mammalian vascular tissues. Although signi®cant species/anatomical variations exist, the data support the hypothesis that U-II in¯uences the physiological regulation of mammalian cardiovascular function. British Journal of Pharmacology (2000) 131, 1262 ± 1274 Keywords: Urotensin-II; GPR14; SENR; endothelin-1; somatostatin; vascular reactivity; spasmogen; coronary artery; endothelium; vasoconstriction Abbreviations: FLIPR,¯uorescent imaging plate reader; GPCR, guanosine triphosphate-binding protein [G-protein]-coupled receptor; LAD coronary artery, left anterior descending coronary artery; LCX, left circum¯ex coronary artery; SENR, sensory epithelial neuropeptide-like receptor; U-II, Urotensin-II IntroductionThe integrated control of cardiovascular homeostasis is achieved through a combination of direct neuronal control and systemic activation of the neurohumoral axis. The principal mammalian vasoactive factors of this axis (angiotensin-II, endothelin [ET]-1, noradrenaline) exert their haemodynamic e ects exclusively via interactions with speci®c seven transmembrane heterotrimeric G-protein-coupled receptors (GPCRs). Drugs which antagonize such interactions constitute one of the most successful classes of therapeutic agents identi®ed to date (Stadel et al., 1997; Wilson et al., 1998). Nowhere is this more evident than within the vasculature where numerous agents have been developed successfully for the clinical ...

Opiate tolerance and dependence are major clinical and social problems. The anti-opiate neuropeptides FF and AF (NPFF and NPAF) have been implicated in pain modulation as well as in opioid tolerance and may play a critical role in this process, although their mechanism of action has remained unknown. Here we describe a cDNA encoding a novel neuropeptide Y-like human orphan G protein-coupled receptor (GPCR), referred to as HLWAR77 for which NPAF and NPFF have high affinity. Cells transiently or stably expressing HLWAR77 bind and respond in a concentration-dependent manner to NPAF and NPFF and are also weakly activated by FMRF-amide (Phe-Met-Arg-Phe-amide) and a variety of related peptides. The high affinity and potency of human NPFF and human NPAF for HLWAR77 strongly suggest that these are the cognate ligands for this receptor. Expression of HLWAR77 was demonstrated in brain regions associated with opiate activity, consistent with the pain-modulating activity of these peptides, whereas the expression in adipose tissue suggests other physiological and pathophysiological activities for FMRF-amide neuropeptides. The discovery that the anti-opiate neuropeptides are the endogenous ligands for HL-WAR77 will aid in defining the physiological role(s) of these ligands and facilitate the identification of receptor agonists and antagonists.

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...