Throughout this application various publications are referred to by partial citations within parenthesis. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications, in their entireties, are hereby incorporated by reference into this application in order to more fully describe the state of the art to which the invention pertains.
Neuroregulators comprise a diverse group of natural products that subserve or modulate communication in the nervous system. They include, but are not limited to, neuropeptides, amino acids, biogenic amines, lipids and lipid metabolites, and other metabolic byproducts. Many of these neuroregulator substances interact with specific cell surface receptors which transduce signals from the outside to the inside of the cell. G-protein coupled receptors (GPCRs) represent a major class of cell surface receptors with which many neurotransmitters interact to mediate their effects. GPCRs are characterized by seven membrane-spanning domains and are coupled to their effectors via G-proteins linking receptor activation with intracellular biochemical sequelae such as stimulation of adenylyl cyclase. This application describes the identification of two GPCRs, SNORF62 and SNORF72, as receptors for neuromedin U (NMU) neuropeptides.
Neuropeptides are synthesized and released from neurons to mediate their effects on cells within the nervous system or on peripheral targets. NMU-25 and NMU-8 are bioactive peptides originally isolated from porcine spinal cord (Minamino, N. et al. 1985a and 1985b). NMU-8 corresponds to the C-terminus of porcine NMU-25 preceded by Arg-Arg residues and may therefore be generated by enzymatic cleavage. NMU homologues have been identified in many species including human (25 amino acids) and rat (23 amino acids).
The amino acid sequence for human NMU-25 is as follows:                F R V D E E F Q S P F A S Q S R G Y F L F R P R N—NH2 (SEQ ID NO: 5).        
The amino acid sequence for porcine NMU-25 is as follows:                F K V D E E F Q G P I V S Q N R R Y F L F R P R N—NH2 (SEQ ID NO: 6).        
The amino acid sequence for rat NMU-23 is as follows:                Y K V N E-Y Q G P-V A P S G G F F L F R P R N—NH2         
(SEQ ID NO: 7)(—indicates gaps in rat NMU-23 sequence to demonstrate optimum alignment).
The amino acid sequence for NMU-8 is Y F L F R P R N—NH2 (SEQ ID NO: 8). All of the preceding sequences were taken from Nandha and Bloom 1993 and Austin, et al. 1995.
Interestingly, the 8 carboxy-terminal residues of human NMU-25 are identical to those in porcine, rabbit and guinea pig NMU and differ only by one residue from the C-terminus of frog, rat, dog and chicken NMU (Austin et al. 1995). C-terminal NMU peptides (8–9 amino acids) have also been identified in guinea pig, chicken and dog tissue extracts (Minamino et al. 1985a and 1985b, Domin et al. 1989, O'Harte et al. 1991). Indeed, the region of rat NMU-23 critical for smooth muscle contractile activity was found to reside between residues 17–22 (C-terminal region) (Hashimoto et al. 1991, Sakura et al. 1991). However, other groups have demonstrated the necessity of the amidated C-terminal asparagine (Asn23) for activity as well (Nandha and Bloom 1993). Full length NMU is approximately 3-fold more potent than NMU-8 in smooth muscle contraction assays suggesting that the N-terminal region of the peptide also contributes to the activity (Nandha and Bloom, 1993). Several residues in the middle region of the peptide are conserved between species including Glu5, Gln8 and Pro10 supporting the functional importance of this region of the peptide (Nandha and Bloom, 1993). The C-terminus of NMU shares some homology with rat pancreatic polypeptide (PP): Leu-X-Arg-Pro-Arg-X-amide and contains a terminal asparaginamide also present in vasoactive intestinal polypeptide (VIP) (Nandha and Bloom 1993). However, the structure of NMU is unrelated to the other neuromedin peptides isolated by Minamino et al. (1985a,b).
A profound effect of NMU has been observed in rats on the in vivo release of stress-related modulators from the anterior pituitary and adrenal glands (reviewed in Malendowicz and Markowska 1994). Following a single subcutaneous injection of NMU-8 (6 μg/100 g body weight), adrenocorticotropic hormone (ACTH) blood concentrations are elevated transiently (3–12 hours) and plasma corticosterone levels remain elevated for 24 hours (Malendowicz et al., 1993). In addition, the stress-evoked rise in corticosterone was absent in rats treated for 6 days with NMU-8 (Malendowicz et al. 1994a). Since corticosterone exerts both mineralocorticoid and glucocorticoid effects, regulation of its release by NMU ligands would be expected to modulate fluid homeostasis, ionic balance and metabolism. Although the mechanisms that mediate these effects remain unclear, identification of NMU-like immunoreactivity in nerve fibers in the rat hypothalamic paraventricular and supraoptic nuclei suggest a potential role for NMU in the hypothalamic regulation of pituitary function (Steel et al. 1988).
The corticosterone releasing effects of NMU may be mediated in part by direct effects on the adrenal gland. In rat adrenal gland slices, NMU-8 markedly increased basal corticosterone and pregnenolone steroid secretion (Malendowicz et al. 1994a and 1994b). These effects require the presence of adrenal medulla suggesting that NMU-8 acts on medullary chromaffin cells which may stimulate cells of the cortex through a paracrine mechanism. On the other hand, rat NMU-23 directly decreased basal corticosterone secretion from isolated rat inner adrenocortical cells (in the absence of medullary cells) while NMU-8 was without effect (Malendowicz and Nussdorfer 1993). This discrepancy between NMU-23 and NMU-8 effects on adrenal cortical cells suggests that the NMU receptor in these cells differs from that responsible for smooth muscle contraction. Repeated NMU-8 administration also decreased adrenal weight and the number of cells in the zona reticularis, further suggesting a stimulatory role for NMU on adrenal gland (Malendowicz et al. 1994a). NMU ligands may therefore be useful for directly regulating secretion from the adrenal gland.
Although NMU-like immunoreactivity has not been demonstrated within the adrenal gland or circulating in plasma, corticotrophs within the anterior lobe of rat and human pituitary gland contain high levels of NMU-like immunoreactivity (Steel et al. 1988) suggesting a possible hormonal role for NMU. Co-release of NMU with other bioactive peptides is likely to occur since NMU was observed by electron microscopy to be present in the same secretory granules as ACTH and galanin (Cimini et al. 1993).
Furthermore, both ACTH and NMU are present in human pituitary corticotropinomas as well as in ACTH expressing tumors from a variety of other tissue sources (Steel et al. 1988). Supporting a potential hormonal role of NMU, is the identification of a small population of NMU positive parafollicular C-cells in rat thyroid gland (Domin et al. 1990 and Lo et al. 1992).
Activities of this peptide also include a hypertensive effect when given intravenously to rats at a high dose (1 nmole; Gardiner et al. 1990). However, at a lower dose (0.1 nmole), NMU caused potent constrictor effects on the superior mesenteric vascular bed reducing mesenteric blood flow without changing systemic blood pressure. The NMU-induced reduction in mesenteric blood flow was also demonstrated in dog (Sumi et al. 1987). In addition, a slight increase in blood flow to the pancreas was measured in these experiments. Such actions suggest the involvement of NMU in the regulation of blood flow to the digestive tract and subsequent effects on digestion.
NMU was originally isolated based on its potent uterine contractile activity in vitro and has contractile activity on other smooth muscle preparations including chicken crop (Minamino, N. et al. 1985a, 1985b). Isolated muscle strips from the dome of the human urinary bladder were also contracted by NMU (Maggi et al. 1990) suggesting a role for this peptide in urinary control. NMU-like immunoreactivity has been identified in high levels in the rat genito-urinary systems including vas deferens, prostate, fallopian tube, urethra, vagina, ovary and uterus (reviewed in Nandha and Bloom 1993). Smooth muscle contractile or other hormonal effects of NMU in these tissues may regulate urinary control and/or reproductive functions.
Along with many other neuropeptides, NMU is present in nerves throughout the gastrointestinal tract (reviewed in Nandha and Bloom 1993). NMU stimulates contraction of isolated longitudinal muscle of human ileum (Maggi et al. 1990) and rat stomach circular muscle (Benito-Orfila et al. 1991) suggesting a role for NMU in gastric emptying and intestinal motility. Interestingly, porcine jejunum (Brown and Quito 1988) and guinea pig small intestine (Minamino et al. 1985b) are not contracted by NMU indicating species differences in gut regulation by this peptide. However, ion transport is modulated by NMU-8 in isolated porcine jejunal mucosa (Brown and Quito 1988). NMU-like immunoreactivity in the intestine has been localized to both the submucosal and myenteric ganglion cells (Ballesta et al. 1988) consistent with the observed effects on contractility, blood flow and absorptive/secretory functions (Ballesta et al. 1988).
Although higher concentrations of NMU are found in the periphery than in the central nervous system (CNS), immunocytochemical analysis demonstrated the presence of NMU in nerve fibers in many CNS regions with concentrations in discrete functional systems (Honzawa et al. 1987, Ballesta et al. 1988 and reviewed in Domin et al. 1987). For example, NMU-like immunoreactivity was identified in all of the cranial nuclei associated with somato-motor function (Ballesta et al. 1988). Several structures associated with sensory processing are also rich in NMU containing fibers including spinal cord (dorsal horn>ventral horn), trigeminal sensory nuclei, vestibular nuclei and other nuclei associated with descending spinal pathways (Honzawa et al. 1987). This localization suggests a role for NMU in perception and processing of sensory stimuli including pain.
Three cerebellar nuclei (nucleus medialis, interpostitus and lateralis) also demonstrated NMU-like immunoreactivity, consistent with the potential importance of NMU in sensory processing. Neuronal cell bodies containing NMU-like immunoreactivity have been identified in the arcuate nucleus of the hypothalamus, an area identified as important for the regulation of food intake and neuroendocrine control.
Relatively high levels of NMU-like immunoreactivity were also detected in the nucleus accumbens (Domin et al. 1987), an area where dopaminergic transmission is involved in reward and reinforcement of learned behaviors. The presence of NMU in another area important in dopaminergic transmission, the substantia nigra, (Domin et al. 1987) suggests a role for NMU in the modulation of dopaminergic actions in movement control as well. NMU-like immunoreactivity is also found in the hippocampus, amygdala and other portions of the limbic system suggesting a role for NMU ligands in affective disorders, psychosis and cognition.
G-protein coupled receptors (GPCR's) activated by this peptide and related analogues were postulated to exist based on binding of [125I]rat NMU in rat uterus membranes (Nandha K. A. et al. 1993). The binding is saturable and of high affinity (Kd=0.35 nM, maximal binding capacity (Bmax)=580 fmol/mg protein). This affinity corresponds to the EC50 of contractile activity in this tissue, 0.2 nM, consistent with the involvement of this binding site in NMU-induced uterine contraction. The GTP analogue, GTPγS, inhibited binding of [125I]rat NMU-23 suggesting that the binding site is a GPCR. In addition, chemical cross-linking identified the binding protein as having an apparent Mr of 48,500 which is consistent with the expected size of a GPCR protein.
Nandha et al. (1994) also identified [125I]rat NMU-23 binding sites in rat uterus tissue slices and in the indusium griseum by autoradiography.