Fusion of diphtheria toxin and urotensin II produces a neurotoxin selective for cholinergic neurons in the rat mesopontine tegmentum.

Urotensin II is a neuropeptide first isolated from fish and later found in mammals: where it has potent cardiovascular, endocrine and behavioral effects. In rat brain the urotensin II receptor (UII-R) is predominately expressed in the cholinergic neurons of the pedunculopontine (PPTg) and laterodorsal tegmental nuclei. Typically, the function of the PPTg has been examined using excitotoxins, destroying both cholinergic and non-cholinergic neurons, which confounds interpretation. We took advantage of UII-R's unique expression profile, by combining UII with diphtheria toxin, to engineer a toxin specific for cholinergic neurons of the PPTg. In vitro, two different toxin constructs were shown to selectively activate UII-R (average EC50 approximately 30 nmol/L; calcium mobility assay) and to be 10,000-fold more toxic to UII-R expressing CHO cells, than wildtype cells (average LD50 approximately 2 nmol/L; cell viability). In vivo, pressure injection into the PPTg of rats, resulted in specific loss of choline transporter and NADPH diaphorase positive neurons known to express the UII-R. The lesions developed over time, resulting in the loss of over 80% of cholinergic neurons at 21 days, with little damage to surrounding neurons. This is the first highly selective molecular tool for the depletion of mesopontine cholinergic neurons. The toxin will help to functionally dissect the pedunculopontine and laterodorsal tegmental nuclei, and advance the understanding of the functions of these structures.

The urotensin II receptor is expressed in the cholinergic mesopontine tegmentum of the rat.

Urotensin II (UII) is a peptide known to be a potent vasoconstrictor. The urotensin II receptor (UII-R) is expressed not only in peripheral tissues but also in the brain of rodents. As a basis for studies of UII central nervous system actions, UII-R localization in the rat brain was analyzed by in situ hybridization and by in situ binding. UII-R mRNA was found in the mesopontine tegmental area colocalizing with choline acetyltransferase. Binding sites were detected throughout the brain with the highest levels found in the pedunculopontine tegmental area, the lateral dorsal tegmental area, and the lateral septal, medial habenular, and interpeduncular nuclei. The majority of these brain nuclei are sites of axonal termination originating from the mesopontine areas, suggesting that UII-R is a presynaptic receptor. This distribution of UII-R in the cholinergic mesopontine area indicates that the UII system may be involved in sensory-motor integration and perhaps in central nervous system blood flow.

The carboxyl terminus of the prolactin-releasing peptide receptor interacts with PDZ domain proteins involved in alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor clustering.

PDZ domain proteins use the PDZ domain binding motif to bind to the C-terminal sequence of membrane proteins to help scaffold them and spatially organize the components of the intracellular signaling machinery. We have identified a sequence at the C terminus of a G protein-coupled receptor, the PrRP receptor, that shares similarities with the C-terminal sequence of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPA-R) subunits that interact with PDZ domain proteins. When coexpressed in human embryonic kidney 293 cells, PrRP receptor was able to coimmunoprecipitate the three PDZ domain proteins known to interact with AMPA receptors: glutamate receptor interacting protein (GRIP), AMPA binding protein (ABP), and protein that interacts with C-kinase (PICK1), but not the PDZ domain protein PSD-95, which does not interact with AMPA receptors. These interactions are sequence-selective as determined by mutagenesis. Furthermore, we show that PrRP receptor forms intracellular clusters when coexpressed with PICK1, and that this clustering effect is dependent on the interaction between the PICK1 PDZ domain and the last four amino acids of PrRP receptor. We found that PrRP receptor interaction with GRIP is not protein kinase C-regulated but may be regulated by other unidentified kinase because okadaic acid dramatically reduced GRIP interaction. By in situ hybridization, we show that the PrRP receptor is expressed in neurons that also express these PDZ domain proteins. We thus demonstrate that PrRP receptor interacts with the same PDZ domain proteins as the AMPA-Rs, raising the possibility that these two proteins could be scaffolded together at the synapse. These results may help to gain important insights into PrRP functions within the central nervous system.

Novel neurotransmitters as natural ligands of orphan G-protein-coupled receptors.

The "orphan" G-protein-coupled receptors (GPCRs) are cloned GPCRs that bind unknown ligands. Since 1995, nineteen orphan GPCRs have been used as targets to identify and isolate their natural ligands via the application of the "orphan receptor strategy". These ligands are peptides, lipids or biogenic amines, and act as transmitter molecules. One nucleotide-sugar derivative and six peptides or peptide families identified through this strategy are novel and have already enriched our understanding of various brain functions.

Molecular cloning and characterization of a second human cysteinyl leukotriene receptor: discovery of a subtype selective agonist.

The cysteinyl leukotrienes (CysLTs) are potent biological mediators in the pathophysiology of inflammatory diseases, in particular of airway obstruction in asthma. Pharmacological studies have suggested the existence of at least two types of CysLT receptors, designated CysLT(1) and CysLT(2). The CysLT(1) receptor has been cloned recently. Here we report the molecular cloning, expression, localization, and functional characterization of a human G protein-coupled receptor that has the expected characteristics of a CysLT(2) receptor. This new receptor is selectively activated by nanomolar concentrations of CysLTs with a rank order potency of LTC(4) = LTD(4) >> LTE(4). The leukotriene analog BAY u9773, reported to be a dual CysLT(1)/CysLT(2) antagonist, was found to be an antagonist at CysLT(1) sites but acted as a partial agonist at this new receptor. The structurally different CysLT(1) receptor-selective antagonists zafirlukast, montelukast, and MK-571 did not inhibit the agonist-mediated calcium mobilization of CysLT(2) receptors at physiological concentrations. Localization studies indicate highest expression of CysLT(2) receptors in adrenal glands, heart, and placenta; moderate levels in spleen, peripheral blood leukocytes, and lymph nodes; and low levels in the central nervous system and pituitary. The human CysLT(2) receptor gene is located on chromosome 13q14.12-21.1. The new receptor exhibits all characteristics of the thus far poorly defined CysLT(2) receptor. Moreover, we have identified BAY u9773 as a CysLT(2) selective agonist, which could prove to be of immediate use in understanding the functional roles of the CysLT(2) receptor.

Human urotensin II mediates vasoconstriction via an increase in inositol phosphates.

The cyclic peptide urotensin II has recently been cloned from human and reported to potently constrict primate blood vessels. To elucidate the cellular signalling mechanisms of this peptide, we investigated a possible relationship of vasomotor effects of human urotensin II and phosphoinositide turnover in isolated rabbit thoracic aorta. Human urotensin II produced a slowly developing increase in isometric contractile force (pEC(50)=9.0) that was endothelium-independent. The contractile effect of urotensin II was significantly inhibited by the phospholipase C inhibitor, 2-nitro-4-carboxyphenyl-N,N,-diphenylcarbamate (NCDC), but not by the cyclooxygenase inhibitor, indomethacin. In slices of rabbit thoracic aorta, human urotensin II increased phosphoinositide hydrolysis, and this effect was also inhibited by NCDC. The potency of urotensin II (pEC(50)=8.6) was similar to that found in the contractile studies. Thus, vasoconstrictor effects of human urotensin II appear to be mediated by a phospholipase C-dependent increase in inositol phosphates, suggesting that the peptide acts via a G(q) protein-coupled receptor.

Melanin-concentrating hormone receptor: an orphan receptor fits the key.

The melanin-concentrating hormone (MCH), a hypothalamic peptide, was identified initially in teleost fish as a regulator of pigmentary changes in background adaptation, and was later also found, in mammals, to be a regulator of feeding and energy homeostasis. Its specific receptor remained an enigma until very recently when it was identified as the orphan G-protein-coupled receptor SLC-1. This review focuses on the identification, structure and signaling of the MCH receptor and discusses some of the implications of its discovery.

The orphanin FQ/nociceptin gene: structure, tissue distribution of expression and functional implications obtained from knockout mice.

Like other neuropeptides, orphanin FQ/nociceptin (OFQ/N) is encoded by a larger precursor protein. The cDNA for the OFQ/N precursor has been cloned from human, rat, mouse and bovine tissue demonstrating that this peptidergic system serves important functions that have been conserved during evolution. The structural organization of the precursor protein is similar to opioid peptide precursors, supporting the view of a common origin for the opioid systems and the OFQ/N system. In addition to OFQ/N, the precursor may encode two other biologically active peptides. Anatomic studies have revealed high levels of expression of the OFQ/N messenger RNA in brain structures involved in sensory, emotional and cognitive processing. In particular, high levels of OFQ/N mRNA were detected in the limbic system, underlining the stress attenuating activities that have been described as an important function of OFQ/N. Recently, mutant mice have been generated that lack the precursor protein of OFQ/N to further define the physiological functions of the OFQ/N system. The OFQ/N-deficient mice are characterized by an increased sensitivity to stressful stimuli and a lack of habituation to chronic and repeated stress. This review will summarize recent findings on the molecular biology of the OFQ/N precursor and relate it to possible physiological functions of this newly discovered neuropeptide system.

Orphan receptors, novel neuropeptides and reverse pharmaceutical research.

By the beginning of the next millennium, the search for the natural ligands of the orphan G-protein-coupled receptors will lead to the discovery of so many new neuropeptides that it may well double their present number. This bounty of new tools will direct us to new insights in brain function and to better understanding of brain disorders. It is expected that the novel neuropeptides will have a particular impact on molecular psychiatry. In view of their potential, the novel neuropeptides should also become the focus of drug discovery programs. It is hoped that these programs will be initiated at an early stage, when understanding of novel neuropeptide function has not necessarily been reached, to allow for the design of neuropeptide chemical surrogates that are crucial to the study of the novel neuropeptide system and may serendipitously develop into highly successful drugs.

Targeted disruption of the orphanin FQ/nociceptin gene increases stress susceptibility and impairs stress adaptation in mice.

The neuropeptide orphanin FQ (also known as nociceptin; OFQ/N) has been implicated in modulating stress-related behavior. OFQ/N was demonstrated to reverse stress-induced analgesia and possess anxiolytic-like activity after central administration. To further study physiological functions of OFQ/N, we have generated OFQ/N-deficient mice by targeted disruption of the OFQ/N gene. Homozygous mice display increased anxiety-like behavior when exposed to a novel and threatening environment. OFQ/N-null mice show elevated basal pain threshold but develop normal stress-induced analgesia. Interestingly, these mice show impaired adaptation to repeated stress when compared with wild-type mice, whereas their performance in spatial learning remained unaffected. Basal and poststress plasma corticosterone levels were found to be elevated in OFQ/N-deficient animals. Thus, OFQ/N appears to be crucially involved in the neurobiological regulation of stress-coping behavior and fear.

Orphan receptors and the concept of reverse physiology: discovery of the novel neuropeptide orphanin FQ/nociceptin.

The cloning of numerous orphan members from the supergene family of G protein-coupled receptors implies the existence of many as yet undiscovered neurotransmitters and neuropeptides. Recently, new technologies were developed to isolate natural ligands for orphan receptors, using the receptor as a biological sensor during the purification process. This manuscript will present the concept and technology of an approach which starts from a cloned receptor to ultimately describe the physiological functions of the transmitter system. This strategy inverts the classical order of biomedical research and was thus termed "reverse physiology". The first natural ligand isolated by this strategy is a peptide with significant similarity to the opioid peptides and has been named orphanin FQ or nociceptin (OFQ/NOC). Evidence for characterizing OFQ/NOC as a genuine neuropeptide will be reviewed. OFQ/NOC is biosynthetically derived from a larger precursor protein which may encode additional bioactive peptides. Since its discovery, a large number of studies have described numerous physiological functions of OFQ/NOC. Because of its relation to the opioid system, much attention has been focused on the involvement of OFQ/NOC in nociception, sometimes with controversial results. However, the pharmacological profile of the OFQ/NOC system suggests a clear separation from the opioids. The discovery of OFQ/NOC and the subsequent analyses of its physiological functions is an example which has already been followed by the identification of two other novel neuropeptides. The orphan receptor strategy holds a lot of promises for the postgenomic era, helping to fill the vast amount of sequence data with life.

Opioid receptor-like (ORL1) receptor distribution in the rat central nervous system: comparison of ORL1 receptor mRNA expression with (125)I-[(14)Tyr]-orphanin FQ binding.

The recently discovered neuropeptide orphanin FQ (OFQ), and its opioid receptor-like (ORL1) receptor, exhibit structural features suggestive of the micro, kappa, and delta opioid systems. The anatomic distribution of OFQ immunoreactivity and mRNA expression has been reported recently. In the present analysis, we compare the distribution of orphanin receptor mRNA expression with that of orphanin FQ binding at the ORL1 receptor in the adult rat central nervous system (CNS). By using in vitro receptor autoradiography with (125)I-[(14)Tyr]-OFQ as the radioligand, orphanin receptor binding was analyzed throughout the rat CNS. Orphanin binding sites were densest in several cortical regions, the anterior olfactory nucleus, lateral septum, ventral forebrain, several hypothalamic nuclei, hippocampal formation, basolateral and medial amygdala, central gray, pontine nuclei, interpeduncular nucleus, substantia nigra, raphe complex, locus coeruleus, vestibular nuclear complex, and the spinal cord. By using in situ hybridization, cells expressing ORL1 mRNA were most numerous throughout multiple cortical regions, the anterior olfactory nucleus, lateral septum, endopiriform nucleus, ventral forebrain, multiple hypothalamic nuclei, nucleus of the lateral olfactory tract, medial amygdala, hippocampal formation, substantia nigra, ventral tegmental area, central gray, raphe complex, locus coeruleus, multiple brainstem motor nuclei, inferior olive, deep cerebellar nuclei, vestibular nuclear complex, nucleus of the solitary tract, reticular formation, dorsal root ganglia, and spinal cord. The diffuse distribution of ORL1 mRNA and binding supports an extensive role for orphanin FQ in a multitude of CNS functions, including motor and balance control, reinforcement and reward, nociception, the stress response, sexual behavior, aggression, and autonomic control of physiologic processes.

Molecular characterization of the melanin-concentrating-hormone receptor.

Orphan G-protein-coupled receptors (GPCRs) are cloned proteins with structural characteristics common to the GPCRs but that bind unidentified ligands. Orphan GPCRs have been used as targets to identify novel transmitter molecules. Here we describe the isolation from brain extracts and the characterization of the natural ligand of a particular orphan GPCR (SLC-1) that is sequentially homologous to the somatostatin receptors. We show that the natural ligand of this receptor is the neuropeptide melanin-concentrating hormone (MCH). MCH is a cyclic peptide that regulates a variety of functions in the mammalian brain, in particular feeding behaviour. We demonstrate that nanomolar concentrations of MCH strongly activate SLC-1-related pathways through G(alpha)i and/or G(alpha)q proteins. We have analysed the tissue localization of the MCH receptor and find that it is expressed in several brain regions, in particular those involved in olfactory learning and reinforcement mechanisms, indicating that therapies targeting the MCH receptor should act on the neuronal regulation of food consumption.

Localization of orphanin FQ (nociceptin) peptide and messenger RNA in the central nervous system of the rat.

Orphanin FQ (OFQ) is the endogenous agonist of the opioid receptor-like receptor (ORL-1). It and its precursor, prepro-OFQ, exhibit structural features suggestive of the opioid peptides. A cDNA encoding the OFQ precursor sequence in the rat recently has been cloned, and the authors recently generated a polyclonal antibody directed against the OFQ peptide. In the present study, the authors used in situ hybridization and immunohistochemistry to examine the distribution of OFQ peptide and mRNA in the central nervous system of the adult rat. OFQ immunoreactivity and prepro-OFQ mRNA expression correlated virtually in all brain areas studied. In the forebrain, OFQ peptide and mRNA were prominent in the neocortex endopiriform nucleus, claustrum, lateral septum, ventral forebrain, hypothalamus, mammillary bodies, central and medial nuclei of the amygdala, hippocampal formation, paratenial and reticular nuclei of the thalamus, medial habenula, and zona incerta. No OFQ was observed in the pineal or pituitary glands. In the brainstem, OFQ was prominent in the ventral tegmental area, substantia nigra, nucleus of the posterior commissure, central gray, nucleus of Darkschewitsch, peripeduncular nucleus, interpeduncular nucleus, tegmental nuclei, locus coeruleus, raphe complex, lateral parabrachial nucleus, inferior olivary complex, vestibular nuclear complex, prepositus hypoglossus, solitary nucleus, nucleus ambiguous, caudal spinal trigeminal nucleus, and reticular formation. In the spinal cord, OFQ was observed throughout the dorsal and ventral horns. The wide distribution of this peptide provides support for its role in a multitude of functions, including not only nociception but also motor and balance control, special sensory processing, and various autonomic and physiologic processes.

Functional genomics and the discovery of new drug targets.

Functional genomics can be defined as the search for the physiological role of a gene for which only its primary sequence is known. One example of a successful functional genomics adventure is the search for the natural ligands of orphan G protein-coupled receptors (GPCRs). GPCRs are proteins containing 7 hydrophobic domains that are the recognition sites of neurotransmitters and neuropeptides. Although many of these have been shown to interact with known natural ligands, several bind ligands that have not been thus far isolated. These are the so-called "orphan" GPCRs. As an example of functional genomics, an "orphan receptor strategy" has been developed to identify the natural ligands of orphan GPCRs. We describe that the application of this strategy has already led to the identification of 4 new neuropeptides and report on what has been learned about these neuropeptides. We finally discuss the importance of the application of the orphan receptor strategy to the development of novel drugs.

Reverse physiology: discovery of the novel neuropeptide, orphanin FQ/nociceptin.

The search for novel neurotransmitters and neuropeptides has been recently revolutionized by the development of a purification strategy based on orphan G protein-coupled receptors, cloned receptors for which no natural ligands are known. This strategy uses the orphan receptor as bait to identify its natural ligand. This article will review the discovery of the first natural ligand isolated following this strategy. This ligand is a peptide that shares some striking sequence similarity to the opioid peptides and has been named Orphanin FQ or Nociceptin (OFQ/NOC). The discovery of OFQ/NOC will be described as one example of the use of orphan receptors in identifying novel neurotransmitters and neuropeptides, an example that has already been followed in the identification of other novel neuropeptides. After reviewing the conceptual and technological basis of the strategy and its successful first application, we discuss the criteria used to validate OFQ/NOC as the natural ligand of the orphan receptor and as a genuine neuropeptide. We also discuss the importance and implications of discovering OFQ/NOC mode of synthesis, which is synthesized as expected in the form of a larger polypeptide precursor, which in turn raises the question of the existence of other OFQ/NOC-related peptides. We then present an overview of the numerous studies that have blossomed after the OFQ/NOC discovery and describe the numerous physiological roles that have already been attributed to OFQ/NOC, and in particular the controversy regarding its involvement in pain perception. Because of the similarities between the OFQ/NOC and opioid systems, we also discuss overlaps between these systems and present evidence favoring a pharmacological separation between these systems. We finish by outlining the power of the orphan receptor strategy and by discussing some of its pitfalls.

Three different prohormones yield a variety of Hydra-RFamide (Arg-Phe-NH2) neuropeptides in Hydra magnipapillata.

The freshwater polyp Hydra is the most frequently used model for the study of development in cnidarians. Recently we isolated four novel Arg-Phe-NH2 (RFamide) neuropeptides, the Hydra-RFamides I-IV, from Hydra magnipapillata. Here we describe the molecular cloning of three different preprohormones from H. magnipapillata, each of which gives rise to a variety of RFamide neuropeptides. Preprohormone A contains one copy of unprocessed Hydra-RFamide I (QWLGGRFG), II (QWFNGRFG), III/IV [(KP)HLRGRFG] and two putative neuropeptide sequences (QLMSGRFG and QLMRGRFG). Preprohormone B has the same general organization as preprohormone A, but instead of unprocessed Hydra-RFamide III/IV it contains a slightly different neuropeptide sequence [(KP)HYRGRFG]. Preprohormone C contains one copy of unprocessed Hydra-RFamide I and seven additional putative neuropeptide sequences (with the common N-terminal sequence QWF/LSGRFGL). The two Hydra-RFamide II copies (in preprohormones A and B) are preceded by Thr residues, and the single Hydra-RFamide III/IV copy (in preprohormone A) is preceded by an Asn residue, confirming that cnidarians use unconventional processing signals to generate neuropeptides from their precursor proteins. Southern blot analyses suggest that preprohormones A and B are each coded for by a single gene, whereas one or possibly two closely related genes code for preprohormone C. Northern blot analyses and in situ hybridizations show that the gene coding for preprohormone A is expressed in neurons of both the head and foot regions of Hydra, whereas the genes coding for preprohormones B and C are specifically expressed in neurons of different regions of the head. All of this shows that neuropeptide biosynthesis in the primitive metazoan Hydra is already rather complex.

Molecular cloning, genomic organization, and developmental regulation of a novel receptor from Drosophila melanogaster structurally related to members of the thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone/choriogonadotropin receptor family from mammals.

Using oligonucleotide probes derived from consensus sequences for glycoprotein hormone receptors, we have cloned an 831-amino acid residue-long receptor from Drosophila melanogaster that shows a striking structural homology with members of the glycoprotein hormone (thyroid-stimulating hormone (TSH); follicle-stimulating hormone (FSH); luteinizing hormone/choriogonadotropin (LH/CG)) receptor family from mammals. This homology includes a very large, extracellular N terminus (20% sequence identity with rat TSH, 19% with rat FSH, and 20% with the rat LH/CG receptor) and a seven-transmembrane region (53% sequence identity with rat TSH, 50% with rat FSH, and 52% with the rat LH/CG receptor). The Drosophila receptor gene is >7.5 kilobase pairs long and contains 17 exons and 16 introns. Seven intron positions coincide with introns in the mammalian glycoprotein hormone receptor genes and have the same intron phasing. This indicates that the Drosophila receptor is evolutionarily related to the mammalian receptors. The Drosophila receptor gene is located at position 90C on the right arm of the third chromosome. The receptor is strongly expressed starting 8-16 h after oviposition, and the expression stays high until after pupation. Adult male flies express high levels of receptor mRNA, but female flies express about 6 times less. The expression pattern in embryos and larvae suggests that the receptor is involved in insect development. This is the first report on the molecular cloning of a glycoprotein hormone receptor family member from insects.