RESUMO
A heptadecapeptide was identified and purified from porcine brain tissue as a ligand for an orphan heterotrimeric GTP-binding protein (G protein)-coupled receptor (LC132) that is similar in sequence to opioid receptors. This peptide, orphanin FQ, has a primary structure reminiscent of that of opioid peptides. Nanomolar concentrations of orphanin FQ inhibited forskolin-stimulated adenylyl cyclase activity in cells transfected with LC132. This inhibitory activity was not affected by the addition of opioid ligands, nor did the peptide activate opioid receptors. Orphanin FQ bound to its receptor in a saturable manner and with high affinity. When injected intracerebroventricularly into mice, orphanin FQ caused a decrease in locomotor activity but did not induce analgesia in the hot-plate test. However, the peptide produced hyperalgesia in the tail-flick assay. Thus, orphanin FQ may act as a transmitter in the brain by modulating nociceptive and locomotor behavior.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Peptídeos Opioides/isolamento & purificação , Peptídeos Opioides/metabolismo , Receptores de Neuropeptídeos/metabolismo , Receptores Opioides/metabolismo , Inibidores de Adenilil Ciclases , Sequência de Aminoácidos , Analgésicos/farmacologia , Animais , Células CHO , Colforsina/farmacologia , Cricetinae , Hipotálamo/química , Injeções Intraventriculares , Injeções Espinhais , Ligantes , Camundongos , Dados de Sequência Molecular , Atividade Motora/efeitos dos fármacos , Peptídeos Opioides/química , Peptídeos Opioides/farmacologia , Medição da Dor , Suínos , Transfecção , NociceptinaRESUMO
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.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Ligantes , Neurotransmissores/metabolismo , Receptores Acoplados a Proteínas G , Sistemas do Segundo Mensageiro/fisiologia , Animais , Humanos , Proteínas Imediatamente Precoces/efeitos dos fármacos , Proteínas Imediatamente Precoces/fisiologia , Neuropeptídeos/efeitos dos fármacos , Neuropeptídeos/metabolismo , Neuropeptídeos/farmacologia , Neurotransmissores/farmacologia , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/metabolismo , Receptores de Lisofosfolipídeos , Sistemas do Segundo Mensageiro/efeitos dos fármacosAssuntos
Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G , Urotensinas/metabolismo , Vasoconstrição/fisiologia , Animais , Células CHO , Cricetinae , Humanos , Hormônios Hipotalâmicos/metabolismo , Ligantes , Melaninas/metabolismo , Neuropeptídeos/metabolismo , Peptídeos/metabolismo , Hormônios Hipofisários/metabolismo , Receptores de Superfície Celular/genética , Somatostatina/metabolismo , Urotensinas/fisiologiaRESUMO
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.
Assuntos
Hormônios Hipotalâmicos/fisiologia , Melaninas/fisiologia , Hormônios Hipofisários/fisiologia , Sequência de Aminoácidos , Animais , Peixes , Humanos , Hormônios Hipotalâmicos/genética , Melaninas/genética , Dados de Sequência Molecular , Hormônios Hipofisários/genéticaRESUMO
Exposure of Ehrlich ascites tumor cells to 5-azacytidine for 5 h resulted in a partial loss of ability of DNA to stimulate ADP-ribosyltransferase activity, as assessed in a reconstituted in vitro enzyme system consisting of purified calf thymus enzyme, calf thymus whole histone and DNA isolated from the cells. The degree of suppression in vitro varied depending on the amount of histone and DNA added and it reached a maximum with a value of 83% and 62% of control for DNAs from cells exposed to 10 microM and 30 microM 5-azacytidine, respectively, at a histone/DNA mass ratio of 0.4. In the absence of histone (conditions of auto-ADP-ribosylation of the enzyme), no suppression was detectable.
Assuntos
Azacitidina/farmacologia , Carcinoma de Ehrlich/enzimologia , DNA de Neoplasias/efeitos dos fármacos , Inibidores de Poli(ADP-Ribose) Polimerases , Animais , Azacitidina/metabolismo , Núcleo Celular/enzimologia , DNA de Neoplasias/metabolismo , Histonas/farmacologia , Magnésio/farmacologiaRESUMO
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.
Assuntos
Sistema Nervoso Central/química , Peptídeos Opioides/análise , RNA Mensageiro/análise , Receptores Opioides/agonistas , Animais , Autorradiografia , Gânglios da Base/química , Tronco Encefálico/química , Colchicina/farmacologia , Imuno-Histoquímica , Hibridização In Situ , Masculino , Peptídeos Opioides/genética , Prosencéfalo/química , Ratos , Ratos Sprague-Dawley , Septo Pelúcido/química , Medula Espinal/química , NociceptinaRESUMO
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.
Assuntos
Sistema Nervoso Central/química , RNA Mensageiro/biossíntese , Receptores Opioides/análise , Animais , Química Encefálica/fisiologia , Sistema Nervoso Central/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Radioisótopos do Iodo , Masculino , Peptídeos Opioides/metabolismo , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Receptores Opioides/genética , Medula Espinal/química , Receptor de Nociceptina , NociceptinaRESUMO
Antho-KAamide (L-3-phenyllactyl-Phe-Lys-Ala-NH2) and Antho-RIamide (L-3-phenyllactyl-Tyr-Arg-Ile-NH2) are novel neuropeptides isolated from the sea anemone Anthopleura elegantissima. They both inhibited spontaneous contractions of isolated muscle preparations from a wide variety of anemone species (threshold around 10(-7) M). Their actions were universal in that they inhibited every muscle preparation tested, regardless of whether the muscle group was located in the ectoderm or endoderm, or was oriented in a circular or longitudinal direction. Injection of Antho-KAamide or Antho-RIamide into the coelenteron of intact sea anemones resulted in a marked expansion of the animals. Similar shape changes followed feeding or exposure to soluble food extracts. Therefore, we hypothesize that nerve cells that release Antho-KAamide and Antho-RIamide are involved in the expansion phase of feeding behaviour in sea anemones.
Assuntos
Contração Muscular/efeitos dos fármacos , Músculos/fisiologia , Neuropeptídeos/farmacologia , Oligopeptídeos/farmacologia , Anêmonas-do-Mar/fisiologia , Animais , Estimulação Elétrica , Técnicas In Vitro , Músculos/efeitos dos fármacos , Neuropeptídeos/isolamento & purificação , Oligopeptídeos/isolamento & purificação , Anêmonas-do-Mar/efeitos dos fármacos , Especificidade da EspécieRESUMO
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.
Assuntos
Peptídeos Opioides/fisiologia , Fisiologia/métodos , Animais , Comportamento Animal/fisiologia , Endorfinas/fisiologia , Vias Neurais/fisiologia , Neuropeptídeos/fisiologia , NociceptinaRESUMO
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.
Assuntos
Peptídeos Opioides/química , Peptídeos Opioides/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Clonagem Molecular , DNA Complementar/metabolismo , Humanos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Peptídeos Opioides/biossíntese , Peptídeos/química , Peptídeos/genética , RNA Mensageiro/biossíntese , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Vasodilatadores/química , NociceptinaRESUMO
Using a radioimmunoassay for the carboxyl-terminal sequence Arg-Val-NH2, two novel peptides were purified from extracts of the sea anemone Anthopleura elegantissima. These peptides were L-3-phenyllactyl-Tyr-Arg-Ile-NH2 (name: Antho-RIamide I) and its des-phenyllactyl fragment Tyr-Arg-Ile-NH2 (Antho-RIamide II). Immunocytochemical staining showed that these peptides were localized in neurons of sea anemones. Application of low concentrations (10(-8) M) of Antho-RIamide I inhibited spontaneous contractions in several muscle groups of sea anemones, whereas Antho-RIamide II was inactive. Antho-RIamide I is the second neuropeptide from sea anemones that bears the unusual, amino-terminal L-3-phenyllactyl blocking group. We suggest that this group renders the peptide resistant agaist degradation by nonspecific aminopeptidases. In addition, the L-3-phenyllactyl residue might also play a role in receptor binding.
Assuntos
Neuropeptídeos/isolamento & purificação , Oligopeptídeos/isolamento & purificação , Anêmonas-do-Mar/química , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Contração Muscular/efeitos dos fármacos , Neuropeptídeos/química , Neuropeptídeos/farmacologia , Oligopeptídeos/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/farmacologiaRESUMO
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.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Neuropeptídeos/metabolismo , Receptores de Superfície Celular/metabolismo , Desenho de Fármacos , HumanosRESUMO
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.
Assuntos
Acetilcolina/fisiologia , Ponte/fisiologia , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G , Tegmento Mesencefálico/fisiologia , Animais , Colina O-Acetiltransferase/genética , Expressão Gênica/fisiologia , Habenula/fisiologia , Hibridização In Situ , Radioisótopos do Iodo , Masculino , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Receptores Pré-Sinápticos/fisiologia , Núcleos Septais/fisiologiaRESUMO
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.
Assuntos
Fenilcarbamatos , Fosfatidilinositóis/metabolismo , Receptores Acoplados a Proteínas G , Urotensinas/farmacologia , Vasoconstrição/efeitos dos fármacos , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/fisiologia , Carbamatos/farmacologia , Humanos , Hidrólise , Técnicas In Vitro , Masculino , Coelhos , Receptores de Superfície Celular/fisiologia , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/fisiologiaRESUMO
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.
Assuntos
Desenho de Fármacos , Genômica , Receptores Citoplasmáticos e Nucleares/fisiologia , Receptores de Neurotransmissores/fisiologia , Animais , Proteínas de Ligação ao GTP , Humanos , Ligantes , Neuropeptídeos/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Neurotransmissores/genéticaAssuntos
Cnidários/fisiologia , Fenômenos Fisiológicos do Sistema Nervoso , Neurônios/fisiologia , Neuropeptídeos/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Cnidários/genética , Dados de Sequência Molecular , Neuropeptídeos/biossíntese , Neuropeptídeos/genética , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
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.
Assuntos
Toxina Diftérica/química , Toxina Diftérica/toxicidade , Neurônios/efeitos dos fármacos , Síndromes Neurotóxicas/patologia , Neurotoxinas/química , Neurotoxinas/toxicidade , Sistema Nervoso Parassimpático/efeitos dos fármacos , Ponte/patologia , Tegmento Mesencefálico/patologia , Urotensinas/química , Urotensinas/toxicidade , Animais , Células CHO , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Toxina Diftérica/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Masculino , NADPH Desidrogenase/metabolismo , NADPH Desidrogenase/fisiologia , Neurônios/patologia , Sistema Nervoso Parassimpático/patologia , Plasmídeos/genética , RatosRESUMO
Using oligonucleotide probes derived from consensus sequences of known vertebrate and invertebrate G protein-coupled receptors, we have cloned the cDNA for a presumed G protein-coupled receptor from sea anemones. This receptor shows a striking structural homology with members of the glycoprotein hormone (FSH, TSH, LH/CG) receptor family from mammals, including a very large, extracellular N terminus (18-25% sequence identity) and a 7 transmembrane region (44-48% sequence identity). As with the mammalian glycoprotein hormone receptor genes, the sea anemone receptor gene yields transcripts which can be alternatively spliced, thereby yielding a shortened receptor variant only containing the large extracellular (soluble) N terminus. All this is strong evidence that the putative glycoprotein hormone receptor from sea anemones is evolutionarily related to those from mammals. This is the first report showing that a putative glycoprotein hormone receptor occurs in invertebrates.
Assuntos
Proteínas de Ligação ao GTP/biossíntese , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/química , Receptores do FSH/química , Receptores do LH/química , Receptores da Tireotropina/química , Anêmonas-do-Mar/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Primers do DNA , Éxons , Proteínas de Ligação ao GTP/química , Biblioteca Gênica , Mamíferos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase/métodos , Ratos , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência de AminoácidosRESUMO
We have isolated and sequenced the neuropeptide L-3-phenyllactyl-Phe-Lys-Ala-NH2 from the sea anemone Anthopleura elegantissima. This neuropeptide (named Antho-KAamide) has the unusual N-terminal L-3-phenyllactyl blocking group which has recently also been discovered in 2 other neuropeptides from sea anemones. We propose that the L-3-phenyllactyl residue renders Antho-KAamide resistant to nonspecific aminopeptidases, thereby increasing the stability of the neuropeptide after neuronal release. The existence of the L-3-phenyllactyl residue in 3 neuropeptides isolated so far suggests that this blocking group is more generally occurring.
Assuntos
Neuropeptídeos/isolamento & purificação , Sequência de Aminoácidos , Animais , Cnidários , Imuno-Histoquímica , Dados de Sequência Molecular , Neuropeptídeos/química , Radioimunoensaio , Anêmonas-do-Mar , Homologia de Sequência do Ácido Nucleico , Espectrometria de Massas de Bombardeamento Rápido de ÁtomosRESUMO
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.