RESUMO
GPR10 is a novel G-protein coupled receptor that is the human orthologue of rat Unknown Hypothalamic Receptor-1 (UHR-1). Human prolactin-releasing peptide (PrRP) has been identified as an endogenous ligand for GPR10, and occurs as 31 and 20 amino acid forms. The present study characterizes the binding of [(125)I]-PrRP-20 to HEK293 cells stably expressing GPR10 receptors. Specific binding of [(125)I]-PrRP-20 was saturable, and analysis suggested evidence of both high and low affinity sites, with K:(D:) values of 0.026+/-0.006 and 0.57+/-0.14 nM respectively, and B(max) values of 3010+/-400 and 8570+/-2240 fmol mg protein(-1) respectively. Kinetic studies were unable to distinguish two sites, but single site analysis of association and dissociation data produced a K:(D:) of 0.012 nM. Competition studies revealed that human and rat PrRP-20 and PrRP-31 all display high affinity for GPR10. A range of other drugs which are known ligands at receptors which share limited homology with GPR10 were also tested. None of the drugs tested, including the RF-amide neuropeptide FF, demonstrated any affinity for GPR10. Human PrRP-20 failed to alter basal or forskolin-stimulated levels of intracellular cyclic AMP in HEK293-GPR10 cells, suggesting that GPR10 does not couple via either G(s) or G(i). Functional studies using measurements of intracellular calcium confirmed that human and rat PrRP-20 and PrRP-31 are all potent, full agonists at the GPR10 receptor. The response was blocked both by thapsigargin, indicating mobilization of intracellular Ca(2+) stores. These studies indicate that [(125)I]-PrRP-20 is a specific, high affinity radioligand for GPR10. The availability of this radioligand binding assay will be a valuable tool for the investigation of the key features involved in PrRP binding and studies on the localization and function of GPR10.
Assuntos
Hormônios Hipotalâmicos/metabolismo , Neuropeptídeos/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G , Sequência de Aminoácidos , Animais , Ligação Competitiva , Cálcio/metabolismo , Células Cultivadas , AMP Cíclico/biossíntese , Humanos , Hormônios Hipotalâmicos/farmacologia , Radioisótopos do Iodo , Cinética , Dados de Sequência Molecular , Neuropeptídeos/farmacologia , Hormônio Liberador de Prolactina , RatosRESUMO
Using a homology-based bioinformatics approach we have analysed human genomic sequence and identified the human and rodent orthologues of a novel putative seven transmembrane G protein coupled receptor, termed GABA(BL). The amino acid sequence homology of these cDNAs compared to GABA(B1) and GABA(B2) led us to postulate that GABA(BL) was a putative novel GABA(B) receptor subunit. The C-terminal sequence of GABA(BL) contained a putative coiled-coil domain, di-leucine and several RXR(R) ER retention motifs, all of which have been shown to be critical in GABA(B) receptor subunit function. In addition, the distribution of GABA(BL) in the central nervous system was reminiscent of that of the other known GABA(B) subunits. However, we were unable to detect receptor function in response to any GABA(B) ligands when GABA(BL) was expressed in isolation or in the presence of either GABA(B1) or GABA(B2). Therefore, if GABA(BL) is indeed a GABA(B) receptor subunit, its partner is a potentially novel receptor subunit or chaperone protein which has yet to be identified.
Assuntos
Encéfalo/metabolismo , Proteínas de Ligação ao GTP/isolamento & purificação , Subunidades Proteicas/isolamento & purificação , Receptores de GABA-B/isolamento & purificação , Sequência de Aminoácidos/genética , Animais , Sequência de Bases/genética , Células Cultivadas , Mapeamento Cromossômico , Cromossomos Humanos Par 3/genética , Clonagem Molecular , DNA Complementar/análise , DNA Complementar/genética , Proteínas de Ligação ao GTP/genética , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Dados de Sequência Molecular , Estrutura Molecular , Filogenia , Estrutura Terciária de Proteína/genética , Subunidades Proteicas/genética , Ratos , Receptores de GABA-B/genéticaRESUMO
Agonist stimulation causes the endocytosis of many G protein-coupled receptors, including muscarinic acetylcholine receptors. In this study we have investigated the agonist-triggered trafficking of the M3 muscarinic receptor expressed in SH-SY5Y human neuroblastoma cells. We have compared the ability of a series of agonists to generate the second messenger Ins(1,4,5)P3 with their ability to stimulate receptor endocytosis. We show that there is a good correlation between the intrinsic activity of the agonists and their ability to increase the rate constant for receptor endocytosis. Furthermore, on the basis of our results, we predict that even very weak partial agonists should under some circumstances be able to cause substantial receptor internalization. Receptor endocytosis occurs too slowly to account for the rapid desensitization of the Ca2+ response to carbachol. Instead, receptor endocytosis and recycling appear to play an important role in resensitization. After an initial agonist challenge, the response to carbachol is fully recovered when only about half of the receptors have been recycled to the cell surface, suggesting that there is a receptor reserve of about 50%. Removal of this reserve by receptor alkylation significantly reduces the extent of resensitization. Resensitization is also reduced by inhibitors of either endocytosis alone (concanavalin A) or of endocytosis and recycling (nigericin). Finally, the protein phosphatase inhibitor calyculin A also reduces resensitization, possibly by blocking the dephosphorylation of the receptors in an endosomal compartment.
Assuntos
Endocitose/efeitos dos fármacos , Agonistas Muscarínicos/farmacologia , Neurônios/metabolismo , Receptores Muscarínicos/metabolismo , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Sítios de Ligação , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Relação Dose-Resposta a Droga , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Ligantes , Agonistas Muscarínicos/metabolismo , Antagonistas Muscarínicos/metabolismo , Antagonistas Muscarínicos/farmacologia , N-Metilescopolamina/metabolismo , Neurônios/efeitos dos fármacos , Mostarda de Propilbenzililcolina/metabolismo , Receptor Muscarínico M3 , Fatores de Tempo , Células Tumorais CultivadasRESUMO
The authors treated their patients, who had fractures of the calcaneus body in association with dislocation, by means of a new method: repositioning was effected by means of a special apparatus, retention via percutaneous screw osteosynthesis. Previously, they had used a Kirschner wire drilled from the tuber calcanei. The types of fracture differ very widely; the authors subdivide them into three groups, taking into consideration the involvement of the posterior talocalcaneal joint surfaces. The patients were subsequently followed up, and the results of 182 fractures of the calcaneum were evaluated, taking numerous different aspects into consideration. The authors were thus able to establish that the results of the treatment are greatly improved by accurate repositioning followed by percutaneous screw osteosynthesis.
Assuntos
Calcâneo/lesões , Fixação Interna de Fraturas/métodos , Fraturas Expostas/cirurgia , Parafusos Ósseos , Feminino , Humanos , Masculino , Complicações Pós-Operatórias/etiologia , CicatrizaçãoRESUMO
The ability of the delta opioid agonist DPDPE ([D-Pen2, D-Pen4]enkephalin) to stimulate binding of the GTP analog guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) to pertussis toxin-sensitive G proteins has been characterized in membranes from NG108-15 mouse neuroblastoma X rat glioma cells. The presence of GDP, or its hydrolysis-resistant analog GDPbetaS, and Mg++ ions was essential to observe agonist-mediated stimulation of [35S]GTPgammaS binding, although the guanine dinucleotides alone had complex inhibitory and stimulatory effects on [35S]GTPgammaS binding. The relative ability of the delta antagonists benzylidenenaltrexone and naltriben to inhibit DPDPE-stimulated [35S]GTPgammaS binding suggested the opioid receptor involved was of the delta-2 subtype. Ligand binding assays demonstrated biphasic binding of these antagonists to this single receptor type. [35S]GTPgammaS binding was also stimulated by [D-Ser2,Leu5,Thr6]enkephalin > deltorphin II = DPDPE = etorphine > levallorphan = diprenorphine = nalorphine = naltrindole. The delta antagonists benzylidenenaltrexone, TIPP (Tyr-Tic-Phe-Phe) and naltriben had no effect, but ICI 174864 (N, N-diallyl-Tyr-Aib-Phe-Leu-OH) acted as an inverse agonist and inhibited [35S]GTPgammaS binding. Pertussis toxin pretreatment blocked agonist stimulation of [35S]GTPgammaS binding and also reduced basal binding, thus confirming the presence of constitutively active delta receptors. Replacement of Na+ in the assay buffer with K+ afforded an increased level of basal [35S]GTPgammaS binding and an apparent increase in both the inverse agonist activity of ICI 174864 and the agonist activity of the partial agonist diprenorphine relative to the full agonist [D-Ser2, Leu5,Thr6]enkephalin. The stimulation of [35S]GTPgammaS binding to NG108-15 cell membranes allows a functional measure of delta opioid activity that can provide systems of differing relative efficacy.
Assuntos
Encefalinas/farmacologia , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Receptores Opioides delta/agonistas , Animais , Compostos de Benzilideno/farmacologia , Células CHO , Cricetinae , Relação Dose-Resposta a Droga , D-Penicilina (2,5)-Encefalina , Encefalina Leucina/análogos & derivados , Encefalina Leucina/farmacologia , Camundongos , Naltrexona/análogos & derivados , Naltrexona/farmacologia , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Ratos , Sódio/farmacologiaRESUMO
A 13-year-old boy had his right second toe transplanted to his right hand to replace a hypoplastic thumb. The free transfer included the second metatarsal bone and the vascular connection was by a dorsalis pedis flap.
Assuntos
Polegar/anormalidades , Dedos do Pé/transplante , Adolescente , Humanos , Masculino , Métodos , Tendões/transplanteRESUMO
Authors performed plastic operation using lower arm insulated flap with distal pedicle in 2 cases for covering traumatic skin defects on the dorsal side of the hand. Attention is called to the advantages of this method. Beside the shorter time of treatment in many cases a series of operations can be avoided by the primary covering with an insulated flap.
Assuntos
Traumatismos da Mão/cirurgia , Retalhos Cirúrgicos/métodos , Adolescente , Procedimentos Cirúrgicos Dermatológicos , Humanos , Masculino , Artéria Radial , Pele/irrigação sanguínea , Pele/lesões , Cirurgia Plástica/métodosRESUMO
The molecular mechanisms underlying the internalization of G protein-coupled receptors are still poorly understood. Normally agonists but not antagonists cause internalization (defined here as a reduction in the number of receptors at the cell surface), suggesting a functional relationship between agonist activity and internalization. In this study we investigated the effects of eight muscarinic ligands on the rate constants for endocytosis and recycling of m3 muscarinic acetylcholine receptors in human SH-SY5Y neuroblastoma cells. We found that there was a linear correlation between the intrinsic activity of the ligand and its ability to increase the rate constant for endocytosis, suggesting that the same active conformation of the receptor is responsible for stimulating both second messenger generation and receptor endocytosis. In contrast, the rate constant for recycling did not depend on which agonist had triggered receptor endocytosis, suggesting that recycling is a purely constitutive process. Because receptor internalization depends on the rate constants for both endocytosis and recycling, the relationship between internalization and intrinsic activity is nonlinear. In particular, mathematical modeling of receptor trafficking revealed that under certain conditions very small (3% or less) increases in the rate constant for endocytosis are sufficient to cause substantial receptor internalization. An important implication of this analysis is that extremely weak partial agonists (which may in practice be indistinguishable from antagonists) may produce significant receptor internalization.
Assuntos
Endocitose/efeitos dos fármacos , Agonistas Muscarínicos/farmacologia , Neuroblastoma/metabolismo , Receptores Muscarínicos/metabolismo , Arecolina/farmacologia , Betanecol/farmacologia , Carbacol/farmacologia , Humanos , Cinética , Ligantes , Cloreto de Metacolina/farmacologia , Oxotremorina/farmacologia , Pilocarpina/farmacologia , Receptor Muscarínico M3 , Receptores Muscarínicos/efeitos dos fármacos , Receptores Muscarínicos/fisiologia , Células Tumorais CultivadasRESUMO
Preexposure of SH-SY5Y cells to the muscarinic agonist carbachol caused a rapid desensitization of subsequent carbachol-stimulated intracellular Ca2+ responses and a slower decrease in the number of receptors at the plasma membrane. Desensitization (to 30% of the control response) was maximal after 1 min of exposure to agonist, whereas the number of cell surface receptors reached a minimum (33% of control) only after 5 min. Following agonist washout, the recovery of response was complete within 12 min, whereas the recovery of surface receptor number reached a plateau at 65% of control after 30 min. Treatment with inhibitors of endocytosis (concanavalin A) or recycling (nigericin) did not affect rapid desensitization but did decrease resensitization, suggesting that receptor cycling is involved in resensitization. Experiments with the irreversible antagonist propylbenzilylcholine mustard demonstrated that the receptor reserve for the Ca2+ response to 1 mM carbachol is approximately 50%. Removal of this receptor reserve led to a decrease in the rate of resensitization. We propose that the existence of a receptor reserve might explain the poor correlation between functional response and surface receptor number, and that one of its roles might be to permit rapid resensitization after a significant agonist-induced decrease in surface receptor number. The purpose of receptor cycling might be to allow dephosphorylation (and reactivation) of receptors that have become phosphorylated (and inactivated) in response to agonist stimulation, because the protein phosphatase inhibitor calyculin A significantly reduced resensitization.
Assuntos
Receptores Muscarínicos/fisiologia , Cálcio/metabolismo , Carbacol/farmacologia , Humanos , Membranas Intracelulares/metabolismo , Agonistas Muscarínicos/farmacologia , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Concentração Osmolar , Fosforilação , Células Tumorais CultivadasRESUMO
NG108-15 cells express predominantly the M4 subtype of the muscarinic receptor for acetylcholine. Stimulation of these receptors by the agonist carbachol causes an inhibition of cellular adenylyl cyclase and a consequent fall in the intracellular cyclic AMP concentration. Pretreatment of the cells with carbachol caused both internalization and desensitization of the M4 receptor. Overexpression of G protein-coupled receptor kinase (GRK) 2 caused an increase in the rate constant for receptor endocytosis (from 0.06 to 0.18 min(-1)) and a decrease in the EC50 for carbachol stimulation of internalization (from 15 to 3 microM). Overexpression of a dominant negative form of GRK2 had more modest effects, reducing the rate constant for endocytosis (from 0.11 to 0.07 min(-1)) and increasing the EC50 for carbachol stimulation of internalization (from 8 to 17 microM). Neither GRK2 nor dominant negative GRK2 overexpression had any effect on the rate constant for receptor recycling following agonist removal. The time course and extent of receptor desensitization in control cells were identical to the corresponding values for receptor internalization, and the rate and extent of desensitization were again increased by GRK2 overexpression. Exposure of the cells to hyperosmolar sucrose (0.6 M) almost completely blocked agonist-induced receptor internalization in both control and GRK2-overexpressing cells. Sucrose treatment also blocked agonist-induced desensitization. We conclude that the internalization and desensitization of the M4 muscarinic receptor in NG108-15 cells can be modulated in response to changes in GRK2 activity and also that internalization plays a key role in desensitization.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Agonistas Muscarínicos/farmacologia , Receptores Muscarínicos/metabolismo , Inibidores de Adenilil Ciclases , Algoritmos , Animais , Carbacol/farmacologia , Bovinos , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/biossíntese , Endocitose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Soluções Hipertônicas , Modelos Biológicos , Ensaio Radioligante , Receptor Muscarínico M4 , Receptores Muscarínicos/efeitos dos fármacos , Sacarose/farmacologia , Quinases de Receptores Adrenérgicos betaRESUMO
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 () (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.
Assuntos
Hormônios Hipotalâmicos/metabolismo , Melaninas/metabolismo , Hormônios Hipofisários/metabolismo , Receptores do Hormônio Hipofisário/genética , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Clonagem Molecular , DNA Complementar , Humanos , Dados de Sequência Molecular , Receptores Acoplados a Proteínas G , Receptores do Hormônio Hipofisário/química , Receptores do Hormônio Hipofisário/metabolismo , Homologia de Sequência de AminoácidosRESUMO
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 protein-coupled 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.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Proteínas de Membrana , Neuropeptídeos/farmacologia , Receptores de Superfície Celular/agonistas , Receptores de Neurotransmissores , Cálcio/metabolismo , Linhagem Celular , Clonagem Molecular , Regulação da Expressão Gênica , Humanos , Fosfatos de Inositol/metabolismo , Neuropeptídeos/genética , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
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 HLWAR77 will aid in defining the physiological role(s) of these ligands and facilitate the identification of receptor agonists and antagonists.
Assuntos
Neuropeptídeos/metabolismo , Oligopeptídeos/metabolismo , Receptores de Neuropeptídeos/metabolismo , Sequência de Aminoácidos , Arrestinas/metabolismo , Sequência de Bases , Cálcio/metabolismo , Linhagem Celular , FMRFamida/farmacologia , Humanos , Ligantes , Dados de Sequência Molecular , Receptores de Neuropeptídeos/genética , beta-ArrestinasRESUMO
A novel human G protein-coupled receptor named AXOR12, exhibiting 81% homology to the rat orphan receptor GPR54, was cloned from a human brain cDNA library. Heterologous expression of AXOR12 in mammalian cells permitted the identification of three surrogate agonist peptides, all with a common C-terminal amidated motif. High potency agonism, indicative of a cognate ligand, was evident from peptides derived from the gene KiSS-1, the expression of which prevents metastasis in melanoma cells. Quantitative reverse transcriptase-polymerase chain reaction was used to study the expression of AXOR12 and KiSS-1 in a variety of tissues. The highest levels of expression of AXOR12 mRNA were observed in brain, pituitary gland, and placenta. The highest levels of KiSS-1 gene expression were observed in placenta and brain. A polyclonal antibody raised to the C terminus of AXOR12 was generated and used to show localization of the receptor to neurons in the cerebellum, cerebral cortex, and brainstem. The biological significance of these expression patterns and the nature of the putative cognate ligand for AXOR12 are discussed.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Proteínas/genética , Proteínas/metabolismo , Receptores de Superfície Celular/genética , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Células CHO , Cricetinae , Feminino , Genes Supressores de Tumor , Humanos , Cinética , Kisspeptinas , Ligantes , Melanoma/genética , Dados de Sequência Molecular , Nephropidae , Neurônios/metabolismo , Especificidade de Órgãos , Fragmentos de Peptídeos/farmacologia , Hipófise/metabolismo , Placenta/metabolismo , Gravidez , Proteínas/química , Ratos , Receptores de Superfície Celular/química , Receptores Acoplados a Proteínas G , Receptores de Kisspeptina-1 , Receptores de Neuropeptídeos/química , Receptores de Neuropeptídeos/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Anêmonas-do-Mar , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transfecção , Proteínas Supressoras de TumorRESUMO
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.