RESUMEN
BACKGROUND AND PURPOSE: Opioid receptor function is modulated by post-activation events such as receptor endocytosis, recycling and/or degradation. While it is generally understood that the peptide ligand gets co-endocytosed with the receptor, relatively few studies have investigated the role of the endocytosed peptide and peptide processing enzymes in regulating receptor function. In this study, we focused on endothelin-converting enzyme 2 (ECE2), a member of the neprilysin family of metallopeptidases that exhibits an acidic pH optimum, localizes to an intracellular compartment and selectively processes neuropeptides including opioid peptides in vitro, and examined its role in modulating µ receptor recycling and resensitization. EXPERIMENTAL APPROACH: The effect of ECE2 inhibition on hydrolysis of the endocytosed peptide was examined using thin-layer chromatography and on µ opioid receptor trafficking using either elisa or microscopy. The effect of ECE2 inhibition on receptor signalling was measured using a cAMP assay and, in vivo, on antinociception induced by intrathecally administered opioids by the tail-flick assay. KEY RESULTS: The highly selective ECE2 inhibitor, S136492, significantly impaired µ receptor recycling and signalling by only those ligands that are ECE2 substrates and this was seen both in heterologous cells and in cells endogenously co-expressing µ receptors with ECE2. We also found that ECE2 inhibition attenuated antinociception mediated only by opioid peptides that are ECE2 substrates. CONCLUSIONS AND IMPLICATIONS: These results suggest that ECE2, by selectively processing endogenous opioid peptides in the endocytic compartment, plays a role in modulating opioid receptor activity. LINKED ARTICLES: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.
Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Metaloendopeptidasas/metabolismo , Péptidos Opioides/farmacología , Receptores Opioides mu/metabolismo , Animales , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Células CHO , Cricetulus , AMP Cíclico/metabolismo , Endocitosis , Enzimas Convertidoras de Endotelina , Concentración de Iones de Hidrógeno , Hidrólisis , Masculino , Metaloendopeptidasas/antagonistas & inhibidores , Ratones Endogámicos C57BL , Transporte de Proteínas , Receptores Opioides mu/agonistas , Transducción de SeñalRESUMEN
The effect of background derivatization on the signal enhancement of pesticide residues extracted from edible oil samples was studied by GC with negative chemical ionization MS. The analytes were extracted by a solvent extraction process, and the extract was subjected to rapid low-temperature fat precipitation. The residual fatty acids were silylated by derivatization with N,O-bis(trimethylsilyl)trifluoroacetamide. The chromatograms obtained from the derivatized samples showed higher signal intensity and lower detection levels when compared to the direct analysis without derivatization. The sensitivity levels of the method are either better or comparable to that of previously reported methodologies. The LODs of the analyzed organochlorine, organophosphorus, and synthetic pyrethroid residues in sunflower, rice bran, and ground oil samples were in the range of 0.020.5 ng/g, and the LOQs were in the range of 0.12 ng/g. The intraday and interday accuracies were in the range of 81116% with RSDs less than 14%. The recoveries obtained were in the range of 5389% with the RSD values less than 13% for all the studied pesticide residues.
Asunto(s)
Residuos de Plaguicidas/análisis , Residuos de Plaguicidas/aislamiento & purificación , Aceites de Plantas/química , Plantas Comestibles/química , Cromatografía de Gases y Espectrometría de Masas , Residuos de Plaguicidas/químicaRESUMEN
The major cellular event in the development and progression of liver fibrosis is the activation of hepatic stellate cells (HSCs). Activated HSCs proliferate and produce excess collagen, leading to accumulation of scar matrix and fibrotic liver. As such, the induction of activated HSC death has been proposed as a means to achieve resolution of liver fibrosis. Here we demonstrate that cannabidiol (CBD), a major non-psychoactive component of the plant Cannabis sativa, induces apoptosis in activated HSCs through a cannabinoid receptor-independent mechanism. CBD elicits an endoplasmic reticulum (ER) stress response, characterized by changes in ER morphology and the initiation of RNA-dependent protein kinase-like ER kinase-, activating transcription factor-6-, and inositol-requiring ER-to-nucleus signal kinase-1 (IRE1)-mediated signaling cascades. Furthermore, CBD induces downstream activation of the pro-apoptotic IRE1/ASK1/c-Jun N-terminal kinase pathway, leading to HSC death. Importantly, we show that this mechanism of CBD-induced ER stress-mediated apoptosis is specific to activated HSCs, as it occurs in activated human and rat HSC lines, and in primary in vivo-activated mouse HSCs, but not in quiescent HSCs or primary hepatocytes from rat. Finally, we provide evidence that the elevated basal level of ER stress in activated HSCs has a role in their susceptibility to the pro-apoptotic effect of CBD. We propose that CBD, by selectively inducing death of activated HSCs, represents a potential therapeutic agent for the treatment of liver fibrosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Cannabidiol/farmacología , Retículo Endoplásmico/efectos de los fármacos , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Retículo Endoplásmico/metabolismo , Células Estrelladas Hepáticas/metabolismo , Humanos , Estrés Oxidativo/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Adolescence is a critical phase of active brain development often characterized by the initiation of marijuana (Cannabis sativa) use. Limited information is known regarding the endogenous cannabinoid system of the adolescent brain as well as related neurotransmitters that appear sensitive to cannabis exposure. We recently observed that adult rats pre-exposed to Delta-9-tetrahydrocannabinol (THC) during adolescence self-administered higher amounts of heroin and had selective impairments of the enkephalin opioid system within the nucleus accumbens (NAc) implicated in reward-related behavior. To explore the ontogeny of the cannabinoid and opioid neuronal systems in association with adolescence THC exposure, rats were examined at different adolescent stages during an intermittent THC paradigm (1.5 mg/kg i.p. every third day) from postnatal days (PNDs) 28-49. Rat brains were examined 24 h after injection at PND 29 (early adolescence), PND 38 (mid adolescence) and PND 50 (late adolescence) and analyzed for endocannabinoids (anandamide and 2-arachidonoylglycerol), Met-enkephalin, cannabinoid CB(1) receptors and micro opioid receptors (microOR) in the NAc, caudate-putamen and prefrontal cortex (PFC). Of the markers studied, the endocannabinoid levels had the most robust alterations throughout adolescence and were specific to the PFC and NAc. Normal correlations between anandamide and 2-arachidonoylglycerol concentrations in the NAc (positive) and PFC (negative) were reversed by THC. Other significant THC-induced effects were confined to the NAc - increased anandamide, decreased Met-enkephalin and decreased microORs. These findings emphasize the dynamic nature of the mesocorticolimbic endocannabinoid system during adolescence and the selective mesocorticolimbic disturbance as a consequence of adolescent cannabis exposure.
Asunto(s)
Moduladores de Receptores de Cannabinoides/fisiología , Corteza Cerebral/crecimiento & desarrollo , Corteza Cerebral/fisiología , Dronabinol/farmacología , Sistema Límbico/crecimiento & desarrollo , Sistema Límbico/fisiología , Péptidos Opioides/fisiología , Psicotrópicos/farmacología , Animales , Ácidos Araquidónicos/metabolismo , Corteza Cerebral/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Endocannabinoides , Encefalina Metionina/metabolismo , Glicéridos/metabolismo , Sistema Límbico/efectos de los fármacos , Masculino , Espectrometría de Masas , Neostriado/crecimiento & desarrollo , Neostriado/metabolismo , Vías Nerviosas/crecimiento & desarrollo , Vías Nerviosas/metabolismo , Núcleo Accumbens/crecimiento & desarrollo , Núcleo Accumbens/metabolismo , Alcamidas Poliinsaturadas/metabolismo , Corteza Prefrontal/crecimiento & desarrollo , Corteza Prefrontal/metabolismo , Radioinmunoensayo , Ratas , Ratas Long-Evans , Receptor Cannabinoide CB1/efectos de los fármacos , Receptor Cannabinoide CB1/fisiología , Receptores Opioides mu/efectos de los fármacos , Receptores Opioides mu/fisiologíaRESUMEN
Endothelin-converting enzyme (ECE)-2 is a metalloprotease that possesses many properties consistent with it being a neuropeptide-processing enzyme. This protease is found primarily in neural tissues, with high levels of expression in midbrain, cerebellum, hypothalamus, frontal cortex and spinal cord and moderate levels in hippocampus and striatum. To evaluate its role in neural function, mice have been generated lacking this enzyme. Physical appearance, autonomic reflexes, motor co-ordination, balance, locomotor activity and spontaneous emotional responses appear normal in these knockout (KO) mice. However, these mutants display deficits in learning and memory as evidenced by marked impairment in the Morris water maze. Knockout mice are also deficient in object recognition memory where they show delays in discerning changes in object location and in recognizing the introduction of a novel object. In this study, perseveration appears to interfere with learning and memory. Finally, mutants are impaired in social transmission of food preference where they show poor short-term memory and perturbations in long-term memory; the latter can be ameliorated by reminder cues. As ECE-2 has been implicated in Alzheimer's disease, the deficits in learning and memory in the KO mice may provide unique insights into processes that may contribute to this disease and possible other disorders of cognition.
Asunto(s)
Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/fisiología , Discapacidades para el Aprendizaje/genética , Discapacidades para el Aprendizaje/psicología , Trastornos de la Memoria/genética , Trastornos de la Memoria/psicología , Metaloendopeptidasas/genética , Metaloendopeptidasas/fisiología , Animales , Ácido Aspártico Endopeptidasas/deficiencia , Conducta Animal/fisiología , Emociones/fisiología , Enzimas Convertidoras de Endotelina , Conducta Exploratoria/fisiología , Femenino , Preferencias Alimentarias/fisiología , Masculino , Aprendizaje por Laberinto/fisiología , Metaloendopeptidasas/deficiencia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora/fisiología , Neuropéptidos/metabolismo , Sistemas Neurosecretores/fisiología , ARN/genética , ARN/aislamiento & purificación , Reconocimiento en Psicología/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Filtrado Sensorial/genética , Filtrado Sensorial/fisiología , Conducta SocialRESUMEN
We previously showed that prolonged morphine treatment and chronic inflammation both enhanced delta opioid receptor (deltaOR) cell surface density in lumbar spinal cord neurons. Here, we sought to determine whether administration of morphine to rats with chronic inflammation would further increase the bio-availability of deltaOR, and thereby the analgesic properties of the deltaOR agonist deltorphin, over that produced by inflammation alone. We found that chronic inflammation produced by injection of complete Freund's adjuvant (CFA) into the hind paw resulted in a bilateral increase in the binding and internalization of fluorescent deltorphin in neurons of the lumbar spinal cord as did prolonged morphine treatment [Morinville A, Cahill CM, Aibak H, Rymar VV, Pradhan A, Hoffert C, Mennicken F, Stroh T, Sadikot AF, O'Donnell D, Clarke PB, Collier B, Henry JL, Vincent JP, Beaudet A (2004a) Morphine-induced changes in delta opioid receptor trafficking are linked to somatosensory processing in the rat spinal cord. J Neurosci 24:5549-5559]. This effect was accompanied by an increase in the antinociceptive efficacy of intrathecal deltorphin as measured using the tail-flick test. Treatment of CFA-injected rats with morphine decreased the cell surface availability of deltaOR in neurons of the dorsal horn of the lumbar spinal cord as compared with treatment with CFA alone. Behaviorally, it significantly enhanced the antihyperalgesic effects of deltorphin (plantar test; % maximum possible antihyperalgesic effect (MPAHE)=113.5%+/-32.4% versus 26.1%+/-11.6% in rats injected with CFA alone) but strongly reduced the antinociceptive efficacy of the drug (tail-flick test; % maximum possible antinociceptive effect (MPE)=29.6%+/-3.6% versus 66.6%+/-6.3% in rats injected with CFA alone) suggesting that the latter, but not the former, is linked to the deltaOR trafficking events observed neuroanatomically. These results demonstrate that in chronic inflammation, the antihyperalgesic effects of deltaOR agonists may be enhanced by morphine pre-treatment. They also reveal a dichotomy between mechanisms underlying antihyperalgesic and antinociceptive effects of deltaOR agonists.
Asunto(s)
Analgésicos Opioides/farmacología , Analgésicos , Hiperalgesia/tratamiento farmacológico , Inflamación/complicaciones , Morfina/farmacología , Oligopéptidos/farmacología , Animales , Conducta Animal/efectos de los fármacos , Enfermedad Crónica , Relación Dosis-Respuesta a Droga , Encefalina Metionina/metabolismo , Adyuvante de Freund , Hiperalgesia/etiología , Hiperalgesia/psicología , Inflamación/inducido químicamente , Masculino , Oligopéptidos/administración & dosificación , Umbral del Dolor/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Tiempo de Reacción/efectos de los fármacos , Receptores de Superficie Celular/efectos de los fármacos , Receptores Opioides delta/efectos de los fármacos , Receptores Opioides delta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Adrenergic and opioid receptors belong to the rhodopsin family of G-protein coupled receptors, couple to analogous signal transduction pathways, and affect the nociceptive system. Although a number of previous studies have reported functional interactions between these two receptors, the basis for this has not been well explored. We propose that direct receptor-receptor interactions could account, in part, for opioid-adrenergic cross-talk. In this report, we have addressed this using biophysical, biochemical, and pharmacological studies. We show that mu opioid and alpha2A adrenergic receptors reside in close proximity in live cells using the bioluminescence resonance energy transfer assay. These receptors colocalize to proximal dendrites in primary hippocampal neurons. mu-alpha2A Receptor complexes can be isolated from heterologous cells or primary neurons coexpressing these receptors. In these cells, the activation of either mu or alpha2A receptor leads to a significant increase in the level of immunoprecipitable mu-alpha2A complexes, whereas activation of both receptors leads to a significant decrease. The implications of these effects on signaling were examined using the agonist-mediated increase in G-protein activity and mitogen-activated protein kinase activity. We find that activation of either mu or alpha2A receptors leads to an increase in the extent of signaling, whereas activation of both receptors leads to a decrease. The increase in signaling by individual ligands and decrease by a combination of ligands is also seen in primary spinal cord neurons endogenously expressing these receptors. Taken together, these results suggest that physical associations between mu and alpha2A receptors could play a role in the functional interactions between these receptors.
Asunto(s)
Receptores Adrenérgicos alfa 2/metabolismo , Receptores Opioides mu/metabolismo , Agonistas de Receptores Adrenérgicos alfa 2 , Animales , Línea Celular , Clonidina/metabolismo , Clonidina/farmacología , Perros , Relación Dosis-Respuesta a Droga , Humanos , Morfina/metabolismo , Morfina/farmacología , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos alfa 2/genética , Receptores Opioides mu/agonistas , Receptores Opioides mu/genéticaRESUMEN
Although classical models predict that G-protein-coupled receptors (GPCRs) function as monomers, several recent studies acknowledge that GPCRs exist as dimeric or oligomeric complexes. In addition to homodimers, heterodimers between members of the GPCR family (both closely and distantly related) have been reported. In some cases heterodimerization is required for efficient agonist binding and signaling, and in others heterodimerization appears to lead to the generation of novel binding sites. In this article, the techniques used to study GPCR heterodimers, and the 'novel pharmacology' and functional implications resulting from heterodimerization will be discussed.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal/fisiología , Animales , Dimerización , Humanos , Neurotransmisores/farmacología , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Receptores de Superficie Celular/fisiología , Transducción de Señal/efectos de los fármacosRESUMEN
Cpe(fat)/Cpe(fat) mice have a naturally occurring point mutation within the carboxypeptidase E gene that inactivates this enzyme, leading to an accumulation of many neuroendocrine peptides containing C-terminal basic residues. These processing intermediates can be readily purified on an anhydrotrypsin affinity resin. Using MS to obtain molecular mass and partial sequence information, more than 100 peptides have been identified. These peptides represent fragments of 16 known secretory pathway proteins, including proenkephalin, proopiomelanocortin, protachykinins A and B, chromogranin A and B, and secretogranin II. Many of the identified peptides represent previously uncharacterized fragments of the precursors. For example, 12 of the 13 chromogranin B-derived peptides found in the present study have not been previously reported. Of these 13 chromogranin B-derived peptides, only five contain consensus cleavage sites for prohormone convertases at both the C and N termini. Two distinct chromogranin B-derived peptides result from cleavage at Trp-Trp bonds, a site not typically associated with neuropeptide processing. An RIA was used to confirm that one of these peptides, designated WE-15, exists in wild-type mouse brain, thus validating the approach to identify peptides in Cpe(fat)/Cpe(fat) mice. These "orphan" peptides are candidate ligands for orphan G protein-coupled receptors. In addition, the general technique of using affinity chromatography to isolate endogenous substrates from a mutant organism lacking an enzyme should be applicable to a wide range of enzyme-substrate systems.
Asunto(s)
Química Encefálica , Carboxipeptidasas/deficiencia , Cromatografía de Afinidad/métodos , Ratones Mutantes/metabolismo , Neuropéptidos/metabolismo , Fragmentos de Péptidos/análisis , Hipófisis/química , Precursores de Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Carboxipeptidasa H , Carboxipeptidasas/genética , Cromatografía Líquida de Alta Presión , Secuencia de Consenso , Furina , Ligandos , Ratones , Ratones Mutantes/genética , Datos de Secuencia Molecular , Neuropéptidos/química , Neuropéptidos/aislamiento & purificación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Mutación Puntual , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Subtilisinas/metabolismo , Técnica de Sustracción , Tripsina/químicaRESUMEN
Protein-protein interactions are involved in the regulation of a large number of biological processes. It is well established that a variety of cell surface receptors interact with each other to form dimers, and that this is essential for their activation. Although the existence of G protein coupled receptor dimers was predicted from early pharmacological and biochemical analysis, solid evidence supporting dimerization has come within the past few years following the cloning of G protein coupled receptor cDNAs. Using differential epitope tagging and selective immunoisolation of receptor complexes, dimerization of a number of G protein coupled receptors including members of the rhodopsin, secretin, and metabotropic glutamate receptor families have been reported. More recently fluorescence or bioluminescence resonance energy transfer techniques have been used to examine dimerization of these receptors in live cells. These studies have found that whereas in some cases there is an agonist induced increase in the level of dimers, in others there is a decrease or no change in dimer levels. Several recent studies have also reported the ability of related members of G protein coupled receptors to heterodimerize. These heterodimers exhibit distinct physical and functional properties. Examination of possible sites of interactions between receptors has implicated a role for extracellular, transmembrane and/or C-terminal region in dimerization. The functional consequences of dimerization, explored mainly using mutant receptors, have demonstrated a role in modulating agonist affinity, efficacy, and/or trafficking properties. Thus dimerization appears to be a universal phenomenon that provides an additional mechanism for modulation of receptor function as well as cross-talk between G protein coupled receptors.
Asunto(s)
Proteínas de Unión al GTP Heterotriméricas/metabolismo , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Animales , Dimerización , Humanos , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Receptores de Superficie Celular/agonistas , Transducción de Señal , Relación Estructura-ActividadRESUMEN
Prohormone convertase 1 (PC1), mediating the proteolytic processing of neural and endocrine precursors, is thought to be regulated by the neuroendocrine protein proSAAS. The PC1 inhibitory sequence is mostly confined within a 10-12-amino acid segment near the C terminus of the conserved human proSAAS and contains the critical KR(244) dibasic motif. Our results show that the decapeptide proSAAS-(235-244)( 235)VLGALLRVKR(244) is the most potent reversible competitive PC1-inhibitor (K(i) approximately 9 nm). The C-terminally extended proSAAS-(235-246) exhibits a 5-6-fold higher K(i) ( approximately 51 nm). The additional LE sequence at P1'-P2', resulted in a competitive substrate cleaved by PC1 at KR(244) downward arrowLE(246). Systematic alanine scanning and in some cases lysine scanning tested the contribution of each residue within proSAAS-(235-246) toward the PC1-inhibition's specificity and potency. The amino acids P1 Arg, P2 Lys, and P4 Arg are all critical for inhibition. Moreover, the aliphatic P3 Val and P5, P6, and P1' Leu significantly affect the degree of enzyme inactivation and PC1 specificity. Interestingly, a much longer N- and C-terminally extended endogenous rat proSAAS-(221-254) called little PenLen, was found to be a 3-fold less potent PC1 inhibitor with reduced selectivity but a much better substrate than proSAAS-(235-246). Molecular modeling studies and circular dichroism analysis indicate an extended and poly-l-proline II type structural conformation for proSAAS-(235-244), the most potent PC1 inhibitor, a feature not present in poor PC1 inhibitors.
Asunto(s)
Precursores Enzimáticos/antagonistas & inhibidores , Neuropéptidos/química , Neuropéptidos/farmacología , Fragmentos de Péptidos/farmacología , Precursores de Proteínas/química , Precursores de Proteínas/farmacología , Subtilisinas/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Unión Competitiva , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Secuencia Conservada , Humanos , Cinética , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Conformación Proteica , Ratas , Proteínas Recombinantes/antagonistas & inhibidores , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Relación Estructura-ActividadRESUMEN
G-protein-coupled receptors (GPCRs) have recently joined the list of cell surface receptors that dimerize. Dimerization has been shown to alter the ligand-binding, signaling, and trafficking properties of these receptors. Recent studies have shown that GPCRs heterodimerize with closely related members, resulting in the modulation of their function. In this study, we have attempted to determine whether members of GPCR superfamilies that couple to different families of G-proteins can associate and form oligomers. We chose the beta2 adrenergic receptor that couples to stimulatory G-proteins and delta & kappa opioid receptors that couple to inhibitory G-proteins. beta2 and delta receptors undergo robust agonist-mediated endocytosis, whereas kappa receptors do not. We find that when coexpressed, beta2 receptors can form heteromeric complexes with both delta and kappa receptors. This heterooligomerization does not significantly alter the ligand binding or coupling properties of the receptors. However, it affects the trafficking properties of the receptors. For example, we find that delta receptors, when coexpressed with beta2 receptors, undergo isoproterenol-mediated endocytosis. Conversely, beta2 receptors in these cells undergo etorphine-mediated endocytosis. However, beta2 receptors, when coexpressed with kappa receptors, undergo neither opioid- nor isoproterenol-mediated endocytosis. Moreover, these cells exhibit a substantial decrease in the isoproterenol-induced phosphorylation of mitogen-activated protein kinases. Taken together, these results provide direct evidence of heteromerization of GPCRs that couple to different types of G-proteins, which results in the modulation of receptor trafficking and signal transduction.
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Proteínas Quinasas Activadas por Mitógenos/metabolismo , Agregación de Receptores , Receptores Adrenérgicos beta 2/metabolismo , Receptores Opioides/metabolismo , Agonistas de Receptores Adrenérgicos beta 2 , Agonistas Adrenérgicos beta/farmacología , Animales , Línea Celular , AMP Cíclico/metabolismo , Dimerización , Relación Dosis-Respuesta a Droga , Endocitosis/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Etorfina/farmacología , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Humanos , Isoproterenol/farmacología , Ligandos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Narcóticos/farmacología , Fosfatidiletanolaminas/metabolismo , Fosforilación , Pruebas de Precipitina , Transporte de Proteínas/efectos de los fármacos , Receptores Adrenérgicos beta 2/genética , Receptores Opioides/agonistas , Receptores Opioides/genética , Proteínas Recombinantes de Fusión , TransfecciónRESUMEN
A spontaneous point mutation in the coding region of the carboxypeptidase E (CPE) gene results in a loss of CPE activity that correlates with the development of late onset obesity (Nagert, J. K., Fricker, L. D., Varlamov, O., Nishina, P. M., Rouille, Y., Steiner, D. F., Carroll, R. J., Paigen, B. J., and Leiter, E. H. (1995) Nat. Genet. 10, 135-142). Examination of the level of neuropeptides in these mice showed a decrease in mature bioactive peptides as a result of a decrease in both carboxypeptidase and prohormone convertase activities. A defect in CPE is not expected to affect endoproteolytic processing. In this report we have addressed the mechanism of this unexpected finding by directly examining the expression of the major precursor processing endoproteases, prohormone convertases PC1 and PC2 in Cpe(fat) mice. We found that the levels of PC1 and PC2 are differentially altered in a number of brain regions and in the pituitary. Since these enzymes have been implicated in the generation of neuroendocrine peptides (dynorphin A-17, beta-endorphin, and alpha- melanocyte-stimulating hormone) involved in the control of feeding behavior and body weight, we compared the levels of these peptides in Cpe(fat) and wild type animals. We found a marked increase in the level of dynorphin A-17, a decrease in the level of alpha-melanocyte-stimulating hormone, and an alteration in the level of C-terminally processed beta-endorphin. These results suggest that the impairment in the level of these and other peptides involved in body weight regulation is mainly due to an alteration in carboxypeptidase and prohormone convertase activities and that this may lead to the development of obesity in these animals.
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Ácido Aspártico Endopeptidasas/metabolismo , Encéfalo/enzimología , Carboxipeptidasas/genética , Carboxipeptidasas/metabolismo , Hipófisis/enzimología , Proproteína Convertasa 1 , Subtilisinas/metabolismo , Secuencia de Aminoácidos , Animales , Ácido Aspártico Endopeptidasas/genética , Peso Corporal , Carboxipeptidasa H , Carboxipeptidasas/deficiencia , Endorfinas/química , Endorfinas/farmacología , Encefalinas/metabolismo , Conducta Alimentaria , Ratones , Ratones Noqueados , Especificidad de Órganos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Mutación Puntual , Proopiomelanocortina/metabolismo , Proproteína Convertasa 2 , Proproteína Convertasas , Precursores de Proteínas/metabolismo , Valores de Referencia , Subtilisinas/genéticaRESUMEN
ProSAAS is a newly discovered protein with a neuroendocrine distribution generally similar to that of prohormone convertase 1 (PC1), a peptide-processing endopeptidase. Several proSAAS-derived peptides were previously identified in the brain and pituitary of the Cpe(fat)/Cpe(fat) mouse based on the accumulation of C-terminally extended peptides due to the absence of enzymatically active carboxypeptidase E, a peptide-processing exopeptidase. In the present study, antisera against different regions of proSAAS were used to develop radioimmunoassays and examine the processing profile of proSAAS in wild type and Cpe(fat)/Cpe(fat) mouse tissues following gel filtration and reverse phase high performance liquid chromatography. In wild type mouse brain and pituitary, the majority of proSAAS is processed into smaller peptides. These proSAAS-derived peptides elute from the reverse-phase column in the same positions as synthetic peptides that correspond to little SAAS, PEN, and big LEN. Mass spectrometry revealed the presence of peptides with the expected molecular masses of little SAAS and big LEN in the fractions containing immunoreactive peptides. The processing of proSAAS is slightly impaired in Cpe(fat)/Cpe(fat) mice, relative to wild-type mice, leading to the accumulation of partially processed peptides. One of these peptides, the C-terminally extended form of PEN, is known to inhibit PC1 activity and this could account for the reduction in enzymatically active PC1 seen in Cpe(fat)/Cpe(fat) mice. The observation that little SAAS and big LEN are the major forms of these peptides produced in mouse brain and pituitary raises the possibility that these peptides function as neurotransmitters or hormones.
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Encéfalo/metabolismo , Carboxipeptidasas/fisiología , Neuropéptidos/metabolismo , Hipófisis/metabolismo , Precursores de Proteínas/metabolismo , Animales , Carboxipeptidasa H , Carboxipeptidasas/genética , Cromatografía Líquida de Alta Presión , Sueros Inmunes/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neuropéptidos/análisis , Fragmentos de Péptidos/análisis , Precursores de Proteínas/análisisRESUMEN
G-protein-coupled receptors (GPCRs) comprise the largest family of transmembrane receptors in the human genome that respond to a plethora of signals, including neurotransmitters, peptide hormones, and odorants, to name a few. They couple to second messenger signaling cascade mechanisms via heterotrimeric G-proteins. Recently, many studies have revealed that GPCRs exist as dimers, which may be present as homo- or heterodimers/oligomers. These recent findings have been met with skepticism, since they are contradictory to the dogma that GPCRs function as monomers. Although the existence of GPCR dimers/oligomers was predicted from early pharmacological and biochemical studies, further studies to critically evaluate this phenomenon were impeded by the lack of appropriate reagents. The availability of cDNAs for GPCRs, of highly selective ligands and of antibodies for these receptors has made it possible to visualize and investigate the functional effects of GPCR oligomers. Pharmacological studies, along with biochemical techniques, such as cross-linking and immunoprecipitation with differentially epitope-tagged receptors, have been employed to demonstrate the oligomerization of a number of GPCRs. Moreover, recent biophysical techniques, such as bioluminescence and fluorescence resonance energy transfer, now make it possible to examine GPCR dimerization/oligomerization in living cells. In this review, we provide a brief overview of some of the techniques employed to describe GPCR dimers, and we discuss their respective limitations. We also examine the implications of dimerization/oligomerization on GPCR function. In addition, we discuss domains of the receptors that are thought to facilitate dimerization/oligomerization. Finally, we consider recent evidence for the subcellular localization of the dimer/oligomer assembly.
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Proteínas de Unión al GTP/farmacología , Receptores de Superficie Celular/fisiología , Dimerización , Endocitosis , Humanos , Ligandos , Narcóticos/farmacología , Transducción de SeñalRESUMEN
Opiate analgesics are widely used in the treatment of severe pain. Because of their importance in therapy, different strategies have been considered for making opiates more effective while curbing their liability to be abused. Although most opiates exert their analgesic effects primarily via mu opioid receptors, a number of studies have shown that delta receptor-selective drugs can enhance their potency. The molecular basis for these findings has not been elucidated previously. In the present study, we examined whether heterodimerization of mu and delta receptors could account for the cross-modulation previously observed between these two receptors. We find that co-expression of mu and delta receptors in heterologous cells followed by selective immunoprecipitation results in the isolation of mu-delta heterodimers. Treatment of these cells with extremely low doses of certain delta-selective ligands results in a significant increase in the binding of a mu receptor agonist. Similarly, treatment with mu-selective ligands results in a significant increase in the binding of a delta receptor agonist. This robust increase is also seen in SKNSH cells that endogenously express both mu and delta receptors. Furthermore, we find that a delta receptor antagonist enhances both the potency and efficacy of the mu receptor signaling; likewise a mu antagonist enhances the potency and efficacy of the delta receptor signaling. A combination of agonists (mu and delta receptor selective) also synergistically binds and potentiates signaling by activating the mu-delta heterodimer. Taken together, these studies show that heterodimers exhibit distinct ligand binding and signaling characteristics. These findings have important clinical ramifications and may provide new foundations for more effective therapies.
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Receptores Opioides delta/metabolismo , Receptores Opioides mu/metabolismo , Somatostatina/análogos & derivados , Analgésicos Opioides/farmacología , Animales , Células CHO , Línea Celular , Cricetinae , Detergentes/química , Dimerización , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Encefalina D-Penicilamina (2,5)/deficiencia , Encefalina D-Penicilamina (2,5)/farmacología , Humanos , Ligandos , Ratones , Antagonistas de Narcóticos/farmacología , Oligopéptidos/farmacología , Pruebas de Precipitina , Receptores Opioides delta/agonistas , Receptores Opioides delta/antagonistas & inhibidores , Receptores Opioides mu/agonistas , Receptores Opioides mu/antagonistas & inhibidores , Somatostatina/farmacologíaRESUMEN
Agonist-induced receptor phosphorylation is an initial step in opioid receptor desensitization, a molecular mechanism of opioid tolerance and dependence. Our previous research suggested that agonist-induced delta-opioid receptor (DOR) phosphorylation occurs at the receptor carboxyl terminal domain. The current study was carried out to identify the site of DOR phosphorylation during agonist stimulation and the kinases catalyzing this reaction. Truncation (Delta15) or substitutions (T358A, T361A, and S363G single or triple mutants) at the DOR cytoplasmic tail caused 80 to 100% loss of opioid-stimulated receptor phosphorylation, indicating that T358, T361, and S363 all contribute and are cooperatively involved in agonist-stimulated DOR phosphorylation. Coexpression of GRK2 strongly enhanced agonist-stimulated phosphorylation of the wild-type DOR (WT), but Delta15 or mutant DOR (T358A/T361A/S363G) failed to show any detectable phosphorylation under these conditions. These results demonstrate that T358, T361, and S363 are required for agonist-induced and GRK-mediated receptor phosphorylation. Agonist-induced receptor phosphorylation was severely impaired by substitution of either T358 or S363 with aspartic acid residue, but phosphorylation of the T361D mutant was comparable with that of WT. In the presence of exogenously expressed GRK2, phosphorylation levels of T358D and S363D mutants were approximately half of that of WT, whereas significant phosphorylation of the T358/S363 double-point mutant was not detected. These results indicate that both T358 and S363 residues at the DOR carboxyl terminus are capable of serving cooperatively as phosphate acceptor sites of GRK2 in vivo. Taken together, we have demonstrated that agonist-induced opioid receptor phosphorylation occurs exclusively at two phosphate acceptor sites (T358 and S363) of GRK2 at the DOR carboxyl terminus. These results represent the identification of the GRK phosphorylation site on an opioid receptor for the first time and demonstrate that GRK is the prominent kinase responsible for agonist-induced opioid receptor phosphorylation in vivo.
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Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Receptores Opioides delta/metabolismo , Secuencia de Aminoácidos , Células Cultivadas , Humanos , Datos de Secuencia Molecular , Fosforilación , Quinasas de Receptores Adrenérgicos betaRESUMEN
No field more eagerly awaits a molecular clarification for G-protein coupled receptor (GPCR) dimerization than the opioid receptor field. Extensive evidence of pharmacological and functional interactions between opioid receptor types has primed this field for such a resolution. In retrospect, much of the data collected on synergy between different opioid receptor types may represent the functional correlate for the newly found opioid receptor dimerization. While previous reports of functional synergy have been, for the most part, consistent in demonstrating cross-regulation between two receptor types, the lack of highly receptor-selective ligands allowed skeptics to remain doubtful over the interpretations of these results. Today, two important developments in the opioid receptor field help reinvigorate the hypothesis of functional, cross-modulating opioid receptor complexes: (1) The existence of highly selective ligands which eliminate any possibility of cross-reactivity between receptor types, and (2) the discovery that opioid receptors and a number of other GPCRs exist as dimers in biochemical, functional and pharmacological assays. It is with these new tools that we seek to understand the mechanisms and implications of dimerization. Initial results of these studies have demonstrated that the dimerization of opioid receptors may help consolidate several pharmacological findings that have remained unanswered. In this review we present biochemical, pharmacological and functional evidence for opioid receptor complexes and add evidence from our recent studies on opioid receptor dimerization. We believe a thorough understanding of receptor dimerization is crucial in clarifying the mechanism of action of opioids and other drugs and may serve a more practical purpose in aiding the development of novel therapeutic drugs.