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1.
Traffic ; 19(1): 58-82, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29044966

RESUMO

The signaling pathway of G protein-coupled receptors is strongly linked to their trafficking profile. Little is known about the molecular mechanisms involved in the vasopressin receptor V1b subtype (V1b R) trafficking and its impact on receptor signaling and regulation. For this purpose, we investigated the role of ß-arrestins in receptor desensitization, internalization and recycling and attempted to dissect the V1b R-mediated MAP kinase pathway. Using MEF cells Knocked-out for ß-arrestins 1 and 2, we demonstrated that both ß-arrestins 1 and 2 play a fundamental role in internalization and recycling of V1b R with a rapid and transient V1b R-ß-arrestin interaction in contrast to a slow and long-lasting ß-arrestin recruitment of the V2 vasopressin receptor subtype (V2 R). Using V1b R-V2 R chimeras and V1b R C-terminus truncations, we demonstrated the critical role of the V1b R C-terminus in its interaction with ß-arrestins thereby regulating the receptor internalization and recycling kinetics in a phosphorylation-independent manner. In parallel, V1b R MAP kinase activation was dependent on arrestins and Src-kinase but independent on G proteins. Interestingly, Src interacted with hV1b R at basal state and dissociated when receptor internalization occurred. Altogether, our data describe for the first time the trafficking profile and MAP kinase pathway of V1b R involving both arrestins and Src kinase family.


Assuntos
Sistema de Sinalização das MAP Quinases , Receptores de Vasopressinas/metabolismo , beta-Arrestinas/metabolismo , Animais , Sítios de Ligação , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Camundongos , Ligação Proteica , Transporte Proteico , beta-Arrestinas/química , Quinases da Família src/metabolismo
2.
Gen Comp Endocrinol ; 258: 15-32, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29155265

RESUMO

It is now accepted that vasopressin, through V1A/V1B receptors, centrally regulates cognitive functions such as memory, affiliation, stress, fear and depression. However, the respective roles of these receptor isoforms and their contribution to stress-related pathologies remain uncertain. The development of new therapeutic treatments requires a precise knowledge of the distribution of these receptors within the brain, which has been so far hampered by the lack of selective V1B markers. In the present study, we have determined the pharmacological properties of three new potent rat V1B fluorescent ligands and demonstrated that they constitute valuable tools for simultaneous visualization and activation of native V1B receptors in living rat brain tissue. Thus, d[Leu4,Lys-Alexa 647)8]VP (analogue 3), the compound with the best affinity-selectivity/fluorescence ratio for the V1B receptor emerged as the most promising. The rat brain regions most concerned by stress such as hippocampus, olfactory bulbs, cortex and amygdala display the highest V1B fluorescent labelling with analogue 3. In the hippocampus CA2, V1B receptors are located on glutamatergic, not GABAergic neurones, and are absent from astrocytes. Using AVP-EGFP rats, we demonstrate the presence of V1B autoreceptors on AVP-secreting neurones not only in the hypothalamus, but also sparsely in the hippocampus. Finally, using both electrophysiology and visualization of ERK phosphorylation, we show analogue 3-induced activation of the V1B receptor in situ. This will help to analyse expression and functionality of V1B receptors in the brain and contribute to further explore the AVPergic circuitry in normal and pathological conditions.


Assuntos
Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Corantes Fluorescentes/metabolismo , Receptores de Vasopressinas/metabolismo , Animais , Arginina Vasopressina/metabolismo , Astrócitos/metabolismo , Células CHO , Cricetinae , Cricetulus , Células HEK293 , Humanos , Hipotálamo/metabolismo , Ligantes , Masculino , Neuroanatomia , Neurônios/metabolismo , Hipófise/citologia , Ratos Sprague-Dawley , Receptores de GABA/metabolismo , Coloração e Rotulagem , Vasopressinas/metabolismo
3.
Am J Physiol Endocrinol Metab ; 312(3): E127-E135, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-27998960

RESUMO

Recent epidemiological studies have revealed novel relationships between low water intake or high vasopressin (AVP) and the risk of hyperglycemia and diabetes. AVP V1A and V1B receptors (R) are expressed in the liver and pancreatic islets, respectively. The present study was designed to determine the impact of different levels of circulating AVP on glucose homeostasis in normal Sprague-Dawley rats, as well as the respective roles of V1AR and V1BR. We showed that acute injection of AVP induces a dose-dependent increase in glycemia. Pretreatment with a selective V1AR antagonist, but not a V1BR antagonist, dose-dependently prevented the rise in glycemia. V1BR antagonism did not modify the hyperinsulinemic response, resulting from AVP-induced hyperglycemia, but enhanced the fall in glucagonemia. Acute administration of selective V1AR or V1BR agonists confirmed the involvement of V1AR in the hyperglycemic effect of AVP. In chronic experiments, AVP levels were altered in both directions. Sustained AVP infusion through implantable minipumps induced a time-dependent increase in fasting glycemia, whereas lowering endogenous AVP by increasing water intake had no effect. After 4 wk of AVP infusion, the rise in glycemia amounted to 1.1 mmol/l (P < 0.01) without significant change in insulinemia. This effect was attenuated by cotreatment with a V1AR antagonist. Similar results were observed in lean Zucker rats. These findings demonstrate for the first time a causal link between chronic high AVP and hyperglycemia through V1AR activation and, thus, provide a pathophysiological explanation for the relationship observed in human cohorts between the AVP-hydration axis and the risk of diabetes.


Assuntos
Arginina Vasopressina/farmacologia , Glicemia/efeitos dos fármacos , Glucagon/efeitos dos fármacos , Hiperglicemia/sangue , Receptores de Vasopressinas/efeitos dos fármacos , Animais , Antagonistas dos Receptores de Hormônios Antidiuréticos/farmacologia , Glicemia/metabolismo , Técnicas de Introdução de Genes , Glucagon/sangue , Hiperinsulinismo/sangue , Indóis/farmacologia , Insulina/sangue , Masculino , Imagem Óptica , Pâncreas/metabolismo , Peptídeos Cíclicos/farmacologia , Pirrolidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Ratos Zucker , Receptores de Vasopressinas/agonistas , Receptores de Vasopressinas/metabolismo
4.
Pharmacol Res ; 113(Pt A): 257-264, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27586252

RESUMO

Terlipressin is recommended as a gold standard to treat hepatorenal syndrome complicating liver cirrhosis. It is presented as a specific V1A receptor agonist, beyond its enzymatic conversion into lysine8-Vasopressin (LVP), able to counteract the splanchnic vasodilation. However, the complete pharmacological characterization of this drug with respect to the different vasopressin receptor subtypes is missing. We studied terlipressin intrinsic properties, focusing not only on V1A, but also on other vasopressin receptor subtypes. The experimental studies were conducted on rat and human cellular models. Binding experiments were performed on rat liver membranes and CHO cells transfected with the different human vasopressin receptor subtypes. Agonist status was assessed from inositol phosphate or cyclic AMP assays, and measurement of intracellular calcium variations, performed on cultured vascular smooth muscle cells from rat aorta and human uterine artery and CHO cells. Terlipressin binds to the rat and human V1A receptors with an affinity in the micromolar range, a value 120 fold lower than that of LVP. It induces a rapid and transient intracellular calcium increase, a robust stimulation of phospholipase C but with reduced maximal efficiencies as compared to LVP, indicating a partial V1A agonist property. In addition, terlipressin is also a full agonist of human V2 and V1B receptors, with also a micromomolar affinity. CONCLUSIONS: Terlipressin is a non-selective vasopressin analogue, exhibiting intrinsic agonist properties. Its full V2 receptor agonism may result in renal effects potentially aggravating water retention and hyponatremia of cirrhosis.


Assuntos
Síndrome Hepatorrenal/tratamento farmacológico , Lipressina/análogos & derivados , Pró-Fármacos/farmacologia , Receptores de Vasopressinas/agonistas , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Síndrome Hepatorrenal/metabolismo , Humanos , Fosfatos de Inositol/metabolismo , Cirrose Hepática/metabolismo , Lipressina/farmacologia , Masculino , Ratos , Ratos Wistar , Terlipressina , Transfecção/métodos , Vasopressinas/efeitos dos fármacos , Vasopressinas/metabolismo
5.
Mol Cell Biol ; 36(6): 1019-31, 2016 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-26787837

RESUMO

Palmitoylation is involved in several neuropsychiatric and movement disorders for which a dysfunctional signaling of the dopamine D3 receptor (Drd3) is hypothesized. Computational modeling of Drd3's homologue, Drd2, has shed some light on the putative role of palmitoylation as a reversible switch for dopaminergic receptor signaling. Drd3 is presumed to be palmitoylated, based on sequence homology with Drd2, but the functional attributes afforded by Drd3 palmitoylation have not been studied. Since these receptors are major targets of antipsychotic and anti-Parkinsonian drugs, a better characterization of Drd3 signaling and posttranslational modifications, like palmitoylation, may improve the prospects for drug development. Using molecular dynamics simulations, we evaluated in silico how Drd3 palmitoylation could elicit significant remodeling of the C-terminal cytoplasmic domain to expose docking sites for signaling proteins. We tested this model in cellulo by using the interaction of Drd3 with the G-alpha interacting protein (GAIP) C terminus 1 (GIPC1) as a template. From a series of biochemical studies, live imaging, and analyses of mutant proteins, we propose that Drd3 palmitoylation acts as a molecular switch for Drd3-biased signaling via a GIPC1-dependent route, which is likely to affect the mode of action of antipsychotic drugs.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Palmitatos/metabolismo , Receptores de Dopamina D3/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas Adaptadoras de Transdução de Sinal/genética , Membrana Celular/metabolismo , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Mapas de Interação de Proteínas , Transporte Proteico , Receptores de Dopamina D3/análise , Receptores de Dopamina D3/genética , Transdução de Sinais
6.
Artigo em Inglês | MEDLINE | ID: mdl-24947211

RESUMO

Endogenous opioids are derived from four related polypeptide precursors: proenkephalin (PENK), prodynorphin (PDYN), pronociceptin (PNOC) and proopiomelanocortin (POMC). In mammals PENK encodes for four copy of Met-enkephalin, one octapeptide Met-enkephalin-Arg-Gly-Leu, one heptapeptide Met-enkephalin-Arg-Phe and a single copy of Leu-enkephalin. Our detailed bioinformatic search on the existing PENK sequences revealed several atypical hexapeptide Met-enkephalins in different vertebrate animals. They are located either in the second enkephalin unit or in the seventh enkephalin core position at the C-terminus. Altogether four different hexapeptide sequences were obtained representing eleven animal species: Met-enkephalin-Arg(6) (YGGFMR) in the bird zebra finch, Met-enkephalin-Asp(6) (YGGFMD), Met-enkephalin-Ile(6) (YGGFMI) in zebrafish; and Met-enkephalin-Ser(6) (YGGFMS) in two pufferfish species. All novel peptides were chemically synthesized and studied in receptor binding and G-protein activation assays performed on rat brain membranes. The four novel enkephalins were equipotent in stimulating G-proteins. Affinities of the peptides determined by equilibrium competition assays in receptor binding experiments were statistically different. At the MOP receptors the highest affinity (Ki 4nM) was obtained with the zebra finch peptide Met-enkephalin-Arg(6). The pufferfish Met-enkephalin-Ser(6) exhibited the highest affinity (Ki 6.7nM) at the DOP receptor. Phylogenetic neuropeptide libraries, defined here as a collection of mutationally different species variants of orthologous and paralogous peptide sequences, represent the natural molecular diversity of the neuropeptides. Such libraries can provide a wide range of structural information establishing comparative functional analyses. Since DNA sequencing data are rapidly increasing, more development in the natural peptide library concept is expected.


Assuntos
Encefalinas/metabolismo , Peptídeos Opioides/metabolismo , Precursores de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Encefalinas/química , Dados de Sequência Molecular , Peptídeos Opioides/química , Filogenia , Precursores de Proteínas/química , Ratos Wistar , Receptores Opioides/metabolismo
7.
PLoS One ; 7(12): e49708, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23236353

RESUMO

Growing evidence points to vasopressin (AVP) as a social behavior regulator modulating various memory processes and involved in pathologies such as mood disorders, anxiety and depression. Accordingly, AVP antagonists are actually envisaged as putative treatments. However, the underlying mechanisms are poorly characterized, in particular the influence of AVP on cellular or synaptic activities in limbic brain areas involved in social behavior. In the present study, we investigated AVP action on the synapse between the entorhinal cortex and CA2 hippocampal pyramidal neurons, by using both field potential and whole-cell recordings in mice brain acute slices. Short application (1 min) of AVP transiently reduced the synaptic response, only following induction of long-term potentiation (LTP) by high frequency stimulation (HFS) of afferent fibers. The basal synaptic response, measured in the absence of HFS, was not affected. The Schaffer collateral-CA1 synapse was not affected by AVP, even after LTP, while the Schaffer collateral-CA2 synapse was inhibited. Although investigated only recently, this CA2 hippocampal area appears to have a distinctive circuitry and a peculiar role in controlling episodic memory. Accordingly, AVP action on LTP-increased synaptic responses in this limbic structure may contribute to the role of this neuropeptide in controlling memory and social behavior.


Assuntos
Região CA2 Hipocampal/efeitos dos fármacos , Potenciação de Longa Duração/efeitos dos fármacos , Células Piramidais/efeitos dos fármacos , Vasopressinas/farmacologia , Animais , Região CA2 Hipocampal/fisiologia , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Potenciação de Longa Duração/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Patch-Clamp , Células Piramidais/fisiologia , Sinapses/efeitos dos fármacos , Sinapses/fisiologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia
8.
Proc Natl Acad Sci U S A ; 109(17): E1028-37, 2012 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-22493236

RESUMO

G protein-coupled receptors (GPCRs) have been shown to activate the mitogen-activated protein kinases, ERK1/2, through both G protein-dependent and -independent mechanisms. Here, we describe a G protein-independent mechanism that unravels an unanticipated role for ß-arrestins. Stimulation of the V2 vasopressin receptor (V2R) in cultured cells or in vivo in rat kidney medullar collecting ducts led to the activation of ERK1/2 through the metalloproteinase-mediated shedding of a factor activating the insulin-like growth factor receptor (IGFR). This process was found to be both Src- and ß-arrestin-dependent. Whereas Src was found to act upstream of the metalloproteinase activation and be required for the release of the IGFR-activating factor, ß-arrestins were found to act downstream of the IGFR transactivation. Unexpectedly, the engagement of ß-arrestins by the IGFR but not by the V2R was needed to promote the vasopressin-stimulated ERK1/2 activation, indicating that a pool of ß-arrestins distinct from those ß-arrestins recruited to the V2R acts downstream of the receptor tyrosine kinase to activate ERK1/2. Such a dual site of action for ß-arrestins helps explain the pleiotropic actions of this scaffolding protein. Given the role that V2R-stimulated ERK1/2 plays in kidney cell proliferation, this transactivation mechanism may have important implications for renal pathophysiology. Still, the role of ß-arrestins downstream of a transactivation event is not limited to the V2R, because we observed a similar involvement for an unrelated GPCR (the platelet-activating factor receptor), indicating that it may be a general mechanism shared among GPCRs.


Assuntos
Arrestinas/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Receptores de Somatomedina/metabolismo , Receptores de Vasopressinas/metabolismo , Ativação Transcricional , Animais , Células Cultivadas , Ativação Enzimática , Medula Renal/citologia , Medula Renal/metabolismo , Ratos , Receptores de Somatomedina/genética , beta-Arrestinas
9.
Mol Endocrinol ; 26(3): 502-20, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22301784

RESUMO

Vasopressin (AVP) and CRH synergistically regulate adrenocorticotropin and insulin release at the level of the pituitary and pancreas, respectively. Here, we first extended these AVP and CRH coregulation processes to the adrenal medulla. We demonstrate that costimulation of chromaffin cells by AVP and CRH simultaneously induces a catecholamine secretion exceeding the one induced by each hormone alone, thus demonstrating a net potentiation. To further elucidate the molecular mechanisms underlying this synergism, we coexpressed human V1b and CRH receptor (CRHR)1 receptor in HEK293 cells. In this heterologous system, AVP also potentiated CRH-stimulated cAMP accumulation in a dose-dependent and saturable manner. This effect was only partially mimicked by phorbol ester or inhibited by a phospholipase C inhibitor respectively. This finding suggests the existence of an new molecular mechanism, independent from second messenger cross talk. Similarly, CRH potentiated the AVP-induced inositol phosphates production. Using bioluminescence resonance energy transfer, coimmunoprecipitation, and receptor rescue experiments, we demonstrate that V1b and CRHR1 receptors assemble as heterodimers. Moreover, new pharmacological properties emerged upon receptors cotransfection. Taken together, these data strongly suggest that direct molecular interactions between V1b and CRHR1 receptors play an important role in mediating the synergistic interactions between these two receptors.


Assuntos
Hormônio Liberador da Corticotropina/fisiologia , Multimerização Proteica , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Receptores de Vasopressinas/metabolismo , Vasopressinas/fisiologia , Glândulas Suprarrenais/citologia , Animais , Antagonistas dos Receptores de Hormônios Antidiuréticos , Catecolaminas/metabolismo , Bovinos , Células Cultivadas , Células Cromafins/metabolismo , Hormônio Liberador da Corticotropina/farmacologia , AMP Cíclico/metabolismo , Ativação Enzimática , Estrenos/farmacologia , Células HEK293 , Humanos , Hidrocarbonetos Halogenados/farmacologia , Indóis/farmacologia , Inositol Polifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/metabolismo , Cultura Primária de Células , Ligação Proteica , Pirrolidinas/farmacologia , Pirrolidinonas/farmacologia , Receptor Cross-Talk , Receptores de Hormônio Liberador da Corticotropina/agonistas , Receptores de Hormônio Liberador da Corticotropina/antagonistas & inibidores , Receptores de Vasopressinas/agonistas , Proteínas Recombinantes de Fusão/agonistas , Proteínas Recombinantes de Fusão/metabolismo , Sistemas do Segundo Mensageiro , Tiazinas/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismo , Vasopressinas/farmacologia
10.
J Med Chem ; 54(8): 2864-77, 2011 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-21428295

RESUMO

Among the four known vasopressin and oxytocin receptors, the specific localization of the V1b isoform is poorly described because of the lack of selective pharmacological tools. In an attempt to address this need, we decided to design, synthesize, and characterize fluorescent selective V1b analogues. Starting with the selective V1b agonist [deamino-Cys(1),Leu(4),Lys(8)]vasopressin (d[Leu(4),Lys(8)]VP) synthesized earlier, we added blue, green, or red fluorophores to the lysine residue at position 8 either directly or by the use of linkers of different lengths. Among the nine analogues synthesized, two exhibited very promising properties. These are d[Leu(4),Lys(Alexa 647)(8)]VP (3) and d[Leu(4),Lys(11-aminoundecanoyl-Alexa 647)(8)]VP (9). They remained full V1b agonists with nanomolar affinity and specifically decorated the plasma membrane of CHO cells stably transfected with the human V1b receptor. These new selective fluorescent peptides will allow the cellular localization of V1b or OT receptor isoforms in native tissues.


Assuntos
Desenho de Drogas , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Receptores de Ocitocina/metabolismo , Receptores de Vasopressinas/metabolismo , Corantes Fluorescentes/síntese química , Humanos , Peptídeos/síntese química
11.
J Biol Chem ; 285(19): 14514-20, 2010 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-20197280

RESUMO

Techniques for analyzing the membrane diffusion of molecules are the most promising methods for investigating the compartmentalization of G-protein-coupled receptors, particularly as relevant to receptor signaling processes. Here, we report fluorescence recovery after photobleaching (FRAP) measurements performed at variable spot radius for human mu opioid (hMOP) receptors on SH-SY5Y neuroblastoma cells in the presence of ligands. Although an antagonist did not affect the behavior of the receptors compared with the basal state, two different agonists, DAMGO and morphine, caused markedly different changes to receptor diffusion. Like receptors in the absence of ligand, receptors bound to morphine exhibited diffusion confined to joined semipermeable domains, but with smaller domain size and diffusion coefficient. This effect was inhibited by pertussis toxin, strongly suggesting that this dynamic behavior is associated with early steps of signaling. In the presence of DAMGO, half of the receptors displayed free long-range diffusion and the other half were confined to smaller isolated domains. Hypertonic sucrose buffer suppressed this effect, which we attribute to receptor entry into clathrin-coated pits. It is likely that the observation of distinct receptor dynamics in the presence of DAMGO and morphine involves the agonist-selective phosphorylation of the receptor.


Assuntos
Recuperação de Fluorescência Após Fotodegradação , Neuroblastoma/metabolismo , Receptores Opioides mu/agonistas , Receptores Opioides mu/metabolismo , Analgésicos Opioides/farmacologia , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Morfina/farmacologia , Toxina Pertussis/farmacologia , Fosforilação/efeitos dos fármacos , Células Tumorais Cultivadas
12.
Neurochem Int ; 55(7): 458-66, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19414055

RESUMO

The synthetic hexapeptide Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-ol (Ac-RYYRIK-ol) represents a highly potent and selective partial agonist ligand for the nociceptin/orphanin FQ (N/OFQ) peptide receptor (nociceptin receptor, NOPr). Ac-RYYRIK-ol has been labeled with tritium yielding [(3)H]Ac-RYYRIK-ol with exceptionally high specific radioactivity of 94Ci/mmol. The radioprobe is chemically stable even at 24 degrees C in ethanol solution for at least 4 days. No significant decomposition of the [(3)H]ligand occurred under the condition of the binding experiments indicating a fine enzymatic stability of the peptide. Radioreceptor binding studies were conducted using native neuronal NOPr preparation of rat brain membrane fractions and recombinant human nociceptin receptor ((h)NOPr) preparations from cultured Chinese Hamster Ovary (CHO) cells stably expressing (h)NOPr. Specific binding of the compound was reversible, saturable and of high affinity. No cross-reaction with the opioid receptors was observed suggesting superior NOPr selectivity of the ligand. Monophasic isotherm curves obtained in radioligand binding saturation and homologous displacement experiments indicated the presence of single binding sites in both preparations. Average densities of the [(3)H]Ac-RYYRIK-ol recognition sites were 237 and 749fmol/mg protein in rat brain and transfected cells, respectively. Equilibrium affinity values (K(d)s) were determined by three independent way providing identical results. In rat brain membranes K(d)s of 0.3-1.3nM were found depending upon the assay type. In homologous competition studies performed on (h)NOP-CHO cell membranes almost the same binding affinities were measured for Ac-RYYRIK-ol either with [(3)H]Ac-RYYRIK-ol (K(i) 2.8nM) or with [(3)H](Leu(14))nociceptin (2.3nM). A number of NOPr and opioid ligands were screened in heterologous displacement experiments and displayed a rank order of affinity profile being consistent with fairly good NOPr selectivity of the sites labeled by [(3)H]Ac-RYYRIK-ol. Taken together, the high molar activity, improved chemical and biological stability and the capability of the selective and high affinity labeling make this novel radioprobe available for further exploring the biochemical pharmacology and receptor-ligand interaction of the NOP receptor.


Assuntos
Neurônios/metabolismo , Oligopeptídeos , Receptores Opioides/metabolismo , Marcadores de Afinidade , Algoritmos , Animais , Ligação Competitiva/efeitos dos fármacos , Células CHO , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Humanos , Técnicas In Vitro , Cinética , Ligantes , Ensaio Radioligante , Compostos Radiofarmacêuticos , Ratos , Ratos Wistar , Receptores Opioides/efeitos dos fármacos , Receptores Opioides/genética
13.
Mol Pharmacol ; 68(2): 467-76, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15901846

RESUMO

The initial aim of this study was to identify protein changes associated with long-term morphine treatment in a recombinant human neuroblastoma SH-SY5Y clone (sc2) stably overexpressing the human mu-opioid (MOP) receptor. In MOP receptor-overexpressing sc2 cells, short-term morphine exposure was found to be much more potent and efficacious in inhibiting forskolin-elicited production of cAMP, and long-term morphine exposure was shown to induce a substantially higher degree of opiate dependence, as reflected by adenylate cyclase sensitization, than it did in wild-type neuroblastoma cells. Differential proteomic analysis of detergent-resistant membrane rafts isolated from untreated and chronically morphine-treated sc2 cells revealed long-term morphine exposure to have reliably induced a 30 to 40% decrease in the abundance of five proteins, subsequently identified by mass spectrometry as G protein subunits alphai(2), alphai(3), beta(1), and beta(2), and prohibitin. Quantitative Western blot analyses of whole-cell extracts showed that long-term morphine treatment-induced down-regulation of Gbeta but not of the other proteins is highly correlated (r(2) = 0.96) with sensitization of adenylate cyclase. Down-regulation of Gbeta and adenylate cyclase sensitization elicited by long-term morphine treatment were suppressed in the presence of carbobenzoxy-l-leucyl-l-leucyl-l-norvalinal (MG-115) or lactacystin. Thus, sustained activation of the MOP receptor by morphine in sc2 cells seems to promote proteasomal degradation of Gbeta to sensitize adenylate cyclase. Together, our data suggest that the long-term administration of opiates may elicit dependence by altering the neuronal balance of heterotrimeric G proteins and adenylate cyclases, with the ubiquitin-proteasome pathway playing a pivotal role.


Assuntos
Adenilil Ciclases/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Morfina/administração & dosagem , Neuroblastoma/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Adenilil Ciclases/genética , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Subunidades beta da Proteína de Ligação ao GTP/genética , Humanos , Neuroblastoma/genética , Complexo de Endopeptidases do Proteassoma/genética , Fatores de Tempo
14.
Biochem Biophys Res Commun ; 325(3): 915-21, 2004 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-15541377

RESUMO

A lipid rafts/detergent-resistant membrane (DRM) fraction was prepared from recombinant HEK293 cells stably expressing the human NOP receptor fused to the green fluorescent protein EGFP (hNOPr-EGFP), and probed for the presence and functionality of the fusion protein. Fluorescence detection as well as immunoblotting with an anti-GFP antibody revealed that most of the fusion protein was recovered in the DRM fraction, wherein it mediated efficiently NOP-induced stimulation of GTPgamma(35)S binding. Recovery of hNOPr-EGFP in the DRM fraction was not affected had the cells been acutely or chronically exposed to NOP prior to detergent treatment. Therefore, in HEK cells, the NOP receptor localizes constitutively to DRMs wherein it retains ability to couple with hetero-trimeric G protein.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Rim/metabolismo , Microdomínios da Membrana/metabolismo , Octoxinol/farmacologia , Receptores Opioides/metabolismo , Linhagem Celular/efeitos dos fármacos , Detergentes/farmacologia , Resistência a Medicamentos/fisiologia , Humanos , Rim/efeitos dos fármacos , Rim/ultraestrutura , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/ultraestrutura , Receptores Opioides/efeitos dos fármacos , Proteínas Recombinantes/metabolismo
15.
Endocrinology ; 145(6): 2876-85, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15016723

RESUMO

To better understand opioid receptor-like 1 (ORL1) internalization, we fused the C terminus of ORL1, the nociceptin (noc) receptor, to the N terminus of a green fluorescent protein and used the fusion protein to characterize receptor endocytosis in live human embryonic kidney cells. The fusion altered neither the affinity of the receptor for noc or other ORL1 receptor ligands nor the ability of the receptor to mediate agonist-induced binding of GTPgamma(35)S, i.e. coupling with heterotrimeric G protein. Confocal microscopy showed that the fluorescent receptor was mostly associated (>75%) with the periplasmic membrane. In the presence of 0.1 microm noc, approximately 80% of receptors were internalized, and half-maximum internalization was reached in approximately 12 min at 22 C and approximately 6 min at 37 C. After washing, a normal receptor level was recovered within 70 min at 22 C. The lack of internalization in the presence of 0.45 m sucrose suggests that noc-induced receptor endocytosis mainly occurred via clathrin-coated pits. Coincubation of the recombinant cells with noc and tetramethylrhodamine-transferrin showed that ORL1 was mainly internalized through the endosome compartment. Lofentanil and Ro64-6198 ([(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one]) promoted endocytosis of the fluorescent receptor as efficiently as noc. Among the two ORL1 receptor antagonists, J-113397 (1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one), but not III-BTD, blocked the noc-induced internalization of the fluorescent receptor. Two partial agonists were dramatically less efficient than noc to promote ORL1 internalization. They recruited very little (the pseudopeptide [Phe(1)psi(CH(2)-NH)Gly(2)]-noc-(1-13)NH(2)) or no (the hexapeptide Ac-Arg-Tyr-Tyr-Lys-Trp-Arg-NH(2)) G protein receptor kinase type 2 coupled to red fluorescent protein 1 at the membrane, suggesting that subsequent receptor phosphorylation necessary for internalization via coated pits is altered. Thus, partial agonists that induce a prolonged cell response without causing substantial receptor internalization may be good tools for further clinical treatments.


Assuntos
Rim/embriologia , Receptores Opioides/metabolismo , Transporte Biológico , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Embrião de Mamíferos/metabolismo , Endocitose , Proteínas de Fluorescência Verde , Humanos , Indicadores e Reagentes , Ligantes , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Antagonistas de Entorpecentes , Peptídeos Opioides/genética , Transporte Proteico , Receptores Opioides/agonistas , Proteínas Recombinantes de Fusão/metabolismo , Quinases de Receptores Adrenérgicos beta
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