Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 8 de 8
Filtrar
Más filtros

Banco de datos
Tipo del documento
Asunto de la revista
Intervalo de año de publicación
1.
Mol Pharmacol ; 91(2): 75-86, 2017 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-27895162

RESUMEN

Cannabinoid receptor interacting protein 1a (CRIP1a) is a CB1 receptor (CB1R) distal C-terminal-associated protein that alters CB1R interactions with G-proteins. We tested the hypothesis that CRIP1a is capable of also altering CB1R interactions with ß-arrestin proteins that interact with the CB1R at the C-terminus. Coimmunoprecipitation studies indicated that CB1R associates in complexes with either CRIP1a or ß-arrestin, but CRIP1a and ß-arrestin fail to coimmunoprecipitate with each other. This suggests a competition for CRIP1a and ß-arrestin binding to the CB1R, which we hypothesized could attenuate the action of ß-arrestin to mediate CB1R internalization. We determined that agonist-mediated reduction of the density of cell surface endogenously expressed CB1Rs was clathrin and dynamin dependent and could be modeled as agonist-induced aggregation of transiently expressed GFP-CB1R. CRIP1a overexpression attenuated CP55940-mediated GFP-CB1R as well as endogenous ß-arrestin redistribution to punctae, and conversely, CRIP1a knockdown augmented ß-arrestin redistribution to punctae. Peptides mimicking the CB1R C-terminus could bind to both CRIP1a in cell extracts as well as purified recombinant CRIP1a. Affinity pull-down studies revealed that phosphorylation at threonine-468 of a CB1R distal C-terminus 14-mer peptide reduced CB1R-CRIP1a association. Coimmunoprecipitation of CB1R protein complexes demonstrated that central or distal C-terminal peptides competed for the CB1R association with CRIP1a, but that a phosphorylated central C-terminal peptide competed for association with ß-arrestin 1, and phosphorylated central or distal C-terminal peptides competed for association with ß-arrestin 2. Thus, CRIP1a can compete with ß-arrestins for interaction with C-terminal CB1R domains that could affect agonist-driven, ß-arrestin-mediated internalization of the CB1R.


Asunto(s)
Proteínas Portadoras/metabolismo , Receptor Cannabinoide CB1/metabolismo , beta-Arrestinas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteínas de la Membrana , Péptidos/química , Fosforilación , Unión Proteica , Ratas
2.
J Neurochem ; 139(3): 396-407, 2016 11.
Artículo en Inglés | MEDLINE | ID: mdl-27513693

RESUMEN

Cannabinoid receptor interacting protein 1a (CRIP1a) is a CB1 receptor (CB1 R) distal C-terminus-associated protein that modulates CB1 R signaling via G proteins, and CB1 R down-regulation but not desensitization (Blume et al. [2015] Cell Signal., 27, 716-726; Smith et al. [2015] Mol. Pharmacol., 87, 747-765). In this study, we determined the involvement of CRIP1a in CB1 R plasma membrane trafficking. To follow the effects of agonists and antagonists on cell surface CB1 Rs, we utilized the genetically homogeneous cloned neuronal cell line N18TG2, which endogenously expresses both CB1 R and CRIP1a, and exhibits a well-characterized endocannabinoid signaling system. We developed stable CRIP1a-over-expressing and CRIP1a-siRNA-silenced knockdown clones to investigate gene dose effects of CRIP1a on CB1 R plasma membrane expression. Results indicate that CP55940 or WIN55212-2 (10 nM, 5 min) reduced cell surface CB1 R by a dynamin- and clathrin-dependent process, and this was attenuated by CRIP1a over-expression. CP55940-mediated cell surface CB1 R loss was followed by a cycloheximide-sensitive recovery of surface receptors (30-120 min), suggesting the requirement for new protein synthesis. In contrast, WIN55212-2-mediated cell surface CB1 Rs recovered only in CRIP1a knockdown cells. Changes in CRIP1a expression levels did not affect a transient rimonabant (10 nM)-mediated increase in cell surface CB1 Rs, which is postulated to be as a result of rimonabant effects on 'non-agonist-driven' internalization. These studies demonstrate a novel role for CRIP1a in agonist-driven CB1 R cell surface regulation postulated to occur by two mechanisms: 1) attenuating internalization that is agonist-mediated, but not that in the absence of exogenous agonists, and 2) biased agonist-dependent trafficking of de novo synthesized receptor to the cell surface.


Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/metabolismo , Animales , Benzoxazinas/farmacología , Línea Celular , Membrana Celular/metabolismo , Ciclohexanoles/farmacología , Endocannabinoides/fisiología , Dosificación de Gen , Técnicas de Silenciamiento del Gen , Ratones , Morfolinas/farmacología , Naftalenos/farmacología , Piperidinas/farmacología , Transporte de Proteínas , Pirazoles/farmacología , ARN Interferente Pequeño , Receptor Cannabinoide CB1/genética , Receptores de Superficie Celular/efectos de los fármacos , Rimonabant , Transducción de Señal/genética
3.
Mol Pharmacol ; 87(4): 747-65, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25657338

RESUMEN

Cannabinoid CB1 receptors (CB1Rs) mediate the presynaptic effects of endocannabinoids in the central nervous system (CNS) and most behavioral effects of exogenous cannabinoids. Cannabinoid receptor-interacting protein 1a (CRIP1a) binds to the CB1R C-terminus and can attenuate constitutive CB1R-mediated inhibition of Ca(2+) channel activity. We now demonstrate cellular colocalization of CRIP1a at neuronal elements in the CNS and show that CRIP1a inhibits both constitutive and agonist-stimulated CB1R-mediated guanine nucleotide-binding regulatory protein (G-protein) activity. Stable overexpression of CRIP1a in human embryonic kidney (HEK)-293 cells stably expressing CB1Rs (CB1-HEK), or in N18TG2 cells endogenously expressing CB1Rs, decreased CB1R-mediated G-protein activation (measured by agonist-stimulated [(35)S]GTPγS (guanylyl-5'-[O-thio]-triphosphate) binding) in both cell lines and attenuated inverse agonism by rimonabant in CB1-HEK cells. Conversely, small-interfering RNA-mediated knockdown of CRIP1a in N18TG2 cells enhanced CB1R-mediated G-protein activation. These effects were not attributable to differences in CB1R expression or endocannabinoid tone because CB1R levels did not differ between cell lines varying in CRIP1a expression, and endocannabinoid levels were undetectable (CB1-HEK) or unchanged (N18TG2) by CRIP1a overexpression. In CB1-HEK cells, 4-hour pretreatment with cannabinoid agonists downregulated CB1Rs and desensitized agonist-stimulated [(35)S]GTPγS binding. CRIP1a overexpression attenuated CB1R downregulation without altering CB1R desensitization. Finally, in cultured autaptic hippocampal neurons, CRIP1a overexpression attenuated both depolarization-induced suppression of excitation and inhibition of excitatory synaptic activity induced by exogenous application of cannabinoid but not by adenosine A1 agonists. These results confirm that CRIP1a inhibits constitutive CB1R activity and demonstrate that CRIP1a can also inhibit agonist-stimulated CB1R signaling and downregulation of CB1Rs. Thus, CRIP1a appears to act as a broad negative regulator of CB1R function.


Asunto(s)
Proteínas Portadoras/metabolismo , Receptor Cannabinoide CB1/metabolismo , Animales , Proteínas Portadoras/genética , Línea Celular , Cerebelo/metabolismo , Endocannabinoides/metabolismo , Proteínas de Unión al GTP/metabolismo , Hipocampo/citología , Hipocampo/metabolismo , Humanos , Masculino , Ratones , Neuronas/metabolismo , Ensayo de Unión Radioligante , Ratas Sprague-Dawley , Receptor Cannabinoide CB1/agonistas , Transducción de Señal
4.
J Neurochem ; 124(6): 808-20, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23286559

RESUMEN

Although biochemical and physiological evidence suggests a strong interaction between striatal CB1 cannabinoid (CB1 R) and D2 dopamine (D2 R) receptors, the mechanisms are poorly understood. We targeted medium spiny neurons of the indirect pathway using shRNA to knockdown either CB1 R or D2 R. Chronic reduction in either receptor resulted in deficits in gene and protein expression for the alternative receptor and concomitantly increased expression of the cannabinoid receptor interacting protein 1a (CRIP1a), suggesting a novel role for CRIP1a in dopaminergic systems. Both CB1 R and D2 R knockdown reduced striatal dopaminergic-stimulated [(35) S]GTPγS binding, and D2 R knockdown reduced pallidal WIN55212-2-stimulated [(35) S]GTPγS binding. Decreased D2 R and CB1 R activity was associated with decreased striatal phosphoERK. A decrease in mRNA for opioid peptide precursors pDYN and pENK accompanied knockdown of CB1 Rs or D2 Rs, and over-expression of CRIP1a. Down-regulation in opioid peptide mRNAs was followed in time by increased DOR1 but not MOR1 expression, leading to increased [D-Pen2, D-Pen5]-enkephalin-stimulated [(35) S]GTPγS binding in the striatum. We conclude that mechanisms intrinsic to striatal medium spiny neurons or extrinsic via the indirect pathway adjust for changes in CB1 R or D2 R levels by modifying the expression and signaling capabilities of the alternative receptor as well as CRIP1a and the DELTA opioid system.


Asunto(s)
Proteínas Portadoras/biosíntesis , Cuerpo Estriado/metabolismo , Receptor Cannabinoide CB1/fisiología , Receptores de Dopamina D2/fisiología , Receptores Opioides delta/biosíntesis , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Antagonistas de los Receptores de Dopamina D2 , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen/métodos , Masculino , Ratones , Ratas , Ratas Sprague-Dawley , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptores Opioides delta/genética
5.
Front Behav Neurosci ; 10: 248, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-28154530

RESUMEN

Recent studies have used conditional knockout mice to selectively delete the D2 autoreceptor; however, these approaches result in global deletion of D2 autoreceptors early in development. The present study takes a different approach using RNA interference (RNAi) to knockdown the expression of the D2 receptors (D2R) in the substantia nigra (SN), including dopaminergic neurons, which project primarily to the dorsal striatum (dStr) in adult rats. This approach restricts the knockdown primarily to nigrostriatal pathways, leaving mesolimbic D2 autoreceptors intact. Analyses of dopamine (DA) kinetics in the dStr reveal a decrease in DA transporter (DAT) function in the knockdown rats, an effect not observed in D2 autoreceptor knockout mouse models. SN D2 knockdown rats exhibit a behavioral phenotype characterized by persistent enhancement of locomotor activity in a familiar open field, reduced locomotor responsiveness to high doses of cocaine and the ability to overcome haloperidol-induced immobility on the bar test. Together these results demonstrate that presynaptic D2R can be depleted from specific neuronal populations and implicates nigrostriatal D2R in different behavioral responses to psychotropic drugs.

6.
Cell Signal ; 27(3): 716-726, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25446256

RESUMEN

CB1 cannabinoid receptors (CB1R) are one of the most abundantly expressed G protein coupled receptors (GPCR) in the CNS and regulate diverse neuronal functions. The identification of GPCR interacting proteins has provided additional insight into the fine-tuning and regulation of numerous GPCRs. The cannabinoid receptor interacting protein 1a (CRIP1a) binds to the distal carboxy terminus of CB1R, and has been shown to alter CB1R-mediated neuronal function [1]. The mechanisms by which CRIP1a regulates CB1R activity have not yet been identified; therefore the focus of this investigation is to examine the cellular effects of CRIP1a on CB1R signaling using neuronal N18TG2 cells stably transfected with CRIP1a over-expressing and CRIP1a knockdown constructs. Modulation of endogenous CRIP1a expression did not alter total levels of CB1R, ERK, or forskolin-activated adenylyl cyclase activity. When compared to WT cells, CRIP1a over-expression reduced basal phosphoERK levels, whereas depletion of CRIP1a augmented basal phosphoERK levels. Stimulation of phosphoERK by the CB1R agonists WIN55212-2, CP55940 or methanandamide was unaltered in CRIP1a over-expressing clones compared with WT. However, CRIP1a knockdown clones exhibited enhanced ERK phosphorylation efficacy in response to CP55940. In addition, CRIP1a knockdown clones displayed a leftward shift in CP55940-mediated inhibition of forskolin-stimulated cAMP accumulation. CB1R-mediated Gi3 and Go activation by CP99540 was attenuated by CRIP1a over-expression, but robustly enhanced in cells depleted of CRIP1a. Conversely, CP55940-mediated Gi1 and Gi2 activation was significant enhanced in cells over-expressing CRIP1a, but not in cells deficient of CRIP1a. These studies suggest a mechanism by which endogenous levels of CRIP1a modulate CB1R-mediated signal transduction by facilitating a Gi/o protein subtype preference for Gi1 and Gi2, accompanied by an overall suppression of G-protein-mediated signaling in neuronal cells.


Asunto(s)
Proteínas Portadoras/metabolismo , Receptor Cannabinoide CB1/metabolismo , Animales , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Línea Celular Tumoral , AMP Cíclico/metabolismo , Ciclohexanoles/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación/efectos de los fármacos , Piperidinas/farmacología , Pirazoles/farmacología , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/genética , Rimonabant , Transducción de Señal
7.
Cell Rep ; 5(2): 508-20, 2013 Oct 31.
Artículo en Inglés | MEDLINE | ID: mdl-24095738

RESUMEN

The serine hydrolase α/ß hydrolase domain 6 (ABHD6) has recently been implicated as a key lipase for the endocannabinoid 2-arachidonylglycerol (2-AG) in the brain. However, the biochemical and physiological function for ABHD6 outside of the central nervous system has not been established. To address this, we utilized targeted antisense oligonucleotides (ASOs) to selectively knock down ABHD6 in peripheral tissues in order to identify in vivo substrates and understand ABHD6's role in energy metabolism. Here, we show that selective knockdown of ABHD6 in metabolic tissues protects mice from high-fat-diet-induced obesity, hepatic steatosis, and systemic insulin resistance. Using combined in vivo lipidomic identification and in vitro enzymology approaches, we show that ABHD6 can hydrolyze several lipid substrates, positioning ABHD6 at the interface of glycerophospholipid metabolism and lipid signal transduction. Collectively, these data suggest that ABHD6 inhibitors may serve as therapeutics for obesity, nonalcoholic fatty liver disease, and type II diabetes.


Asunto(s)
Síndrome Metabólico/enzimología , Monoacilglicerol Lipasas/metabolismo , Secuencia de Aminoácidos , Animales , Dieta Alta en Grasa , Endocannabinoides/metabolismo , Ácidos Grasos/biosíntesis , Humanos , Hígado/enzimología , Hígado/metabolismo , Masculino , Síndrome Metabólico/metabolismo , Síndrome Metabólico/patología , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Monoacilglicerol Lipasas/antagonistas & inhibidores , Monoacilglicerol Lipasas/genética , Obesidad/prevención & control , Oligonucleótidos Antisentido/metabolismo , Receptor Cannabinoide CB1/metabolismo , Alineación de Secuencia , Transducción de Señal
8.
Curr Med Chem ; 17(14): 1382-93, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-20166926

RESUMEN

CB1 receptors are G-protein coupled receptors (GPCRs) abundant in neurons, in which they modulate neurotransmission. The CB(1) receptor influence on memory and learning is well recognized, and disease states associated with CB(1) receptors are observed in addiction disorders, motor dysfunction, schizophrenia, and in bipolar, depression, and anxiety disorders. Beyond the brain, CB(1) receptors also function in liver and adipose tissues, vascular as well as cardiac tissue, reproductive tissues and bone. Signal transduction by CB(1) receptors occurs through interaction with Gi/o proteins to inhibit adenylyl cyclase, activate mitogen-activated protein kinases (MAPK), inhibit voltage-gated Ca(2+) channels, activate K(+) currents (K(ir)), and influence Nitric Oxide (NO) signaling. CB(1) receptors are observed in internal organelles as well as plasma membrane. beta-Arrestins, adaptor protein AP-3, and G-protein receptor-associated sorting protein 1 (GASP1) modulate cellular trafficking. Cannabinoid Receptor Interacting Protein1a (CRIP1a) is an accessory protein whose function has not been delineated. Factor Associated with Neutral sphingomyelinase (FAN) regulates ceramide signaling. Such diversity in cellular signaling and modulation by interacting proteins suggests that agonists and allosteric modulators could be developed to specifically regulate unique, cell type-specific responses.


Asunto(s)
Receptor Cannabinoide CB1/metabolismo , Complejo 3 de Proteína Adaptadora/metabolismo , Adenilil Ciclasas/metabolismo , Arrestinas/metabolismo , Canales de Calcio/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Óxido Nítrico/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Proteínas de Transporte Vesicular/metabolismo
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA