RESUMEN
Beta-arrestins-1 and 2 are known to play important roles in desensitization of membrane receptors and facilitation of signal transduction pathways. It has been previously shown that beta-arrestins are required for signal termination, internalization, and ERK1/2 activation downstream of protease-activated-receptor-2 (PAR-2), but it is unclear whether they are functionally redundant or mediate specific events. Here, we demonstrate that in mouse embryonic fibroblasts (MEFs) from beta-arrestin-1/2 knockout mice, G alpha q signaling by PAR-2, as measured by mobilization of intracellular Ca(2+), is prolonged. Only expression of beta-arrestin-1 shortened the signal duration, whereas either beta-arrestin-1 or 2 was able to restore PKC-induced receptor desensitization. Beta-arrestin-1 also mediated early, while beta-arrestin-2 mediated delayed, receptor internalization and membrane-associated ERK1/2 activation. While beta-arrestin-1 colocalized with a lysosomal marker (LAMP-1), beta-arrestin-2 did not, suggesting a specific role for beta-arrestin-1 in lysosomal receptor degradation. Together, these data suggest distinct temporal and functional roles for beta-arrestins in PAR-2 signaling, desensitization, and internalization.
Asunto(s)
Arrestinas/metabolismo , Señalización del Calcio , Endocitosis , Fibroblastos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Receptor PAR-2/metabolismo , Animales , Arrestinas/deficiencia , Arrestinas/genética , Células CHO , Señalización del Calcio/efectos de los fármacos , Línea Celular Tumoral , Cricetinae , Cricetulus , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Endocitosis/efectos de los fármacos , Activación Enzimática , Exocitosis , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Cinética , Proteína 1 de la Membrana Asociada a los Lisosomas/metabolismo , Lisosomas/metabolismo , Ratones , Ratones Noqueados , Oligopéptidos/farmacología , Isoformas de Proteínas/metabolismo , Proteína Quinasa C/metabolismo , Receptor PAR-2/agonistas , Receptor PAR-2/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Tripsina/metabolismo , beta-Arrestina 1 , Arrestina beta 2 , beta-ArrestinasRESUMEN
An understanding of the mechanisms that regulate signaling by the substance P (SP) or neurokinin 1 receptor (NK1-R) is of interest because of their role in inflammation and pain. By using activators and inhibitors of protein kinase C (PKC) and NK1-R mutations of potential PKC phosphorylation sites, we determined the role of PKC in desensitization of responses to SP. Activation of PKC abolished SP-induced Ca(2+) mobilization in cells that express wild-type NK1-R. This did not occur in cells expressing a COOH-terminally truncated NK1-R (NK1-Rdelta324), which may correspond to a naturally occurring variant, or a point mutant lacking eight potential PKC phosphorylation sites within the COOH tail (NK1-R Ser-338, Thr-339, Ser-352, Ser-387, Ser-388, Ser-390, Ser-392, Ser-394/Ala, NK1-RKC4). Compared with wild-type NK1-R, the t(1/2) of SP-induced Ca(2+) mobilization was seven- and twofold greater in cells expressing NK1-Rdelta324 and NK1-RKC4, respectively. In cells expressing wild-type NK1-R, inhibition of PKC caused a 35% increase in the t(1/2) of SP-induced Ca(2+) mobilization. Neither inhibition of PKC nor receptor mutation affected desensitization of Ca(2+) mobilization to repeated challenge with SP or SP-induced endocytosis of the NK1-R. Thus PKC regulates SP-induced Ca(2+) mobilization by full-length NK1-R and does not regulate a naturally occurring truncated variant. PKC does not mediate desensitization to repeated stimulation or endocytosis of the NK1-R.
Asunto(s)
Señalización del Calcio/fisiología , Proteína Quinasa C/metabolismo , Receptores de Neuroquinina-1/fisiología , Sustancia P/farmacología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Señalización del Calcio/efectos de los fármacos , Línea Celular , Cartilla de ADN , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Indoles , Riñón , Maleimidas , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Forbol 12,13-Dibutirato/farmacología , Fosforilación , Fosfoserina/metabolismo , Fosfotreonina/metabolismo , Conformación Proteica , Ratas , Receptores de Neuroquinina-1/química , Receptores de Neuroquinina-1/efectos de los fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , TransfecciónRESUMEN
A requirement for scaffolding complexes containing internalized G protein-coupled receptors and beta-arrestins in the activation and subcellular localization of extracellular signal-regulated kinases 1 and 2 (ERK1/2) has recently been proposed. However, the composition of these complexes and the importance of this requirement for function of ERK1/2 appear to differ between receptors. Here we report that substance P (SP) activation of neurokinin-1 receptor (NK1R) stimulates the formation of a scaffolding complex comprising internalized receptor, beta-arrestin, src, and ERK1/2 (detected by gel filtration, immunoprecipitation, and immunofluorescence). Inhibition of complex formation, by expression of dominant-negative beta-arrestin or a truncated NK1R that fails to interact with beta-arrestin, inhibits both SP-stimulated endocytosis of the NK1R and activation of ERK1/2, which is required for the proliferative and antiapoptotic effects of SP. Thus, formation of a beta-arrestin-containing complex facilitates the proliferative and antiapoptotic effects of SP, and these effects of SP could be diminished in cells expressing truncated NK1R corresponding to a naturally occurring variant.
Asunto(s)
Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Arrestinas/fisiología , Receptores de Neuroquinina-1/fisiología , Transducción de Señal/efectos de los fármacos , Sustancia P/farmacología , Animales , División Celular/efectos de los fármacos , División Celular/fisiología , Línea Celular , Sistema de Señalización de MAP Quinasas , Ratas , beta-ArrestinasRESUMEN
Recently, a requirement for beta-arrestin-mediated endocytosis in the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by several G protein-coupled receptors (GPCRs) has been proposed. However, the importance of this requirement for function of ERK1/2 is unknown. We report that agonists of Galphaq-coupled proteinase-activated receptor 2 (PAR2) stimulate formation of a multiprotein signaling complex, as detected by gel filtration, immunoprecipitation and immunofluorescence. The complex, which contains internalized receptor, beta-arrestin, raf-1, and activated ERK, is required for ERK1/2 activation. However, ERK1/2 activity is retained in the cytosol and neither translocates to the nucleus nor causes proliferation. In contrast, a mutant PAR2 (PAR2deltaST363/6A), which is unable to interact with beta-arrestin and, thus, does not desensitize or internalize, activates ERK1/2 by a distinct pathway, and fails to promote both complex formation and cytosolic retention of the activated ERK1/2. Whereas wild-type PAR2 activates ERK1/2 by a PKC-dependent and probably a ras-independent pathway, PAR2(deltaST363/6A) appears to activate ERK1/2 by a ras-dependent pathway, resulting in increased cell proliferation. Thus, formation of a signaling complex comprising PAR2, beta-arrestin, raf-1, and activated ERK1/2 might ensure appropriate subcellular localization of PAR2-mediated ERK activity, and thereby determine the mitogenic potential of receptor agonists.
Asunto(s)
Arrestinas/fisiología , Endocitosis , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Receptores de Trombina/fisiología , Animales , Calcio/metabolismo , División Celular , Línea Celular , Núcleo Celular/fisiología , Núcleo Celular/ultraestructura , Citosol/fisiología , Citosol/ultraestructura , Activación Enzimática , Humanos , Cinética , Microscopía Confocal , Proteína Quinasa 3 Activada por Mitógenos , Modelos Biológicos , Mutagénesis , Ratas , Receptor PAR-2 , Receptores de Trombina/genética , Proteínas Recombinantes/metabolismo , Transfección , beta-ArrestinasRESUMEN
At the endoplasmic reticulum membrane, the prion protein (PrP) can be synthesized in several topological forms. The role of these different forms was explored with transgenic mice expressing PrP mutations that alter the relative ratios of the topological forms. Expression of a particular transmembrane form (termed CtmPrP) produced neurodegenerative changes in mice similar to those of some genetic prion diseases. Brains from these mice contained CtmPrP but not PrPSc, the PrP isoform responsible for transmission of prion diseases. Furthermore, in one heritable prion disease of humans, brain tissue contained CtmPrP but not PrPSc. Thus, aberrant regulation of protein biogenesis and topology at the endoplasmic reticulum can result in neurodegeneration.