{"id":101886,"date":"2018-03-11T10:23:52","date_gmt":"2018-03-11T10:23:52","guid":{"rendered":"https:\/\/www.deberes.net\/tesis\/sin-categoria\/nuevas-funciones-celulares-de-grks-implicaciones-fisio-patologicas\/"},"modified":"2018-03-11T10:23:52","modified_gmt":"2018-03-11T10:23:52","slug":"nuevas-funciones-celulares-de-grks-implicaciones-fisio-patologicas","status":"publish","type":"post","link":"https:\/\/www.deberes.net\/tesis\/biologia-molecular\/nuevas-funciones-celulares-de-grks-implicaciones-fisio-patologicas\/","title":{"rendered":"Nuevas funciones celulares de grks: implicaciones fisio-patologicas"},"content":{"rendered":"<h2>Tesis doctoral de <strong> Mar\u00eda Jurado Pueyo <\/strong><\/h2>\n<p>En los \u00faltimos a\u00f1os, se est\u00e1n descubriendo un n\u00famero creciente de funciones celulares adicionales para las quinasas de receptores acoplados a prote\u00ednas g (grks), inicialmente conocidas por su participaci\u00f3n en la desensibilizaci\u00f3n de estos receptores. en este trabajo, aportamos nuevos datos sobre interacciones funcionales entre grks y mapks. En primer lugar, describimos que grk5 es capaz de regular funciones celulares dependientes de la subunidad \u00c2\u00bf12\/13 de las prote\u00ednas g heterotrim\u00e9ricas, a trav\u00e9s de su interacci\u00f3n con esta prote\u00edna preferentemente en su estado no activo. la fosforilaci\u00f3n de grk5 en s114, por erk mapk u otras quinasas, parece aumentar la afi nidad de grk5 por la forma activa de g\u00c2\u00bf12. por otro lado, al inicio de esta tesis nuestro grupo describi\u00f3 que grk2 fosforila a la mapk p38, inhibiendo su activaci\u00f3n y actividad. En el presente trabajo, detectamos cambios en la fosforilaci\u00f3n inhibitoria de p38 por grk2 en procesos como la diferenciaci\u00f3n de c\u00e9lulas adipoc\u00edticas y en la secreci\u00f3n de citoquinas infl amatorias por macr\u00f3fagos en respuesta a lps. Hemos generado nuevos fosfoanticuerpos para mejorar la detecci\u00f3n de esta fosforilaci\u00f3n y explorar otros procesos en los que podr\u00eda estar implicada. En general, no hemos hallado correspondencia entre los niveles de grk2 o est\u00edmulos que activan esta quinasa y la se\u00f1al detectada por los fosfo-anticuerpos anti-pt123-p38, indicando que este proceso debe de ser muy din\u00e1mico, muy espec\u00edfi co de contexto celular o modulado por factores adicionales. sin embargo, s\u00ed hemos detectado diferencias en el nivel de pt123-p38 en cardiomiocitos estimulados con isoproterenol, as\u00ed como en determinados tipos celulares en situaci\u00f3n \u00c2\u00bfbasal\u00c2\u00bf, aunque nos queda por defi nir el signifi cado biol\u00f3gico de estas diferencias. por \u00faltimo, hemos descrito que grk2 interacciona directamente y en c\u00e9lulas con mek, y que esta asociaci\u00f3n es m\u00e1s estable cuando mek est\u00e1 activa. El efecto inhibidor de grk2 sobre erk es principalmente independiente de la actividad quinasa de grk2 en varios tipos celulares tales como c\u00e9lulas epiteliales, esplenocitos y astrocitos. Sin embargo, grk2 no altera signifi cativamente la fosforilaci\u00f3n de erk por mek in vitro. Estos resultados, junto con otros descritos en esta memoria, parecen indicar que grk2 puede estar secuestrando a mek en determinadas localizaciones subcelulares. por otro lado, hemos analizado el efecto de la reducci\u00f3n cr\u00f3nica a nivel sist\u00e9mico de los niveles de la prote\u00edna grk2 en la patofi siolog\u00eda card\u00edaca, utilizando como modelo ratones hemicigotos grk2+\/-. La disminuci\u00f3n en los niveles de grk2 induce el desarrollo de una hipertrofi a cardiaca temprana con la edad, que parece ser de tipo compensado y no patol\u00f3gico, dado que no existe presencia de fi brosis o disfunci\u00f3n cardiaca aparente seg\u00fan un estudio ecocardiogr\u00e1fi co. adem\u00e1s, detectamos a los 9 meses una reprogramaci\u00f3n de la expresi\u00f3n g\u00e9nica en ratones grk2+\/-, con una reducci\u00f3n en la expresi\u00f3n de genes fetales indicativos de remodelado patol\u00f3gico, as\u00ed como un aumento en la expresi\u00f3n de marcadores de funcionalidad cardiaca, de tipo cardioprotector. la v\u00eda de se\u00f1alizaci\u00f3n cardioprotectora de mek-erk se encuentra sobre-activada a nivel basal en ratones hemicigotos para grk2 a los 9 meses. Adem\u00e1s, los ratones grk2+\/- muestran hipersensibilidad a insulina con un mayor incremento en la fosforilaci\u00f3n de componentes de esta ruta a diferentes niveles en coraz\u00f3n en respuesta a un tratamiento agudo con esta hormona. por el contrario, los niveles de grk2 est\u00e1n aumentados en muestras de pacientes con s\u00edndrome metab\u00f3lico. estos datos apoyan la existencia de una inhibici\u00f3n de la v\u00eda de insulina por grk2 a distintos niveles. Puesto que la v\u00eda de insulina media crecimiento tisular y es cardioprotectora, y dado que grk2 inhibe la ruta de erk tanto a nivel basal como inducida por insulina, el conjunto de nuestros datos puede explicar que en animales con menores niveles de grk2 la hiperactivaci\u00f3n de estas rutas promueva el desarrollo de hipertrofi a cardiaca y que \u00e9sta sea de tipo compensado. resumen\/summary an increasing number of cellular functions have been proposed in the last few years for g protein-coupled receptor kinases (grks), a family of proteins that were initially described by their role in gpcr desensitization. in the present work, we provide new insights into the functional interactions between grks and mitogen activated protein kinases (mapks). First, we describe that grk5 is able to modulate certain g\u00c2\u00bf12\/13-dependent cellular functions through the interaction of grk5 with this heterotrimeric g protein subunit, which is stabilized by the inactive state of g\u00c2\u00bf12\/13. Grk5 phosphorylation in s114, by erk mapk or other kinases, seems to increase the affi nity of grk5 for the active state of g\u00c2\u00bf12. in addition, in the beginning of this thesis, we described a new modulatory phosphorylation of p38 mapk in thr123, which was carried out by grk2, that inhibits both p38 activation and activity. In this work, we have detected changes is the inhibitory phosphorylation of p38 by grk2 in some cellular processes such as adipocytic cell differentiation or in the secretion of infl ammatory cytokines by macrophages in response to lipopolysaccaryde. We have generated a new pool of phospho-antibodies, in order to improve the detection of this phosphorylation, and to explore other cellular processes in which it may be involved. In the majority of the conditions tested, we have not been able to detect any correlation between grk2 levels, or grk2 stimulation by different means, and the detection of phospho-t123-p38, which indicates that this phosphorylation might be extremely dynamic, context-specifi c, or modulated by additional factors. Nevertheless, we have detected differences in pt123-p38 levels in isoproterenol-stimulated cardiomyocytes, and also basally in some other cellular types, although the biological meaning of these differences needs to be defi ned in more depth. we have also described that grk2 interacts with mek directly, and that this association is stabilized by the active state of mek. Through this mechanism, grk2 inhibits the activation of erk in different cell types such as epithelial cells, splenocytes or astrocytes, mainly in a kinase activityindependent manner. However, grk2 does not affect erk phosphorylation in vitro, which together with other results described in this thesis suggests that grk2 might sequester mek in particular subcellular localizations. on the other hand, we have analyzed the effect of a chronic and systemic reduction of grk2 levels in cardiac patho-physiology, using grk2+\/- hemizygous mice as a model. A decrease in grk2 levels induces the development of an early hypertrohic phenotype, which seems to be of a compensated non-pathological type, since no fi brosis or cardiac dysfunction, as analyzed by echocardiography, were detected. in addition, at the age of 9 months we observe a genetic reprogramming in grk2+\/- mice, with a reduction of some fetal genes indicative of pathological remodelling, and an increase in the transcription of cardioprotective markers. the cardio-protective signalling pathway mek-erk is basally over-activated in grk2+\/- mice at the age of 9 months. These mice also show insulin hypersensibility in cardiac tissue, with a higher increase of the phosphorylation of insulin pathway components at different levels in response to an acute insulin treatment. On the contrary, grk2 levels are increased in blood samples obtained from metabolic syndrome patients. in sum our data support the existence of an inhibition of the insulin pathway by grk2 at different levels. As this hormone mediates tissue growth and has cardio-protective effects, and taking into account grk2 inhibits erk pathway both at a basal state and after insulin stimulation, altogether these fi ndings might explain the fact that in these animals, which express lower grk2 levels, over-activation of these pathways promotes the development of a compensated heart hypertrophy.<\/p>\n<p>&nbsp;<\/p>\n<h3>Datos acad\u00e9micos de la tesis doctoral \u00ab<strong>Nuevas funciones celulares de grks: implicaciones fisio-patologicas<\/strong>\u00ab<\/h3>\n<ul>\n<li><strong>T\u00edtulo de la tesis:<\/strong>\u00a0 Nuevas funciones celulares de grks: implicaciones fisio-patologicas <\/li>\n<li><strong>Autor:<\/strong>\u00a0 Mar\u00eda Jurado Pueyo <\/li>\n<li><strong>Universidad:<\/strong>\u00a0 Aut\u00f3noma de Madrid<\/li>\n<li><strong>Fecha de lectura de la tesis:<\/strong>\u00a0 21\/06\/2010<\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n<h3>Direcci\u00f3n y tribunal<\/h3>\n<ul>\n<li><strong>Director de la tesis<\/strong>\n<ul>\n<li>Federico Mayor Menendez<\/li>\n<\/ul>\n<\/li>\n<li><strong>Tribunal<\/strong>\n<ul>\n<li>Presidente del tribunal: jorgina Satr\u00fastegui gil-delgado <\/li>\n<li>almudena Porras gallo (vocal)<\/li>\n<li>Jos\u00e9 Mar\u00eda Carrascosa baeza (vocal)<\/li>\n<li>sonia Fernandez veledo (vocal)<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Tesis doctoral de Mar\u00eda Jurado Pueyo En los \u00faltimos a\u00f1os, se est\u00e1n descubriendo un n\u00famero creciente de funciones celulares adicionales 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