{"id":107309,"date":"2011-11-03T00:00:00","date_gmt":"2011-11-03T00:00:00","guid":{"rendered":"https:\/\/www.deberes.net\/tesis\/sin-categoria\/estudio-de-la-regulacion-del-trafico-trafico-intracelular-del-transportador-de-glutamato-glt1\/"},"modified":"2011-11-03T00:00:00","modified_gmt":"2011-11-03T00:00:00","slug":"estudio-de-la-regulacion-del-trafico-trafico-intracelular-del-transportador-de-glutamato-glt1","status":"publish","type":"post","link":"https:\/\/www.deberes.net\/tesis\/biologia-molecular\/estudio-de-la-regulacion-del-trafico-trafico-intracelular-del-transportador-de-glutamato-glt1\/","title":{"rendered":"Estudio de la regulaci\u00f3n del tr\u00e1fico tr\u00e1fico intracelular del transportador de glutamato glt1."},"content":{"rendered":"<h2>Tesis doctoral de <strong> Noem\u00ed Garc\u00eda Tard\u00f3n <\/strong><\/h2>\n<p>El glutamato liberado sin\u00e1pticamente se inactiva por acci\u00f3n de sus transportadores, lo que impide la excesiva estimulaci\u00f3n de los receptores de glutamato que producen neurotoxicidad. Glt1 es el principal transportador de glutamato en el snc y su actividad y su tr\u00e1fico intracelular puede ser regulado por diferentes efectores y quinasas. Se sabe que la activaci\u00f3n de la pkc por \u00e9steres de forbol promueve la internalizaci\u00f3n del transportador mediante un sistema dependiente de clatrina. Sin embargo, los mecanismos moleculares por los que la activaci\u00f3n de pkc induce la internalizaci\u00f3n de glt1 no se conocen. En este trabajo, se muestra que el proceso de internalizaci\u00f3n es dependiente de la ubiquitinaci\u00f3n de los residuos de lisina localizados en el extremo carboxilo  de glt1. La exposici\u00f3n a pma aumenta la ubiquitinaci\u00f3n de glt1 en c\u00e9lulas transfectadas y la glt1 ubiquitinada se acumula en el compartimiento intracelular. La eliminaci\u00f3n de las siete lisinas de la c-terminal del transportador bloquea la ubiquinaci\u00f3n de glyt1 y su endocitosis.  Sin embargo, la reintroducci\u00f3n de la lisina 517 en este mutante era suficiente para restablecer la ubiquitinaci\u00f3n dependiente de pma y la internalizaci\u00f3n de glt1. Tambi\u00e9n se ha demostrado que la pkc fosforila glt1 en la serina 520 de su c-terminal en respuesta al tratamiento de pma, aunque esta fosforilaci\u00f3n no es necesaria para la ubiquitinaci\u00f3n de glt1 y posterior endocitosis. Adem\u00e1s, la ligasa e3 nedd4-2 parece participar en este proceso. Nuestros datos tambi\u00e9n demuestran que la ubiquitinaci\u00f3n juega un papel importante no s\u00f3lo en la endocitosis sino tambi\u00e9n en la regulaci\u00f3n de otros aspectos del tr\u00e1fico intracelular de glt1, como salida desde el ret\u00edculo endopl\u00e1smico y el tr\u00e1nsito a trav\u00e9s del aparato de golgi. Adem\u00e1s, la ubiquitinaci\u00f3n de glt1 podr\u00eda desempe\u00f1ar un papel en la regulaci\u00f3n de esta prote\u00edna observada en diversas patolog\u00edas como la esclerosis lateral amiotr\u00f3fica (als). En otra serie de experimentos se ha demostrado la interacci\u00f3n de ambas isoformas de glt1, glt1a y glt1b, con los mals, una prote\u00edna peque\u00f1a de andamiaje, en c\u00e9lulas cos7, aunque fuimos incapaces de detectar esta interacci\u00f3n en las neuronas. Finalmente, se ha desarrollado un sistema de expresi\u00f3n para glt1 usando el virus sindbis para estudiar el tr\u00e1fico intracelular in vivo de este transportador en cultivos primarios de neuronas de rata. Los resultados obtenidos por la t\u00e9cnica de frap indican que las isoformas a y b de glt1 no son est\u00e1ticas en las espinas dendr\u00edticas de las neuronas primarias, sin no que  se mueven r\u00e1pidamente dentro de ellas.  synaptically released glutamate is inactivated by glutamate transporters, thereby preventing the excessive stimulation of glutamate receptors that would produce neurotoxicity. Glt1 is the main glutamate transporter in the cns and its activity and intracellular traffic can be regulated by different effectors and kinases. It is known that activation of pkc by phorbol esters promotes the clathrin-dependent internalization of the transporter. However, the molecular mechanisms that link pkc activation and the internalization of glt1 are not fully understood. In this work, we show that this internalization process is dependent on the ubiquitination of lysine residues located in the c-terminal tail of glt1. Exposure to pma increases the ubiquitination of glt1 in transfected cells and this ubiquitinated glt1 accumulates in the intracellular compartment. The elimination of the seven lysines from the c-terminus of the transporter blocked glt1 ubiquitination and endocytosis. However, reintroduction of lysine 517 alone into this mutant was sufficient to restore pma dependent ubiquitination and internalization of glt1. We also have demonstrated that pkc phosphorylates glt1 in the serine 520 of its c-terminal tail in response to the treatment of pma, although this phosphorylation is not necessary for glt1 ubiquitination and subsequent endocytosis. Moreover, the e3 ligase nedd4-2 seems to participate in this process. We also provide evidences that ubiquitination plays an important role not only in endocytosis but also in the regulation of other aspects of the intracellular traffic of glt1, including exit from the endoplasmic reticulum and transit through the golgi apparatus. Furthermore, ubiquitination of glt1 might play a role in the down regulation of this protein observed in diverse pathologies like amyotrophic lateral sclerosis (als). In another series of experiments we have shown the interaction of both isoforms of glt1, glt1a y glt1b, with the small scaffold protein mals in cos7 cells, although we were unable to detect such an interaction in neurons. Finally, we have developed an expression system for glt1 using the sindbis virus to study the intracellular traffic in vivo of this transporter in primary cultures of rat neurons culture. The results obtained by frap technique indicate that the isoforms a and b of glt1 are not static at dendritic spines of primary neurones, but they move fast within them.<\/p>\n<p>&nbsp;<\/p>\n<h3>Datos acad\u00e9micos de la tesis doctoral \u00ab<strong>Estudio de la regulaci\u00f3n del tr\u00e1fico tr\u00e1fico intracelular del transportador de glutamato glt1.<\/strong>\u00ab<\/h3>\n<ul>\n<li><strong>T\u00edtulo de la tesis:<\/strong>\u00a0 Estudio de la regulaci\u00f3n del tr\u00e1fico tr\u00e1fico intracelular del transportador de glutamato glt1. <\/li>\n<li><strong>Autor:<\/strong>\u00a0 Noem\u00ed Garc\u00eda Tard\u00f3n <\/li>\n<li><strong>Universidad:<\/strong>\u00a0 Aut\u00f3noma de Madrid<\/li>\n<li><strong>Fecha de lectura de la tesis:<\/strong>\u00a0 11\/03\/2011<\/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>Cecilio Gimenez Martin<\/li>\n<\/ul>\n<\/li>\n<li><strong>Tribunal<\/strong>\n<ul>\n<li>Presidente del tribunal: Antonio Zorzano olarte <\/li>\n<li>Mar\u00eda ar\u00e1nzazu Tabernero urbieta (vocal)<\/li>\n<li>Jos\u00e9 Antonio Esteban garcia (vocal)<\/li>\n<li>Jos\u00e9 S\u00e1nchez-prieto borja (vocal)<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Tesis doctoral de Noem\u00ed Garc\u00eda Tard\u00f3n El glutamato liberado sin\u00e1pticamente se inactiva por acci\u00f3n de sus transportadores, lo que impide [&hellip;]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"site-sidebar-layout":"default","site-content-layout":"","ast-site-content-layout":"","site-content-style":"default","site-sidebar-style":"default","ast-global-header-display":"","ast-banner-title-visibility":"","ast-main-header-display":"","ast-hfb-above-header-display":"","ast-hfb-below-header-display":"","ast-hfb-mobile-header-display":"","site-post-title":"","ast-breadcrumbs-content":"","ast-featured-img":"","footer-sml-layout":"","theme-transparent-header-meta":"","adv-header-id-meta":"","stick-header-meta":"","header-above-stick-meta":"","header-main-stick-meta":"","header-below-stick-meta":"","astra-migrate-meta-layouts":"default","ast-page-background-enabled":"default","ast-page-background-meta":{"desktop":{"background-color":"var(--ast-global-color-4)","background-image":"","background-repeat":"repeat","background-position":"center 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