title: Activity dependent internalization of the glutamate transporter GLT-1 mediated by β-arrestin 1 and ubiquitination.
abstract: GLT-1 is the main glutamate transporter in the brain and undergoes trafficking processes that control its concentration on the cell surface thereby shaping glutamatergic neurotransmission. We have investigated how the traffic of GLT-1 is regulated by transporter activity. We report that internalization of GLT-1 from the cell surface is accelerated by transportable substrates like glutamate or aspartate, as well as by the transportable inhibitor L-trans-2,4-PDC, but not by the non-substrate inhibitor WAY 213613 in primary mixed cultures and in transiently transfected HEK293 cells. Analysis of the mechanism of endocytosis in HEK293 cells revealed that glutamate promoted the association with the transporter of the adaptor protein β-arrestin and the ubiquitin ligase Nedd4-2. The addition of glutamate is accompanied by an increase in the transporter ubiquitination, and the internalization is suppressed by an ubiquitination inhibitor (PYR41), and in a mutant defective in C-terminal lysines. The glutamate triggered endocytosis was also suppressed by siRNA for β-arrestin. This regulatory mechanism might be relevant in controlling the amount of transporter on the cell surface in conditions such as ischemia or traumatic brain injury, where extracellular concentrations of glutamate are persistently elevated.
authors.affiliation: Centro de Biología Molecular Severo Ochoa, Facultad de Ciencias, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Madrid, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras, Spain; IdiPAZ, Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Ibáñez
authors.affiliation: Centro de Biología Molecular Severo Ochoa, Facultad de Ciencias, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Madrid, Spain.
authors.forename: F Javier
authors.initials: FJ
authors.lastname: Díez-Guerra
authors.affiliation: Centro de Biología Molecular Severo Ochoa, Facultad de Ciencias, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Madrid, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras, Spain; IdiPAZ, Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: Cecilio
authors.initials: C
authors.lastname: Giménez
authors.affiliation: Centro de Biología Molecular Severo Ochoa, Facultad de Ciencias, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Madrid, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras, Spain; IdiPAZ, Instituto de Salud Carlos III, Madrid, Spain. Electronic address: fzafra@cbm.csic.es.
authors.forename: Francisco
authors.initials: F
authors.lastname: Zafra
mesh_terms: D000818:Animals; D002478:Cells, Cultured / Q000648:ultrastructure; D002540:Cerebral Cortex / Q000187:drug effects / Q000502:physiology; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D027342:Excitatory Amino Acid Transporter 2 / Q000378:metabolism*; D018698:Glutamic Acid / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D051381:Rats; D017208:Rats, Wistar; D054875:Ubiquitination / Q000187:drug effects / Q000502:physiology*; D000071559:beta-Arrestin 1 / Q000502:physiology*
chemical_list: D027342:Excitatory Amino Acid Transporter 2; C493395:Slc1a2 protein, rat; D000071559:beta-Arrestin 1; D018698:Glutamic Acid
keywords: Endocytosis; Glutamate; Intracellular trafficking; Transport; Ubiquitination