4.7 Article

GRASP1 ubiquitination regulates AMPA receptor surface expression and synaptic activity in cultured hippocampal neurons

Journal

FASEB JOURNAL
Volume 35, Issue 8, Pages -

Publisher

WILEY
DOI: 10.1096/fj.201903061RR

Keywords

AMPA receptors; GRASP1; receptor trafficking; synaptic expression; ubiquitination

Funding

  1. Portuguese Science and Technology Foundation (FCT) [POCI-01-0145-FEDER-028656, UIDB/04539/2020, UIDP/04539/2020, SFRH/BPD/115546/2016, PD/BD/135498/2018, IF/01302/2012]
  2. European Regional Development Fund (ERDF), Centro 2020 Regional Operational Program, Operational Programme for Competitiveness and Internationalization-COMPETE 2020 [POCI-01-0145-FEDER-028656, CENTRO-01-0145-FEDER-000008:BrainHealth 2020]
  3. European Social Fund (ESF) [IF/01302/2012, BPD/115546/2016, BD/135498/2018]
  4. Fundação para a Ciência e a Tecnologia [PD/BD/135498/2018] Funding Source: FCT

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This study investigated the regulation of the synaptic protein GRASP1 by ubiquitin-dependent mechanisms, revealing an impact on the surface distribution and activity of AMPA receptors. Ubiquitination was found to decrease dendritic levels of GRASP1 and surface expression of GluA1 subunits, leading to a reduction in mEPSC amplitudes, effects that were partially reversed by cotransfection with GRASP1.
The synaptic expression of glutamate receptors of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) type is dynamically controlled by interaction with binding partners and auxiliary proteins. These proteins can be regulated by posttranslational modifications, including ubiquitination. In this work, we investigated the regulation of glutamate receptor interacting protein-associated protein 1 (GRASP1) by ubiquitin-dependent mechanisms and its impact on surface expression and activity of synaptic AMPA receptors. Cotransfection of GFP-ubiquitin decreased myc-GRASP1 protein levels in HEK293T cells, and this effect was inhibited upon transfection of an ubiquitin mutant that cannot be ubiquitinated on Lys48. In addition, transfection of cultured hippocampal neurons with GFP-ubiquitin reduced the dendritic levels of endogenous GRASP1 and decreased the surface expression of GluA1 AMPA receptor subunits, an effect that was partly reversed by cotransfection with GRASP1. Similarly, transfection of hippocampal neurons with GFP-ubiquitin decreased the amplitude of miniature excitatory postsynaptic currents (mEPSCs) mediated by Ca2+-impermeable AMPA receptors, and this effect was abrogated by cotransfection of GRASP1. Together, the results show a role for ubiquitination in the regulation of the postsynaptic protein GRASP1, which has an impact on the surface distribution of AMPA receptors and on their activity at the synapse.

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