Identification of novel regulatory partners of the glutamate transporter GLT‐1

We used proximity‐dependent biotin identification (BioID) to find proteins that potentially interact with the major glial glutamate transporter, GLT‐1, and we studied how these interactions might affect its activity. GTPase Rac1 was one protein identified, and interfering with its GTP/GDP cycle in m...

Full description

Saved in:
Bibliographic Details
Published in:Glia 2018-12, Vol.66 (12), p.2737-2755
Main Authors: Piniella, Dolores, Martínez‐Blanco, Elena, Ibáñez, Ignacio, Bartolomé‐Martín, David, Porlan, Eva, Díez‐Guerra, Javier, Giménez, Cecilio, Zafra, Francisco
Format: Article
Language:English
Subjects:
Actin
Animals
Astrocytes
Biological Transport - drug effects
Biological Transport - genetics
Biotin
Biotinylation
Brain
Cdc42 protein
Cell surface
Cells, Cultured
Cercopithecus aethiops
Cerebral Cortex - cytology
Clustering
COS Cells
Cytoskeleton
Embryo, Mammalian
Excitatory Amino Acid Transporter 2 - genetics
Excitatory Amino Acid Transporter 2 - metabolism
glutamate transporters
Glutamic Acid
Glutamic acid receptors (metabotropic)
Glutamic acid transporter
Glutathione Transferase - genetics
Glutathione Transferase - metabolism
Guanosine diphosphate
Guanosine triphosphatases
Guanosine triphosphate
HEK293 Cells
Homeostasis
Humans
Immunoprecipitation
Kinetics
Models, Molecular
Neuroglia - drug effects
Neuroglia - metabolism
Neurons - metabolism
Photobleaching
Proteins
proteomics
rac1 GTP-Binding Protein - metabolism
Rac1 protein
Rats
Receptors
Rodents
signaling
Transfection
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:We used proximity‐dependent biotin identification (BioID) to find proteins that potentially interact with the major glial glutamate transporter, GLT‐1, and we studied how these interactions might affect its activity. GTPase Rac1 was one protein identified, and interfering with its GTP/GDP cycle in mixed primary rat brain cultures affected both the clustering of GLT‐1 at the astrocytic processes and the transport kinetics, increasing its uptake activity at low micromolar glutamate concentrations in a manner that was dependent on the effector kinase PAK1 and the actin cytoskeleton. Interestingly, the same manipulations had a different effect on another glial glutamate transporter, GLAST, inhibiting its activity. Importantly, glutamate acts through metabotropic receptors to stimulate the activity of Rac1 in astrocytes, supporting the existence of cross‐talk between extracellular glutamate and the astrocytic form of the GLT‐1 regulated by Rac1. CDC42EP4/BORG4 (a CDC42 effector) was also identified in the BioID screen, and it is a protein that regulates the assembly of septins and actin fibers, influencing the organization of the cytoskeleton. We found that GLT‐1 interacts with septins, which reduces its lateral mobility at the cell surface. Finally, the G‐protein subunit GNB4 dampens the activity of GLT‐1, as revealed by its response to the activator peptide mSIRK, both in heterologous systems and in primary brain cultures. This effect occurs rapidly and thus, it is unlikely to depend on cytoskeletal dynamics. These novel interactions shed new light on the events controlling GLT‐1 activity, thereby helping us to better understand how glutamate homeostasis is maintained in the brain. Main Points By using a proximity labelling technique, we identified potential interactors of GLT‐1. Regulators of the cytoskeleton (Rac1 or BORG4) and signaling proteins (βγ subunits of heteromeric G‐proteins) might regulate the activity glutamate transporters.
ISSN:0894-1491
1098-1136
DOI:10.1002/glia.23524