Reticulon RTN2B Regulates Trafficking and Function of Neuronal Glutamate Transporter EAAC1

Abstract

Excitatory amino acid transporters (EAATs) are the primary regulators of extracellular glutamate concentrations in the central nervous system. Their dysfunction may contribute to several neurological diseases. To date, five distinct mammalian glutamate transporters have been cloned. In brain, EAAC1 (excitatory amino acid carrier 1) is the primary neuronal glutamate transporter, localized on the perisynaptic membranes that are near release sites. Despite its potential importance in synaptic actions, little is known concerning the regulation of EAAC1 trafficking from the endoplasmic reticulum (ER) to the cell surface. Previously, we identified an EAAC1-associated protein, GTRAP3-18, an ER protein that prevents ER exit of EAAC1 when induced. Here we show that RTN2B, a member of the reticulon protein family that mainly localizes in the ER and ER exit sites interacts with EAAC1 and GTRAP3-18. EAAC1 and GTRAP3-18 bind to different regions of RTN2B. Each protein can separately and independently form complexes with EAAC1. RTN2B enhances ER exit and the cell surface composition of EAAC1 in heterologous cells. Expression of short interfering RNA-mediated knockdown of RTN2B decreases the EAAC1 protein level in neurons. Overall, our results suggest that RTN2B functions as a positive regulator in the delivery of EAAC1 from the ER to the cell surface. These studies indicate that transporter exit from the ER controlled by the interaction with its ER binding partner represents a critical regulatory step in glutamate transporter trafficking to the cell surface.

Early-onset epileptic encephalopathy as the initial clinical presentation of WDR45 deletion in a male patient

“…In fact, the PRAF2 gene encodes a member of the PRA1 domain-containing protein family. This family is already involved in the regulation of glutamate transport 7,8 whose defects are implicated in epilepsy. 9 The function of PRAF2 is unknown, and the corresponding protein has a brain expression and is enriched in synaptic vesicles.…”

Regulation of Neuronal Glutathione Synthesis

“…GTRAP3-18 was identified by a yeast two-hybrid screening system using the carboxyl-terminal intracellular domain (arginine 438 -phenylalanine 524) of EAAC1 (73,82). A recent study using GTRAP3-18 transfected cell lines demonstrated GTRAP3-18 to be an integral ER membrane protein necessary for retaining EAAC1 at the ER as a trafficking regulator (89,92). Another EAAC1-regulating mechanism is phosphorylation of the carboxylterminal.…”

“…RTN2B, a member of the reticulon family of proteins, interacts with EAAC1 to enhance the endoplasmic reticulum (ER) exit process and cell surface expression of EAAC1 with increased glutamate transport activity (89). Rab1 is a GTPase, which is activated at the ER exit site, to support ER-Golgi trafficking of EAAC1 (90).…”

“…GTRAP3-18 is located in the ER and prevents EAAC1 maturation by restricting EAAC1 exit from the ER (89,92). An earlier investigation showed GTRAP3-18 to reduce EAAC1-mediated glutamate transport with no effect on translocation to the cell surface (82), while recent studies demonstrated an inhibitory effect on EAAC1 trafficking from the ER (89,90). Chronic morphine administration leads to a 3 -4 fold increase in GTRAP3-18 mRNA (93).…”

Location matters: the endoplasmic reticulum and protein trafficking in dendrites

“…For example, reticulons are a family of eukaryotic proteins associated to ER membranes that are responsible for keeping the ER's tubular shape (Sparkes et al, 2009;Voeltz et al, 2006). Interestingly, reticulon proteins RTN2B and RTN3 are distributed along neurites (Hu et al, 2007;Liu et al, 2008), and overexpression of RTN3 causes aggregation and neuritic dystrophy (Hu et al, 2007). In addition, a dynaminlike GTPase called atlastin-1 interacts directly with reticulon proteins promoting fusion and the formation of the tubular ER network.…”