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. 2008 Mar 7;283(10):6561-71.
doi: 10.1074/jbc.M708096200. Epub 2007 Dec 20.

Reticulon RTN2B regulates trafficking and function of neuronal glutamate transporter EAAC1

Yiting Liu  1 , Svetlana Vidensky, Alicia M Ruggiero, Susanne Maier, Harald H Sitte, Jeffrey D Rothstein
Affiliations

Reticulon RTN2B regulates trafficking and function of neuronal glutamate transporter EAAC1

Yiting Liu et al. J Biol Chem. .

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.

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Figures

FIGURE 1
FIGURE 1. Interactions of RTN2B with GTRAP3-18 and EAAC1
A and B, interaction of RTN2B and GTRAP3-18 in living cells as shown by FRET. Three-filter FRET microscopy was performed on COS7 cells transfected with equal amounts of CFP-RTN2B and YFP-GTRAP3-18. A representative single experiment taken in the CFP, YFP, or FRET channel is shown (A). B, a summary of 6 experimental days with 25 to 35 evaluated regions. NFRET was calculated as described under “Experimental Procedures” (n = 6, *, p <0.0005). C and D, co-immunoprecipitation of GTRAP3-18, RTN2B, and EAAC1 from transfected HEK293 cells. Lysates from HEK 293 cells transfected with HA-GTRAP3-18, RTN2B-V5, and/or Myc-EAAC1, as indicated were immunoprecipitated with an HA (C) or a V5 (D) antibody. Cell lysates (Input: 10% of the total lysates) and precipitates (IP) were immunoblotted for HA, V5, and Myc. The complex oligosaccharide form and high mannose oligosaccharide form of EAAC1 were marked with open and filled arrowheads, respectively. E, immunoprecipitation of RTN2B, GTRAP3-18, and EAAC1 with anti-RTN2B or anti-EAAC1 antibodies from adult rat brain extracts. Blots were probed with RTN2B, GTRAP3-18 (arrow), EAAC1, and RTN1A antibodies.
FIGURE 2
FIGURE 2. EAAC1 and GTRAP3-18 bind to different regions of RTN2B
A, diagram of truncation constructs used for the co-immunoprecipitation experiments to identify the binding domains to EAAC1 and GTRAP3-18. All the constructs, including the full-length RTN2B were COOH terminally tagged with V5 epitopes. The Y-2-H fragment represented the construct obtained from the yeast two-hybrid screen for interaction with GTRAP3-18. Co-immunoprecipitation of HA-GTRAP3-18 (B) or Myc-EAAC1 (C) with truncation mutants of RTN2B-V5 from transfected HEK 293 cells. Cell lysates were precipitated with a V5 antibody. Input: 10% of the total lysates. Input and immunoprecipitated samples were analyzed by SDS-PAGE followed by Western blot. The bands migrating at the molecular weights corresponding to the possible dimers of RTN2B truncation mutants were marked with an * in red in the immunoprecipitation blots. Open and filled arrowheads indicated the complex oligosaccharide (mature) form and high mannose oligosaccharide (immature) form of EAAC1.
FIGURE 3
FIGURE 3. Expression of RTN2B in brain and neurons
A, Western blot analyses of RTN2B protein expression in HEK 293 cells transfected with RTN2B-V5 and multiple tissues, using a chicken anti-RTN2B against the N-terminal peptide of RTN2B. As estimated by molecular weight, the bands marked with arrows likely represented dimer and multimers of RTN2B. The band marked with * in white in the skeleton muscle lane was determined as a cross-reaction band by peptide block blots. B, immunostaining of RTN2B and GTRAP3-18 proteins in primary cortex neuron and astrocyte mixed culture from E16 rat brain. Neurons were labeled with the neuronal specific β-tubulin antibodies. Surrounding astrocytes were stained with the astrocyte marker glial fibrillary acidic protein. Scale bars represented 20 μm. C, immunofluorescence microscopy of primary cortex neuronal culture. Neurons were stained with anti-RTN2B, anti-EAAC1, and anti-GTRAP3-18 antibodies. Scale bar, 20 μm. D, immunoblots of total (T), intracellular (I), and plasma membrane (M) fractions of cell surface biotinylation of primary neurons. Actin was used as total protein and intracellular controls.
FIGURE 4
FIGURE 4. Subcellular localization of RTN2B in transfected cells
Confocal immunofluorescence microscopy of COS7 cells transiently transfected with RTN2B-V5. A, fixed cells were stained with anti-Calnexin (ER marker), anti-GM130 (Golgi marker, indicated by arrows), and anti-V5 antibodies. B, a higher magnification view of the RTN2B ER association. The representative examples of ER exit sites were marked with dotted circles and shown in the inset panel. Arrows indicate the putative Golgi localization. Scale bars, 20 μm.
FIGURE 5
FIGURE 5. Effect of RTN2B on EAAC1 trafficking in transfected cells
A, cell surface biotinylation of HEK 293 cells transfected with Myc-EAAC1 alone or together with RTN2B-V5 and GTRAP3-18. Total (T), intracellular (I), and biotinylated plasma membrane (M) fractions were blotted with a Myc antibody. Actin was used as total and intracellular controls. B, quantification of immunoblots for cell surface biotinylation. Ratio of cell surface EAAC1 was compared (*, p < 0.05; **, p < 0.01). Cell surface (mature form in the M fraction) EAAC1 was normalized with total EAAC1 (mature and immature forms in the T fraction) and calculated as the ratio of cell surface EAAC1. C, glutamate uptake assay in HEK 293 cells transfected with the indicated plasmids. Each + indicates equal amount, and ++ represents double amount of DNA added in the transfection reaction (*, p < 0.05; **, p < 0.001). D, immunofluorescence microscopy of COS7 cells transfected with GFP-EAAC1 alone or together with RTN2B-V5. Fixed cells were stained with anti-Calnexin (ER marker) and anti-GM130 (cis-Golgi marker) antibodies. Scale bars, 10 μm.
FIGURE 6
FIGURE 6. Knock down of RTN2B in neurons results in reduced total expression of EAAC1
A, immunoblot analysis of heterologously expressed RTN2B in the HEK 293 cell co-transfected with negative (Neg) or Rtn2B siRNA for 24 h. B, Rtn2B siRNA knocks down endogenous RTN2B in neurons. Hippocampal neurons at 5 days in vitro were transfected with GFP plus negative (Neg) or RTN2B siRNA for 48 h. Neurons were fixed and stained with antibodies against RTN2B (red), EAAC1 (blue), or NogoA (blue). Soma and processes of GFP positive neurons were marked with arrows and shown with neighboring GFP negative counterparts. Scale bars, 10 μm. C, quantification of the relative immunostaining intensity for RTN2B (a), EAAC1 (b), and NogoA (c) on the soma and processes in neurons transfected with siRNA and GFP (n = 5– 8, *, p < 0.0001). The staining intensity of neighboring GFP negative neurons was used as control to calculate the percentage.
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