Identification of RTN2B as an Interacting Protein of EAAC1 and Its Binding Partner GTRAP3-18
Previous studies showed that the ER protein GTRAP3-18 associated with EAAC1 and negatively regulated EAAC1 glutamate uptake activity on the cell surface via a retention of EAAC1 in the ER (13
To further reveal the regulatory mechanism of EAAC1 trafficking between the ER and Golgi complex, we initiated a program to identify proteins interacting with GTRAP3-18. The 188 amino acids of full-length GTRAP3-18 were used as bait to probe a yeast two-hybrid rat brain cDNA library. Two of the clones isolated in this screen contained the sequences highly homologous to the COOH-terminal coding region of mouse brain reticulon Rtn2
. Full-length rat Rtn2
cDNA (GI: 33415442) was isolated from rat brain cDNA. It encodes a protein of 469 aa, with a calculated relative molecular mass of 52 kDa.
Like other reticulon genes, the Rtn2
gene is transcribed into different mRNA variants, which share a common carboxyl-terminal segment. Three RTN2 proteins have been identified in human: RTN2A (545 aa, translated from mRNA contains exon 1 to 11), RTN2B (472 aa, translated from mRNA without exon 5), and RTN2C (205 aa, translated from mRNA initiated from exon 5 to 11) (22
). In mouse, two major transcripts encode a short (RTN2C, 204 aa) and a long (RTN2B, 471 aa, mRNA contains no exon 5) protein, which has been found to be enriched in muscle and brain, respectively (23
). The rat RTN2 cDNA we cloned shared 95.3% identity to the long mouse RTN2 brain transcript, so it was named RTN2B.
To confirm the yeast two-hybrid results, we first examined the interaction of RTN2B with GTRAP3-18 by FRET analysis in living mammalian cells. Three-filter FRET analysis revealed a statistically significant higher FRET signal of CFP-RTN2B with YFP-GTRAP3-18 compared with the negative control (), indicating that these two proteins interact in living cells.
Interactions of RTN2B with GTRAP3-18 and EAAC1
The interactions between RTN2B and GTRAP3-18, as well as EAAC1, were further tested by in vitro
and in vivo
co-immunoprecipitation. Full-length Rtn2B
was subcloned in-frame with a V5 tag at its COOH terminus. GTRAP3-18 and EAAC1 were NH2
terminally tagged with an HA and a Myc epitope, respectively. These constructs were cloned into mammalian expression vectors and expressed the expected protein products after transfection into HEK 293 cells (). On gradient gels, as other transporter isoforms (e.g.
GLT1), EAAC1 migrated as double bands with close molecular mass, representing two oligosaccharide states (24
). The upper band (marked with open arrowhead
) was the complex oligosaccharide form (the mature form). When co-expressed with GTRAP3-18, EAAC1 was retained in the ER thus enriched in the high mannose oligosaccharide form (the immature form, marked with filled arrowhead
, ). In all Western blots shown in this report, more than 80% of EAAC1 migrates as dimers, which were presented in the figures. This is a well known observation in the electrophoretic studies of glutamate transporters (25
). Monomer and multioligomers have the same results as the dimer. Transfected GTRAP3-18-HA displayed a double band pattern on 15% gel. We prepared lysates from the transfected cells, and precipitated GTRAP3-18 with an HA antibody and RTN2B with a V5 antibody. As shown in , RTN2B-V5 and Myc-EAAC1 were co-immunoprecipitated with HA-GTRAP3-18 (, lanes 4
). HA-GTRAP3-18 and Myc-EAAC1 were also found in RTN2B-V5 precipitates (, lanes 7–9
). At a similar expression level, the binding of RTN2B to an HA-tagged actin-binding protein, Spinophilin fragment (1–221 aa), our negative control, was not detected (, lane 6
, and D
, lane 10
). Thus, RTN2B specifically interacted with GTRAP3-18 and transporter EAAC1 in transfected cells. Notably, the interactions of GTRAP3-18 and EAAC1 with RTN2B were independent of each other (, lane 4
, and D
, lanes 7
To determine whether RTN2B associates with transporter EAAC1 in vivo, co-immunoprecipitation experiments were performed using detergent extracts from adult rat brain. EAAC1 was immunoprecipitated with an anti-EAAC1 antibody, along with RTN2B and GTRAP3-18. Meanwhile, using a chicken antibody raised against residues 30 – 48 on the NH2 terminus of RTN2B, we were able to co-immunoprecipitate EAAC1 and GTRAP3-18 with RTN2B. Specifically, RTN1A was not present in the EAAC1 or RTN2B immunoprecipitates (). These data indicate that RTN2B associated with both EAAC1 and GTRAP3-18 in brain tissue. The consistent results of in vitro and in vivo immunoprecipitation also demonstrated that the epitope tags added to RTN2B, GTRAP3-18, and EAAC1 did not have an effect on their interaction.
The First Transmembrane Domain of RTN2B Binds to GTRAP3-18
Having established that GTRAP3-18 and EAAC1 interacted with RTN2B independently, we next investigated whether GTRAP3-18 and EAAC1 competed for the binding to RTN2B or they associated with different domains of RTN2B. RTN2B was predicted to have two transmembrane domains. The transmembrane domains and the 66-aa loop region in between, called reticulon-homology domain, shared homologies with other reticulon family members (26
). The NH2
-terminal domain had three hydrophilic regions. To determine the region(s) on RTN2B responsible for EAAC1 and GTRAP3-18 binding, we generated NH2
-terminal, COOH-terminal, or transmembrane domain-truncated constructs of RTN2B tagged with V5 epitopes (). All of the RTN2B mutants were expressed at their expected sizes although some at apparently reduced levels compared with the full-length protein. In the immunoprecipitation blot using V5 antibodies, besides the bands migrating at the corresponding molecular weights, extra bands were observed in the full-length and some truncated RTN2B, except ΔC. As estimated by molecular weights, these bands likely represented the dimers of the RTN2B proteins (marked with * in red
in ). This observation is consistent with the previous reports that RTNs can form homo-oligomers (28
EAAC1 and GTRAP3-18 bind to different regions of RTN2B
GTRAP3-18 displayed a significantly reduced expression level when co-expressed with RTN2B-ΔN2 (, lane 3). It co-immunoprecipitated with all the RTN2B mutant proteins, except ΔTM1, demonstrating that the first transmembrane domain was required for GTRAP3-18 association. Also, RTN2B-ΔN (residues 239 – 469) co-precipitated with GTRAP3-18 (, lane 11), consistent with the yeast two-hybrid screen result. Considering that both RTN2B-ΔC (residues 1–320) and RTN2B-ΔN (residues 239 – 469) bound to GTRAP3-18 (, lanes 10 and 11), we concluded that the first transmembrane domain included in both constructs was sufficient for GTRAP3-18 interaction. Therefore, the first transmembrane domain was responsible for GTRAP3-18 binding.
NH2 Terminus of RTN2B Is Required for EAAC1 Binding
EAAC1 was co-precipitated with full-length RTN2B, as well as ΔTM1 (, lanes 7 and 12). Whereas RTN2B-ΔN1, −ΔN2, and ΔN proteins were all readily recovered from the immunoprecipitation, we failed to detect EAAC1 protein in these precipitates. This suggested that the NH2-terminal domain of RTN2B was required for its association with EAAC1 (, lanes 8, 9, and 11). There was a much smaller amount of RTN2B-ΔC expressed and recovered from the anti-V5 immunoprecipitation compared with the full-length and NH2-terminal-truncated RTN2B, making it difficult to draw a clear conclusion on whether the NH2 terminus (RTN2B-ΔC) was sufficient for EAAC1 interaction (, lane 10). Nevertheless, RTN2B-ΔTM, which failed to bind to GTRAP3-18, retained the interaction with EAAC1, indicating this trans-membrane domain was not involved in EAAC1 binding (, lane 12). Under a closer examination, 90% of EAAC1 was present in the mature form (open arrowhead) in the total cell lysates, whereas in the precipitates, this percentage decreased to 40%, indicating RTN2B bound to both mature and immature forms (filled arrowhead) of EAAC1 with a preference to the immature one enriched in the ER (, comparing lanes 1 and 6 with 7 and 12).
The results of the truncation studies were summarized in . Taken together, it showed that the first transmembrane domain of RTN2B was required and sufficient to bind to GTRAP3-18. For EAAC1 association, the NH2 terminus of RTN2B, more specifically the first hydrophilic peak was required. Because EAAC1 and GTRAP3-18 interacted with different domains of RTN2B, they did not compete for the binding sites. Thus, it was likely that all three proteins interacted in one complex.
Summary of RTN2B truncation studies
Expression and Localization of RTN2B in Neurons
Our data showed that RTN2B interacted with neuronal glutamate transporter EAAC1 and the ER protein GTRAP3-18. We expected to find RTN2B protein in neurons and associated with the ER. To further verify this hypothesis, we examined the expression profile and localization of RTN2B.
Two specific immunoreactive bands, migrating at 52 and 18 kDa, were detected in Western blot analysis of rat tissue homogenates, using a rabbit antibody against the last 19 aa on the COOH-terminal region of RTN2, where the two RTN2 isoforms shared identity. The long isoform was present in the brain and spinal cord, whereas the short one was expressed in the skeleton muscle and heart (data not shown). The distribution of the long isoform of RTN2 (i.e. RTN2B) in all regions of the brain and the spinal cord was further confirmed in a Western blot, using a chicken antibody to the NH2 terminus of RTN2B. This antibody specifically recognized the 52-kDa RTN2B. The transfected HEK sample was loaded to verify that the transfected RTN2B-V5 was recognized by this antibody as well. A little higher molecular weight of RTN2B-V5 was caused by the tag (). In a primary neuron and astrocyte mixed culture from embryonic rat cortex, the anti-RTN2B antibody reacted only with neurons, which was co-labeled with neuronal specific β-tubulin III. In contrast, GTRAP3-18 had a broader distribution than RTN2B and was found in both cultured neurons and astrocytes (). In addition, using reverse transcriptase-PCR, RTN2B transcripts were detected only in neuronal culture, but not in astrocyte culture (data not shown). Co-staining the primary cultured neurons for RTN2B, EAAC1, and GTARP3–18 showed that these three proteins, which interacted with each other as demonstrated in the whole brain co-immunoprecipitation (), co-expressed in neurons ().
Expression of RTN2B in brain and neurons
Reticulon family members were generally thought to be located in the ER (hence the name), according to the immunocytochemical localization of RTN1A in transfected COS cells (26
). However, it has been reported that Nogo/RTN4-A, -B, and -C are present on the cell surface of fibroblasts, cultured neurons, and muscle cells (31
). In the primary neuronal cultures, using a cell surface biotinylation technique, we found that a small fraction (17 ± 4%) of RTN2B was present on the cell surface compared with total protein, similar to NogoA (23 ± 3% on the plasma membrane), whereas RTN1A was exclusively distributed in the intracellular fraction (). This finding suggests that RTN2B function might not be restricted to the ER.
Consistent with the nomenclature, we observed ER localization of RTN2B in transfected COS7 cells (). Immunostaining showed that transfected RTN2B displayed a combined pattern of reticular filaments and punctuate perinuclear structures. The vast majority of the RTN2B staining co-localized with Calnexin, showing the typical distribution of an ER protein with the reticular network around the nuclear envelope. In addition to the prominent peripheral tubular distribution, some RTN2B exhibited a tight and punctuate perinuclear staining, suggesting a Golgi localization shown by cis-Golgi marker GM130 staining, where Calnexin was essentially absent (, arrows
). The stable and stationary RTN2B puncta distributed on the ER membrane (, circles
) identified the ER exit sites, specialized subdomains of ER membrane where cargo is concentrated into vesicles that bud from the ER and are transported to the Golgi (32
). This distribution pattern suggested that at least a portion of RTN2B may exit out of the ER and traffic to the Golgi apparatus.
Subcellular localization of RTN2B in transfected cells
RTN2B Increases EAAC1 Trafficking from ER to the Cell Surface
Having established that RTN2B is localized in neurons and associated with EAAC1, we next investigated the biological effects of this interaction on EAAC1. We first examined the cell surface distribution of EAAC1. We used a cell membrane impermeant biotinylation reagent to selectively label cell surface protein. The densitometry analysis of the following Western blot showed that co-expression of RTN2B with EAAC1 resulted in a 26% increase in the percentage of EAAC1 protein distributed on the cell surface. Co-expression of GTRAP3-18 increased the ER form of EAAC1, and reduced its cell surface distribution (). In parallel, we measured the sodium-dependent glutamate transport activity of HEK cells that had been transfected either with EAAC1 alone or together with RTN2B or GTRAP3-18. Consistent with previous reports (13
glutamate transport activity decreased progressively with increasing expression of the GTRAP3-18 protein. As co-expression of RTN2B modestly increased the cell surface level of EAAC1, it also resulted in a concomitant 27% increase in Na+
-dependent glutamate transport activity. Notably, expression of RTN2B did not alter the inhibitory effect of GTRAP3-18 on transport activity ().
Effect of RTN2B on EAAC1 trafficking in transfected cells
To determine whether the increased cell surface EAAC1 was the result of up-regulated ER exit of EAAC1, we compared the intracellular localization of GFP-EAAC1 in the presence or absence of RTN2B. As shown in , we observed a strong but not complete co-localization of intracellular GFP-EAAC1 with the ER marker Calnexin in the perinuclear region and tubuloreticular network. Some GFP-EAAC1 displayed a punctuated staining pattern that overlapped with the cis-Golgi marker GM130. Because only a small amount of EAAC1 (about 25%) was delivered to the plasma membrane in transfected cells, according to surface biotinylation (), a cell surface GFP signal was difficult to detect in this imaging paradigm, because of the presence of strong intracellular signals. Co-expression of RTN2B caused an apparent shift of localization of GFP-EAAC1 signals with a reduced ER co-localization and an increased punctuated Golgi staining ().
In summary, these results demonstrated that RTN2B protein positively regulated EAAC1 trafficking to the plasma membrane by facilitating its ER exit. In our transient transfection conditions, RTN2B was expressed at a level 5–10 times higher than the endogenous protein, whereas EAAC1 was overexpressed more than 100-fold (data not shown). This extremely low ratio of RTN2B to EAAC1 may be the reason of the modest effects observed in the heterologous cell expression system. To justify these observations from the overexpressed heterologous system, we next studied primary cortical neurons where RTN2B and EAAC1 are endogenously expressed.
Knocking Down RTN2B Reduces EAAC1 Expression in Neurons
To test if the endogenous RTN2B affects the trafficking of EAAC1 in neurons, we used an RNA interference approach to knock-down Rtn2B
mRNA in cultured primary cortical neurons. The specificity and efficacy of siRNAs were tested in HEK 293 cells. The Rtn2B
siRNA almost completely abolished transfected RTN2B protein () and was used for our loss-of-function studies in rat neurons. A GFP plasmid was co-transfected with the siRNA in the primary neuronal cultures to serve as a marker of the transfected cells. Under normal conditions, RTN2B and EAAC1 were present in all cultured neurons (33
). Recently, Horton and Ehlers and other groups (32
) demonstrated that ER and ER exit sites were distributed throughout neurons, including the dendrites. Consistent with their reports and our immunocyto-chemistry result obtained from COS7 cells, which showed that RTN2B localized in the ER and ER exit sites (), RTN2B was found abundant in neurites (). There was no change in RTN2B and EAAC1 staining intensity in neurons transfected with negative control siRNA (, panels a
, and C
, panels a
). Transfection of Rtn2B
siRNA resulted in a significant reduction of endogenous RTN2B staining (red
) on the soma and processes of transfected neurons to 26 ± 7 and 22 ± 11%, respectively, relative to the untransfected ones (, panels d
, and C
, panel a
). Meanwhile, the intensity of EAAC1 in the soma and process domains in these neurons was remarkably decreased to 49 ± 11 (soma) and 40 ± 15% (processes) of that in the untransfected neurons (, panels d
, and C
, panel b
). The unaltered NogoA staining intensity indicated that Rtn2B
siRNA specifically knocked down RTN2B, and did not cause nonspecific protein degradation as observed in unhealthy neurons (, panels g
, and C
, panel c
). Corroborating our results obtained from the transfected COS cells, we conclude that the EAAC1 expression level was reduced if its trafficking from the ER was compromised through loss of RTN2B. These findings were consistent with those reported for another transporter subtype, GLT1 (23
). In this case, mutation of the leucine-based ER export motif resulted in retention of GLT1 in the ER and a reduced expression level. In a different scenario, we observed the reduced expression of EAAC1, versus
an accumulation/build-up in the ER when we inhibited protein trafficking from the ER in the primary cultured neurons (data not shown). This also suggested that the ER form of EAAC1 was rapidly degraded when its trafficking out of ER was compromised.
Knock down of RTN2B in neurons results in reduced total expression of EAAC1