PSD-95 Interacts with RalBP1
Yeast two-hybrid screens with PSD-95 identified RalBP1 as a novel PSD-95-interacting protein, which is coupled to other endocytic proteins including POB1, RalA, AP2, epsin, and Eps15 () 
. The PDZ-binding motif at the RalBP1 C-terminus interacted with the PDZ domains of PSD-95 (). RalBP1 binding to PSD-95 was further confirmed by in vitro and in vivo pull down and coimmunoprecipitation assays (). In addition to interacting with PSD-95, RalBP1 formed an in vivo complex with PSD-93/chapsyn-110 (a PSD-95 relative), but no associations of other PSD-95 family proteins (SAP97 and SAP102) with RalBP1 were detected. RalBP1 also formed a complex with POB1 and α-adaptin (a subunit of the AP2 complex) in the brain (). Notably, RalBP1 tightly associated with POB1, but relatively weakly with α-adaptin and PSD-95.
Expression Patterns of RalBP1, RalA, and POB1 in the Brain
In situ hybridization revealed that mRNAs of RalBP1, RalA, and POB1 are widely expressed in various brain regions (Figure S1
). The three different mRNAs showed overlapping as well as distinct distribution patterns.
In the brain, RalBP1 and POB1 antibodies recognized a single and double band, respectively (Figure S2A and S2B
). RalBP1, RalA, and POB1 proteins were most abundant in the brain, compared to other tissues (Figure S2C
). RalBP1, RalA, and POB1 proteins were detected in various brain regions (Figure S2D
), consistent with the in situ results. Expression levels of RalBP1 and RalA proteins remained largely unchanged during postnatal brain development, whereas POB1 and α-adaptin showed age-dependent increases (Figure S2E
In subcellular brain fractions, RalA was mainly detected in crude synaptosomal (P2) and synaptic membrane (LP1) fractions (P21 and 6 wk), similar to PSD-95 (Figure S2F
). This is consistent with the previous reports that RalA is present in postsynaptic protein complexes 
. In contrast, RalBP1 and POB1 were largely found in cytosolic (S3) and microsomal (P3) fractions. RalA was detected in the PSD I fraction and weakly in PSD II and III fractions (Figure S2G
), suggesting that RalA is not tightly associated with the PSD, although it is mainly detected in synaptic fractions. RalBP1 was minimally detected in PSD fractions.
Phosphorylation of RalBP1 at C-terminal Thr 645 Inhibits PSD-95 Binding
The limited subcellular overlap between RalBP1 and PSD-95 (Figure S2F
) suggests that their interaction might be regulated. Because PDZ interactions can be regulated by phosphorylation 
, we tested if Thr 645 (−2 position) at the RalBP1 C-terminus can be phosphorylated (). To this end, we generated a RalBP1 antibody that selectively recognizes RalBP1 proteins with phosphorylated Thr 645. This antibody could detect wild-type (WT) RalBP1 but not a RalBP1 T645A mutant (). λ-Phosphatase digestion of RalBP1 proteins expressed in vitro and in vivo substantially weakened protein detection by phospho-specific antibodies (). An upstream mutation in RalBP1 (R642A; −5 position) abolished the phosphorylation (), indicating that R642 is important for RalBP1 phosphorylation. Of note, the RKET sequence in the RalBP1 C-terminus (−5 to −2 positions) matches the consensus sequence for protein kinase A (PKA) phosphorylation. Consistent with this possibility, PKA could directly phosphorylate RalBP1 in vitro ().
Phosphorylation of RalBP1 at C-terminal Thr 645 inhibits PSD-95 binding.
A phosphomimetic RalBP1 mutant (T645E) failed to interact with PSD-95 in yeast two-hybrid and in vitro coprecipitation assays (). RalBP1 proteins phosphorylated at T645 showed reduced biochemical association with PSD-95 in HEK293T cells, compared to total (phosphorylated+non-phosphorylated) RalBP1 (). These results suggest that RalBP1 is phosphorylated at Thr 645 in vivo, and this inhibits PSD-95 binding.
RalBP1 Is Dephosphorylated by NMDAR Activation via PP1 and Rephosphorylated by PKA
We next tested if NMDAR activation, via protein phosphatases, dephosphorylates RalBP1. NMDAR activation by NMDA treatment induces AMPAR endocytosis in cultured neurons 
and LTD in slices 
. NMDA treatment (20 µM for 3 min) of cultured hippocampal neurons induced a rapid and significant (~50%) dephosphorylation of RalBP1 at Thr 645 (). As metabotropic glutamate receptor (mGluR)–dependent LTD also involves AMPAR endocytosis 
, we tested if mGluR activation leads to the dephosphorylation of RalBP1. DHPG, a group I mGluR agonist, however, did not induce RalBP1 dephosphorylation ().
RalBP1 is dephosphorylated by NMDAR activation through PP1 and rephosphorylated by PKA.
RalBP1 dephosphorylation induced by NMDA treatment was blocked by APV, an NMDAR antagonist (). RalBP1 dephosphorylation was blocked by okadaic acid (1 µM), which inhibits both protein phosphatase 1 and 2A (PP1 and PP2A) (). In contrast, RalBP1 dephosphorylation was not affected by low-concentration okadaic acid (10 nM), which inhibits only PP2A, or FK506, an inhibitor of PP2B (calcineurin), indicating that PP1 is important. Notably, okadaic acid (1 µM) increased RalBP1 phosphorylation before NMDA treatment (), indicating that PP1 also mediates basal dephosphorylation of RalBP1.
After NMDA washout, RalBP1 was rephosphorylated to near-normal levels in ~1 h (). This rephosphorylation was blocked by KT5720 (1 µM), a PKA inhibitor, but not by KN-93 (10 µM) and PD98059 (25 µM), which inhibit CaM kinases 
and MEK, respectively (). These results suggest that RalBP1 is rapidly dephosphorylated by NMDAR activation and relatively slowly rephosphorylated by PKA.
NMDA Treatment Induces Activation of RalA, and Activated RalA Binds and Translocates RalBP1 to Dendritic Spines
Because RalA activation is regulated by Ras, Rap, and calcium/calmodulin 
, which act downstream of NMDAR activation 
, we tested if NMDAR activation leads to the activation of RalA. NMDA treatment of cultured neurons induced RalA activation, measured by pull down assays (). The RalA activation consisted of two phases; a rapid (<1 min) and small increase followed by a slow and bigger increase.
NMDAR activation induces RalA activation, and activated RalA and PSD-95 act together to bind and translocate dephosphorylated RalBP1 to spines.
In cultured neurons, RalBP1 expressed alone showed a widespread distribution pattern in dendrites (). Interestingly, coexpression of a constitutively active form of RalA (G23V) with RalBP1 induced a marked translocation of RalBP1 to dendritic spines, whereas WT and dominant negative RalA (S28N; constitutively in the GDP-bound state) had no effect (). RalBP1-enriched spines were positive for PSD-95 (Figure S3
), indicating that RalBP1 was translocated to synaptic sites. RalBP1 ΔCC, which lacks the CC domain that is involved in RalA binding, showed no significant RalA-dependent spine translocation, indicating that the direct binding of RalBP1 to RalA is important. A RalA G23V mutant with weakened RalBP1 binding (G23VD49N; termed GVDN) induced a spine translocation of RalBP1 that is smaller than that of RalBP1 coexpressed with RalA G23V, further suggesting that RalA directly recruits RalBP1 to spines. Notably, RalBP1 ΔC, which lacks PSD-95 binding, showed a RalA-dependent spine translocation similar to that of WT RalBP1, indicating that activated RalA alone is sufficient to induce spine translocation of RalBP1. These results suggest that NMDAR activation induces RalA activation and that activated RalA binds and translocates RalBP1 to dendritic spines.
RalBP1 Dephosphorylation Combined with RalA Activation Further Promotes Spine Translocation of RalBP1
We next tested whether RalBP1 dephosphorylation induced by NMDAR activation affects RalA-induced spine translocation of RalBP1. NMDA treatment of neurons coexpressing RalA G23V and RalBP1 further increased RalA-dependent spine translocation of RalBP1 (). In contrast, such increases were not observed in control neurons expressing RalA G23V and RalBP1 ΔC, indicating that NMDA-induced RalBP1 binding to PSD-95 is important. RalBP1 that was transfected alone was not translocated to spines upon NMDA treatment, indicating that dephosphorylation of RalBP1 alone is not sufficient to induce RalBP1 translocation to spines. Spine morphology, as measured by spine head area, was not changed by overexpression of RalA and RalBP1 constructs (WT and mutants), or by NMDA treatment of the transfected neurons (Figure S4
). These results suggest that binding of dephosphorylated RalBP1 to PSD-95, combined with RalBP1 binding to activated RalA, further promotes synaptic localization of RalBP1.
Biochemically, NMDA treatment of cultured neurons significantly increased coimmunoprecipitation of RalBP1 and PSD-95, but not of RalBP1 and POB1 (). Whether the association between RalBP1 and RalA is affected by NMDA treatment could not be determined because RalBP1 did not coprecipitate with RalA under our experimental conditions, possibly owing to the transient nature of RalA binding to RalBP1. In support of this interpretation, RalBP1 did not form a complex with RalA WT, in contrast to the strong association of RalBP1 with RalA G23V (Figure S5A
The results described thus far indicate that a ternary complex containing RalA, RalBP1, and PSD-95 might be formed in a regulated manner. Because the formation of a RalA-RalBP1 complex could not be demonstrated in vivo, we tested this possibility in heterologous cells using RalA G23V. In HEK293T cells, RalA G23V formed a ternary complex with RalBP1 and PSD-95 (Figure S5B
). In addition, RalA, which is mainly associated with the plasma membrane by geranyl-geranylation, induced translocation of PSD-95 to the plasma membrane in HEK293T cells coexpressing RalBP1 WT, but not in those coexpressing RalBP1 ΔC that lacks PSD-95 binding ability (Figure S5C
). Collectively, these results indicate that RalA and PSD-95 act together to bind and translocate RalBP1 to synapses.
NMDAR Activation by Low-Frequency Electrical Stimulation (LFS) Induces RalBP1 Dephosphorylation
The results described thus far are based on experiments using NMDA treatment to induce RalA activation and RalBP1 dephosphorylation. Bath application of NMDA leads to activation of both synaptic and extrasynaptic NMDARs, which can be coupled to different signal transduction pathways 
; therefore, we attempted NMDAR activation by LFS (1 Hz, 900 pulses), which likely enhances activation of synaptic NMDARs 
. The levels of RalBP1 phosphorylation at T645 were significantly decreased by LFS given to the CA1 region of hippocampal slices, an effect that was blocked by the NMDAR antagonist APV (). RalBP1 phosphorylation levels returned to a normal range 60 min after LFS (), a result similar to that obtained in NMDA-treated cultured neurons. In contrast, neither induction of LTP by theta-burst stimulation (TBS) nor induction of LTD by paired-pulse LFS (PP-LFS, 50 ms interstimulus interval) in the presence of APV induced RalBP1 dephosphorylation (). Although there was a tendency for LFS to increase coimmunoprecipitation of RalBP1 and PSD-95 compared with that of LFS and APV (), this difference did not reach statistical significance (p
6). This result is in contrast to the enhanced coprecipitation of RalBP1 and PSD-95 observed in NMDA-treated cultured neurons (). This discrepancy might be attributable to the fact that PSD-95 proteins in slices are more difficult to extract than those in cultured neurons, leading to a decrease in the efficiency of coprecipitation between PSD-95 and RalBP1.
NMDAR activation by low-frequency electrical stimulation induces RalBP1 dephosphorylation.
RalBP1 and RalA Are Required for NMDA-induced AMPAR Endocytosis and LTD
RalBP1, an endocytic adaptor, translocated to synapses by NMDAR activation might regulate AMPAR endocytosis. To test this hypothesis, we attempted knockdown of RalBP1 and RalA by shRNA constructs, which reduced expression of exogenous RalBP1 and RalA by 78% and 86%, respectively, in HEK293T cells, and by 90% and 77%, respectively, in cultured neurons (Figure S6
). Knockdown of endogenous proteins could not be observed due to the lack of suitable antibodies.
In cultured neurons, knockdown of RalBP1 and RalA significantly reduced NMDA-induced endocytosis of the GluR2 subunit of AMPARs (). A scrambled version of RalA shRNA had no effect. shRNA-resistant expression constructs of RalBP1 and RalA coexpressed with RalBP1 and RalA shRNAs, respectively, rescued the knockdown effects (Figure S7
). Overexpression of RalBP1 TE, a phosphomimetic RalBP1 mutant that lacks PSD-95 binding, inhibited NMDA-induced GluR2 endocytosis, while WT RalBP1 did not (), suggesting that RalBP1 binding to PSD-95 is important. In addition, the CC domain of POB1 (POB1 CC), which binds and inhibits RalBP1, significantly reduced NMDA-induced GluR2 endocytosis ().
RalBP1 and RalA are required for NMDA-induced AMPAR endocytosis and LTD.
Further supporting the importance of RalA, the Ral binding domain of RalBP1 (RalBD), which binds and inhibits only active RalA, significantly reduced NMDA-induced GluR2 endocytosis (). Furthermore, RalA S28N (dominant negative) and RalA GVDN (a RalA G23V mutant with weakened RalBP1 binding) inhibited NMDA-induced GluR2 endocytosis, whereas RalA WT and RalA G23V had no effect (Figure S8A
). These results suggest that RalBP1 and RalA are required for NMDA-induced endocytosis of GluR2.
In hippocampal slice culture, RalBP1 knockdown in CA1 pyramidal neurons significantly reduced paring-induced LTD at Schaffer collateral (SC)–CA1 pyramidal cell (CA1) synapses (~81% of baseline; p
0.12 compared to before paring; Student's unpaired t
-test), relative to untransfected control neurons (~52% of baseline; *** p
<0.001) (). The LTD magnitudes from neurons expressing RalBP1 shRNA and untransfected control neurons (~81% and ~52% of baseline, respectively) were significantly different (** p
<0.01, Student's paired t
-test). In contrast, neurons transfected with empty shRNA vector showed an LTD magnitude comparable to that of untransfected neurons (). RalBP1 knockdown did not affect basal synaptic transmission, as measured by amplitudes of evoked excitatory postsynaptic currents (EPSCs) ().
Supporting the role of RalA in LTD regulation, the RalA-inhibiting construct RalBD significantly reduced paring-induced LTD (~71% of baseline; * p<0.05 compared to before paring; Student's unpaired t-test), relative to untransfected control neurons (~46% of baseline; *** p<0.001). LTD magnitudes observed in RalBD overexpressing and untransfected neurons were significantly different (* p<0.05; Student's paired t-test; ). Basal transmission was unaffected by RalBD overexpression (). These results suggest that RalBP1 and RalA are required for LTD induction.
RalA, but not RalBP1, Inhibits Basal AMPAR Endocytosis in a GTP-independent Manner
We next tested whether RalA and RalBP1 regulate AMPAR endocytosis under basal conditions. Intriguingly, basal GluR2 endocytosis in the absence of NMDAR activation was enhanced by the knockdown of RalA, but not RalBP1 (), suggesting that RalA, but not RalBP1, inhibits GluR2 endocytosis under basal conditions. Inhibition of RalBP1 by overexpression of RalBP1 TE and POB1 CC had no effect on basal GluR2 endocytosis (), further suggesting that RalBP1 does not regulate basal GluR2 endocytosis. Intriguingly, basal GluR2 endocytosis was not affected by overexpression of RalBD (), RalA S28N, or RalA GVDN (Figure S8B
). RalBD, RalA S28N, and RalA GVDN commonly interfere with GTP-dependent actions of RalA by trapping activated RalA, blocking RalA activation, and suppressing the binding of activated RalA to RalBP1, respectively. Considering that RalA knockdown reduces total RalA (both active and inactive), these results suggest that RalA inhibits basal AMPAR endocytosis in a GTP-independent (or RalA activation-independent) manner.
RalA, but not RalBP1, inhibits basal AMPAR endocytosis in a GTP-independent manner and is required for the maintenance of surface AMPARs.
RalA, but not RalBP1, Is Required for the Maintenance of Surface AMPAR Levels
RalA inhibits basal AMPAR endocytosis, so we reasoned that RalA might regulate surface AMPAR levels. Indeed, knockdown of RalA, but not RalBP1, significantly reduced surface levels of GluR2 (), suggesting that surface GluR2 levels are maintained by RalA but not RalBP1. Consistent with this, inhibition of RalBP1 by RalBP1 TE or POB1 CC had no effect on surface GluR2 levels ().
Interestingly, surface GluR2 levels were reduced by overexpression of RalBD () or RalA S28N (Figure S8C
), which inhibits active RalA, indicating that active RalA is important for the maintenance of surface GluR2 levels. Collectively, these results indicate that both active and inactive RalA are involved in maintaining surface GluR2 levels. Inactive RalA may help maintain surface GluR2 levels by inhibiting basal GluR2 endocytosis (, and S8B). How then might active RalA contribute to the maintenance of surface GluR2 levels? One possibility is that active RalA might help internalized GluR2 recycle back to the plasma membrane. However, inhibition of active RalA by overexpression of RalA S28N had no effect on GluR2 recycling (Figure S9A
). In addition, knockdown of RalA, which reduces total (active+inactive) RalA levels, did not affect GluR2 recycling (Figure S9B
), indicating that neither active nor inactive RalA regulate GluR2 recycling. An alternative possibility is that active RalA might regulate synaptic delivery of GluR2 from a cytoplasmic, non-recycling pool, perhaps via the interaction of RalA with the exocyst complex. In support of this possibility, two components of the exocyst complex (Sec8 and Exo70), which interact with active RalA, have been shown to promote surface insertion and synaptic targeting of AMPARs 
Constitutive RalA Activation Combined with RalBP1 Binding to PSD-95 Reduces Surface AMPAR Levels and Occludes NMDA-Induced AMPAR Endocytosis
The results described thus far suggest that two molecular mechanisms, RalA activation and RalBP1 binding to PSD-95, are important for NMDAR-dependent AMPAR endocytosis. We next reasoned that these two mechanisms might be sufficient to induce AMPAR endocytosis in the absence of NMDAR activation. To this end, we transfected cultured neurons with constitutively active RalA (G23V) and RalBP1 (YFP-tagged) and monitored surface levels of endogenous AMPARs, using surface GluR2 antibodies. Intriguingly, surface AMPAR levels in these neurons were significantly reduced in the absence of NMDA treatment, compared to those expressing RalA G23V alone (without RalBP1 coexpression) or those coexpressing WT RalA (not G23V) and RalBP1 (). In contrast, coexpression of RalA G23V and a mutant RalBP1 (ΔC) that lacks PSD-95 binding did not induce a reduction in surface AMPAR levels, relative to the coexpression of RalA G23V and WT RalBP1. These results suggest that RalA activation combined with RalBP1 binding to PSD-95 are sufficient to induce a reduction in surface AMPAR levels in the absence of NMDAR activation, likely through a constitutive endocytosis of AMPARs.
Constitutive RalA activation combined with RalBP1 binding to PSD-95 reduces surface AMPAR levels and occludes NMDA-induced AMPAR endocytosis.
The results described above () also suggest that a fraction of exogenously expressed RalBP1 proteins is basally dephosphorylated (in the absence of NMDAR activation), and the amount of dephosphorylated RalBP1 proteins is sufficient to bind to both RalA G23V and PSD-95 and induce significant AMPAR endocytosis. In support of this possibility, NMDA treatment of the neurons coexpressing RalA G23V and RalBP1 did not induce AMPAR endocytosis (), suggesting NMDA-induced AMPAR endocytosis was occluded. In contrast, neurons coexpressing RalA G23V alone (without RalBP1 coexpression) showed an NMDA-induced reduction in surface AMPAR levels. It is conceivable that the amount of endogenous RalBP1 proteins, although a fraction of them is dephosphorylated, may not be sufficient to induce AMPAR endocytosis, unless a significant fraction of them is dephosphorylated by NMDAR activation.
Generation and Characterization of RalBP1−/− Mice
To investigate the role of RalBP1 in AMPAR endocytosis and LTD in vivo, we generated RalBP1−/− mice using an ES cell line gene-trapped in the intron between exons 3 and 4 of the RalBP1 gene (). PCR genotyping was used to identify WT and gene-trapped RalBP1 alleles (). Expression levels of RalBP1 proteins in RalBP1−/− brain was ~18.1%±3.1% (n
8) of WT mice (), likely due to incomplete gene trapping. The gene trapping generated a small amount of fusion proteins containing RalBP1 (first 235 aa) and β-geo (), which were detected in various brain regions including hippocampus (Figure S10
Generation and characterization of RalBP1−/− mice.
No abnormalities were observed in gross morphology of RalBP1−/− brain or in the cellular architecture of RalBP1−/− neurons, as determined by staining for NeuN and MAP2, respectively (). There were no changes in expression levels of RalBP1-interacting proteins such as RalA, α-adaptin, and PSD-95, as well as subunits of AMPARs and NMDARs in RalBP1−/− brain (). Interestingly, however, POB1 expression was significantly decreased by 43.8%±10.9% (n
8), suggesting that RalBP1 is important for the stability of POB1.
Selective Impairment of NMDAR-Dependent LTD at RalBP1−/− CA1 Synapses
We investigated synaptic plasticity at RalBP1−/− hippocampal SC-CA1 synapses. LFS for LTD induction (1 Hz, 900 stimulations) induced robust synaptic depression in WT slices (17–21 d) that averaged 71.6%±0.9% (n
25 slices, 10 animals) (). In contrast, LTD induction in RalBP1−/− mice was significantly attenuated (87.6%±1.1%; n
24, 10 animals; *** p
<0.001), despite that ~18% of RalBP1 proteins are still expressed. Inhibition of NMDAR by APV during LFS abolished the difference between the two genotypes (unpublished data).
Impaired NMDAR-dependent LTD at RalBP1−/− SC-CA1 pyramidal synapses.
Hippocampal SC-CA1 synapses also exhibited mGluR-dependent LTD, which does not require protein phosphatase 
. Bath application of DHPG (mGluR agonist) induced stable depression in both WT and RalBP1−/− slices (8 wk), with magnitude of depression essentially identical throughout the recording (). These results suggest that the reduced expression of RalBP1 selectively impairs NMDAR-dependent LTD.
LTD deficits give rise to corresponding enhancement in potentiation, a metaplastic shift 
. However, LTP induced by TBS in RalBP1−/− slices (4–7 wk) was not substantially different from that of WT littermates throughout the recording ().
Homosynaptic LTD and depotentiation have many common properties 
. To induce depotentiation, LFS (1 Hz, 900 stimulations) was delivered to slices (4–7 wk) 5 min after TBS. In contrast to de novo LTD, synaptic depression by LFS after TBS was not different in WT and RalBP1−/− mice (). These results suggest that RalBP1 is involved selectively in NMDAR-dependent de novo LTD, but not in LTP or depotentiation.
Normal Excitatory Synaptic Transmission at RalBP1−/− CA1 Synapses
To test whether RalBP1 deficiency affects presynaptic functions at hippocampal SC-CA1 synapses, we examined paired-pulse facilitation (PPF), known to be inversely related to presynaptic release probability. PPF at all interstimulus intervals tested was not changed at RalBP1−/− SC-CA1 synapses (). In addition, post-tetanic potentiation, another form of short-term plasticity, measured after TBS also appeared normal in RalBP1−/− mice ().
Normal basal synaptic transmission at RalBP1−/− SC-CA1 synapses.
We next examined the synaptic input-output relationship and spontaneous miniature EPSCs (mEPSCs) to test if RalBP1 deficiency affected excitatory synaptic functions. The relationship between the number of stimulated axons (presynaptic fiber volley) and the slope of postsynaptic fEPSPs at different stimulus intensities was not changed in RalBP1−/− slices (). Furthermore, we observed no significant difference in the amplitudes or frequencies of mEPSCs between RalBP1−/− and WT mice (). Because mEPSCs and fEPSPs are mainly mediated by AMPARs, we examined NMDAR functionality by measuring the ratio of AMPAR and NMDAR currents (AMPA/NMDA ratio). The stimulation intensity was adjusted to achieve an AMPA-mediated current of ~150 pA at the holding potential of −70 mV. The AMPA/NMDA ratios measured in RalBP1−/− slices were not different from those of WT littermates (). Pipette solutions used to measure mEPSCs and AMPA/NMDA ratios contained high concentrations of EGTA (10 mM) to inhibit possible contamination of recordings by currents activated by increases in intracellular Ca2+. This would not be expected to affect our interpretations because RalBP1 selectively regulates NMDA-induced AMPAR endocytosis and LTD but not surface AMPAR levels associated with mEPSCs and AMPA/NMDA ratios ( and ). Together, these results suggest that neither AMPA- nor NMDA-mediated excitatory synaptic transmission was affected by the reduced expression of RalBP1 under basal conditions.