Regulated RalBP1 Binding to RalA and PSD-95 Controls AMPA Receptor Endocytosis and LTD

Yeast two-hybrid screens with PSD-95 identified RalBP1 as a novel PSD-95-interacting protein.

“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 (Figure 1A) [34],[36].”

• cites [34] M Ikeda, 1998, Identification and characterization of a novel protein interacting with Ral-binding protein 1, a putative effector protein of Ral., J Biol Chem sourceDisputed

  The Experimental Procedures describe a yeast two-hybrid screen using RalBP1, not PSD-95. The Isolation of RalBP1-interacting Protein section says the newly identified partner was POB1, not RalBP1. (LLM, conf=0.88)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured
• cites [36] T Itoh, 2006, BAR, F-BAR (EFC) and ENTH/ANTH domains in the regulation of membrane-cytosol interfaces and membrane curvature., Biochim Biophys Acta sourceNot checked — no full text of the source

PSD-95 is a key determinant of synaptic levels of AMPARs.

“Accordingly, PSD-95 is a key determinant of synaptic levels of AMPARs [21],[22].”

• cites [21] G. M Elias, 2007, Synaptic trafficking of glutamate receptors by MAGUK scaffolding proteins., Trends Cell Biol sourceNot checked — no full text of the source
• cites [22] S. M Fitzjohn, 2006, Promiscuous interactions between AMPA-Rs and MAGUKs., Neuron sourceSupported

Acute knockdown of PSD-95 in brain slices impairs LTD.

“In addition, acute knockdown of PSD-95 in brain slices impairs LTD [25],[26].”

• cites [25] I Ehrlich, 2007, PSD-95 is required for activity-driven synapse stabilization., Proc Natl Acad Sci U S A sourceSupported
• cites [26] W Xu, 2008, Molecular dissociation of the role of PSD-95 in regulating synaptic strength and LTD., Neuron sourceSupported

Active RalA associates with RalBP1/RLIP76 to promote endocytosis.

“Endocytosis occurs when active RalA associates with RalBP1/RLIP76 [31],[32], an endocytic adaptor that directly interacts with the μ2 subunit of the endocytic AP2 complex [33] and EH domain proteins POB1 and Reps1 [34],[35].”

• cites [31] V Jullien-Flores, 1995, Bridging Ral GTPase to Rho pathways. RLIP76, a Ral effector with CDC42/Rac GTPase-activating protein activity., J Biol Chem sourcePartly supported

  The Results show that RLIP1 binds GTP-bound RalA, and the Abstract identifies the full-length protein as RLIP76. The paper does not report that this association promotes endocytosis; its Discussion instead examines a possible role in Rho-pathway signaling. The indexed passages provide section labels but no page numbers. (LLM, conf=0.90)

  Experimental — accuracy not yet measured
• cites [32] S. B Cantor, 1995, Identification and characterization of Ral-binding protein 1, a potential downstream target of Ral GTPases., Mol Cell Biol sourcePartly supported

  The Abstract and “Binding of RalBP1 to Ral in vitro” section report that RalBP1 binds active, GTP-bound RalA. The Discussion notes Ral’s presence in clathrin-coated vesicles and speculates about where it might recruit RalBP1, but does not show that their association promotes endocytosis. Page numbers are unavailable in our copy. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

RalBP1 and POB1 were largely found in cytosolic (S3) and microsomal (P3) fractions.

“In contrast, RalBP1 and POB1 were largely found in cytosolic (S3) and microsomal (P3) fractions.”

A phosphomimetic RalBP1 mutant (T645E) failed to interact with PSD-95 in yeast two-hybrid and in vitro coprecipitation assays.

“A phosphomimetic RalBP1 mutant (T645E) failed to interact with PSD-95 in yeast two-hybrid and in vitro coprecipitation assays (Figure 2G and 2H).”

• AssumptionThe authors state this as an assumption: The T645E mutant is a phosphomimetic that mimics phosphorylation of RalBP1 at Thr 645.

RalBP1 dephosphorylation was not affected by low-concentration okadaic acid (10 nM) or FK506.

“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.”

RalBP1 ΔCC showed no significant RalA-dependent spine translocation.

“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.”

• AssumptionThe authors state this as an assumption: RalBP1 ΔCC lacks the CC domain that is involved in RalA binding.

Coexpression of constitutively active RalA (G23V) with RalBP1 induced a marked translocation of RalBP1 to dendritic spines.

“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 (Figure 4B).”

• AssumptionThe authors state this as an assumption: RalA G23V is a constitutively active form of RalA.
• AssumptionThe authors state this as an assumption: RalA S28N is a dominant negative RalA that is constitutively in the GDP-bound state.

NMDA treatment of cultured neurons induced RalA activation, measured by pull down assays.

“NMDA treatment of cultured neurons induced RalA activation, measured by pull down assays (Figure 4A).”

Such increases were not observed in control neurons expressing RalA G23V and RalBP1 ΔC.

“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.”

RalA G23V formed a ternary complex with RalBP1 and PSD-95 in HEK293T cells.

“In HEK293T cells, RalA G23V formed a ternary complex with RalBP1 and PSD-95 (Figure S5B).”

• AssumptionThe authors state this as an assumption: RalA G23V is a constitutively active form of RalA.

The effect of LFS on RalBP1 phosphorylation was blocked by APV.

“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 (Figure 5A and 5B).”

No associations of SAP97 and SAP102 with RalBP1 were detected.

“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.”

RalA was mainly detected in crude synaptosomal (P2) and synaptic membrane (LP1) fractions, similar to PSD-95.

“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).”

RalA induced translocation of PSD-95 to the plasma membrane in HEK293T cells coexpressing RalBP1 WT, but not RalBP1 ΔC.

“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).”

RalBP1 formed an in vivo complex with PSD-93/chapsyn-110.

“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.”

A RalA G23V mutant with weakened RalBP1 binding (G23VD49N; GVDN) induced a spine translocation of RalBP1 that is smaller than that of RalBP1 coexpressed with RalA G23V.

“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.”

• AssumptionThe authors state this as an assumption: RalA GVDN (G23VD49N) is a RalA G23V mutant with weakened RalBP1 binding.

RalBP1 dephosphorylation was blocked by okadaic acid (1 μM).

“RalBP1 dephosphorylation was blocked by okadaic acid (1 µM), which inhibits both protein phosphatase 1 and 2A (PP1 and PP2A) (Figure 3D).”

RalBP1 ΔC showed a RalA-dependent spine translocation similar to that of WT RalBP1.

“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.”

NMDA treatment of neurons coexpressing RalA G23V and RalBP1 further increased RalA-dependent spine translocation of RalBP1.

“NMDA treatment of neurons coexpressing RalA G23V and RalBP1 further increased RalA-dependent spine translocation of RalBP1 (Figure 4C).”

LFS (1 Hz, 900 pulses) given to the CA1 region of hippocampal slices significantly decreased levels of RalBP1 phosphorylation at T645.

“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 (Figure 5A and 5B).”

After NMDA washout, RalBP1 was rephosphorylated to near-normal levels in ~1 h.

“After NMDA washout, RalBP1 was rephosphorylated to near-normal levels in ∼1 h (Figure 3E).”

RalBP1 was minimally detected in PSD fractions.

“RalBP1 was minimally detected in PSD fractions.”

NMDA treatment (20 μM for 3 min) of cultured hippocampal neurons induced a rapid and significant (~50%) dephosphorylation of RalBP1 at Thr 645.

“NMDA treatment (20 µM for 3 min) of cultured hippocampal neurons induced a rapid and significant (∼50%) dephosphorylation of RalBP1 at Thr 645 (Figure 3A).”

RalBP1 did not coprecipitate with RalA under our experimental conditions.

“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.”

The PDZ-binding motif at the RalBP1 C-terminus interacted with the PDZ domains of PSD-95.

“The PDZ-binding motif at the RalBP1 C-terminus interacted with the PDZ domains of PSD-95 (Figure 1B).”

RalBP1 binding to PSD-95 was confirmed by in vitro and in vivo pull down and coimmunoprecipitation assays.

“RalBP1 binding to PSD-95 was further confirmed by in vitro and in vivo pull down and coimmunoprecipitation assays (Figure 1C–1H).”

PKA could directly phosphorylate RalBP1 in vitro.

“Consistent with this possibility, PKA could directly phosphorylate RalBP1 in vitro (Figure 2F).”

λ-Phosphatase digestion of RalBP1 proteins expressed in vitro and in vivo substantially weakened protein detection by phospho-specific antibodies.

“λ-Phosphatase digestion of RalBP1 proteins expressed in vitro and in vivo substantially weakened protein detection by phospho-specific antibodies (Figure 2D).”

RalBP1 phosphorylation levels returned to a normal range 60 min after LFS.

“RalBP1 phosphorylation levels returned to a normal range 60 min after LFS (Figure 5C), a result similar to that obtained in NMDA-treated cultured neurons.”

RalBP1 is phosphorylated at Thr 645 in heterologous cells.

“RalBP1 is phosphorylated at Thr 645 in heterologous cells.”

RalBP1 dephosphorylation induced by NMDA treatment was blocked by APV.

“RalBP1 dephosphorylation induced by NMDA treatment was blocked by APV, an NMDAR antagonist (Figure 3C).”

RalBP1 proteins phosphorylated at T645 showed reduced biochemical association with PSD-95 in HEK293T cells, compared to total RalBP1.

“RalBP1 proteins phosphorylated at T645 showed reduced biochemical association with PSD-95 in HEK293T cells, compared to total (phosphorylated+non-phosphorylated) RalBP1 (Figure 2I).”

Okadaic acid (1 μM) increased RalBP1 phosphorylation before NMDA treatment.

“Notably, okadaic acid (1 µM) increased RalBP1 phosphorylation before NMDA treatment (Figure 3D), indicating that PP1 also mediates basal dephosphorylation of RalBP1.”

RalBP1 formed a complex with POB1 and α-adaptin in the brain.

“RalBP1 also formed a complex with POB1 and α-adaptin (a subunit of the AP2 complex) in the brain (Figure 1I and 1J).”

DHPG, a group I mGluR agonist, did not induce RalBP1 dephosphorylation.

“DHPG, a group I mGluR agonist, however, did not induce RalBP1 dephosphorylation (Figure 3B).”

An upstream mutation in RalBP1 (R642A; −5 position) abolished the phosphorylation.

“An upstream mutation in RalBP1 (R642A; −5 position) abolished the phosphorylation (Figure 2E), indicating that R642 is important for RalBP1 phosphorylation.”

RalBP1 transfected alone was not translocated to spines upon NMDA treatment.

“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.”

There was no significant difference in the amplitudes or frequencies of mEPSCs between RalBP1−/− and WT mice.

“we observed no significant difference in the amplitudes or frequencies of mEPSCs between RalBP1−/− and WT mice (Figure 11E–11H).”

Inhibition of active RalA by overexpression of RalA S28N had no effect on GluR2 recycling.

“However, inhibition of active RalA by overexpression of RalA S28N had no effect on GluR2 recycling (Figure S9A).”

• AssumptionThe authors state this as an assumption: RalA S28N is a dominant negative RalA that is constitutively in the GDP-bound state.

RalA G23V alone does not occlude the NMDA-induced reduction in surface AMPAR levels.

“In addition, RalA G23V alone does not occlude the NMDA-induced reduction in surface AMPAR levels.”

RalBD significantly reduced NMDA-induced GluR2 endocytosis.

“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 (Figure 6C).”

• AssumptionThe authors state this as an assumption: RalBD binds and inhibits only active RalA.

Coexpression of RalA G23V and RalBP1 ΔC did not induce a reduction in surface AMPAR levels, relative to coexpression of RalA G23V and WT 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.”

• AssumptionThe authors state this as an assumption: RalA G23V is a constitutively active form of RalA.

Knockdown of RalBP1 and RalA significantly reduced NMDA-induced endocytosis of the GluR2 subunit of AMPARs in cultured neurons.

“In cultured neurons, knockdown of RalBP1 and RalA significantly reduced NMDA-induced endocytosis of the GluR2 subunit of AMPARs (Figure 6A).”

Knockdown of RalA did not affect GluR2 recycling.

“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.”

RalBP1 forms a complex with POB1 and AP2 (α-adaptin) in the brain.

“we demonstrated that RalBP1 forms a complex with POB1 and AP2 (α-adaptin) in the brain.”

Basal GluR2 endocytosis was not affected by overexpression of RalBD, RalA S28N, or RalA GVDN.

“Intriguingly, basal GluR2 endocytosis was not affected by overexpression of RalBD (Figure 7C), RalA S28N, or RalA GVDN (Figure S8B).”

• AssumptionThe authors state this as an assumption: RalA S28N is a dominant negative RalA that is constitutively in the GDP-bound state.
• AssumptionThe authors state this as an assumption: RalA GVDN (G23VD49N) is a RalA G23V mutant with weakened RalBP1 binding.
• AssumptionThe authors state this as an assumption: RalBD binds and inhibits only active RalA.

NMDAR activation rapidly induces RalA activation.

“NMDAR activation rapidly induces RalA activation.”

The gene trapping generated a small amount of fusion proteins containing RalBP1 (first 235 aa) and β-geo.

“The gene trapping generated a small amount of fusion proteins containing RalBP1 (first 235 aa) and β-geo (Figure 9C)”

Neurons transfected with empty shRNA vector showed an LTD magnitude comparable to that of untransfected neurons.

“In contrast, neurons transfected with empty shRNA vector showed an LTD magnitude comparable to that of untransfected neurons (Figure 6F and 6G).”

NMDA-induced AMPAR endocytosis is suppressed by inhibition of RalA by overexpression of RalBD.

“NMDA-induced AMPAR endocytosis is suppressed by knockdown of RalA, and inhibition of RalA by overexpression of RalBD and RalA (S28N).”

Inhibition of RalBP1 by overexpression of RalBP1 TE and POB1 CC had no effect on basal GluR2 endocytosis.

“Inhibition of RalBP1 by overexpression of RalBP1 TE and POB1 CC had no effect on basal GluR2 endocytosis (Figure 7B and 7C), further suggesting that RalBP1 does not regulate basal GluR2 endocytosis.”

• AssumptionThe authors state this as an assumption: The T645E mutant is a phosphomimetic that mimics phosphorylation of RalBP1 at Thr 645.
• AssumptionThe authors state this as an assumption: POB1 CC binds and inhibits RalBP1.

RalBP1 protein expression in RalBP1−/− brain is ∼18.1%±3.1% of WT mice.

“Expression levels of RalBP1 proteins in RalBP1−/− brain was ∼18.1%±3.1% (n = 8) of WT mice (Figure 9C and 9D)”

• Also stated in: § RalBP1 and PSD-95 in NMDAR-Dependent AMPAR Endocytosis and LTD · plain

  “that is, RalBP1−/− mice have ∼18% of residual RalBP1 expression.”

LTP induced by TBS in RalBP1−/− slices (4-7 wk) was not substantially different from that of WT littermates throughout the recording.

“LTP induced by TBS in RalBP1−/− slices (4–7 wk) was not substantially different from that of WT littermates throughout the recording (Figure 10C).”

LFS did not significantly enhance coimmunoprecipitation of RalBP1 and PSD-95 compared with LFS and APV (p = 0.1; n = 6).

“Although there was a tendency for LFS to increase coimmunoprecipitation of RalBP1 and PSD-95 compared with that of LFS and APV (Figure 5G–5H), this difference did not reach statistical significance (p = 0.1; n = 6).”

Surface AMPAR levels in neurons expressing RalA G23V and RalBP1 were significantly reduced in the absence of NMDA treatment, compared to those expressing RalA G23V alone or WT RalA and RalBP1.

“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 (Figure 8A).”

• AssumptionThe authors state this as an assumption: RalA G23V is a constitutively active form of RalA.

RalBP1 knockdown in cultured neurons decreases NMDA-induced GluR2 endocytosis.

“RalBP1 knockdown in cultured neurons, or RalBP1 inhibition by overexpression of POB1 CC or phosphomimetic RalBP1 (TE), decreases NMDA-induced GluR2 endocytosis.”

Neurons coexpressing RalA G23V alone (without RalBP1 coexpression) showed an NMDA-induced reduction in surface AMPAR levels.

“In contrast, neurons coexpressing RalA G23V alone (without RalBP1 coexpression) showed an NMDA-induced reduction in surface AMPAR levels.”

RalBP1 inhibition by overexpression of phosphomimetic RalBP1 (TE) decreases NMDA-induced GluR2 endocytosis.

“RalBP1 knockdown in cultured neurons, or RalBP1 inhibition by overexpression of POB1 CC or phosphomimetic RalBP1 (TE), decreases NMDA-induced GluR2 endocytosis.”

There were no changes in expression levels of RalBP1-interacting proteins RalA, α-adaptin, and PSD-95, as well as subunits of AMPARs and NMDARs in RalBP1−/− brain.

“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 (Figure 9F).”

The CC domain of POB1 (POB1 CC) significantly reduced NMDA-induced GluR2 endocytosis.

“In addition, the CC domain of POB1 (POB1 CC), which binds and inhibits RalBP1, significantly reduced NMDA-induced GluR2 endocytosis (Figure 6C).”

• AssumptionThe authors state this as an assumption: POB1 CC binds and inhibits RalBP1.

Basal GluR2 endocytosis in the absence of NMDAR activation was enhanced by knockdown of RalA, but not RalBP1.

“Intriguingly, basal GluR2 endocytosis in the absence of NMDAR activation was enhanced by the knockdown of RalA, but not RalBP1 (Figure 7A), suggesting that RalA, but not RalBP1, inhibits GluR2 endocytosis under basal conditions.”

The AMPA/NMDA ratios measured in RalBP1−/− slices were not different from those of WT littermates.

“The AMPA/NMDA ratios measured in RalBP1−/− slices were not different from those of WT littermates (Figure 11I and 11J).”

NMDA treatment of neurons coexpressing RalA G23V and RalBP1 did not induce AMPAR endocytosis.

“NMDA treatment of the neurons coexpressing RalA G23V and RalBP1 did not induce AMPAR endocytosis (Figure 8B)”

In slice culture, RalBD overexpression suppresses LTD.

“In slice culture, RalBD overexpression suppresses LTD.”

The relationship between presynaptic fiber volley and slope of postsynaptic fEPSPs at different stimulus intensities was not changed in RalBP1−/− slices.

“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 (Figure 11C and 11D).”

Post-tetanic potentiation measured after TBS appeared normal in RalBP1−/− mice.

“In addition, post-tetanic potentiation, another form of short-term plasticity, measured after TBS also appeared normal in RalBP1−/− mice (Figure 10C and 10D).”

RalBP1 knockdown or inhibition in cultured neurons decreased NMDA-induced GluR2 endocytosis by ∼20%.

“RalBP1 knockdown or inhibition in cultured neurons, however, decreased NMDA-induced GluR2 endocytosis by ∼20%.”

A scrambled version of RalA shRNA had no effect on NMDA-induced GluR2 endocytosis.

“A scrambled version of RalA shRNA had no effect.”

Surface GluR2 levels were reduced by overexpression of RalBD or RalA S28N.

“Interestingly, surface GluR2 levels were reduced by overexpression of RalBD (Figure 7F) or RalA S28N (Figure S8C), which inhibits active RalA, indicating that active RalA is important for the maintenance of surface GluR2 levels.”

• AssumptionThe authors state this as an assumption: RalA S28N is a dominant negative RalA that is constitutively in the GDP-bound state.
• AssumptionThe authors state this as an assumption: RalBD binds and inhibits only active RalA.

LFS for LTD induction (1 Hz, 900 stimulations) induced robust synaptic depression in WT slices (17-21 d) that averaged 71.6%±0.9%.

“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) (Figure 10A).”

Inhibition of NMDAR by APV during LFS abolished the difference in LTD between WT and RalBP1−/− genotypes.

“Inhibition of NMDAR by APV during LFS abolished the difference between the two genotypes (unpublished data).”

Synaptic depression by LFS after TBS (depotentiation) was not different in WT and RalBP1−/− mice.

“In contrast to de novo LTD, synaptic depression by LFS after TBS was not different in WT and RalBP1−/− mice (Figure 10D).”

RalA activation combined with RalBP1 binding to PSD-95 occludes the NMDA-induced reduction in surface AMPAR levels.

“In support of this, RalA activation combined with RalBP1 binding to PSD-95 is sufficient to reduce surface AMPAR levels in the absence of NMDAR activation; it also occludes the NMDA-induced reduction in surface AMPAR levels (Figure 8).”

RalBP1 inhibition by overexpression of POB1 CC decreases NMDA-induced GluR2 endocytosis.

“RalBP1 knockdown in cultured neurons, or RalBP1 inhibition by overexpression of POB1 CC or phosphomimetic RalBP1 (TE), decreases NMDA-induced GluR2 endocytosis.”

RalBP1 knockdown in slice culture reduces LTD.

“RalBP1 knockdown in slice culture reduces LTD.”

LTD induction in RalBP1−/− mice was significantly attenuated (87.6%±1.1%) compared to WT.

“In contrast, LTD induction in RalBP1−/− mice was significantly attenuated (87.6%±1.1%; n = 24, 10 animals; *** p<0.001)”

• Also stated in: § RalBP1 and PSD-95 in NMDAR-Dependent AMPAR Endocytosis and LTD · plain

  “Reduced RalBP1 expression in RalBP1−/− mice suppresses LFS-induced LTD.”

• AssumptionRests on an assumption: The residual RalBP1-β-geo fusion protein in RalBP1−/− mice is non-functional or does not contribute to RalBP1 activity.

RalA activation combined with RalBP1 binding to PSD-95 is sufficient to reduce surface AMPAR levels in the absence of NMDAR activation.

“In support of this, RalA activation combined with RalBP1 binding to PSD-95 is sufficient to reduce surface AMPAR levels in the absence of NMDAR activation; it also occludes the NMDA-induced reduction in surface AMPAR levels (Figure 8).”

Inhibition of RalBP1 by RalBP1 TE or POB1 CC had no effect on surface GluR2 levels.

“Consistent with this, inhibition of RalBP1 by RalBP1 TE or POB1 CC had no effect on surface GluR2 levels (Figure 7E and 7F).”

• AssumptionThe authors state this as an assumption: The T645E mutant is a phosphomimetic that mimics phosphorylation of RalBP1 at Thr 645.
• AssumptionThe authors state this as an assumption: POB1 CC binds and inhibits RalBP1.

NMDA-induced AMPAR endocytosis is suppressed by inhibition of RalA by overexpression of RalA (S28N).

“NMDA-induced AMPAR endocytosis is suppressed by knockdown of RalA, and inhibition of RalA by overexpression of RalBD and RalA (S28N).”

POB1 expression was significantly decreased by 43.8%±10.9% in RalBP1−/− brain.

“POB1 expression was significantly decreased by 43.8%±10.9% (n = 8)”

RalBD significantly reduced pairing-induced LTD (~71% of baseline) relative to untransfected control neurons (~46% of baseline).

“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).”

• AssumptionThe authors state this as an assumption: RalBD binds and inhibits only active RalA.

Knockdown of RalA, but not RalBP1, significantly reduced surface levels of GluR2.

“Indeed, knockdown of RalA, but not RalBP1, significantly reduced surface levels of GluR2 (Figure 7D), suggesting that surface GluR2 levels are maintained by RalA but not RalBP1.”

shRNA-resistant expression constructs of RalBP1 and RalA coexpressed with RalBP1 and RalA shRNAs, respectively, rescued the knockdown effects.

“shRNA-resistant expression constructs of RalBP1 and RalA coexpressed with RalBP1 and RalA shRNAs, respectively, rescued the knockdown effects (Figure S7).”

Bath application of DHPG induced stable depression in both WT and RalBP1−/− slices (8 wk), with magnitude essentially identical throughout the recording.

“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 (Figure 10B).”

NMDA-induced AMPAR endocytosis is suppressed by knockdown of RalA.

“NMDA-induced AMPAR endocytosis is suppressed by knockdown of RalA, and inhibition of RalA by overexpression of RalBD and RalA (S28N).”

PPF at all interstimulus intervals tested was not changed at RalBP1−/− SC-CA1 synapses.

“PPF at all interstimulus intervals tested was not changed at RalBP1−/− SC-CA1 synapses (Figure 11A and 11B).”

RalBP1 knockdown in CA1 pyramidal neurons significantly reduced pairing-induced LTD at SC-CA1 synapses (~81% of baseline) relative to untransfected control neurons (~52% of baseline).

“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) (Figure 6D and 6E).”

Constitutive RalA activation combined with RalBP1 binding to PSD-95 occludes NMDA-induced reduction in levels of surface AMPARs.

“Constitutive RalA activation combined with RalBP1 binding to PSD-95 occludes NMDA-induced reduction in the levels of surface AMPARs.”

• AssumptionThe authors state this as an assumption: RalA G23V is a constitutively active form of RalA.

Reduced RalBP1 expression in transfected slices and mice reduced LTD magnitudes by ∼50%-60%.

“Quantitatively, reduced RalBP1 expression in transfected slices and mice reduced LTD magnitudes by ∼50%–60%”

• AssumptionRests on an assumption: The residual RalBP1-β-geo fusion protein in RalBP1−/− mice is non-functional or does not contribute to RalBP1 activity.

Dephosphorylation of RalBP1 alone is not sufficient to induce RalBP1 translocation to spines.

“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.

“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).”

Activated RalA alone is sufficient to induce spine translocation of RalBP1.

“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.”

PP1 is important for RalBP1 dephosphorylation.

“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.”

Binding of dephosphorylated RalBP1 to PSD-95, combined with RalBP1 binding to activated RalA, further promotes synaptic localization of RalBP1.

“These results suggest that binding of dephosphorylated RalBP1 to PSD-95, combined with RalBP1 binding to activated RalA, further promotes synaptic localization of RalBP1.”

PP1 mediates basal dephosphorylation of RalBP1.

“Notably, okadaic acid (1 µM) increased RalBP1 phosphorylation before NMDA treatment (Figure 3D), indicating that PP1 also mediates basal dephosphorylation of RalBP1.”

The limited subcellular overlap between RalBP1 and PSD-95 suggests that their interaction might be regulated.

“The limited subcellular overlap between RalBP1 and PSD-95 (Figure S2F) suggests that their interaction might be regulated.”

A two-step mechanism regulates receptor endocytosis during the development of long-term depression (LTD).

“A two step mechanism was identified that regulates receptor endocytosis during the development of long-term depression (LTD), a long-lasting decrease in synaptic transmission.”

The study provides the first specific mechanism for how the interaction of activated RalA with RalBP1 is coupled to the endocytosis of target membrane proteins.

“Our study provides the first specific mechanism for the general question of how the interaction of activated RalA with RalBP1 is coupled to the endocytosis of target membrane proteins.”

LTD-inducing NMDAR activation would seem to bring both enzyme (PP1) and substrate (RalBP1) together at synapses, enabling their functional interaction.

“Thus, LTD-inducing NMDAR activation would seem to bring both enzyme (PP1) and substrate (RalBP1) together at synapses, enabling their functional interaction.”

The dual and regulated binding of RalBP1 with RalA and PSD-95 mediates AMPAR endocytosis during NMDAR-dependent LTD.

“In conclusion, our in vitro and in vivo results suggest that the dual and regulated binding of RalBP1 with RalA and PSD-95 mediates AMPAR endocytosis during NMDAR-dependent LTD.”

• AssumptionRests on an assumption: The residual RalBP1-β-geo fusion protein in RalBP1−/− mice is non-functional or does not contribute to RalBP1 activity.

RalBP1 is rephosphorylated by PKA during the recovery phase (∼1 h) after NMDA treatment.

“Our data suggest that RalBP1 is rephosphorylated by PKA during the recovery phase (∼1 h) after NMDA treatment.”

A fraction of exogenously expressed RalBP1 proteins is basally dephosphorylated in the absence of NMDAR activation.

“The results described above (Figure 8A) also suggest that a fraction of exogenously expressed RalBP1 proteins is basally dephosphorylated (in the absence of NMDAR activation)”

The direct interaction of dephosphorylated RalBP1 with a scaffolding protein that is coupled to target membrane proteins mediates this coupling.

“That is, in our case, the direct interaction of dephosphorylated RalBP1 with a scaffolding protein that is coupled to target membrane proteins.”

Neither active nor inactive RalA regulate GluR2 recycling.

“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.”

RalBP1 is important for the stability of POB1.

“suggesting that RalBP1 is important for the stability of POB1.”

• AssumptionRests on an assumption: The residual RalBP1-β-geo fusion protein in RalBP1−/− mice is non-functional or does not contribute to RalBP1 activity.

RalBP1 binding to PSD-95 is important for NMDA-induced GluR2 endocytosis.

“Overexpression of RalBP1 TE, a phosphomimetic RalBP1 mutant that lacks PSD-95 binding, inhibited NMDA-induced GluR2 endocytosis, while WT RalBP1 did not (Figure 6B), suggesting that RalBP1 binding to PSD-95 is important.”

• AssumptionThe authors state this as an assumption: The T645E mutant is a phosphomimetic that mimics phosphorylation of RalBP1 at Thr 645.

AP2 and POB1 further associate with EH domain-containing endocytic proteins epsin and Eps15.

“which further associate with EH domain-containing endocytic proteins epsin and Eps15 [36].”

• cites [36] T Itoh, 2006, BAR, F-BAR (EFC) and ENTH/ANTH domains in the regulation of membrane-cytosol interfaces and membrane curvature., Biochim Biophys Acta sourceNot checked — no full text of the source

The requirement for RalA activation and RalBP1 dephosphorylation in NMDAR-dependent AMPAR endocytosis suggests that these two events may function as a dual-key mechanism that protects against AMPAR endocytosis under conditions in which only a single criterion is fulfilled.

“The requirement for these two mechanisms—RalA activation and RalBP1 dephosphorylation—in NMDAR-dependent AMPAR endocytosis suggests that these two events may function as a dual-key mechanism that protects against AMPAR endocytosis under conditions in which only a single criterion is fulfilled.”

Inactive RalA may help maintain surface GluR2 levels by inhibiting basal GluR2 endocytosis.

“Inactive RalA may help maintain surface GluR2 levels by inhibiting basal GluR2 endocytosis (Figures 7A, 7C, and S8B).”

Active RalA is important for the maintenance of surface GluR2 levels.

“Interestingly, surface GluR2 levels were reduced by overexpression of RalBD (Figure 7F) or RalA S28N (Figure S8C), which inhibits active RalA, indicating that active RalA is important for the maintenance of surface GluR2 levels.”

The residual RalBP1 expression in RalBP1−/− mice is likely due to incomplete gene trapping.

“likely due to incomplete gene trapping.”

RalA and RalBP1 act together to mediate NMDAR-dependent AMPAR endocytosis.

“Our data indicate that RalA and RalBP1 act together to mediate NMDAR-dependent AMPAR endocytosis.”

Reduced expression of RalBP1 selectively impairs NMDAR-dependent LTD.

“These results suggest that the reduced expression of RalBP1 selectively impairs NMDAR-dependent LTD.”

• AssumptionRests on an assumption: The residual RalBP1-β-geo fusion protein in RalBP1−/− mice is non-functional or does not contribute to RalBP1 activity.

RalBP1 and RalA are required for LTD induction.

“These results suggest that RalBP1 and RalA are required for LTD induction.”

Enhanced RalBP1 binding to PSD-95 may bring RalBP1-associated endocytic proteins such as AP2 and POB1 close to PSD-95, which is linked to the complex of TARPs and AMPARs.

“Therefore, enhanced RalBP1 binding to PSD-95 may bring RalBP1-associated endocytic proteins such as AP2 and POB1 close to PSD-95, which is linked to the complex of TARPs and AMPARs [18].”

• cites [18] R. A Nicoll, 2006, Auxiliary subunits assist AMPA-type glutamate receptors., Science sourceNot checked — no full text of the source

Scaffolding proteins can switch their functions from the maintenance to regulated endocytosis of interacting membrane proteins.

“Secondly, our results suggest that scaffolding proteins can switch their functions from the maintenance to regulated endocytosis of interacting membrane proteins.”

Both active and inactive RalA are involved in maintaining surface GluR2 levels.

“Collectively, these results indicate that both active and inactive RalA are involved in maintaining surface GluR2 levels.”

RalBP1 and RalA are required for NMDA-induced endocytosis of GluR2.

“These results suggest that RalBP1 and RalA are required for NMDA-induced endocytosis of GluR2.”

RalA inhibits basal AMPAR endocytosis in a GTP-independent (or RalA activation-independent) manner.

“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.”

NMDAR activation by low-frequency electrical stimulation (LFS) induces RalBP1 dephosphorylation.

“NMDAR activation by low-frequency electrical stimulation (LFS) induces RalBP1 dephosphorylation”

RalBP1 knockdown did not affect basal synaptic transmission, as measured by amplitudes of evoked excitatory postsynaptic currents (EPSCs).

“RalBP1 knockdown did not affect basal synaptic transmission, as measured by amplitudes of evoked excitatory postsynaptic currents (EPSCs) (Figure 6H).”

Basal transmission was unaffected by RalBD overexpression.

“Basal transmission was unaffected by RalBD overexpression (Figure 6K).”