Regulation of {alpha}-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor trafficking through PKA phosphorylation of the Glu receptor 1 subunit

Abstract

alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors mediate the majority of excitatory synaptic transmission in the brain. Recent studies have shown that activation of PKA regulates the membrane trafficking of the AMPA receptor Glu receptor 1 (GluR1) subunit, but the role of direct phosphorylation of GluR1 in regulating receptor redistribution is not clear. Here we show that phosphorylation of the GluR1 subunit on serine 845 by PKA is required for PKA-induced increases in AMPA receptor cell-surface expression because it promotes receptor insertion and decreases receptor endocytosis. Furthermore, dephosphorylation of GluR1 serine 845 triggers NMDA-induced AMPA receptor internalization. These findings strongly suggest that dynamic changes in direct phosphorylation of GluR1 by PKA are crucial in the modulation of AMPA receptor trafficking and synaptic plasticity.

Fig. 1.

PKA activation increases AMPA receptor cell-surface expression in cultured cortical neurons. (A) Cell-surface biotinylation shows an increase in AMPA receptor surface expression (Surf) by using forskolin treatment (20 μM forskolin plus 50 μM 3-isobutyl-1-methylxanthine) (Forsk) compared with control (Con). This effect was abolished with PKA-specific inhibitor H89 (Forsk+H89). No changes were found in the total AMPA receptor protein amount (Total). (B) Densitometric quantitation of Western blots on AMPA receptor surface biotinylation. Data represent means ± SE (n = 4; ∗, P < 0.05 relative to control, t test). (C and D) Forskolin treatments show no effect on AMPA receptor constitutive internalization. Surface biotinylation at 4°C without stripping produced total surface receptors (T-surf), and stripping immediately after biotinylation showed low background (Strip and Con) (n = 3; P > 0.05, t test). (E and F) PKA inhibits NMDA-induced AMPA receptor internalization. Surface-biotinylated neurons were incubated at 37°C in the presence of 30 μM NMDA, with (NMDA+Forsk) or without (NMDA) forskolin treatment, for 15 min to induce receptor endocytosis (n = 4; ∗, P < 0.05, t test). (G and H) Surface biotinylation-based receptor insertion assays. Forskolin treatment decreased the remaining endocytosed receptor amount by using double strips (n = 2), indicating a facilitated receptor reinsertion. (I) AMPA receptor surface insertion by colorimetric assays. Surface AMPA receptors were first blocked with an anti-GluR1 N terminus antibody and a cold (nonconjugated) secondary antibody. After incubation at 37°C with (Forsk) or without (Con) forskolin treatment, newly inserted surface receptors were detected by using a second round of antibody labeling (n = 8; ∗, P < 0.05 compared with control, t test).

Fig. 2.

PKA-dependent increase in AMPA receptor cell-surface expression is subunit-specific. (A) Surface biotinylation assays in transfected HEK cells. Forskolin treatment for 15 min increased the AMPA receptor subunit surface expression (Surf) in HEK cells expressing GluR1-GFP (Left) but not in those expressing GluR2-GFP (Right). (B) Quantitation of cell-surface expression of AMPA receptor subunits in HEK cells. Forskolin treatment (Forsk) increased GluR1-GFP surface expression (n = 3; ∗, P < 0.05, t test) (Left) but did not change the abundance of cell-surface GluR2-GFP (n = 3; P > 0.05) (Right). Con, control.

Fig. 3.

Phosphorylation of GluR1S845 is required in PKA activity-dependent regulation of AMPA receptor surface expression. (A) PKA phosphorylates GluR1S845 in both cortical neurons and GluR1-expressing HEK cells. Cultured cortical neurons (Left) or HEK cells transiently transfected with GluR1-GFP (Right) were treated with forskolin (Forsk) for 10 min, and GluR1S845 phosphorylation was examined by using anti-phospho-GluR1S845 (GluRI p-S845) antibodies. Compared with controls (Con), forskolin treatment greatly increased the phosphorylation level that was blocked by the PKA inhibitor H89 (Forsk+H89) (2 μM), and the PKC activator PMA (1 μM) had no effect. (B and C) PKA increases surface-GluR1 positive rate in transfected HEK cells. (B) In GluR1-GFP-expressing or GluR1S845A-GFP-expressing HEK cells, surface staining (red) revealed that a certain amount of cells had no visible surface labeling. (C) PKA treatment increased the percentage of surface-positive cells in the GluR1-expressing HEK population (Left) (n = 300 transfected cells in three experiments; ∗, P < 0.05 relative to control, t test) but not in cells expressing GluR1S845A (Right) (n = 300 transfected cells in three experiments). (D) Surface biotinylation assays showed no effect of forskolin treatment on GluR1S845A surface expression in transfected HEK cells. (E) Densitometric quantitation of surface biotinylation experiments in D. Forskolin treatment significantly increased the surface expression of GluR1-GFP (Left) (n = 5; ∗, P < 0.05, t test), but not GluR1S845A-GFP (Right) (n = 5; P > 0.05, t test).

Fig. 4.

Forskolin increases GluR1 cell-surface insertion rate in an S845-dependent manner in transfected cortical neurons. (A) Cortical neurons were transfected with GluR1 subunit (green) tagged with a BBS and GFP at its extracellular N terminus (BBS-GFP-GluR1). Rhodamine-Btx surface binding assay demonstrated that BBS-GFP-GluR1 was expressed on cell surface in clusters (Con). Immediately after incubation with free Btx, rhodamine-Btx labeling showed no signal, indicating a complete block of surface GluR1 BBS sites (Block). (B) Forskolin treatment facilitated GluR1 cell-surface insertion rate (BBS-GFP-GluR1) but had no effect on the mutant (GluR1S845A). (C) Quantitation of BBS-GFP-GluR1 plasma membrane insertion. Forskolin treatment increased both the cluster intensity (Left) and cluster size (Right) of the newly inserted surface BBS-GFP-GluR1 (n = 30; P < 0.05, t test).

Fig. 5.

A quick switch of GluR1S845 from a phosphorylated to a nonphosphorylated state is crucial to NMDA-induced AMPA receptor internalization. (A and B) NMDA treatment induces cell-surface GluR1S845 dephosphorylation. Plasma membrane AMPA receptors were isolated by using surface biotinylation and probed by using anti-phospho-GluR1S845 (GluR1 P-S845) antibodies. When receptor internalization was blocked with hypertonic sucrose solution (Sucrose), NMDA still caused a dramatic dephosphorylation (NMDA+Sucrose) of GluR1 (n = 2). (C) GluR1 is dephosphorylated at a rate faster than AMPA receptors are internalized after NMDA application. At 10 min after NMDA treatment, a large amount of AMPA receptors remained on the surface, but almost all of the surface receptors were dephosphorylated. (D) PKA preactivation enhances NMDA-induced AMPA receptor internalization. Cells were surface-biotinylated, incubated with forskolin for 5 min, and treated with NMDA. Endocytosed receptors were collected after surface stripping. PKA pretreatment increased the NMDA-induced AMPA receptor internalization (Pre-forsk), an effect that was blocked by H89 (Pre-forsk+H89). (E) Quantification of D. NMDA-caused AMPAR internalization was increased significantly by preforskolin treatment (n = 3; ∗, P < 0.05, t test).

Fig. 6.

Preventing dynamic GluR1S845 dephosphorylation abolishes NMDA-mediated GluR1 internalization. (A) Surface expression of GluR1-GFP in cortical neurons. Cultured cortical neurons transfected with GluR1-GFP or GluR1S845A-GFP (green) were immunostained with anti-GFP antibodies (red) under nonpermeant conditions. (B) NMDA fails to induce GluR1S845-GFP internalization. Surface receptors were labeled with antibodies against GFP, and receptor endocytosis was induced by using NMDA treatment. The internalized receptors were visualized after surface acid stripping. Note that NMDA treatment enhanced internalization of wild-type GluR1-GFP but had no effect on GluR1S845A. (C) Stripping immediately after surface biotinylation showed complete removal of surface labeling. (D) Quantitation of the internalization assays in B. NMDA treatment significantly increased GluR1-GFP internalization (n = 22; ∗, P < 0.05, t test) but had no effect on the internalization of GluR1S845A-GFP (n = 24; P > 0.05, test). (E and F) In contrast, AMPA treatment (50 μM AMPA for 10 min) increased receptor internalization of both GluR1-GFP (n = 21; ∗, P < 0.05, t test) and GluR1S845A-GFP (n = 23; ∗, P < 0.05, t test).