Tyrosine phosphorylation and regulation of the AMPA receptor by SRC family tyrosine kinases

Abstract

Phosphorylation of AMPA receptors is a major mechanism for the regulation of receptor function and underlies several forms of synaptic plasticity in the CNS. Although serine and threonine phosphorylation of AMPA receptors has been well studied, the potential role of tyrosine phosphorylation of AMPA receptors has not been investigated. Here, we show that the GluR2 subunit of AMPA receptors is tyrosine phosphorylated in vitro and in vivo by Src family tyrosine kinases on tyrosine 876 near its C terminus. In addition, GluR agonist treatment of cultured cortical neurons increased phosphorylation of tyrosine 876. The association with GluR2-interacting molecules GRIP1/2 was decreased by tyrosine phosphorylation of GluR2, whereas PICK1 interaction was not influenced. Moreover, mutation of tyrosine 876 eliminated AMPA- and NMDA-induced internalization of the GluR2 subunit. These data indicate that tyrosine phosphorylation of tyrosine 876 on the GluR2 C terminus by Src family tyrosine kinases is important for the regulation of AMPA receptor function and may be important for synaptic plasticity.

Figure 1.

Tyrosine phosphorylation in the GluR2 C-terminal region in rodent brain lysates. A, Tyrosine phosphorylation of each AMPA receptor subunit. Immunoprecipitates were isolated from mouse whole-brain lysates using rabbit IgG or each anti-GluR subunit antibody and were then immunoblotted with anti-phosphotyrosine antibody (PY20) or anti-GluR antibodies recognizing each subunit. B, C-terminal amino acid sequences of AMPA receptor subunits GluR1-4. The antigen phosphopeptide for anti-GluR2CPY antibodies and its corresponding non-phosphopeptide are shown. C, C-terminal tyrosine-phosphorylated GluR2 in the lysate from mouse cortex (Cx), hippocampus (Hi), or cerebellum (Cb). Brain lysates were immunoblotted with anti-GluR2, anti-GluR2CPY, or anti-tubulin antibodies. Recognition of GluR2 by anti-GluR2CPY was blocked by preabsorption of the antibodies with the antigen phosphopeptide or by λ phosphatase treatment but not by preabsorption with the non-phosphopeptide. Anti-tubulin immunoblot confirmed equal protein loading.

Figure 2.

Tyrosine phosphorylation of the GluR2 C-terminal region by Src family PTKs. A, C-terminal tyrosine-phosphorylated GluR2 in lysates from HEK 293T cells transfected with plasmids encoding GluR2 and plasmids encoding Src family PTK Lyn or a kinase-negative form of Lyn (KN). Cell lysates were immunoblotted with anti-GluR2CPY antibodies. Expression of each protein was confirmed by protein immunoblots of cell lysates probed with anti-GluR2 or anti-Lyn antibodies (top). Preabsorption experiments with antigen GluR2 C-terminal phosphopeptide or non-phosphopeptide (middle) and λ phosphatase treatment experiment (bottom) are shown.B, C-terminal tyrosine phosphorylation of GluR2 and GluR3 in lysates from HEK293T cells transfected with plasmids encoding GluR2 or GluR3 and plasmids encoding Src. Cell lysates were immunoblotted with anti-GluR2CPY antibodies. Expression of each protein was confirmed by protein immunoblots of cell lysates probed with anti-GluR2/3 or anti-Src antibodies.

Figure 3.

Phosphorylated tyrosine residues in the GluR2 C terminus by Src family PTKs. A, Three single-point mutant GluR2 subunits containing tyrosine to phenylalanine mutations (Y869F, Y873F, and Y876F) were generated. B, C-terminal tyrosine phosphorylation of the GluR2 wild type (WT) and the three GluR2 YF mutants in anti-GluR2 immunoprecipitates from lysates of HEK 293T cells transfected with wild-type or mutant GluR2 with plasmids encoding the Src family PTK Lyn. Cell lysates were immunoprecipitated with anti-GluR2 antibody, followed by immunoblotting with anti-GluR2CPY or anti-GluR2 antibodies.

Figure 4.

GluR2 C-terminal tyrosine phosphorylation differently regulated its interaction with GRIP1/2 and PICK1. A-F, The regulation of the association of GluR2 with its interacting proteins GRIP1, GRIP2, and PICK1 by tyrosine phosphorylation was analyzed in transfected HEK cells. GluR2 was immunoprecipitated with anti-GluR2 antibodies from lysates of HEK 293T cells transfected with plasmids encoding GluR2 wild type (A-C) or Y876F mutant (D-F), wild-type or kinase-negative (KN) form of Src family PTK Lyn, and GRIP1 (A, D), GRIP2 (B, E), or PICK1 (C, F). Anti-GluR2 coimmunoprecipitates from cell lysates were immunoblotted with each antibody (top). Expression of each protein was confirmed by protein immunoblots of cell lysates probed with anti-GluR2, anti-GRIP1 (A, D), anti-GRIP2 (B, E), anti-PICK1 (C, F) or anti-Lyn antibodies (middle). Tyrosine phosphorylation of the GluR2 C terminus was checked by protein immunoblots of cell lysates probed with anti-GluR2CPY antibodies (bottom). G-I, The equal binding of GluR2 wild type and Y876F mutant with GRIP1 (G), GRIP2 (H), and PICK1 (I) was analyzed in transfeceted HEK cells. Anti-GluR2 coimmunoprecipitation (top) and protein expression (bottom) are shown.

Figure 5.

Regulation of GluR2 C-terminal tyrosine phosphorylation in cultured cortical neurons. A, C-terminal tyrosine phosphorylated GluR2 in lysates from primary cortical cultures (culture DIV, 3-4 weeks) stimulated with 100 μm glutamate, 100 μm AMPA, or 50 μm NMDA for 10 min. Cell lysates were immunoblotted with anti-GluR2CPY or anti-GluR2 antibodies [n = 8 (control), n =8 (+glutamate), n = 8 (+AMPA), n = 5 (+NMDA), respectively]. B, The effect of Src family PTK-specific inhibitor PP2 (20 μm) or the inactive structural analog PP3 (20 μm) on GluR agonist-induced GluR2 C-terminal tyrosine phosphorylation. Cortical cultured neurons were stimulated with 100 μm glutamate, 100 μm AMPA, or 50 μm NMDA for 10 min (n = 4, respectively). C, The effect of PP2 (20 μm) or PP3 (20 μm) on surface and total GluR2 C-terminal tyrosine phosphorylation. Cortical culture neurons were treated with PP2 or PP3 for 1 hr, and surface-expressed proteins were biotinylated and precipitated with NeutrAvidin beads (n = 4, respectively). Ratios of GluR2 C-terminal tyrosine phosphorylation to total GluR2 are shown. Data are mean ± SEM; t test; ^*p < 0.05, ^***p < 0.001, compared with control.

Figure 6.

Phosphorylation of tyrosine 876 regulated clustering and surface expression of GFP-GluR2 in cortical neurons. A, Colocalization of GFP-GluR2 and GFP-GluR2YF mutant with synaptophysin in dendrites of transfected low-density hippocampal neurons. The ratio of GFP in synapses was shown (n = 4, respectively). B, Typical patterns of GFP-GluR2 clusters in cortical neurons. GFP fluorescences are shown in red (top). Cluster numbers along the dendrites (n = 10, respectively) and total GFP expressions in the dendrites (n = 10, respectively; t test; p > 0.5) of GFP-GluR2 and GFP-GluR2YF mutant in transfected cortical neurons are shown (bottom). C, Ratio of surface to total GFP expressions (left; n = 10, respectively) and total GFP expression (right; n = 10, respectively; t test; p > 0.5) in GFP-GluR2- or GFP-GluR2YF-transfected cortical neurons. Data are mean ± SEM; t test; ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, compared with control.

Figure 7.

Phosphorylation of tyrosine 876 is required for regulated internalization of GFP-GluR2 in cultured neurons. A, AMPA- and NMDA-induced internalization of GFP-GluR2 and GFP-GluR2YF. Typical patterns of GluR agonist stimulation induced internalization of surface GFP in GFP-GluR2- or GFP-GluR2YF-transfected cortical neurons (left). The ratio of fluorescence intensities of internalized anti-GFP antibodies (visualized by Cy3-conjugated anti-rabbit IgG) to total GFP expression (green fluorescent signal of GFP) are shown (middle) (left three bars: n = 7, n = 6, n = 8, respectively; F = 4.4; p < 0.05; right three bars: n = 8, n = 8, n = 8, respectively; F = 0.011; p > 0.98). Total GFP expressions in the dendrites of GFP-GluR2 and GFP-GluR2YF mutant in transfected cortical neurons are shown (right) (left three bars: n = 7, n = 6, n = 8, respectively; F = 0.523; p > 0.6; right three bars: n = 8, n = 8, n = 8, respectively; F = 0.34; p > 0.7). B, The effect of Src family PTK-specific inhibitor PP2 (20 μm) or inactive structural analog PP3 (20 μm) on AMPA- and NMDA-induced internalization of GFP-GluR2. The ratio of fluorescence intensities of internalized anti-GFP antibodies (visualized by Cy3-conjugated anti-rabbit IgG) and total GFP expression (green fluorescent signal of GFP) are shown (left) (six bars; n = 5, n = 10, n = 9, n = 5, n = 9, n = 11, respectively; F = 2.7; p < 0.05). Total GFP expression in the dendrites of GFP-GluR2 in transfected cortical neurons are shown (right) (six bars; n = 4, n = 12, n = 10, n = 6, n = 9, n = 11, respectively; F = 0.44; p > 0.8). C, The effect of PP2 (20 μm) or PP3 (20 μm) on AMPA- and NMDA-induced internalization of endogenous GluR1 in hippocampal neurons. The ratios of fluorescence intensities of internalized anti-GluR1 N terminus antibodies (visualized by Cy3-conjugated anti-rabbit IgG) are shown (AMPA: four bars; n = 9, n = 9, n = 7, n = 9, respectively; F = 6.344; p < 0.01; NMDA: four bars; n = 7, n = 6, n = 9, n = 7, respectively; F = 5.618; p < 0.01). Data are mean ± SEM; ANOVA; ^*p < 0.05, ^**p < 0.01, compared with control.