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AMPA-receptor stabilization

GRIP1 co-localizes together with GluR2 and ephrinB2 in hippocampal neurons. A further question that needed to be addressed was whether this interaction was important for ephrinB2 mediated AMPA-receptor stabilization at the cell surface

5.2.4.1 Interference of GRIP binding to ephrinB2 by a GRIP-peptide (PDZ6) GRIP proteins are PDZ-domain-containing proteins which bind via their PDZ6-domain to ephrinBs (Bruckner et al., 1999; Torres et al., 1998) and via their PDZ4 and PDZ5- domains to AMPA receptors (Dong et al., 1997). Here, we performed an interference

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approach by over-expressing the PDZ6-domain of GRIP in hippocampal neurons. This PDZ-domain acts as a dominant negative molecule binding to ephrinBs and thereby preventing endogenous GRIP1 to bind (Figure 5-16, a+b).

Figure 5-16: Interference with GRIP-ephrinB2 interaction impairs AMPA-receptor stabilization. (a) Hippocampal neurons transfected with the dominant negative molecule GRIP1-PDZ6- CFP were assayed for AMPA-receptor internalization by the antibody feeding assay. Neurons were stimulated as indicated with pre-clustered Fc (control) or EphB4-Fc alone or together with 100 µM AMPA. Internalized receptors appear in red, surface remaining receptors in green. Scale bars, 20 µm whole neurons, 5 µm enlargements. (b) Quantification of AMPA-receptor internalization pictured as percentage of internalized GluR2 versus total GluR2 in transfected neurons and control cells under the conditions presented in a. (SEM, *** P < 0.0001). (c) Over-expression of GRIP1-PDZ7-CFP in hippocampal neurons did not affect AMPA-receptor internalization when assayed under the conditions as in (a). (d) Quantification of GluR2 internalization of PDZ7 transfected neurons and control cells as in b. Scale bars, 20 µm whole neurons, 5 µm enlargements. (SEM, *** P < 0.0001).

82 Hippocampal neurons were transfected with PDZ6-CFP at 11 days in culture and analyzed 2-4 days later by the aforementioned antibody feeding assay. Under control conditions (Fc) transfected neurons already showed a slight increase in AMPA-receptor internalization compared to untransfected control cells (33.1 % ± 1.8 internalization in GRIP1 PDZ6 transfected neurons compared to 19.5 % ± 1.3 in control transfected neurons). AMPA-receptor endocytosis was successfully induced by 100 µM AMPA in both the PDZ6 over-expressing and control cells. Importantly, AMPA-induced receptor endocytosis was no longer inhibited by ephrinB2 activation with pre-clustered EphB4-Fc in the PDZ6-expressing cells. Therefore, interfering with the binding of GRIP1 to ephrinB2 seemed to disrupt AMPA-receptor stabilization via ephrinB2.

To verify, whether the effect seen in PDZ6-over-expressing neurons was specific, we transfected neurons with a different PDZ-domain of GRIP (PDZ7), unable to bind to ephrinB2, and determined AMPA-receptor endocytosis in these neurons (Figure 5-16,

c+d). The analysis of PDZ7-CFP expressing cells revealed no differences in AMPA- receptor internalization compared to control cells. The same levels of AMPA-receptor endocytosis were observed under control conditions (Fc) (33.8 % ± 1 transfected compared to 34.3 % ± 1.2 untransfected neurons) and ephrinB2 activation successfully inhibited AMPA-induced receptor internalization in control and PDZ7-expressing cells (32.5 % ± 1.2 to 32.6 % ± 1.4 respectively).

5.2.4.2 Interference of GRIP binding to ephrinB2 by ephrinB2-peptide A second way to interfere with GRIP binding to ephrinB2 is to over-express an ephrinB2 peptide that contains the GRIP-binding sequence. The sequence of the last 22 amino acids of ephrinB2 was cloned into a CFP-N-terminal-fusion plasmid to generate B2-pepWT CFP. Hippocampal neurons (11DIV) were transfected with B2-pepWT CFP and analyzed for AMPA-receptor internalization by the antibody feeding assay method 2-4 days later. This treatment increased AMPA-receptor internalization even without AMPA stimulation (45.7 % ± 1.9 compared to to 23.2 % ± 1.3 in the untransfected controls) (Figure 5-17). AMPA treatment resulted in an increase of AMPA-receptor endocytosis in both transfected (56.2 % ± 2.0) and untransfected neurons (56.3 % ± 2.0). Simultaneous

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activation of ephrinB2 effectively blocked AMPA-induced receptor endocytosis in control cells (29.5 % ± 1.6). In B2-pepWT CFP-expressing neurons a smaller reduction (45.7 % ± 2.1) was observed. Most probably the peptide was not able to completely inhibit endogenous ephrinB2-GRIP interaction.

Figure 5-17: Interference with GRIP-ephrinB2 interaction with an ephrinB2 peptide. (a) Hippocampal neurons transfected with the ephrinB2 peptide B2-pepWT CFP were assayed for AMPA- receptor internalization by the antibody feeding assay. Neurons were stimulated as indicated with pre- clustered Fc (control) or EphB4-Fc alone or together with 100 µM AMPA. Internalized receptors appear in red, surface remaining receptors in green. (b) Quantification of AMPA receptor internalization as in Figure 5-16 (SEM, *** P < 0.0001).

5.2.4.3 EphrinB2 activation decreased GluR2 phosphorylation levels

The binding of GRIP to AMPA receptors is known to be regulated by the phosphorylation of Serine880 (ser880) in GluR2. GRIP1 stabilizes AMPA receptors at membranes while binding to the unphosphorylated GluR2 subunit (Matsuda et al., 1999). Upon receptor activation, the GluR2 subunit is phosphorylated at ser880. This event leads to the dissociation of GRIP1 and enables PICK1 to bind to GluR2 thus initiating AMPA-receptor internalization (Chung et al., 2000). Furthermore, binding of GRIP to GluR2 has been

84 shown to inhibit ser880 phosphorylation (Fu et al., 2003). Since ephrinB2 seemed to stabilize AMPA receptors at the surface via GRIP1, we wondered whether eprhinB2 activation might have a direct effect on the serine-phosphorylation status of GluR2. Therefore, we stimulated cultured hippocampal neurons (18DIV) with pre-clustered Fc (control), EphB4-Fc alone, or EphB4-Fc together with AMPA and assayed levels of ser880 phosphorylation by Western blot with a phosphorylation-specific antibody. As expected, AMPA-receptor activation with 100 µM AMPA led to high levels of ser880 phosphorylation of the GluR2 subunit. Importantly, co-stimulation together with EphB4- Fc inhibited AMPA-induced serine-phosphorylation, supporting our model that ephrinB2 stabilizes AMPA receptors at the membrane via GRIP1.

Figure 5-18: EphrinB2 inhibits activation induced ser880 phosphorylation in GluR2. (a) Hippocampal neurons (20DIV) were stimulated as indicated with pre-clustered Fc, EphB4-Fc alone or together with 100 µM AMPA, immunoprecipitated (IP) for GluR2 and analyzed by Western blot for phospho-ser880 GluR2 and total GluR2. (b) Lack of ephrinB2 increased phospho-ser880 GluR2 levels in cultured hippocampal neurons. eB2KO and control litter mate neurons were cultured for 16 days and analyzed for levels of ser880-phosphorylation by immunoprecipitation of GluR2 and analysis by Western blot.

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