Volume 27, Issue 2, Pages 313-325 (August 2000) Mutagenesis Reveals a Role for ABP/GRIP Binding to GluR2 in Synaptic Surface Accumulation of the AMPA Receptor  Pavel Osten, Latika Khatri, Joey L Perez, Georg Köhr, Guenter Giese, Christopher Daly, Torsten W Schulz, Allen Wensky, Laveria M Lee, Edward B Ziff  Neuron  Volume 27, Issue 2, Pages 313-325 (August 2000) DOI: 10.1016/S0896-6273(00)00039-8

Figure 1 Characterization of MycGluR2 Constructs for Assembly of AMPA Receptors (A) Schematic drawing of the GluR2 C-terminal mutations. Top, wt C terminus (GluR2 aa 834–883) shows sites for NSF (aa 844–853) and PDZ proteins ABP, GRIP, and PICK1 (aa 879–883). Deletion 1–10 lacks the last 10 aa, deletion 5–10 has the last 5 aa replaced to the 10 aa truncation. Positions of single residue substitutions are shown in red: S880A in GluR2(EAVKI) and I883E in GluR2(ESVKE) sequence. (B) Immunostaining and electrophysiology of HEK-293 cells transfected with (a) GluR2, (b) MycGluR2, and (c) MycGluR2Δ1-10 (all R607Q form and flop [Sommer et al. 1990; Burnashev et al. 1992]). Intracellulary and surface-expressed receptors (red and green channel, respectively) are shown in overlay images of single confocal z planes. Bar in (Bc) = 10 μm. On the right are corresponding electrophysiological recordings for each construct. Glutamate responses (1000 μM, 100 ms) were fast desensitizing, and kainate responses (300 μM, 100 ms) were nondesensitizing at −60 mV. Steady-state I–V relations during kainate application (−120 to +60 mV, voltage ramps) and peak I−V relations of glutamate-activated currents (−120 to +80 mV; data not shown) were inwardly rectifying. (C) Western blot of HEK-293 cell lysates transfected as indicated. The lysates were precipitated with anti-GluR1 specific Ab. Top blot shows precipitated proteins detected by anti-GluR1 Ab; bottom blot is reprobed with anti-GluR2 Ab recognizing the juxtamembrane region of the C terminus. Neuron 2000 27, 313-325DOI: (10.1016/S0896-6273(00)00039-8)

Figure 2 Expression of MycGluR2 in Cultured Hippocampal Neurons (A) Immunostaining of neurons infected with Sindbis virus expressing MycGluR2. Twenty-four hours postinfection, living neurons were immunolabeled with monoclonal anti-Myc Ab (green channel) to detect surface-inserted receptors. After fixation and permeabilization, polyclonal anti-Myc Ab (red channel) was used to detect intracellulary expressed receptors. (Aa) shows cell body area, (Ab) distal dendrite; arrows point to examples of spine-like structures with MycGluR2 surface labeling. (B) Immunostaining of neurons infected as in (A). After fixation and permeabilization, the cells were stained with anti-Myc Ab to detect MycGluR2 receptors (green channel, [a]) and anti-GluR1 Ab to detect endogenous GluR1 receptors (red channel, [b]). Projection of the combined green and red fluorescence is shown in (c). Arrows point to examples of MycGluR2 and GluR1 colocalization in glutamatergic spines. The images in (A) and (B) are simulated fluorescence process (SFP) projections of a stack of 0.08 μm separated confocal z planes processed by deconvolution (see Experimental Procedures). Neuron 2000 27, 313-325DOI: (10.1016/S0896-6273(00)00039-8)

Figure 3 Surface Expression of MycGluR2 and the C-terminal Truncated Mutants in Cultured Hippocampal Neurons (A–C) Immunostaining of neurons infected with (A) MycGluR2, (B) MycGluR2Δ1-10, and (C) MycGluR2Δ5-10. Living neurons were immunolabeled 24 hr postinfection with monoclonal anti-Myc Ab (green channel) to detect surface-inserted receptors. After fixation and permeabilization, polyclonal anti-Myc Ab (red channel) was used to detect intracellulary expressed receptors. Asterisk in each insert marks position from which the main image was enlarged. Bar in (A) = 10 μm. Images are single confocal z planes, taken with the same laser settings. (D) The GluR2 C-terminal 5 aa are sufficient for PDZ binding. GST protein alone (lane 1), GST-R2C (lane 2), GST-R2CΔ1-10 (lane 3), and GST-R2CΔ5-10 (lane 4) were bound to glutathione agarose beads and incubated with 35S-labeled ABP, GRIP, or PICK1 as indicated. (E) Quantitation of surface expression of constructs shown in (A)–(C). Bars: 1 = MycGluR2, 2 = MycGluR2Δ1-10, 3 = MycGluR2Δ5-10. (Ea) ASA, average surface area, corresponds to a mean size of the surface cluster staining for each construct. Analysis of variance (ANOVA): DF = 2, mean square = 345.8, F = 71.1, and p < 0.0001. Fisher's PLSD test: bars 1 versus 2, p < 0.0001; bars 2 versus 3, p < 0.0001; and 1 versus 3, p = 0.8. (Eb) Surface/intracellular (S/I) ratio corresponds to the total area of the surface staining divided by total area of intracellular staining, multiplied by 100. ANOVA: DF = 2, mean square = 2279.1, F = 50.2, and p < 0.0001. Fisher's PLSD test: bars 1 versus 2, p < 0.0001; 2 versus 3, p < 0.0001; and 1 versus 3, p = 0.14. Neuron 2000 27, 313-325DOI: (10.1016/S0896-6273(00)00039-8)

Figure 4 Time-Dependent, Increased Surface Accumulation of MycGluR2 Compared to MycGluR2Δ1-10 Receptors in Cultured Hippocampal Neurons Immunostaining analysis of neurons infected with MycGluR2 and MycGluR2Δ1-10 constructs. Selective surface versus intracellular immunostaining (green and red channel, respectively; same as in Figure 3) was done in a time course of 9, 12, 18, and 24 hr after infection. (A) Examples of staining at 9 and 24 hr for MycGluR2 (top 2 images) and MycGluR2Δ1-10 (bottom 2 images). (B) Time course of changes in S/I ratio. MycGluR2, filled triangles; MycGluR2Δ1-10, open circles. Fisher's PLSD test MycGluR2 versus MycGluR2Δ1-10: 9 hr, p = 0.23; 12 hr, p = 0.17; 18 hr, p < 0.0001; 24 hr, p = < 0.0001. (C) Time course of changes in ASA values. MycGluR2, filled triangles; MycGluR2Δ1-10, open circles. Fisher's PLSD test MycGluR2 versus MycGluR2Δ1-10: 9 hr, p = 0.16; 12 hr, p = 0.02; 18 hr, p = 0.0002; 24 hr, p < 0.0001. (D) Immunoblot analysis of neurons infected with MycGluR2 (lanes 1 and 3) or with MycGluR2Δ1-10 (lanes 2 and 4). At 12 or 24 hr postinfection (as indicated), surface proteins of neurons were labeled with NHS-SS-biotin. Cells were then washed, scraped, and triton solubilized. The lysates were precipitated with streptavidin-coated beads to isolate biotinylated proteins. Top panel shows 10% of the input used for the precipitations. Lane 5 shows control noninfected lysate. Western blots with anti-Myc Ab of two independent experiments were scanned and analyzed by densitometry. The input level of MycGluR2Δ1-10 as compared to MycGluR2 was at 12 hr 111% and 83%, and at 24 hr 82% and 96%, while the surface level was at 12 hr 89% and 94%, and at 24 hr 18% and 16%. (E) Immunoblot analysis of neurons infected with MycGluR2 (lanes 1 and 2), Myc-GluR2Δ1-10 (lanes 3 and 4), or mock infected (lanes 5 and 6). At 24 hr postinfection, surface proteins were labeled with biotin as in (D). After washing, the cells were either scraped (lanes 1, 3, and 5) or returned for further 30 min incubation at 37°C and then incubated with glutathione-containing solution to release biotin from noninternalized surface proteins (lanes 2, 4, and 6). The lysates from all experiments were in the end precipitated by streptavidin-coated beads. The rate of surface internalization as measured by the ratio between total and internalized protein detected by anti-Myc Ab (lanes 1–4) or anti-GluR2/3 Ab (lanes 5 and 6) from two independent experiments was as follows: MycGluR2, 15% and 14%; MycGluR2Δ1-10, 53% and 48%; and endogenous GluR2/3 receptors, 23% and 18%. Neuron 2000 27, 313-325DOI: (10.1016/S0896-6273(00)00039-8)

Figure 5 Synaptic Targeting of MycGluR2Δ1-10 Receptors in Cultured Hippocampal Neurons (A) Immunostaining of neurons infected with MycGluR2Δ1-10 construct. Twenty-four hours postinfection, cells were immunolabeled for surface and intracellular MycGluR2Δ1-10 receptors (green and red channel, respectively; same protocol as for MycGluR2 in Figure 2A). Arrows point to examples of spine-like structures with MycGluR2Δ1-10 surface labeling. (B) Immunostaining of neurons infected as in (A). Living cells were stained with anti-Myc Ab to detect surface MycGluR2Δ1-10 receptors (green channel, [a]) and anti-GluR1 Ab to detect surface endogenous GluR1 receptors (red channel, [b]). Overlay of the two channels is shown in (Bc). Arrows point to examples of surface colocalization of MycGluR2Δ1-10 and GluR1. (C) Immunostaining of neurons infected as in (A). Living cells were stained with anti-Myc Ab to detect surface MycGluR2Δ1-10 receptors (green channel, [a]). After fixation, the cells were stained with anti-synaptophysin Ab to label presynaptic terminals (red channel, [b]). Projection of the combined channels is shown in (Cc). Arrows point to examples of colocalization of MycGluR2Δ1-10 surface clusters and synaptophysin-labeled terminals. (A–C) Images are projections of a stack of 0.08 μm separated confocal z sections. Images in (A) and (C) were processed by deconvolution (see Experimental Procedures). Neuron 2000 27, 313-325DOI: (10.1016/S0896-6273(00)00039-8)

Figure 6 Evidence for Homomeric Surface-Expressed MycGluR2Δ1-10 Receptors in Hippocampal Neurons Western blot of neurons infected with MycGluR2Δ1-10 (lanes 1, 3, 5, and 7) or mock infected (lanes 2, 4, 6, and 8). Twenty-four hours postinfection, surface proteins of living neurons were labeled with NHS-SS-biotin. Cells were then washed, scraped, and solubilized. The lysates were divided into two equal aliquots, and the first aliquot was precipitated with anti-GluR1 Ab (IP-R1, lanes 3 and 4), followed by anti-GluR2/3 combined with anti-GluR4 Ab (IP-R2/3 + R4, lanes 5 and 6), followed by pulldown with streptavidin-coated beads (strept., lanes 7 and 8). The second aliquot was twice mock immunoprecipitated with unrelated Ab (lanes 3b–6b), followed by streptavidin pulldown (lanes 7b–8b). Antibody used for Western blotting is indicated on the left of each panel. Lanes 1 and 2 show 10% of the input used for the precipitations. Homomeric MycGluR2Δ1-10 is detected in the fraction of surface biotinylated proteins that does not contain endogenous GluR subunit (lane 7, anti-Myc Ab Western blot). Total surface MycGluR2Δ1-10 is shown in lane 7b, anti-Myc Ab Western blot. The densitometric values from two independent experiments were as follows: MycGluR2Δ1-10 coprecipitated with GluR1, 4.7% and 3.6%; MycGluR2Δ1-10 coprecipitated with GluR2/3 and R4 subunits, 4.3% and 3.6%; homomeric surface MycGluR2Δ1-10, 4.4% and 3.4%; and total surface MycGluR2Δ1-10, 4.7% and 3.7% of total MycGluR2Δ1-10. Neuron 2000 27, 313-325DOI: (10.1016/S0896-6273(00)00039-8)

Figure 7 GRIP/ABP but Not PICK1 Interaction Is Required for Surface Accumulation of MycGluR2 Receptors in Hippocampal Neurons (A) Substitution of Ser into Ala in position −4 of the GluR2 C terminus inhibits in vitro binding of ABP and GRIP but not PICK1. GST protein alone (lane 1, CTR), GST-R2C (lane 2), GST-R2C(EAVKI) (lane 3), and GST-R2C(ESVKE) (lane 4) were bound to glutathione agarose beads and incubated with 35S-labeled ABP, GRIP, or PICK1. (B) Immunostaining of neurons infected with MycGluR2(EAVKI). Twenty-four hours postinfection, surface versus intracellular immunostaining (green and red channel, respectively) was done as in Figure 3. Asterisk in the insert marks position from which the main image was enlarged. Bar = 10 μm. Image is a single confocal z plane. (C) Quantitation of surface expression of the constructs: 1 = MycGluR2, 2 = Myc-GluR2Δ1-10, 3 = MycGluR2(EAVKI), and 4 = MycGluR2(ESVKE). (Ca) ASA, average surface area, is a mean size of the surface cluster staining for each construct. ANOVA: DF = 3, mean square = 147.4, F = 112.9, and p < 0.0001. Fisher's PLSD test: bars 1 versus 2, p < 0.0001; 1 versus 3, p < 0.0001; 1 versus 4, p < 0.0001; 2 versus 3, p = 0.25; 2 versus 4, p = 0.12; and 3 versus 4, p = 0.7. (Cb) S/I ratio is the total area of the surface staining divided by the total area of intracellular staining, multiplied by 100. ANOVA: DF = 3, mean square = 698.9, F = 47.7, and p < 0.000.1. Fisher's PLSD test: bars 1 versus 2, p < 0.0001; 1 versus 3, p < 0.0001; 1 versus 4, p < 0.0001; 2 versus 3, p = 0.9; 2 versus 4, p = 0.7; and 3 versus 4, p = 0.8. (D) Model for synaptic targeting and insertion of AMPA receptors. (1) AMPA receptor (schematically drawn with only one GluR2 C terminus; red end corresponds to the PDZ binding region) is targeted in a transport vesicle toward insertion at the synaptic plasma membrane by a yet to be identified membrane-fusion mechanism that does not require PDZ-mediated interactions. (2) After synaptic insertion, the receptor recruits stabilizing mechanism mediated by the PDZ binding site, likely interacting with ABP or GRIP but not PICK1, which clusters and/or anchors the receptor at the postsynaptic membrane. ABP/GRIP is schematically drawn as a black oval positioned at the synaptic plasma membrane (see text for possible role of ABP/GRIP binding in reinsertion during synaptic vesicular recycling of the GluR2 receptor). Neuron 2000 27, 313-325DOI: (10.1016/S0896-6273(00)00039-8)