PMC

Ca²⁺-sensitive PICK1 interactions influence GluR1 trafficking to the same extent as GluR2. (A) Overexpression of WT PICK1 in hippocampal neurons enhances NMDA-induced removal of surface GluR1. Dissociated hippocampal cultures were infected with either empty -IRES-EGFP or WT PICK1-IRES-EGFP virus and exposed to drugs as in . Western blots were probed with anti-GluR1 antibody. n=5, ^*P<0.05. (B) Overexpression of ΔNT PICK1 in hippocampal neurons occludes NMDA-induced removal of surface GluR1. As above, except cultures were infected with WT PICK1-IRES-EGFP or ΔNT PICK1-IRES-EGFP virus. n=5, ^*P<0.05.

A model for the role of PICK1 Ca²⁺-sensor in NMDAR-dependent AMPAR endocytosis. (A) Under basal conditions, when NMDARs are inactive, GluR2-containing AMPARs are either weakly bound to PICK1 or anchored by ABP/GRIP, resulting in a low level of AMPAR internalisation, representing constitutive cycling. (B) Activation of NMDARs localised in close proximity to AMPARs results in a Ca²⁺ flux that raises the local [Ca²⁺] to around 15 μM and therefore enhances binding of PICK1 to GluR2. (C) This results in increased AMPAR internalisation and hence a decrease in surface AMPAR.

PICK1 is a Ca²⁺-binding protein. (A) PICK1 is a Ca²⁺-binding protein, and Ca²⁺ binding is reduced in ΔNT PICK1. Equilibrium dialysis using ⁴⁵Ca²⁺ was employed to determine the Ca²⁺ binding to known amounts of his₆WT PICK1^flag (solid line) and his₆ΔNT PICK1^flag (dashed line) in a range of [Ca²⁺]_free buffers. Graph shows nanomoles of Ca²⁺ bound per milligram of protein at each [Ca²⁺]_free. Pooled data, n=4. Diagrams demonstrate domain mutations made in PICK1 protein. (B) Alanine substitutions in the N-terminal acidic region of PICK1 reduce Ca²⁺-binding capacity. His₆PICK1 mutants as shown were subjected to equilibrium dialysis in buffer containing 15 μM [Ca²⁺]_free. Graph shows pooled data relative to Ca²⁺-binding values for his₆WT PICK1, n=4, ^*P<0.05. Diagrams demonstrate mutations made in PICK1 protein. (C) A C-terminal acidic domain is also involved in Ca²⁺ binding to PICK1. His₆ΔCT PICK1^flag (solid line) and his₆ΔNT/ΔCT PICK1^flag (dashed line) were subjected to equilibrium dialysis as in (A). Graph shows nanomoles of Ca²⁺ bound per milligram of protein at each [Ca²⁺]_free. Pooled data, n=4. Diagrams demonstrate domain mutations made in PICK1 protein.

Deletion of the N-terminal acidic region in PICK1 removes Ca²⁺-sensitivity of GluR2–PICK1 interaction. (A) WT PICK1 contains a region of acidic amino acids at the N-terminus, from residues 4–12. A mutant was constructed that lacks this region, termed ΔNT PICK1. (B) ΔNT PICK1 binds GluR2 C-terminus at maximal level in all [Ca²⁺]_free tested. GST-R2C immobilised on glutathione beads was incubated with his₆WT PICK1^flag or his₆ΔNT PICK1^flag in the presence of different [Ca²⁺]_free as shown. After washing in the same [Ca²⁺]_free buffer, bound PICK1 was analysed by Western blotting using anti-flag antibody. (Bottom panel) Graph shows pooled data for WT PICK1 (left) and ΔNT PICK1 (right), n=4. (C) Total levels of his₆WT PICK1^flag, his₆ΔNT PICK1^flag and GST-R2C are stable in the 0–30 μM [Ca²⁺]_free range. His₆PICK1^flag and his₆ΔNT PICK1^flag were incubated in buffer A in the presence of different [Ca²⁺]_free as shown. GST-R2C immobilised on beads was treated in the same way in separate tubes. The levels of PICK1 were analysed by Western blotting using anti-flag and GST-R2C was analysed by Coomassie staining.

PICK1 is a Ca²⁺-sensor for NMDA-induced endocytosis of GluR2-containing AMPARs in hippocampal neurons. (A) Characterisation of NMDA-induced GluR2 internalisation in dissociated hippocampal neurons. Low-density hippocampal cultures, live-labelled with anti-GluR2 antibody, were exposed to drugs as in . After acid stripping of remaining surface antibody, endocytosis of GluR2-containing AMPAR was assayed by immunocytochemistry (visualised by Alexa 568 secondary). Transmission images and internalised GluR2, including a magnified dendritic segment, are shown from representative neurons. Graph shows quantification of internalised AMPAR. Values represent total internalised GluR2 immunoreactivity normalised for cell area (arbitrary units). n=15 cells per condition, ^***P<0.0005. (B) Overexpression of WT PICK1 enhances and ΔNT PICK1 occludes NMDA-induced endocytosis of GluR2 in hippocampal neurons. Neurons were infected with virus encoding -IRES-EGFP alone, WT PICK1-IRES-EGFP or ΔNT PICK1-IRES-EGFP, live-labelled with anti-GluR2 antibody, and exposed to 25 μM NMDA for 3 min followed by 12 min incubation after drug washout or 25 μM MK-801 for 15 min. Images of EGFP fluorescence and internalised GluR2, including a magnified dendritic segment, are shown from representative neurons. Graph shows quantification of internalised AMPAR. Values represent total internalised GluR2 immunoreactivity normalised for cell area (arbitrary units). n=15 cells per condition, ^*P<0.005, ^**P<0.001, ^***P<0.0005.

PICK1–GluR2 binding is Ca²⁺-sensitive. (A) Endogenous PICK1–GluR2 complexes from hippocampal neurons show maximal interaction at 15 μM Ca²⁺. Extracts of rat cultured hippocampal neurons were immunoprecipitated in different [Ca²⁺]_free with rabbit anti-PICK1 antibody and protein A sepharose. After washing beads in the same [Ca²⁺]_free buffers, bound GluR2 was analysed by Western blotting using anti-GluR2 antibody. Graph shows pooled data, n=8. t-tests 0–15 μM: P<0.001; 15–30 μM: P<0.05. (B) Total levels of PICK1 and GluR2 are stable in the 0–30 μM [Ca²⁺]_free range. (Top panel) Immunoprecipitates identical to those analysed in (A), above,were probed for PICK1 with goat anti-PICK1 antibody. (Bottom panel) Extracts of rat hippocampal neurons were prepared as above, and incubated on ice in the presence of different [Ca²⁺]_free, in the absence of antibody. The levels of GluR2 in the extracts were analysed by Western blotting using anti-GluR2 antibody. (C) Endogenous GRIP–GluR2 complexes are not Ca²⁺-sensitive. Experiments were carried out as in (A) above using anti-GRIP antibody for immunoprecipitation. Graph shows pooled data, n=6. (D) GluR2 C-terminus and PICK1 are sufficient for the Ca²⁺-sensitive interaction. GST-R2C immobilised on glutathione beads was incubated with his₆PICK1^flag in the presence of different [Ca²⁺]_free. After washing in the same [Ca²⁺]_free buffer, bound PICK1 was analysed by Western blotting using anti-flag antibody. Graph shows pooled data, n=5. t-tests 0–15 μM: P<0.01; 15–30 μM: P<0.05. (E) Total levels of his₆PICK1^flag and GST-R2C are stable in the 0–30 μM [Ca²⁺]_free range. (Top panel) His₆PICK1^flag was incubated in buffer A in the presence of different [Ca²⁺]_free as shown. The levels of PICK1 were analysed by Western blotting using anti-flag. (Bottom panel) GST-R2C immobilised on beads was treated in the same way in separate tubes and levels of GST-R2C analysed by Coomassie staining. (F) PICK1–GluR2 binding is not sensitive to [Mg²⁺]. GST-R2C immobilised on glutathione beads was incubated with his₆PICK1^flag in the presence of different [Mg²⁺]_free as shown. After washing in the same [Mg²⁺]_free buffer, bound PICK1 was analysed by Western blotting using anti-flag antibody. Graph shows pooled data, n=4.

PICK1 Ca²⁺ sensor regulates GluR2 internalisation in heterologous cells. (A) COS cells coexpressing GluR2^myc and PICK1^flag exhibit Ca²⁺-sensitive GluR2 endocytosis. COS cells cotransfected with ^mycGluR2 and WT PICK1^flag were live-labelled with rabbit anti-myc antibody. After 5 min ionomycin (1 μM) treatment in media containing different [Ca²⁺]_O as shown, cells were incubated in medium lacking Ca²⁺ for 10 min. GluR2 endocytosis was then assayed by acid-stripping immunocytochemistry. Internalised ^mycGluR2 was visualised with Alexa 488 and total PICK1 staining by anti-flag and Alexa 568. Representative confocal images of COS cells are shown for each condition. (B) Quantification of internalised ^mycGluR2. Values represent total internalised GluR2 (anti-myc immunoreactivity) normalised for cell area (arbitrary units). n=26–28 cells per condition, ^*P<0.01; ^**P<0.0001. (C) Expression levels of GluR2^myc and PICK1^flag are unaffected by ionomycin and [Ca²⁺]_O. Cells were treated with ionomycin and Ca²⁺ buffers as in (A), and 1% Triton X-100 extracts were analysed for total GluR2^myc and PICK1^flag expression by Western blotting using anti-myc and anti-flag antibodies, respectively. (D) ^mycGluR2 expressed in the absence of PICK1 does not exhibit Ca²⁺-dependent increases in endocytosis. COS cells transfected with ^mycGluR2 alone were subjected to the same treatment as in (A). Graph shows quantification of internalised GluR2^myc. n=15–20 cells per condition. (E) Ca²⁺-insensitive ΔNT PICK1 stimulates maximal GluR2 endocytosis even in the absence of Ca²⁺ and at the same level for all [Ca²⁺]_O tested. COS cells transfected with ^mycGluR2 and ΔNT PICK1^flag were subjected to the same treatment as in (A). Graph shows quantification of internalised GluR2^myc. n=15–20 cells per condition. (F) COS cells coexpressing GluR1^myc and PICK1^flag show minimal GluR1 endocytosis, which is not Ca²⁺-dependent. Cells cotransfected with^mycGluR1 and WT PICK1^flag were subjected to the same treatment as in (A). Graph shows quantification of internalised GluR1^myc. n=15–20 cells per condition.

PICK1 is a Ca²⁺ sensor for NMDA-induced reduction in surface GluR2-containing AMPAR in hippocampal cultures. (A) Binding of endogenous neuronal GluR2/3 to overexpressed WT PICK1^flag is Ca²⁺-sensitive. Extract of rat hippocampal cultures infected with Sindbis virus encoding WT PICK1^flag-IRES-EGFP was prepared in buffer A and divided into six equal portions with a range of [Ca²⁺]_free. Extracts were immunoprecipitated with anti-flag antibody and protein G sepharose. After washing beads in the same [Ca²⁺]_free buffers, bound GluR2/3 was analysed by Western blotting using anti-GluR2/3 antibody. Graph shows pooled data, n=5. (B) Binding of endogenous neuronal GluR2/3 to overexpressed ΔNT PICK1^flag is Ca²⁺-insensitive. As above, except that cultures were infected with virus encoding ΔNT PICK1^flag-IRES-EGFP. Graph shows pooled data, n=5. (C) Binding of endogenous GluR2/3 to overexpressed GRIP1a^HA is not Ca²⁺-sensitive. Extract of hippocampal cultures infected with Sindbis virus encoding GRIP1a^HA-IRES-EGFP were treated as in (A) above, except that anti-HA antibody was used for immunoprecipitation. Graph shows pooled data, n=4. (D) Characterisation of chem-LTD in uninfected cultures. Hippocampal cultures were treated with combinations of drugs as shown. In all, 50 μM APV or 25 μM MK-801 (MK) were applied for 15 min; 25 μM NMDA (N) was applied for 3 min, followed by 12 min incubation after drug washout. Biotinylation was subsequently used to quantify surface levels of GluR2. Top panel shows representative Western blot of total GluR2 present in lysates and surface (biotinylated) GluR2 after drug treatment. Graph shows pooled data presented as ratios of surface over total GluR2. n=6,^*P<0.05. (E) Overexpression of WT PICK1 in hippocampal cultures enhances NMDA-induced removal of surface GluR2, but only in the presence of NMDAR activity. Dissociated hippocampal cultures were infected with either empty-IRES-EGFP or WT PICK1-IRES-EGFP virus and exposed to 25 μM NMDA for 3 min followed by 12 min incubation after drug washout or 25 μM MK-801 for 15 min. Top panel shows representative Western blot of 25% total GluR2 present in lysates and 100% surface (biotinylated) GluR2 after treatment. Graph shows pooled data presented as ratios of surface over total GluR2. n=7, ^*P<0.05. (F) Overexpression of ΔNT PICK1 in hippocampal cultures occludes NMDA-induced removal of surface GluR2. Dissociated hippocampal cultures were infected with either WT PICK1-IRES-EGFP or ΔNT PICK1-IRES-EGFP virus and exposed to drugs as above. n=7, ^**P<0.001, ^*P<0.05.