PICK1 targets activated protein kinase Calpha to AMPA receptor clusters in spines of hippocampal neurons and reduces surface levels of the AMPA-type glutamate receptor subunit 2

Abstract

The PICK1 protein interacts in neurons with the AMPA-type glutamate receptor subunit 2 (GluR2) and with several other membrane receptors via its single PDZ domain. We show that PICK1 also binds in neurons and in heterologous cells to protein kinase Calpha (PKCalpha) and that the interaction is highly dependent on the activation of the kinase. The formation of PICK1-PKCalpha complexes is strongly induced by TPA, and PICK1-PKCalpha complexes are cotargeted with PICK1-GluR2 complexes to spines, where GluR2 is found to be phosphorylated by PKC on serine 880. PICK1 also reduces the plasma membrane levels of the GluR2 subunit, consistent with a targeting function of PICK1 and a PKC-facilitated release of GluR2 from the synaptic anchoring proteins ABP and GRIP. This work indicates that PICK1 functions as a targeting and transport protein that directs the activated form of PKCalpha to GluR2 in spines, leading to the activity-dependent release of GluR2 from synaptic anchor proteins and the PICK1-dependent transport of GluR2 from the synaptic membrane.

Fig. 1.

PICK1 forms perinuclear clusters after binding activated PKCα and GluR2, whereas the PICK1 mutant Δ121 forms clusters constitutively in 3T3 cells. A, Structure of PICK1 and PDZ mutants of PICK1. PICK1 has an N-terminal PDZ domain, an α-helical coiled coil domain, and a C-terminal acidic domain. The structures of the PDZ mutant KD27,28AA, which does not bind peptide C termini, and mutant Δ121, which lacks the PDZ domain altogether, are shown. In some experiments PICK1 and its mutants were tagged with the FLAG epitope at the PICK1 C terminus, as noted. B, GluR2 was expressed individually or in combination with PICK1 or the PICK1 mutant Δ121 in 3T3 cells. GluR2 on its own displayed a diffuse cytoplasmic pattern, whereas the coexpression of PICK1 induced GluR2–PICK1 complexes that entered perinuclear clusters. Δ121 formed perinuclear clusters in the presence of GluR2 without influencing GluR2 distribution. Scale bar, 25 μm. C, 3T3 cells were transfected singly with vectors expressing PICK1, PKCα, or mutant Δ121, as indicated. Parallel cultures were treated with TPA or were left untreated. Cells were permeabilized and stained by immunocytochemistry for the designated protein, as described in Materials and Methods. PICK1 displayed diffuse staining with or without TPA treatment, whereas PKCα moved to the cell perimeter. Mutant Δ121 was in perinuclear clusters, with or without TPA stimulation. Scale bar, 25 μm. D, 3T3 cells were cotransfected with vectors expressing either PICK1 or mutants KD27,28AA or Δ121 plus a vector expressing PKCα, as noted. Parallel cultures were treated with TPA or were left untreated as a control. Protein localization was visualized by double-immunofluorescent staining, as described in Materials and Methods. TPA induced the movement of coexpressed PICK1 and PKCα from a diffuse distribution to perinuclear clusters. In cells expressing KD27,28AA and PKCα, TPA affected only the localization of PKCα, which moved to the cell perimeter. The mutant Δ121 was constitutively present in clusters around the nucleus, whereas PKCα in the same cell as Δ121 underwent a TPA-induced movement to the cell perimeter. These results demonstrate a TPA-dependent formation of PICK1–PKCα complexes that are targeted to the 3T3 cell perinuclear region.

Fig. 2.

Immunoprecipitation of PICK1 complexes with activated PKCα and with GluR2 and molecular models of the complexes.A, B, The formation of complexes by PICK1 with PKCα and GluR2 was assayed in 293T cells. PICK1 formed complexes with PKCα exclusively in the PKCα-activated form that was induced by TPA, but the formation of complexes with GluR2 was constitutive and depended strongly on V881 in the PDZ binding site of GluR2. A, 293T cells were transfected with plasmids expressing PKCα or PICK1–FLAG as shown or with an empty vector (MOCK). Parallel cultures were treated with TPA or were left untreated. Cell extracts were prepared, and complexes containing PICK1–FLAG were immunoprecipitated with anti-FLAG antibody and assayed for PKCα and PICK1 content by Western blot analysis with anti-PKCα and anti-PICK1 antibodies, respectively. The whole-cell extract (WCE) was Western blotted directly with anti-PKCα to confirm PKCα expression. TPA induced the formation of a complex of PKCα with PICK1. B, PICK1 protein tagged at its C terminus with the FLAG epitope was coexpressed in 293T cells with the GluR2 protein. Complexes containing PICK1 were isolated from detergent extracts by precipitation with anti-FLAG antibody and assayed for GluR2 by Western blot analysis with C-terminal α-GluR2 antibody. The presence of GluR2 in whole-cell extracts (WCE) and PICK1 in anti-FLAG immunoprecipitates was confirmed by Western blotting.C, PICK1 protein expressed by in vitrotranslation in reticulocyte lysate and labeled with [³⁵S]methionine was incubated with wild-type and mutant GST-R2C, GluR2 C-terminus fusion proteins with the indicated Alanine substitution mutations; protein binding was assayed by gel electrophoresis and autoradiography. An aliquot of the translation extract was coelectrophoresed as a migration control. Mutations of three C-terminal amino acids to Alanine decreased PICK1 binding.D, E, PICK1 PDZ–PKCα and PICK1 PDZ–GluR2 complexes were modeled via homology with the three-dimensional crystallographic structure of a complex of peptide with PDZ3 of PSD-95 (Doyle et al., 1996) by the ICM-Homology method (Cardozo et al., 1995). Residues beyond the fourth amino acid of the sixth β-strand of PICK1 PDZ were truncated secondary to significant divergence from the parent structure. The side chains were placed optimally (Cardozo et al., 1995), and the model was refined together with the GluR2 C-terminal SVKI quadrapeptide or with the PKCα C-terminal QSAV quadrapeptide. The peptide backbone was tethered to occupy the same position as the backbone of the AQTSV peptide that was cocrystallized with PDZ3 of PSD-95, whereas the side chains of the peptide were optimized globally together with the surrounding PDZ side chains. D, A model of the PKCα C-terminal peptide QSAV bound to the PICK1 PDZ. Residue Q669 of PKCα can hydrogen-bond with a serine from the PICK1 PDZ β-strand. The inset represents a space-filling model of the interaction. Terminus-specific contacts are described in Results. E, A model of the PICK1 PDZ bound to the GluR2 C-terminal peptide SVKI. Residue V881 can form a hydrophobic contact with the αB1 lysine of PICK1 PDZ. The inset shows a space-filling model of this interaction.

Fig. 3.

PICK1 dimers form via the coiled coil domain and can assemble heterocomplexes by interaction with the PKCα and GluR2 C termini. A, Diagrammatic structures of PICK and PICK1 PDZ truncation mutants. PICK1 peptides were tagged with a FLAG or a Myc epitope at the C terminus, as noted. B, The Δ121–Myc mutant of the PICK1 peptide was coexpressed in 3T3 cells, with wild-type or mutant PICK1 tagged with a FLAG epitope, as noted. Complexes containing the FLAG-tagged species were isolated with anti-FLAG antibody and were analyzed for the Δ121–Myc and the FLAG-tagged species by Western blotting. The expression of Δ121–Myc was confirmed by Western blot analysis of the cell extracts. C, A series of FLAG-tagged mutants of PICK1, shown in A, was expressed in 3T3 cells together with PICK1–Myc, as noted. Complexes containing PICK1–Myc were isolated with anti-Myc antibody and were assayed for FLAG-tagged PICK1 species by Western blotting. Blotting with anti-Myc antibody confirmed the presence of PICK1–Myc in these complexes. The expression of FLAG-tagged PICK1 species was confirmed by Western blotting of the cell extracts with anti-FLAG antibody. D, The peptides PKCα and PICK1 and the PICK1 mutants KD27,28AA and Δ121 were expressed alone or in combination in 3T3 cells; parallel cultures were treated with TPA or left untreated, as noted. Cell extracts were prepared and incubated with GST-R2C, a GST protein fusion to the C-terminal 50 amino acids of GluR2. Complexes containing GST-R2C were isolated on glutathione–Sepharose and were assayed for PICK1, PKCα, and GST-R2C by Western blotting. Protein expression was confirmed by Western blot analysis. TPA induced the formation of a complex containing GST-R2C, PICK1, and PKCα.

Fig. 4.

Interaction with PICK1 targets the activated form of PKCα and GluR2 to spine clusters, whereas mutant Δ121 is targeted constitutively. PICK1, MycGluR2, and PICK1 or its mutants were expressed as noted in cultured embryonic hippocampal neurons 14 DIV from Sindbis virus vectors, and the virally encoded peptides were detected by immunofluorescence and confocal microscopy.A, Western blot analysis of extracts of cultured cortical neurons either uninfected (Control) or infected with Sindbis virus vectors expressing PICK1, KD27,28AA, or Δ121. Virally encoded PICK1 peptides were visualized by blotting with polyclonal anti-PICK1 serum. Peptide bands of the expected sizes confirm faithful peptide expression by the vectors. B, PICK1–FLAG that is expressed on its own in hippocampal neurons is distributed diffusely in the cell body and enters spines. Scale bar, 10 μm. C, D, PICK1–FLAG was expressed from Sindbis virus in cultured hippocampal neurons; 24 hr after infection the cells were treated with TPA (D) or were left untreated as a control (C). Immunofluorescent detection of endogenous PKCα with PKCα antiserum (red) and of PICK1 with anti-FLAG antibody (green) revealed that TPA induced the movement of PKC into spine clusters that contain PKC. The bottom three panels show higher magnification of the merged, the PKCα, and the PICK1 images, respectively.Asterisks in the top panels indicate the positions that have been magnified beneath. Scale bars (for both sets of panels), 10 μm. E–G, PICK1 (E) or mutant KD27,28AA (F) or mutant Δ121 (G) was coexpressed in hippocampal neurons with MycGluR2 by confection with two Sindbis virus vectors. Virally encoded peptides were visualized in permeabilized cells by indirect immunofluorescence and confocal microscopy. PICK1 and its mutants were detected via a FLAG epitope tag at the C terminus with anti-FLAG antiserum (green). MycGluR2 was visualized with anti-Myc polyclonal antiserum (red). PICK1 and MycGluR2 colocalized in spine clusters. When KD27,28AA was coexpressed with MycGluR2, both peptides were distributed diffusely. Δ121 entered spine clusters or other dendritic protrusions, some of which also contained MycGluR2. The bottom three panels show at higher magnification the merged, the MycR2, and the PICK1 images, respectively. Asterisks in the top panels indicate the positions that have been enlarged beneath. Magnification is as in C.

Fig. 5.

Serine 880 phosphorylation of GluR2 in spine clusters with PICK1 and receptor endocytosis. A, Specificity of anti-GluR2Ser880-PO₄ phosphopeptide antiserum. This serum recognizes GST-R2C phosphorylated in vitro by PKC, but not untreated GST-R2C or GST-R2C S880A with or without TPA treatment. This establishes the specificity of the serum for GluR2Ser880-PO₄ C-terminal sequences. B, Cultures of cortical neurons were treated with TPA or were left untreated. Immune precipitates of extracts with anti-GluR2Ser880-PO₄ antiserum were analyzed by Western blotting with the same serum. TPA treatment strongly induced the formation of GluR2Ser880-PO₄. C, Treatment of cultured hippocampal neurons with TPA induced the phosphorylation of endogenous GluR2 to yield GluR2Ser880-PO₄. GluR2Ser880-PO₄ was visualized by immunofluorescence with phosphopeptide antiserum and was abundant in spines. The signal was strongly competed by preincubation of the antibody with phosphopeptide antigen (data not shown). Scale bar, 20 μm. D, Cultured hippocampal neuron infected with Sindbis virus vector expressing PICK1–FLAG and treated with TPA. Shown are GluR2Ser880-PO₄ phosphopeptide antiserum and rhodamine-conjugated 20 antibody, and PICK1–FLAG with anti-FLAG antiserum and fluorescein-conjugated 20 antibody. PICK1 and GluR2Ser880-PO₄ coclustered in spines. Thepanels on the right show color-resolved images of the region that has been indicated by anasterisk. Scale bar, 10 μm.

Fig. 6.

Reduction of plasma membrane levels of MycGluR2 by PICK1. A, Hippocampal neurons were coinfected with Sindbis virus vectors expressing MycGluR2 and PICK1 or MycGluR2 and KD27,28AA. Surface MycGluR2 was stained in living cells. Cells were permeabilized; PICK1 also was stained. Coexpression of MycGluR2 with PICK1, but not with KD27,28AA, reduced MycGluR2 surface levels. Scale bar, 10 μm. B, Hippocampal neurons were coinfected with Sindbis virus expressing MycGluR2 and PICK1. Cells were stained for surface and internal MycGluR2 as described previously (Osten et al., 2000). Shown is a pair of cells, one expressing only MycGluR2 and the other also expressing PICK1. *Cell infected with MycGluR2 expression Sindbis virus alone. **Cell infected with MycGluR2 expression Sindbis virus and with PICK1 expression Sindbis virus. The surface level of MycGluR2 is reduced greatly in the latter cell, but not in the former cell. Doubly infected cells were identified by the presence of characteristic MycGluR2 clusters. Scale bar, 10 μm. C, Model for the targeting of activated PKCα and PICK1 to spines and for the phosphorylation and endocytosis of GluR2. Activation of PKCα (yellow) by TPA exposes the PDZ binding site at the C terminus of PKCα (1). The PDZ binding site binds to a PDZ domain of a PICK1 dimer (red), inducing a structural transition in PICK1. The PICK1–PKCα complex is transported to spines (2). GluR2 (green) that is initially in a complex with ABP or GRIP (gray) is released from this complex and is phosphorylated (purple circle) on serine 880 of the GluR2 C-terminal domain (3). The phosphorylated C terminus of GluR2 binds to PICK (4). The PICK1–GluR2 complex is endocytosed (5). Not shown is the possible involvement of triple complexes in which PKCα and GluR2 are bound to the PDZ domains of a PICK1 dimer (see Discussion). GluR2 in this model is present in AMPA receptor complexes.