PICK1 interacts with and regulates PKC phosphorylation of mGLUR7

Abstract

The G-protein-coupled metabotropic glutamate receptor subtype 7a (mGluR7a) is a member of group III metabotropic glutamate receptors that plays an important role as a presynaptic receptor in regulating transmitter release at glutamatergic synapses. Here we report that the protein interacting with C-kinase (PICK1) binds to the C terminus (ct) of mGluR7a. In the yeast two-hybrid system, the extreme ct of mGluR7a was shown to interact with the PSD-95/Discs large/ZO-1 (PDZ) domain of PICK1. Pull-down assays indicated that PICK1 was retained by a glutathione S-transferase fusion of ct-mGluR7a. Furthermore, recombinant and native PICK1/mGluR7a complexes were coimmunoprecipitated from COS-7 cells and rat brain tissue, respectively. Confocal microscopy showed that both PICK1 and mGluR7a displayed synaptic colocalization in cultured hippocampal neurons. PICK1 has previously been shown to bind protein kinase C alpha-subunit (PKCalpha), and mGluR7a is known to be phosphorylated by PKC. We show a relationship between these three proteins using recombinant PICK1, mGluR7, and PKCalpha, where they were co-immunoprecipitated as a complex from COS-7 cells. In addition, PICK1 caused a reduction in PKCalpha-evoked phosphorylation of mGluR7a in in vitro phosphorylation assays. These results suggest a role for PICK1 in modulating PKCalpha-evoked phosphorylation of mGluR7a.

Fig. 1.

Defining the mGluR7a/PICK1 interaction in the yeast two-hybrid system. A, Site of interaction on ct-mGluR7a. Overlapping deletion mutants were constructed to determine the site of interaction on ct-mGluR7a for PICK1. The importance of the last three amino acids was determined using a series of single point mutations at the residues 0, −1, and −2. Asteriskindicates a termination site; triangles show the last three residues of ct-mGluR7a and indicate the PDZ binding motif important for PICK1 interaction; positive interactions, as defined by filter β-galactosidase assays, are indicated as + and negative as −.B, Site of interaction on PICK1. Full-length PICK1 (residues 1-416) and a long version fragment of PICK1 (13-358) containing the PDZ domain gave a positive interaction with ct-mGluR7a. A shorter PICK1 fragment (1-305) still containing the PDZ domain gave negative results. PICK1 constructs (305-416 and 305-358) lacking the PDZ domain failed to interact with ct-mGluR7a. The three point mutations, K27E, K27A, and KD27/28AA, abolished the PICK1/ct-mGluR7a association. Boxshows the PDZ domain of PICK1 that is important for ct-mGluR7a interaction; a carboxylate-binding domain (cbd) of eight amino acids is located at the N terminus of the PDZ; positive interactions, as defined by filter β-galactosidase assays, are indicated as + and negative as −.

Fig. 2.

In vitro binding of ct-mGluR7a/PICK1 interaction. Shown are GST-ct-mGluR7a pull-down assays. Western blot using anti-flag M2 mAb showing the levels of COS-7 cell expressed flag-PICK1 (top panel) and point mutated flag-PICK1 (K27E) (center panel) retained by GST alone, GST-ct-GluR2, GST-ct-mGluR7. Western blot using anti-PICK1-rab Ab showing the amount of rat brain lysate-derived native PICK1 (bottom panel) retained by GST alone, GST-ct-GluR2, GST-ct-mGluR7. Supernatant indicates the level of PICK1 expression in the input.

Fig. 3.

Co-immunoprecipitation of a ct-mGluR7a/PICK1 complex. A, Co-immunoprecipitation studies in COS-7 cells. COS-7 cells transfected with no cDNA, GFP-PICK1, flag-mGluR7a, GFP-PICK1, and flag-mGluR7a. A GFP-PICK1/flag-mGluR7a complex was co-immunoprecipitated using anti-flag M2-agarose affinity beads. The amount of flag-mGluR7a and GFP-PICK1 retained by the beads was detected by Western blotting using anti-flag M2 mAb (top blot) and anti-GFP Ab (bottom blot), respectively. B, Co-immunoprecipitation from rat brain. Rat brain tissue expressing native PICK1 and mGluR7a was precipitated using protein-G Sepharose beads that had been previously coupled to control rabbit IgG, anti-mGluR7a-rab Ab, anti-PICK-rab Ab. The amount of mGluR7a and PICK1 retained by the beads was detected by Western blotting using anti-mGluR7-gpig Ab (top blot) or anti-PICK-gpig Ab (bottom blot), respectively.

Fig. 4.

PICK1 and mGluR7a distribution in rat hippocampal neurons. Co-staining of cultured hippocampal neurons for PICK1 and mGluR7a, PICK1 and synaptophysin, mGluR7a and synaptophysin. Top panel, Green channel; center panel, red channel; bottom panel, overlay seen inyellow. Immunoreactivity for mGluR7a shows a punctate distribution that is colocalized with immunoreactivity for PICK1 and synaptophysin. A portion of PICK1 immunoreactivity is also found not colocalized with mGluR7a or with synaptophysin. Scale bar is indicated in white.

Fig. 5.

PKCα/PICK1/mGluR7a form a complex in COS-7 cells. Shown are immunoprecipitation studies. Lysates of COS-7 cells co-transfected with HA-PKCα, flag-PICK1, and flag-mGluR7a were precipitated using protein-G Sepharose beads that had been previously coupled to either control rabbit IgG (control Ab) or anti-HA Ab (HA Ab). The amount of HA-PKCα, flag-PICK1, and flag-mGluR7a retained by the beads was detected by Western blotting using anti-HA mAb (top blot), anti-PICK1-gpig Ab (middle blot), or anti-flag M2 mAb (bottom blot), respectively. The schematics next to each blot indicate the number of protein/protein interactions between the anti-HA Ab and the precipitated protein.

Fig. 6.

PICK1 inhibits PKCα phosphorylation of mGluR7a.A, Inhibition of PKCα phosphorylation by PICK1. GST-ct-mGluR7a (15 pmol) was preincubated with 15 pmol MAL-PICK1 or MAL (control) in 10 μl buffer B (20 mm HEPES, pH 7.4, 120 mm NaCl, 1 mm CaCl₂) for 3 hr at 4°C. The phosphorylation reaction was started by adding varying concentrations of PKCα in buffer A (supplemented with 200 μm [γ-³²P]ATP, 50 mCi/mmol) and allowed to proceed for a total of 3 min. When compared with MAL alone, MAL-PICK1 inhibited PKCα-evoked phosphorylation of GST-ct-mGluR7a throughout the range of PKCα concentrations examined. B, Inhibition of PKCα phosphorylation requires PICK1/mGluR7a interaction. GST-ct-mGluR7a (15 pmol) or GST-ct-mGluR7a-mutant (H⁸⁵¹–L⁸⁹², a construct that contains the PKCα phosphorylation sites but not the interaction site for PICK1) was preincubated with 15 pmol MAL-PICK1 and 1.35 μg/ml PKCα in 10 μl buffer A (supplemented with 1 mmCaCl₂) for 3 hr at 4°C. The phosphorylation reaction was started by addition of 10 μl buffer C (40 mmTris-HCl, pH 7.5, 200 μm [γ-³²P]ATP, 50 mCi/mmol) and allowed to proceed for times indicated. The rate of PKC-evoked phosphorylation of GST-ct-mGluR7a was reduced as compared with the GST-ct-mGluR7a-mutant. The mean percentage inhibitions of PKCα phosphorylation of GST-ct-mGluR7a as compared with GST-ct-mGluR7a-mutant were 14.4, 16.4, 15.5, 17.9, and 19.2% at the time points 1, 2, 3, 5, and 10 min, respectively.