The Journal of Neuroscience, March 1, 2006, ():
Lipid Binding Regulates Synaptic Targeting of PICK1, AMPA Receptor Trafficking, and Synaptic Plasticity
J. Neurosci. Jin et al.
26: 2380
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplementary Figure S1. Diagram of PICK1’s domain structure and alignment of PICK1’s BAR domain with amphiphysin and arfaptin.
(A) The domain structure of PICK1. The PDZ domain of PICK1 is responsible for interaction with the C-termini of AMPA receptors and other membrane proteins. The predicted BAR (Bin/amphiphysin/Rvs) domain occupies ~200 residues in the middle of PICK1. The D/E domain is a stretch of 9 acidic amino acids (glutamic acids and aspartic acids).
(B) Sequence alignment of the BAR domains from PICK1, arfaptin2 and amphiphysin1. BAR domains of PICK1 and arfaptin2 are highly homologous but they are only moderately homologous to amphiphysin. However, the crystal structures of arfaptin and amphiphysin revealed that their BAR domain structures are strikingly similar (Peter et al., 2004). The highlighted basic residues in amphiphysin and arfaptin were found to be responsible for lipid binding (Peter et al., 2004). The bold lysine residues in PICK1 are the predicted lipid binding sites being explored in this study.
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Supplemental Figure S2. Lipid binding is critical for PICK1’s subcellular localization.
(A) Wild-type and different mutants of GFP-tagged PICK1 were transfected into 293T cells and visualized by fluorescence microscope. Wild-type PICK1 is shown here to form two clusters in one cell but not the other cell. The lipid binding deficient mutants, PICK1 2K-E, 3K-E and 5K-E, are mainly diffused in the cytosol of 293T cells. Scale bar: 10 μm.
(B) The percentage of clustered cells was calculated by counting the number of cells that had at least one cluster and the total number of transfected cells. Results were averaged from three independent experiments. At least one hundred cells were quantified for each experiment. The lipid binding deficient mutants formed significantly fewer clusters compared with wild-type PICK1 (** p<0.01, t-test).
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Supplementary Figure S3. Lipid binding is required for PICK1-induced clustering of AMPA receptors.
(A) GluR2 and wild-type GFP-PICK1 or mutant PICK1 2K-E were co-transfected into 293T cells. Cells were fixed and stained with the anti-GluR2/3C antibody. PICK1 and GluR2 formed numerous co-clusters in the cells. Scale bar: 10 μm.
(B) The lipid binding deficient mutant PICK1 2K-E did not form any co-cluster with GluR2. Both of them were diffuse in the cytosol. Similar results were obtained with PICK1 3K-E and 5K-E mutants. Scale bar: 10μm.
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Supplementary Figure S4. PICK1 2K-E mutant still interacts with GluR2 and itself in yeast two hybrid studies.
The yeast expression vector pPC86 containing wild-type PICK1, PICK1 2K-E, PICK1 KD-AA or a vector alone were co-transformed with either GluR2 or PICK1 in the pPC97 vector. The interaction was confirmed by both growth (on plates lacking leucine, tryptophan, and histidine) and blue assay (which measures β–galactosidase activity).
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Supplementary Figure S5. The BAR domain mutant PICK1 2K-E interacts with GRIP1.
HEK293T cells were transfected with GRIP and myc-tagged wild type PICK1, PICK1 2K-E or the vector control, as indicated on the top. Cell lysates were immunoprecipitated with anti-myc antibody and immunoblotted with either GRIP1 or PICK1 antibody as indicated. The lipid binding deficient mutant PICK1 2K-E immunoprecipitated with GRIP1 similarly to wild-type PICK1.
