Journal: The Journal of Biological Chemistry

Article Title: PICK1 links KIBRA and AMPA receptor subunit GluA2 in coiled-coil-driven supramolecular complexes

doi: 10.1016/j.jbc.2025.108397

Figure Lengend Snippet: KIBRA promotes PICK1 cluster formation and alters PICK1/GluA2 co-clustering. A , mGFP-PICK1 and Myc-GluA2 were transfected into HEK293T cells. Singly transfected PICK1 and GluA2 show diffuse localization ( top ) whereas co-transfected PICK1 and GluA2 form clusters ( bottom ). B , mCherry-KIBRA and Myc-GluA2 were transfected into HEK293T cells. KIBRA forms puncta whether singly transfected ( top ) or co-transfected with GluA2 (bottom), but does not affect the diffuse localization of GluA2. C , mCherry-KIBRA, mGFP-PICK1 and Myc-GluA2 were transfected into HEK293T cells. Co-transfected KIBRA alters PICK1 and GluA2 co-clusters. D , example line scans of GluA2 and PICK1 in the indicated clusters from panel A . E , example line scans of GluA2, KIBRA, and PICK1 signals from the indicated clusters in panel C . F and G , summary of line scan quantification for HEK293T cells transfected with GluA2 and PICK1 ( F ) or GluA2, PICK1, and KIBRA ( G ). Data are plotted as mean ± 95% CI. Signals were aligned to the edges (x = 0) of each PICK1 ( F ) or KIBRA/PICK1 ( G ) cluster. H , GluA2 signal intensity at the border of each cluster was subtracted from the signal at the cluster center. Dark lines on the violin plot denote median, and dashed lines indicate quartiles. Mann-Whitney test, p < 0.0001; median and 95% CI limits: PICK1 +GluA2: = 106.3, 79.88 to 121.2, PICK1 + GluA2 + KIBRA = 2.447, −6.371 to 12.5. n = 36 cells for PICK1/GluA2-expressing cells and 70 cells for PICK1/GluA2/KIBRA-expressing cells, each from three biological replicates. Signals in D – H were measured following local background subtraction. I , mCherry-PICK1 and mGFP-KIBRA were transfected into wild-type (WT) mouse hippocampal neurons at DIV 10, and the neurons were subsequently immunostained for synaptophysin at DIV 14. Two representative neurons shown from independent cultures. Scale bars for A–C = 10 μm; scale bars for D and E = 5 μm; and scale bars for I = 20 μm. J , mGFP/mCherry-PICK1 and Myc-GluA2 were transfected into HEK293T cells with Myc or Myc-KIBRA. Anti-mCherry antibodies were used to immunoprecipitate mCherry-PICK1, and co-precipitated Myc-KIBRA, Myc-GluA2 and mGFP-PICK1 were detected by immunoblot using mGFP or Myc antibodies. K and L , PICK1 homodimerization ( K , KIBRA+ normalized to KIBRA negative sample within the same gel: 1.15 ± 0.35; p = 0.5287) and PICK1-GluA2 interaction ( L , KIBRA+, normalized to KIBRA-negative sample within the same gel: 1.26 ± 0.39; p = 0.3594) were not affected by KIBRA expression, quantified from three biological replicates. One sample t test versus 1. Data reported as mean ± SD unless otherwise noted.

Article Snippet: 25 to 50 ul Protein G Magnetic Beads (NEB #S1430S) were incubated with 3 μg GFP (D5.1, Rabbit mAb, Cell Signaling Technology #2956S), mCherry (E5D8F, Rabbit mAb, Cell Signaling Technology #43590S) or Myc (9B11, Mouse mAb, Cell Signaling Technology #2276S) antibody at 4 °C for 1 h. Cell lysate was incubated with antibody-conjugated beads at 4 °C overnight.

Techniques: Transfection, MANN-WHITNEY, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: PICK1 links KIBRA and AMPA receptor subunit GluA2 in coiled-coil-driven supramolecular complexes

doi: 10.1016/j.jbc.2025.108397

Figure Lengend Snippet: PICK1 BAR domain facilitates the interaction between KIBRA and PICK1. A , mGFP-PICK1 WT, KD-AA, 5K-E or BAR was transfected with mCherry-KIBRA WT into HEK293T cells. mCherry-KIBRA was immunoprecipitated and co-precipitated mGFP-PICK1 variants were detected by immunoblot using anti-GFP antibodies. B , quantification of six biological replicates for PICK1 WT, KD-AA and 5K-E and five biological replicates for PICK1 BAR as shown in A ; PICK1 KD-AA or 5K-E impair interaction with KIBRA compared with PICK1 WT (PICK1 pull down normalized to input and same-gel WT PICK1; PICK1 KD-AA: 0.81 ± 0.02, ∗∗∗∗ p < 0.0001; PICK1 5K-E: 0.67 ± 0.21, ∗ p = 0.0127; PICK1 BAR: 1.62 ± 0.79, p = 0.1541, one sample t test versus 1). C , mGFP-PICK1 WT or 5K-E and Myc-GluA2 were transfected into HEK293T cells. Immunoprecipitation of PICK1 was performed using a GFP antibody, and the co-precipitated GluA2 was detected using a Myc antibody. GluA2 showed equivalent interaction with mGFP-PICK1 WT and 5K-E. D , mGFP-PICK1 WT or 5K-E was co-transfected with mCherry-KIBRA and Myc-GluA2 into HEK293T cells. mGFP-PICK1 was immunoprecipitated and co-precipitated mCherry-KIBRA and Myc-GluA2 were detected by immunoblot. E and F , quantification of three biological replicates of conditions shown in D ; When PICK1, KIBRA and GluA2 are all present, mutation of the PICK1 BAR domain (PICK1 5K-E) decreases interaction with both KIBRA and GluA2. (All bands were normalized to input. KIBRA signal with PICK1 5K-E normalized to +PICK1 WT: 0.63 ± 0.14, ∗ p = 0.0434; GluA2 signal with PICK1 5K-E normalized to +PICK1 WT: 0.27 ± 0.13, ∗∗ p = 0.0096, one sample t test.) G , structure and domains of PICK1. Predicted structure modified from AlphaFold Protein Structure Database. Data reported as mean ± SD.

Article Snippet: 25 to 50 ul Protein G Magnetic Beads (NEB #S1430S) were incubated with 3 μg GFP (D5.1, Rabbit mAb, Cell Signaling Technology #2956S), mCherry (E5D8F, Rabbit mAb, Cell Signaling Technology #43590S) or Myc (9B11, Mouse mAb, Cell Signaling Technology #2276S) antibody at 4 °C for 1 h. Cell lysate was incubated with antibody-conjugated beads at 4 °C overnight.

Techniques: Transfection, Immunoprecipitation, Western Blot, Mutagenesis, Modification

Journal: The Journal of Biological Chemistry

Article Title: PICK1 links KIBRA and AMPA receptor subunit GluA2 in coiled-coil-driven supramolecular complexes

doi: 10.1016/j.jbc.2025.108397

Figure Lengend Snippet: No single domain of KIBRA is essential for interaction between KIBRA and PICK1 or for KIBRA-KIBRA dimerization. A , mGFP-KIBRA WT or KIBRA mutants missing the WW domain, coiled-coil linker, C2 domain, C-terminal region, or aPKC binding region (ΔWW, ΔCCL, ΔC2, ΔCt, ΔαBR, respectively) were transfected with mCherry-PICK1 WT into HEK293T cells. Anti-mCherry antibodies were used to immunoprecipitate PICK1, and co-precipitated mGFP-KIBRA variants were detected by immunoblot for GFP. B , quantification of five biological replicates under conditions shown in A . None of the KIBRA mutants disrupt interaction between KIBRA and PICK1 (values normalized to WT sample from the same blot; ΔCCL 1.48 ± 1.00, p = 0.3465; ΔC2 1.29 ± 0.39, p = 0.1792; ΔαBR 1.94 ± 0.96, p = 0.0936, one-sample t test versus 1). KIBRA ΔWW and ΔCt show increased interaction with PICK1 (ΔWW 1.79 ± 0.23, ∗∗ p = 0.0016; ΔCt 2.49 ± 0.56, ∗ p = 0.0129, one-sample t test versus 1). C , Myc- and GFP- KIBRA WT, ΔCCL, ΔC2, ΔCt, or ΔαBR were transfected into HEK293T cells. Myc-KIBRA was immunoprecipitated and co-precipitated mGFP-KIBRA was detected by immunoblot. D , quantification of three biological replicates under conditions shown in C . No single KIBRA mutant prevents homodimerization (values normalized to WT sample from the same blot; ΔCCL 1.60 ± 0.59, p = 0.2205; ΔC2 1.36 ± 0.43, p = 0.2853; ΔCt 1.00 ± 0.28, p = 0.9904; ΔαBR 1.55 ± 0.23, p = 0.0560, one-sample t test versus 1). E , structure and domains of KIBRA. Predicted structure modified from AlphaFold Protein Structure Database. Data reported as mean ± SD.

Article Snippet: 25 to 50 ul Protein G Magnetic Beads (NEB #S1430S) were incubated with 3 μg GFP (D5.1, Rabbit mAb, Cell Signaling Technology #2956S), mCherry (E5D8F, Rabbit mAb, Cell Signaling Technology #43590S) or Myc (9B11, Mouse mAb, Cell Signaling Technology #2276S) antibody at 4 °C for 1 h. Cell lysate was incubated with antibody-conjugated beads at 4 °C overnight.

Techniques: Binding Assay, Transfection, Western Blot, Immunoprecipitation, Mutagenesis, Modification