Figure 2

PTCHD1 (Patched domain containing 1) is a synaptic receptor interacting with PSD95 and SAP102 but does not modulate the canonical sonic hedgehog signalling pathway. (a) Subcellular neuronal localization of PTCHD1-GFP (green fluorescent protein) in primary cultured hippocampal neurons. Representative images from confocal microscopy showing mature primary neurons expressing PTCHD1-GFP protein displaying punctate fluorescence throughout dendrites and dendritic spines. Hippocampal neurons were transfected at 12 days in vitro with PTCHD1-GFP (wild type (WT)), PTCHD1-GFP lacking the last 39 amino acids (PTCHD1delCter-GFP) or the last 15 amino acids (PTCHD1del874_888-GFP) and visualized 48 h later. Co-transfection with pDsRed was performed with PTCHD1delCter-GFP to visualize the entire cell. PTCHD1-GFP, n=15 neurons from 4 transfections; PTCHD1delCter-GFP, n=11 neurons from 3 transfections; PTCHD1del874_888-GFP, n=6 neurons from 2 transfections. Scale bars: 50 μm and 10 μm for PTCHD1-GFP and PTCHD1del874_888-GFP, 20 μm for PTCHD1delCter-GFP. (b) PTCHD1-GFP co-localizes with PSD95 in dendritic spines. Co-localization assay was performed using three primary neuronal cultures transfected with PTCHD1wtGFP (green) at 11 days in vitro (DIV11) and fixed at DIV14 and then immunostained with a mouse monoclonal PSD-95 antibody and a secondary Donkey anti-Mouse antibody (Alexa Fluor 594, red). Co-localization signal in spines (yellow) is indicated by white arrowheads. Data were collected from 10 transfected neurons with 31 dendrite sections in total (n=3 independent transfections). Scale bar: 10 μm. (c) Identification of a PDZ-binding motif in the C-terminal tail of PTCHD1 allowing interaction with PSD95 and SAP102 proteins. Predicted structure of PTCHD1 and sequence alignment of the 10 C-terminal amino acids of human proteins PTCHD1, PTCHD2, PTCHD3, PTCHD4, PTCH1 and PTCH2. The PDZ-binding motif is shown in red. PTCHD1 and PTCHD4 C-terminal sequences are identical between human and mouse. GST-pulldown assays using the C-teminal tail (39 amino acids) with or without the predicted PDZ-binding site as bait, and the synaptoneurosomal lysates from adult WT mouse cortex lysates as the prey, evidenced for the presence of a specific interaction between the predicted PDZ-binding motif and PSD95 and SAP102. The presence of a faint band in eluates from GST and from GST-Cter-delITTV using SAP102 antibody was considered as the non-specific background. A total of three independent experiments were performed. (d) PTCHD1 does not repress Gli reporter activity in Ptch1^−/− mouse embryonic fibroblasts (MEFs). Luciferase activity was assessed in Ptch1^−/− MEFs co-transfected with PTCHD1-GFP or PTCH1, and an 8 × Gli Luciferase Reporter Plasmid. A constitutively active renilla luciferase plasmid was also co-transfected to normalize for transfection efficiency. Luciferase values were measured using the Dual Luciferase Reporter Assay System (Promega) and normalized to an empty GFP control vector. Data were evaluated using a one-way analysis of variance test followed by a pairwise comparison of means using a Student’s t-test (with Bonferroni correction for multiple comparisons). Data are displayed as a box and whisker plot with a central line for the median, a box comprised of the first–third quartiles and whiskers showing the minimum and maximum values. n=3 independent experiments, with the displayed value for each being the average of duplicate plates of cells. *P<0.05.

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