Computational protocol: PDZ Domain-Mediated Interactions of G Protein-Coupled Receptors with Postsynaptic Density Protein 95: Quantitative Characterization of Interactions

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[…] HEK293 cells were cultured at 37°C in DMEM supplemented with 2.2% FBS, in a 5% CO2 atmosphere with 95% humidity. Cells were plated on sterile glass cover slips two days before an experiment. Following overnight incubation, adherent cells were transiently transfected with the appropriate plasmids in a 1∶1 ratio, using TurboFect (Fermentas) as transfection reagent, according to the protocol from the manufacturer. Finally, cells were cultured overnight for protein expression.PSD-95-GFP was kindly provided by Philippe Marin (Institut de Génomique Fonctionnelle, Montpellier, France) . Plasmids for SNAP-tagged receptors (5-HTR2C, β1AR, β2AR, κOR, and hSSTR1) were obtained from Cisbio (France). For SNAP-hSSTR1-AAA and SNAP-β1AR-AAA, three alanine residues were added to the C-terminus of the respective wild type plasmids (GenScript, Piscataway, NJ, USA).SNAP-GPCR constructs were fluorescently labeled with fluorescent O6-benzylguanine (BG)-647 (New England Biolabs), according to the protocol from the manufacturer. In short, cells were incubated for 10 min with 5 µM BG-647 at 37°C and then washed. In samples that were not transfected with a receptor, cell membranes were stained with Vybrant DiD cell-labeling solution (Invitrogen). Subsequently, the cells were imaged in serum-free DMEM with HEPES (Invitrogen).Cross section micrographs of cells were acquired on an inverted confocal microscope (TCS SP5, Leica), using a water immersion objective (63x magnification, numerical aperture 1.2). GFP was excited with a wavelength of 488 nm; BG-647 and DiD were excited with a wavelength of 633 nm.Images were digitally processed with ImageJ to quantify the recruitment of PSD-95-GFP to the membrane and the colocalization with GPCRs. Straight line regions of interest perpendicular to the plasma membrane and covering several micrometers inside and outside the cell were selected manually, and the fluorescence intensities along these line segments were measured for both the receptor and PSD-95-GFP. These signals were further treated using Igor Pro (WaveMetrics). In brief, the position of the membrane was determined from the receptor fluorescence and set to 0 µm. At least five traces from the same cell were averaged and normalized for each graph. [...] Primary hippocampal neuronal cell cultures were prepared from 18.5 days post coitum mouse embryos as previously described . Pregnant mothers were sacrificed by an overdose of isoflurane and embryos by decapitation approved by the Guide for the Care and Use of Laboratory Animals of the Government of Upper Bavaria (Germany) as well as by the Animal Care and Use Committee of the Max Planck Institute of Psychiatry (Munich, Germany). Dissociated neurons were grown in Neurobasal-A medium supplemented with B27 Supplement (Invitrogen) and GlutaMAXI (Invitrogen). Neurons were plated on coverslips (Menzel) coated with 50 µg/ml poly-d-lysine (Sigma) and 5 µg/ml laminin (Invitrogen). After 20 days in vitro, neurons were fixed with 4% paraformaldehyde containing 4% sucrose. Immunohistochemistry was carried out as described , using the following antibodies: anti-SSTR1 (1∶50; Novus Biologicals, NB120-2366), anti-PSD-95 (1∶500; Neuromab, 75-028), anti-rabbit conjugated to Alexa-Fluor 594 (1∶1000; Invitrogen, A11037) and anti-mouse conjugated to Alexa-Fluor 488 (1∶1000; Invitrogen, A11029). To confirm the specificity of the antibodies used to detect SSTR1 and PSD-95, immunohistochemistry was performed as above but omitting the first antibody (). Nuclei were counterstained using DAPI. Immunocytochemical analysis was carried out by laser-scanning confocal microscopy. Images were acquired simultaneously in two acquisition channels with the FLUOVIEW FV 1000 (version 2.0a) acquisition analyzer program. Images were digitalized using Image J, Adobe Photoshop CS2, and Adobe Illustrator CS2. […]

Pipeline specifications

Software tools ImageJ, Adobe Illustrator
Databases NeuroMab
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus, Homo sapiens
Chemicals Somatostatin