Fig. 4

Identification of an interfering peptide for restoring MARCH1 induced downregulation of plasma membrane GABA_B receptors. (a) Screening for interfering peptides. HEK 293-cells expressing GABA_B1/GABA_B2 as a control (Ctrl) or GABA_B1/GABA_B2/MARCH1 were treated with the indicated peptides (10 µg/ml) and tested for cell surface expression of receptors using antibodies directed against and GABA_B1 and GABA_B2. Peptides 1–4 and 2–5 restored the cell surface expression of the receptors. Left: sequences of peptides used for screening derived from the C-terminal domains of GABA_B1 and GABA_B2. Right: quantification of fluorescence intensities (mean ± SD of 40–112 cells per condition, 2 independent experiments). Kruskal-Wallis test followed by Dunn’s multiple comparison test (ns, p > 0.05; *, p < 0.05; **, p < 0.01 ***, p < 0.001; ****, p < 0.0001). (b) Dose-response of peptides 1–4 and 2–5. HEK-293 cells expressing GABA_B1/GABA_B2/MARCH1 were treated with increasing concentrations of the respective peptides and were tested the following day for cell surface expression of GABA_B2. The data represents the mean ± SD of 30 cells per condition from 2 independent experiments. One-way ANOVA followed by Dunnett’s multiple comparison test (ns, p > 0.05; ****, p < 0.0001) (c) Peptide 2–5 did not restore the CHOP mediated downregulation of GABA_B receptors. HEK-293 cells expressing GABA_B1/GABA_B2 as a control (Ctrl) or GABA_B1/GABA_B2/CHOP were treated with peptide 2–5 (10 µg) and tested for cell surface expression of GABA_B2 the next day. The data represents the mean ± SD of 93–103 cells per condition from 3 independent experiments. Brown-Forsythe and Welch’s one-way ANOVA followed by Games-Howell’s multiple comparisons test (****, p < 0.0001) (d) Treatment of neurons with M1-Pep does not affect cell surface expression of native GABA_B receptors under physiological conditions. Neuron/glia co-cultures were treated for 16 h with or without M1-Pep or Ctrl-Pep and tested for cell surface (using GABA_B2 antibodies) and total GABA_B receptor (using GABA_B1 antibodies) expression. Top: representative images (scale bar: 10 μm). Bottom: quantification of fluorescence intensities (mean ± SD of 25 cells per condition, 2 independent experiments). One-way ANOVA followed by Tukey’s multiple comparison test (ns, p > 0.05). (e) M1-Pep marginally affects constitutive internalization of GABA_B receptors. Neurons transfected with GABA_B2 tagged at the N-terminus with the minimum binding site for α-bungarotoxin (GABA_B2(BBS)) were labeled with AlexaFluor 555 conjugated α-bungarotoxin and then analyzed by live cell imaging for internalization of labeled receptors. M1-Pep was added 10 min after starting the recording. Left: Representative images (scale bar: 5 μm). Right: Quantification of the immunofluorescence signal and statistical analysis. The statistical evaluation was performed on data recorded 15 min and 20 min after starting the recording. The data represent the mean ± SD of 7–8 neurons derived from three independent experiments. Unpaired t-test (ns, p > 0.05; *p < 0.05).