Figure 7. Unaltered response of β-arrestin-mediated signalling after conformational changes in S1P₁ receptor to α-Syn(A53T).

(a) S1P₁ receptor-YFP-expressing SH-SY5Y cells were preincubated at 37 °C with or without 1 μM α-Syn(A53T) for 18 hr in serum-free DMEM/F-12 and treated with or without 100 nM S1P for 1 hr. Cell surface S1P₁ receptor was then biotinylated, solubilised and pulled-down by streptavidin agarose, followed by measurement of fluorescence of S1P₁ receptor-YFP. Values represent means ± s.e.m. of 3 independent experiments carried out in triplicate. (b) SH-SY5Y cells were treated without or with 1 μM α-Syn(A53T) as in (a) and stimulated with 100 nM S1P for 1 hr as specified. Cells were harvested and incubated with anti-S1P₁ receptor antibody conjugated with Alexa 488, and analysed by flow cytometry. One of the representative histogram plots obtained from three independent experiments is shown. (c) Cell surface S1P₁ receptor was quantitated based on histogram plots in (b) and expressed as % of control. Values represent means ± s.e.m. of 3 independent experiments. Statistical significance was analysed by Student’s t-test (**P < 0.01 versus no stimulation). (d) S1P₁-YFP-transfected SH-SY5Y cells were incubated with or without α-Syn(A53T) as in (a) and stimulated with 100 nM S1P for 1 hr as specified. Cells were fixed with paraformaldehyde. The localisation of S1P₁-YFP was analysed by confocal microscopy. Bars, 10 μm. One of the representative results from three independent experiments is shown.