Figure 2: α-Syn-induced uncoupling of S1P1 receptor from G-protein, while leaving S1P2 receptor unchanged. | Scientific Reports

Figure 2: α-Syn-induced uncoupling of S1P1 receptor from G-protein, while leaving S1P2 receptor unchanged.

From: Extracellular α-synuclein induces sphingosine 1-phosphate receptor subtype 1 uncoupled from inhibitory G-protein leaving β-arrestin signal intact

Figure 2

(a) Expression level of S1P1, S1P2, S1P3, S1P4 and S1P5 receptor mRNAs in SH-SY5Y cells were quantitated by real-time quantitative reverse transcription PCR. Values of mRNA amounts were normalised to GAPDH expression. (b) Cells transiently expressing the S1P1-CFP, Gβ and Gγ-YFP were pretreated without (closed bars) or with 1 μM wild-type α-Syn (grey bars) or α-Syn(A53T) (hatched bars) for 18 hr and then stimulated with 100 nM S1P for 1 min, fixed and analysed for FRET efficiencies. Values represent means ± s.e.m. (n ≥ 50). Statistical significance was analysed by Student’s t-test (*P < 0.05 versus S1P control). (c) SH-SY5Y cells transiently expressing the S1P2 receptor-CFP, Gβ and Gγ-YFP were treated with either buffer (control) or 1 μM wild-type α-Syn (grey bars) or α-Syn(A53T) (hatched bars), stimulated with S1P and analysed for FRET efficiencies as in (b). Values represent means ± s.e.m. (n ≥ 50). (d) Cells transiently expressing the Giα-CFP, Gβ and Gγ-YFP were pretreated without (closed bars) or with 1 μM α-Syn(A53T) (hatched bars) for 18 hr and then stimulated with 100 nM S1P for 1 min, fixed and analysed for FRET efficiencies. Values represent means ± s.e.m. (n ≥ 50). Statistical significance was analysed by Student’s t-test (*P < 0.05). (e) Cells transiently expressing the S1P1-YFP and Giα-CFP were pretreated without or with 1 μM α-Syn(A53T) for 18 hr and then stimulated with 100 nM S1P or 20 ng/ml PDGF for 1 min, fixed and analysed for FRET efficiencies. Values represent means ± s.e.m. (n ≥ 50; *P < 0.05, **P < 0.01 versus control).

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