Fig 1.
G-protein Inhibitor reduces Pls-mediated phosphorylation of Akt and ERK in the neuronal cells.
(A) Pls-mediated phosphorylation of ERK and Akt was cancelled by treatments with the G-protein inhibitor. The neuronal cells (N2A) were cultured in low serum (2% FBS) containing medium for 20 hr and then treated with the G-protein inhibitor GDPβS at 200 μM concentration for 4 hours followed by Pls (500 ng/ml) treatments for 20 minutes. Cell extracts were then subjected to western blotting assays. (B) The quantification data of panel A showed that Pls treatment failed to increase phosphorylation of ERK and Akt in the GDPβS pretreated cells (One way ANOVA followed by Bonferoni’s test, n = 5, *, P<0.05 and **, P<0.01. n.s. stands for not significant). Image-J software was used to quantify the signals. (C) Real-time PCR data shows the orphan GPCRs expression in the primary neurons, astrocytes, microglia and also in the cell lines of N2A (neuronal derived cells), A1 (astrocytes) and MG6 (microglia). The data represent mean ± S.E.M (standard error of mean) (n = 3).
Fig 2.
Knockdown of GPCRs inhibits Pls-mediated activation of ERK.
(A) Total RNAs extracted from the sh-RNA containing lentivirus transfected N2A cells were subjected to the real-time PCR analysis. The relative expression of the target GPCRs was reduced by their respective sh-RNA (Student’s t-test, n = 5, *, P<0.05 and **, P<0.01). (B) Western-blotting assays showed the reduction of Pls-mediated phosphorylation of ERK in the GPR1, GPR19, GPR21, GPR27 and GPR61 knockdown cells. N2A cells were infected by the sh-RNAs lentiviruses followed by the treatments with 2% FBS containing medium for 24 hours. Pls (500 ng/ml) were added 20 minutes before the cells were collected for western blotting assays. The data represent three independent experiments (n = 3). (C) The quantification data of panel B showed a significant reduction in the expression of phosphorylated ERK in the GPR1, GPR19, GPR21, GPR27 and GPR61 knockdown cells treated with the Pls. The data represent mean ± S.E.M and the n.s. indicates not significant (One way ANOVA followed by Dunnett’s post hoc test by considering the sh-Luc with Pls group as reference. n = 5, *, P<0.05 and **, P<0.01). (D) Western-blotting assays showed the difference in the p-ERK expression between Pls-treated and non-treated groups of GPR1, GPR19, GPR21, GPR27, GPR61 and GPR62 knockdown cells. The data represents 3 independent experiments. (E) The quantification data of panel D showed no effect of Pls in the sh-GPR1, sh-GPR19, sh-GPR21, sh-GPR27 and sh-GPR61, but not sh-Luc and sh-GPR62 groups. The data represent mean ± S.E.M and the n.s. indicates not significant (Student`s t-test; n = 3, **, P <0.01). Furthermore, the reduction of p-ERK expression in the Pls treatment groups of GPR1, GPR19, GPR21, GPR27 and GPR61 knockdown cells was significant compared with the control sh-Luc group. (Dunnett’s test. n = 3, *, P < 0.05).
Fig 3.
Overexpression of GPCRs enhances phosphorylation of ERK and Akt in the cells.
(A) Hek293-T cells cultured in 2% FBS containing medium were transfected by the flag tagged GPR1, GPR19, GPR21 and GPR61 expression plasmids along with the control flag tagged empty plasmids (mock group) for 48 hours followed by western blotting assays. Overexpression of the proteins was confirmed by the immunoblotting with anti-Flag antibody. Quantification of p-ERK, p-Akt and the total proteins of ERK and Akt showed the significant increases in the phosphorylation of ERK and Akt by the overexpression of GPCRs compared with mock empty flag tagged plasmid transfected group (Dunnett’s test, n = 5, *, P<0.05 and **, P<0.01). The data represents three independent experiments. (B) Similar to that of the Hek-293T cells of panel A, N2A cells were transfected by the GPCR expressing plasmids along with the control mock empty flag tagged plasmid and phosphorylation of ERK and Akt was investigated by the western blotting assays. The data represents 5 independent experiments. (C) Quantification data of the panel C showed the significant increase in the phosphorylation of ERK and Akt among the GPCRs overexpressed N2A cells compared with control (mock) group. The data represent mean ± S.E.M. (Student’s t-test, n = 5, P<0.05* and 0.01**)
Fig 4.
The GPCRs enhances Pls-mediated signaling in the cells.
(A) N2A cells cultured with low serum (2% FBS) were transfected by the control and GPCRs plasmids for 48 hours followed by the treatments with Pls (500 ng/ml) for 20 minutes. Cell extracts were subjected to western blotting assays. The data represents five independent experiments (n = 3). (B) Quantification data of panel A showed the significant increase in the phosphorylation of ERK and Akt proteins among the GPCRs (GPR1, GPR19, GPR21 and GPR61) transfected and the mock transfected cells by the Pls treatments. The data represent mean ± S.E.M (Student’s t-test, n = 5, *, P<0.05 and **, P<0.01). (C) The fold induction in the level of phosphorylated ERK and Akt compared with the mock transfected cells (considered as one unit) was counted in the GPCRs overexpressed cells. Statistical analysis showed a significant increase in the Pls-mediated induction of ERK and Akt phosphorylation by the GPCRs overexpression in the neuronal cells shown in panel A (Bonferroni’s test, n = 5, *, P<0.05 and **, P<0.01).
Fig 5.
The Pls specific cellular signaling mediated by the GPCRs.
(A) Western blotting assays showed the reduction of p-ERK and p-Akt expression in the N2A cells where the Pls-synthesizing enzyme GNPAT was knocked down by sh-GNPAT lentivirus. The data represent three independent experiments. (B) The quantification data of the panel A showed the significant reduction of phosphorylated ERK and Akt proteins in the GNPAT knockdown cells. The data represent mean ± S.E.M (Student’s t-test, n = 3, **, P < 0.01). (C) Serum deprived N2A cells were treated with Pls (500 ng/ml) and PtdEtn (phosphatidylethanolamine, 500 ng/ml) for 20 minutes. Cells extracts were then subjected to western blotting assays to detect phosphorylation status of ERK and Akt proteins. The data represent three independent experiments. (D) Quantification data of panel C showed that PtdEtn treatments did not significantly increase the phosphorylated ERK and Akt as compared with the Pls treatments group. The data represent mean ± S.E.M (Bonferroni’s test; n = 3, *, P < 0.05). (E) N2A cells were infected by sh-GNPAT and the control sh-Luc lentivirus particles for 24 hours flowed by the overexpression of the GPCRs expressing plasmids for 48 hours. Cell extracts were then subjected to western blotting assays. The data represents three independent experiments (n = 3). (F) Quantification data of the panel E showed that the GPCRs-mediated increase in phosphorylation of ERK in the control lentivirus group (sh-Luc) was abolished in the sh-GNPAT groups. The data represent mean ± S.E.M (Dunnett`s test; n = 3, *, P < 0.05).