Effects of the NAC on the dimerization of PAC1.

<p>(A) BiFC assays. Shown were YFP fluorescence intensity re-produced by the transfection of the receptor constructs as indicated. The cells without transfection were used as negative control and the cells transfected with PAC-YFP as positive control. Exogenous NAC (10 nM) decreased the YFP fluorescence intensity produced by PAC-Y/N+PAC-Y/C significantly (*, P<0.01 PAC-Y/N+PAC-Y/C+NAC vs. PAC-Y/N+PAC-Y/C), while the transfection of M+PAC-Y/N+M+PAC-Y/C produced no YFP fluorescence signals. Data were presented as means ± S.E. of three independent experiments. (B) Saturation BRET. Shown were the BRET saturation curves plotted as a ratio of YFP fluorescence to Rlu luminescence that were observed for tagged receptor constructs studied with a fixed amount of donor and increasing amounts of acceptor. PAC-Rluc/PAC-YFP receptor constructs yielded exponential curves that reached asymptotes indicating significant homo-dimerization of PAC1, while M-PAC-Rluc/M-PAC-YFP yielded curves not different from a straight line, indicating that D-PAC1 lost the ability to form dimers. The addition with NAC (10 nM) at 2 h before the BRET signal assay lowered the curves significantly (*, P<0.01 PAC-Rluc/PAC-YFP+NAC vs. PAC-Rluc/PAC-YFP). The data were represented as the means ± S.E. of three independent experiments. (C) Static BRET. BRET ratios for CHO cells expressing receptor constructs as indicated. For static BRET, a total of 1.0 µg of DNA per well divided equally among the noted constructs in each condition was utilized. The shaded area represents the nonspecific BRET signal generated between PAC-Rlu and soluble YFP protein, with BRET signals above this area considered to be significant. As shown the BRET ratio in PAC-Rluc/PAC-YFP CHO cells incubated with NAC (10 nM) was significantly lower than that in cells without treatment with NAC (*, P<0.01 PAC-Rluc/PAC-YFP+NAC vs. PAC-Rluc/PAC-YFP). The data were presented as the means ± S.E. of three independent experiments. (D) Western blotting analysis with a goat polyclonal IgG against the C-terminus of PAC1 using non-reductive SDS-PAGE. When PAC-YFP expressing cells incubated with exogenous NAC (10 nM), as shown, the band with the molecular weight (about 160 kD) consistent with the molecular weight of the PAC1 dimer was weakened by the presence of NAC (10 nM). All these results showed that NAC was an inhibitor of the dimerization of PAC1, which offered us a tool to analysis the relation of the dimerization of PAC1 with its basal activity.