Morphologic localization of YFP-tagged M-PAC1.

<p>Shown were confocal fluorescence micrographs of CHO cells expressing YFP-tagged M-PAC1 transiently and stably in light field, fluorescent field and their merge. It was observed that in both transient and stable expression,M-PAC1 construct was transported to the cell surface normally. Bar, 5 µm.</p

Effects of the oligopeptide HSDCIF on PAC1 BiFC (A).

<p>Shown were YFP fluorescence intensity produced by the transfection of the receptor constructs as indicated. The cells without transfection were used as negative control and the cells transfected with D-PAC-YFP as positive control. Exogenous HSDCIF decreased the YFP fluorescence intensity produced by PAC-Y/N+PAC-Y/C significantly. (Δ, p<0.01 vs. PAC-Y/N+PAC-Y/C. * p<0.01 vs. negative control). Data were presented as means±S.E. of six independent experiments. Effects of the oligopeptide HSDCIF on PAC1 saturation BRET (B). Shown were the BRET saturation curves plotted as a ratio of YFP fluorescence to Rlu luminescence that were obtained for pairs of PAC-Rluc and PAC-YFP studied with a fixed amount of donor (1.0 µg of DNA/dish) and increasing amounts of acceptor (0.3–6 µg of DNA/dish). The experiments were performed in the absence or presence of the oligopeptide HSDCIF. PAC1 produced a significant saturable exponential curve, while incubation with HSDCIF lowered the curves significantly. The data are represented as the means±S.E. of six independent experiments. Effects of the oligopeptide HSDCIF on PAC1 static BRET (C). BRET ratios for CHO cells expressing receptor constructs as indicated. 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. *,p<0.01, significantly above the background and significantly higher than negative control (PAC-Rluc). The BRET ratio in PAC1-expressing CHO cells incubated with HSDCIF was significantly lower than that in cells without treatment with HSDCIF (Δ, p<0.01 vs. PAC-Rluc/PAC-YFP). The data are presented as the means±S.E. of six independent experiments. Westernblot analysis of CHO cells expresing PAC-YFP, D-PAC-YFP, and PAC-YFP expressing cells incubated with exogenous oligopeptide HSDCIF (D). As shown, the band with the molecular weight (about 160 kD) consistent with the molecular weight of the dimer was absent in D-PAC-CHO, and the exogenous oligopeptide HSDCIF decreased the dimer amount in PAC-CHO significantly.</p

Oligonucleotide primers employed.

<p>Oligonucleotide primers employed.</p

Static BRET assays (A).

<p>Shown were BRET ratios generated from CHO cells expressing Rlu-tagged receptor with YFP-tagged receptor constructs as indicated. For static BRET, a total of 1.0 ug of DNA/5×105 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. Data were presented as means±S.E. of six independent experiments. *p<0.01, significantly above the background and significantly higher than negative control (PAC-Rluc). Saturation BRET assays (B). Shown were the BRET saturation curves plotted as ratios 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 D-PAC-Rluc/D-PAC-YFP and D-PAC-Rluc/ PAC-YFP yielded curves not different from a straight line, indicating that D-PAC1 lost the ability to form dimer with itself and with PAC1. The data are represented as the means±S.E. of six independent experiments.</p

Effects of oligopeptide HSDCIF on the viability of PAC1-CHO cells as measured by MTT assay (A).

<p>Data are presented as means ±S.E. obtained from six independent experiments. *p< 0.01 HSDCIF groups vs. control group, #p< 0.01, PACAP groups vs. control group. As shown, exogenous HSDCIF had contrary effects with PACAP on the cell viability. Effects of oligopeptide HSDCIF (100 nM) on the activity of PACAP with gradient concentrations (B). Data are presented as means ±S.E. obtained from six independent experiments. $p< 0.01 HSDCIF(100 nM)+PACAP groups vs. PACAP groups. Effects of oligopeptide HSDCIF with gradient concentrations on the activity of PACAP(100 nM) (C). Data are presented as means ±S.E. obtained from six independent experiments. &p< 0.01 HSDCIF+PACAP(100 nM) groups vs. PACAP(100 nM) group. As shown, exogenous HSDCIF decreased the effects of PACAP on the cell viability significantly.</p

Bimolecular fluorescence complementation and immunofluorescence (A, B).

<p>Shown were fluorescence images of CHO cells expressing various receptor constructs, as indicated. The positive results of immunofluorescence using antibody against PAC1 showed that all constructs expressed PAC1 or D-PAC1. Cells transfected with alone Y/N- or Y/C- tagged receptors did not produce YFP fluorescence. YFP fluorescence could be visualized in the cells transfected with the PAC-Y/N +PAC-Y/C supporting homo-dimerization of PAC1. The cells transfected with D-PAC-Y/N +D-PAC-Y/C produced no YFP fluorescence. Absence of fluorescence supported no physical interaction between PAC1 and D-PAC1, while D-PAC-YFP was used as a positive control producing YFP fluorescence. Bar, 5 µm. The statistical analysis of the YFP fluorescence intensity (C) showed that only cells tranfected with PAC-Y/N +PAC-Y/C or D-PAC-YFP (positive control) had significantly higher YFP fluorescence intensity than the negative control (cells without transfection) (*p<0.01 vs. no transfection). Data were presented as means±S.E. of six independent experiments.</p

The structure sketch map of PAC1 (A, B).

<p>The HSDCIF motif is indicated as red. The homology analysis between PACAP and PAC1-EC1 (C). The red region showed the HSDCIF motif and its high homologue PACAP (1–6). The construction of D-PAC1 (D). The gene coding D-PAC1 with the deletion of the HSDCIF motif was amplified using over-lap PCR (The arrows represented the primers used to amplify the genes coding D-PAC1 and PAC1.).</p

BiFC and immunofluorescence (A).

<p>Shown were fluorescence images of CHO cells expressing various receptor constructs, as indicated. The positive results of immunofluorescence using antibody against PAC1 showed that all constructs expressed M-PAC1. The cells transfected with M-PAC-Y/N +M-PAC-Y/C produced no YFP fluorescence. Absence of fluorescence supported no physical interaction between PAC1 and M-PAC1, while M-PAC-YFP was used as a positive control producing YFP fluorescence. Bar, 5 µm. The statistical analysis of the YFP fluorescence intensity (B) usied M-PAC-YFP as positive control and cells without transfection as the negative control (*p<0.01 vs. no transfection). Data were presented as means±S.E. of six independent experiments. Static BRET assays (C). Shown were BRET ratios generated from CHO cells expressing Rlu-tagged receptor with YFP-tagged receptor constructs as indicated. 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. Data were presented as means±S.E. of six independent experiments. *p<0.01, significantly above the background and significantly higher than negative control (PAC-Rluc). Saturation BRET assays (D). Shown were the BRET saturation curves plotted as ratios 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. M-PAC-Rluc/M-PAC-YFP and M-PAC-Rluc/ PAC-YFP yielded curves not different from a straight line, indicating that M-PAC1 lost the ability to form dimer with itself and with intact PAC1. The data are represented as the means±S.E. of six independent experiments. Westernblot analysis of CHO cells expresing PAC-YFP and M-PAC-YFP (E). As shown, the band with the molecular weight (about 160 kD) consistent with the molecular weight of the dimer was absent in M-PAC-CHO.</p

Effects of oligopeptide HSDCIF on the traffic of PAC1.

<p>Eight fluorescence confocal images from the animation of 0–1120 s after the addition of oligopeptide indicated that the fluorescence presenting the site of PAC1 detached from the cell-surface and moved to the inside of the cell, especially significantly in the region shown by the arrow. Bar, 5 µm.</p