The N-terminal HSDCIF motif is required for cell surface trafficking and dimerization of family B G protein coupled receptor PAC1

Abstract

PAC1 is PACAP (pituitary adenylate cyclase-activating polypeptide) preferring receptor belonging to class B G protein coupled receptor (GPCR) mediating the most effects of PACAP. The important role of G protein coupled receptor homo/heteromerization in receptor folding, maturation, trafficking, and cell surface expression has become increasingly evident. The bimolecular fluorescence complementation (BiFC) and bioluminescence resonance energy transfer (BRET) assay were used in this research to confirm the dimerization of PAC1 for the first time. The structure-activity relationship focused on the N-terminal HSDCIF motif, which locates behind the signal sequence and has high homology with PACAP (1-6), was assayed using a receptor mutant with the deletion of the HSDCIF motif. The fluorescence confocal microscope observation showed that the deletion of the HSDCIF motif impaired the cell delivery of PAC1. The results of BiFC, BRET and westernblot indicated that the deletion of HSDCIF motif and the replacement of the Cys residue with Ala in HSDCIF motif resulted in the disruption of receptor dimerization. And the exogenous chemically synthesized oligopeptide HSDCIF (100 nmol/L) not only down-regulated the dimerization of PAC1, induced the internalization of PAC1, but also inhibited the proliferation of CHO cells expressing PAC1 stably and decreased the activity of PACAP on the cell viability. All these data suggested that the N-terminal HSDCIF motif played key role in the trafficking and the dimerization of PAC1, and the exogenous oligopeptide HSDCIF had effects on the cell signaling, trafficking and the dimerization of PAC1.

Figure 1. The structure sketch map of PAC1 (A, B).

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.).

Figure 2. Morphologic localization of YFP-tagged PAC1 and YFP-tagged D-PAC1.

Shown were confocal fluorescence micrographs of CHO cells expressing YFP-tagged PAC1 and YFP-tagged D-PAC1 transiently and stably in light field, fluorescent field and their merge. It was observed that in both transient and stable expression, D-PAC1 construct was not able to traffic to cell membrane and accumulated inside the cells,while PAC1 construct was transported to the cell surface normally. Bar, 5 µm.

Figure 3. Bimolecular fluorescence complementation and immunofluorescence (A, B).

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.

Figure 4. Static BRET assays (A).

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.

Figure 5. Effects of the oligopeptide HSDCIF on PAC1 BiFC (A).

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.

Figure 6. Effects of oligopeptide HSDCIF on the traffic of PAC1.

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.

Figure 7. Morphologic localization of YFP-tagged M-PAC1.

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.

Figure 8. BiFC and immunofluorescence (A).

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.

Figure 9. Effects of oligopeptide HSDCIF on the viability of PAC1-CHO cells as measured by MTT assay (A).

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.