The Effects Of The N-terminal Cys/Ala On The Dimerization Of PACAP Preferring Receptor PAC1

Objective:G-protein couple receptors (GPCRs) are the biggest super family ofmembrane receptors, which have the common phenomenon of dimerization oroligomerization. PAC1is PACAP(pituitary adenylate cyclase-activating polypeptide)preferring receptor, belonging to class B GPCR. PAC1mediates the most effects ofPACAP, which is a important target for drug development of the nervous systemdiseases. The dimerization of PAC1has been proven by our previous research and ithas been indicated that the deletion of N-terminal HSDCIF motif resulted in thedisruption of PAC1dimerization. The N-terminal residue cystein (Cys) in theHSDCIF motif is the first single Cys of PAC1, which is not reported to form theknown disulfide bond. It was hypothesized that this Cys residue played a key role inthe PAC1dimerization. In this research, the mutant PAC1with the replacement of theextracellular N-terminal Cys in the HSDCIF motif with Ala (named M-PAC1) wasconstructed. And the effects of the Cys/Ala mutantation on the receptor dimerizationand on the Chinese hamster ovary (CHO) cells viabilities and anti-apoptotic activitiesin serum-withdraw condition were detected.Method: With gene engineering principle and technology, we constructed serieseukaryotic recombinant expression vectors including M-PAC1-EYFP fused M-PAC1with enhanced yellow fluorescent protein (EYFP), M-PAC1-Rluc fused M-PAC1withrenilla luciferase (Rluc) for the assay of bioluminescence resonance energy transfer(BRET), M-PAC1-EYFP/N and M-PAC1-EYFP/C for complements bimolecularfluorescence (BiFC). The stable expression of M-PAC1in CHO cells was achieved.Fluorescence confocal microscope was used to detect the expression of M-PAC1. Thetechniques of BRET, BiFC and Westernblot were used to detect dimerization ofM-PAC1. Meanwhile the techniques of MTT, ELISA and flow cytometry were used todetect the cells viaobilities and the anti-apoptotic abilities against serum-withdrawinduced apoptosis of the cells expressing wild type PAC1or M-PAC1stablyinclucing the levels of anti-apoptotic protein Bcl-2in the cells as well as the plasma apoptotic protein Caspase-3activities.Results: The recombinant vectors inclucing M-PAC1-EYFP, M-PAC1-Rluc,M-PAC1-EYFP/N, M-PAC1-EYFP/C were constructed and identified. And theM-PAC1-CHO cell line stably expressing M-PAC1was obtained. The fluorescencconfocal microscopy showed that M-PAC1trafficked to the cell membrane normally.The results of BRET, BiFC and Westernblot indicated that M-PAC1lost the ability toform dimer with itself and with intact PAC1. In the serum-withdraw culture, theviability of the cells that expressed PAC1was significantly higher than that expressedM-PAC1.The assays results showed that the cells that expressed PAC1had lowercaspase-3activity and significantly higher Bcl-2level than that expressed M-PAC1.Therefore, it was deduced that PAC1had dimer-dependent intrinsic/basal activity,which conferred the cells with anti-apoptotic activity, because M-PAC1withoutdimerization did not endow the cells with anti-apoptotic activity.Conclusion: It was reported for the first time that M-PAC1lost the ability to formdimer with itself and with intact PAC1, which suggested that the N-terminal Cysresidue in the HSDCIF motif played a key role in the dimerization of PAC1. It wasdeduced that in the serum-free condition, PAC1had dimer-dependent intrinsic/basalactivity, which conferred anti-apoptotic activity to the cells in a ligand-independentmanner. This findings may layed a new foundation for the further research on themechanism and the biological role of the dimerization of PAC1.