The Dimerization Of Family B G-protein-couple-receptor PAC1and The Effects Of The N-terminal HSDCIF Motif On The Dimerization

Objetive：G-protein couple receptors (GPCRs) are the biggest super family ofmembrane receptors. PAC1belongs to the B family of GPCRs and is pituitaryadenosine acid cyclization enzyme excited peptide (PACAP) preferring receptors,mediating PACAP neural protection function, which is one of the important targets fordrug development to diseases of the nervous system. Dimerization or oligomerizationis a common phenomenon to GPCRs. But there is no report homologus dimerizationor oligomerization for PAC1at present. The purpose of this paper is to verify thehomologous dimerization of PAC1, and explore the role of the N-terminal motif ofHSDCIF located in the first extracellar domain1(EC1） in the homologousdimerization and the cell surface trafficking using the PAC1deletion mutant namedD-PAC1missing N-terminal motif of HSDCIF. Meanwhile, the effects of thechemically synthesized peptide HSDCIF on the trafficking, the dimerization of PAC1and the cells proliferation were also assayed. The clarification of the mechanism forthe dimerization of PAC1will not only help to understand the physiological andpathological role of PAC1, but also benefit the subsequent drug development targetingPACE, and offer illumination and reference for the similar research on other GPCRs.Method: With gene engineering principle and technology, as well as gene knockouttechnology, we construct eukaryotic series recombinant expression vectors includingPAC1-EYFP and D-PAC1-EYFP, which fused PAC1with enhanced yellowfluorescent protein (EYFP), PAC1-Rluc and D-PAC1-Rluc for the assay ofbioluminescence resonance energy transfer (BRET), PAC1-EYFP/N, PAC1-EYFP/C,D-PAC1-EYFP/N and D-PAC1-EYFP/C for complements bimolecular fluorescence(BiFC). PAC1-EYFP and D-PAC1-EYFP were transfected to CHO (chinese hamsterovary cells) cells respectively to get CHO cells stably expresing PAC1-EYFP namedPAC1-CHO and CHO cells stably expresing D-PAC1-EYFP named D-PAC1-CHO.The techniques including western blot, BRET and BiFC were used to detecthomologous dimerization of PAC1and D-PAC1. The techniques including fluorescence confocal microscope and immunofluorescence were used to detect theeffects of the N-terminal motif HSDCIF on the cell trafficking of PAC1. The effectsof the chemically synthesized HSDCIF on cell surface trafficking and homologousdimerization of PAC1were also detected.Results: The recombinant vectors inclucing PAC1-EYFP, D-PAC1-EYFP, PAC1-Rluc,D-PAC1-Rluc, PAC1-EYFP/N, PAC1-EYFP/C, D-PAC1-EYFP/N, D-PAC1-EYFP/Cwere constructed. And the CHO cell lines of stably expressing PAC1or D-PAC1named PAC1-CHO and D-PAC1-CHO were obtained. BRET, BiFC and western blotconfirmed the homologous dimerization of PAC1. And it was found that the deletionmutant D-PAC1failed to form dimers. Meanwhile the fluorescenc confocalmicroscopy showed that D-PAC1could not traffick to the cell membrane normallyand remained in the endoplasmic reticulum. And the chemically synthesized peptideHSDCIF (10nM) inhibited the proliferation of PAC1-CHO, interfered thedimerization of PAC1, and induced the intracellular translocation of the PAC1.Conclusion: B family of G protein coupled receptor PAC1can produce homologousdimerization, but D-PAC1missing N-terminal HSDCIF motif not noly failed to formdimers but also lost the ability to traffick to the cell membrane. These results indicatedthe important role of the N-terminal HSDCIF motif in the cell trafficking and thedimerization of PAC1. Meanwhile it was first found that the chemically synthesizedpeptide HSDCIF competitively inhibited PAC1homologous dimerizations andinduced the PAC1internalization. The mechanism of the dimerization and the celltrafficking of PAC1may lay the foundation for the drug development targeting PAC1.