Exploration On The Biological Role Of The Free Cysteine In The N-terminal Extracellular Domain Of Class B GPCR VPAC1

Objective: VPAC1 is class B G protein-coupled receptors(GPCR) shared by pituitary adenylate cyclase activating polypeptide(PACAP) and vasoactive intestinal peptide(VIP). The first cysteine(Cys) 37 located in the N-terminal extracellular domain of VPAC1 is a free Cys which has not been involved in the formation of conserved intracellular disulfide bonds. In order to investigate the biological role of this Cys in VPAC1, in this study the N-terminal first Cys of VPAC1 was mutated to Ala, and the wild-type VPAC1 and Cys/Ala mutant(M-VPAC1) were expressed as fusion proteins with enhanced yellow fluorescent protein(EYFP) respectively in Chinese hamster ovary(CHO) cells. The biological role of Cys37 in the function of VPAC1 was explored by comparing the anti-apoptotic ability mediated by VPAC1 with that by M-VPAC1.Method: VPAC1 Cys37/Ala mutation gene named M-VPAC1 was obtained by gene mutations technology, then recombinant vectors expressing the wild-type VPAC1 and Cys/Ala mutant(M-VPAC1) as fusion proteins with EYFP were constructed using recombinant gene technology. Western blotting and quantitative fluorescence assay were used firstly to determine the equal expression levels of VPAC1-EYFP and M-VPAC1-EYFP in CHO cells. Confocal fluorescence microscopy was used to detect the trafficking and the internalization of the receptors in the cells with 0.01 n M VIP(VIP+) or without VIP(VIP-). Using camptothecin(CPT) induced apoptosis model, with the incubation of 0.01 n M VIP, the remaining cell viabilities, the level of intracellular anti-apoptotic protein Bcl-2 and the activity of apoptosis protein Caspase 3 were assayed for the detection of the anti-CPT-induced apoptosis difference between the cells expressing wild-type VPAC1 and the cells expressing mutation M-VPAC1. TUNEL dye was used to visualize the difference of anti-CPT-induced apoptosis activities between VPAC1-EYFP-CHO and M-VPAC1-EYFP-CHO. Pathway inhibitors such as H89(c AMP signaling pathway inhibitor), Nystatin(caveolae inhibitors), NAC(disulfide bond depolymerization agent), XAV939(Wnt / β-catenin signaling pathway inhibitor) and 2-BP(2-bromopalmitate, palmitoylation inhibitor) were used for the exploration the anti-apoptotic mechanisms which Cys37 was involved. Furthermore, the recombinant expression vectors VPAC1-Rluc and M-VPAC1-Rluc were also constructed for the Top flash assay, which was used to determine whether the β-catenin pathway was involved.Results: The recombinant vectors including VPAC1-EYFP, M-VPAC1-EYFP, VPAC1-Rluc and M-VPAC1-Rluc were constructed and identified. And VPAC1-EYFP-CHO cells and M-VPAC1-EYFP-CHO cells with stable high expression of recombinant EYFP fusion receptors were successfully screened. Western blotting and quantitative fluorescence assay confirmed the equal high expression levels of VPAC1-EYFP and MVPAC1-EYFP in CHO cells. Confocal microscope analysis showed that both VPAC1-EYFP and M-VPAC1-EYFP trafficked to the cell membrane without VIP, but with 0.01 n M VIP, VPAC1-EYFP translocated into the nucleus significantly different from the extranuclear location of M-VPAC1-EYFP after the internalization, indicating that Cys37 was involved in the ligand-dependent nuclear translocation of VPAC1. Meanwhile VPAC1-EYFP-CHO cells displayed more effective anti-apoptotic activity than MVPAC1-EYFP-CHO cells against CPT-induced-apoptosis associated with higher remaining cell viabilities, higher anti-apoptotic protein Bcl-2 level and lower Caspase 3 activity, which was significantly inhibited by c AMP-PKA pathway inhibitor H89 and caveolae inhibitor nystatin, but was not involved byβ-catenin pathway and disulfide bone. These results showed that Cys37 played a key role in the formation of the antiapoptotic activity mediated by VPAC1. Further bioinformatics analysis on the predicted modification on the N-terminal Cys37 of VPAC1 showed that Cys37 had high possibility of being submitted to palmitoylation. And the inhibitory effects of palmitoylation inhibitor 2-BP on the ligand-dependent nuclear translocation of VPAC1 and the antiCPT-induced-apoptotic activity induced by VPAC1, which Cys37 was involved in. These results supported Cys37 was involved in the activity of VPAC1 by palmitoylation.Conclusion: This study indicated that the first Cys in the N-terminal extracellular domain of VPAC1 is involved in anti-CPT-induced-apoptotic activity mediated by VPAC1 due to the possible palmitoylation of Cys37, which promotes the cavoelae mediating liganddependent nuclear translocation of VPAC1 and the c AMP pathway signal endowing the cells with anti-apoptotic activity.