The Construction And Application Of Tetracycline-inducibleexpression System For Class B G-protein Coupled Receptor PAC1

Objective: PAC1is the neuropeptide pituitary adenylate cyclase activatingpolypeptide (PACAP) preferring receptor, which belongs to class B G protein-coupledreceptors (GPCRs) family. PAC1mediates the most effects of PACAP asneurotransmitter, neuroregulator and neuroprotectant, while its high expression hasclose relationship with some physiological and pathological processes such asnerve-injury and tumor. Tet (tetracycline)-on gene expression systems is developedquickly in recent years. It becomes the most widely used system of regulating geneexpression in eukaryotes for its efficiency and strict switch function.For furtherunderstanding the functional role of PAC1, a cell line that expressed inducible PAC1was constructed to achieve theDoxycycline (Dox) dependent expression of PAC1inChinese hamster ovary(CHO)cell using the improved Tet-on Advanced System in thisresearch to investigate the correlation between the receptor expression level and itsconstitutive activity.Method: Firstly, the PAC1-EYFP fusion gene composed of PAC1gene and geneencoding EYFP(enhanced yellow fluorescent protein) was sub-cloned to thetetracycline response element pTRE-Tight vector to construct the recombinant vectorpEYFP-PAC1-EYFP by double enzyme digestion. Secondly, the tetracyclineregulation components including pTet-On advanced vector and the response elementpTRE-PAC1-EYFP vector were both introduced into CHO cells successively. Thepositive clones were screened with G418and hygromycin respectively. Thirdly, thecontrolled expression of PAC1-EYFP in CHO was induced by tetracycline analoguesDox in different concentrations and the corresponding levels of receptor PAC1-EYFPwere detected.Meanwhile the proliferative activities and the anti-apoptoticabilitiesagainst serum-withdraw induced apoptosis of the cells with different expression levels of PAC1-EYFP were detected along with some apoptosis relatedindexes, including the levels of the intracellular second messenger cAMP, the levelsof anti-apoptotic protein Bcl-2in the cells as well as the plasma apoptotic proteinCaspase-3activities.Results: With gene engineering principle and technology we constructedaeukaryoticrecombinant expression vector pTRE-PAC1-EYFP. The results of fluorescenceanalysis and westernblot showed that the cell strain displaying the Dox dependentexpression of PAC1-EYFP named PAC1-Tet-CHO was obtained. Moreover, inPAC1-Tet-CHO cells the expression of PAC1-EYFP was induced by Dox in adose-dependent manner. The inducible expression of PAC1-EYFP was stable aftersub-culturing for more than ten passages.It was shown that in normal culturecondition, the higher expressions of PAC1-EYFP confer the cells with the higherproliferative activities;while in serum-withdraw induced apoptosis without theactivation of the ligands for PAC1, the higher expression levels of PAC1-EYFPendowed the cells with the higher intracellular cAMP levels, the higher cellsviabilities and the stronger anti-apoptotic abilities as well as the higher anti-apoptoticprotein Bcl-2levels and the lowerapoptotic protein Caspase-3activitiesin the plasma.These results indicated that PAC1maybe have the ligand-independentanti-apoptoticconstitutive (basal) activities in an expression level dependent manner.Conclusion: In this study the controlled expression system ofPAC1was constructedand PAC1was found to confer the cells with expression level dependent proliferativeviabilities and the anti-apoptotic abilities in ligand independent manner for the firsttime, which indicated that PAC1may have the ligand independent constitutive (basal)activities. The research may not only help to understand the physiological orpathologicalroles of PAC1, but also help to offer a new theoretical basis and researchfoundation for the drug development using PAC1as a drug target.