The Construction And Function Identification Of Stable Transfection C5aR Cell Line Molt-4

BackgroundG protein-coupled receptor is a member of the superfamily of proteins in nature, which essential expresses ranging from yeast to the apparatus in humans,encoded by more than 1% of human genes and they are targets of 30% of all available pharmaceutical drugs. GPCR is a polypeptide chain composition of 400 ~ 600 amino acid, this polypeptide chain has seven transmembrane domains (transmembrane domain, TMD). The complement C5a anaphylatoxin receptor (C5aR;CD88) is a member of the GPCR, which isα-helix transmembrane receptor, At the extracellular face of transmembrane the N-terminal containing N-glycosylation sites, the phosphorylation sites is on the C-terminal intracellular face, Seven middle-peptide formed the seven transmembrane domain, which composition of six hydrophilic amino acid the ring linking process extracellular and intracellular. Extracellular three called e1, e2 and e3; and intracellular called i1, i2, and i3, A disulfide bond connecting the e1 and e2 by two conservative Cys. TMD is the conservative elements which a great similarity in different GPCR, and the two extreme domain and the Central region have variation of amino acids .C5aR expression was originally described on myeloid cells including neutrophils, eosinophils, basophils, and monocytes.More recently, C5aR has also been found expressed on a variety of nonmyeloid cells in many organs, especially in the lung and liver. To further research the bioactivities of C5aR, we established the anaphylatoxin C5aR expressing cell model is need.PurposesThe C5aR cDNA was reversely transcripted from extracted total RNA of human neutrophils and were inserted into the plasmid pcDNA4.0. The single colony of transfected MOLT-4 cells was picked out after Zeocin selecting and subcloning. It provided an excellent model for further research of the downstream signaling events and the biological effects mediated by C5aR.MethodsHuman C5aR gene was amplified by RT-PCR, cloned into pGEM-T Easy vector, and confirmed by sequence analysis. Then the gene was subcloned into expression vector pcDNA4.0 and transfected into MOLT-4 cell line . Finally, Zeocin from 100 to 500μg/ml was used to select the stably transfcted cell line. The single colony of transfected MOLT-4 cells was picked out after subcloning and selecting. RT-PCR and flow cytometry results showed that C5aR could express on the MOLT-4/C5aR cell line . We had established the anaphylatoxin C5aR expressing cell model MOLT-4. C5aR and Gα16 in human neutrophil and Molt-4/C5aR were analyzed by RT-PCR. To test the functional activity of our cell line, we examined the ability of C5a to stimulate the ERK1/2 and PKB/AKT signaling pathways in MOLT-4/C5aR cells. The cells were stimulated by recombined human C5a at dose of 0.002~2000 nM respectively. C5a could induce Ca++ influx in MOLT-4/C5aR cells .The chemotaxis was detected by lung lavement.ResultsThe agarose gel electrophoresis (AGE, 10 g/L) showed the size of the PCR products about 1.1 kp and 400 bp long respectively, which were definitely the same size as C5aR gene and Gα16. We established a C5aR expressing cell line on human MOLT-4 cells, to test the functional activity of our cell line, we examined the ability of C5a to stimulate the ERK1/2 and PKB/AKT signaling pathways in MOLT-4/C5aR cells. To test whether our cell model functionally worked well, the effects of C5a-C5aR interaction on Ca++ influx in MOLT-4/C5aR were detected. The chemotaxis was detected by lung lavement.ConclusionsC5aR expressed highly in neutrophils, macrophages and endothelial cells. C5aR is also expressed. Interaction of C5a-C5aR induces many signaling events which mediate the biological effects of target cells. The function of neutrophils is identified in many infection diseases. However, the isolation procedures of neutrophil are too complicated, time-consuming, and non-specific cell activation always happens. In addition,the survival time of neutrophil in vitro is very short. To solve this problem, we established a cell line which stably and functionally expressed C5aR. The expression of C5aR was detected by RT-PCR and flow cytometry; neutrophils and untransfectied MOLT-4 cells were used as positive and negative controls respectively. The results showed that C5aR expressed on MOLT-4/C5aR cell line but not on untransfected MOLT-4 cells.To further study the function of transfected cell line, C5a was ligated on MOLT-4/C5aR cell and the signaling events were detected. As we know, C5a-C5aR binding could induce the downstream signal transduction which is dependent on Gαi and Gα16. As the MOLT-4 cells involve the C5aR associated Gα16 subunit, it provides a good tool for our study. Western blotting assays showed that ERK1/2 and PKB/AKT phosphorylation could be induced by C5a in MOLT-4/C5aR cells, The chemotaxis was detected by lung lavement. Suggesting that C5a ligation could induce the activation of downstream signaling events in MOLT-4/C5aR cells, so transfected cell line provided a new means for studying neutrophil signaling transduction.