Research On Aggregation Of Human Chemokine Receptor CCR3

G protein-coupled receptors?GPCRs?are one class of membrane protein receptors having seven transmembrane helices.For a long time,GPCRs are considered to function as a monomer,but recent researches suggest that,GPCRs can form dimers or oligomers,and dimeric form plays a key role in its cross membrane signaling processes.At present,whether the aggregation of GPCRs is a general phenomenon,GPCRs aggregation function and its biological significance is still unclear,and the human chemokine receptor type 3?CCR3?aggregation has not been reported.In this paper,we studied CCR3 aggregation phenomenon in vitro and vivo.Firstly,we constructed CCR3-EGFP fluorescent protein expression vector,then the PEI transfection of mammalian cells were optimized,the optimal transient transfection conditions was:using HEK293 cells,cell density is 80%,the 6-well cell culture plate transfected DNA was 4?g?2?g/mL?per well,PEI and DNA mass ratio was 3:1,cells were harvested 48h after transfection.The cell transfection efficiency calculated was about 60%under these conditions.Then we constructed the CCR3-ECFP,CCR3-EYFP fluorescent protein expression vector,and the two recombinant plasmids were transfected and expressed in HEK293 cells.Confocal fluorescence microscopy analysis showed that,CCR3 fluorescent fusion protein uniformly distributed in the cytoplasm and cell membrane.FRET analysis showed that the fluorescence resonance energy transfer did occur between CCR3-ECFP and CCR3-EYFP,it proved the aggregation phenomenon of CCR3 in living cells,when the receptor donor ratio was about 0.6,the FRET ratio got half of the maximum.Then we also examined the impact of CCR3 agonists CCL11 and CCL24,and neutral agonists SB297006 and SB328437 on the aggregation process.The study found that different types of agonists,the agonists concentration and duration of stimulation could not significantly change the aggregation process of CCR3 in living cells levels,which suggested that there is no necessary contact between CCR3 aggregation and ligand binding.We also enlarge cultured the T-REx-293 cell line which stably transfected human CCR3,by using Ni affinity chromatography and size exclusion chromatography we obtained high purity CCR3 protein.Then Alexa Fluor 488 and Alexa Fluor 647 dyes were used to label the N-terminal of purified CCR3 respectively,then the CCR3 aggregation was detected in surfactant solution.We also detected the aggregation of fluorescently labeled CCR3 in surfactant solution.After FRET analysis we obtained kinetic parameters of the aggregation process,the value is 3.96×10^-4 s^-1.In this paper,we studied and proved the human chemokine receptor CCR3 aggregation both in the level of living cells and in vitro surfactant environment,and the aggregation phenomena was preliminary analyzed,thus providing important experimental data for the clearer understanding of the mechanism of GPCRs aggregation and their biological significance,this study also provides an important theoretical basis for further drug design and related disease treatment.