Research On Aggregation Of Human Chemokine Receptor CXCR4

G-protein-coupled receptors?GPCRs?are a large family of seven-helices transmembrane proteins,which play key roles in signal transduction in various processes.Recently,dimerization and oligomerization have been demonstrated to be critical for many GPCRs to realize their functions.Therefore,better understanding on the structure,oligomerization and functions of GPCRs is expected to greatly facilitate rational design of drugs with increased efficacy and selectivity.Biophysical studies on GPCRs have therefore attracted enormous attention.CXCR4 is a member of GPCR A family,which is expressed in a wide variety of cell types.As natural ligand stromal cell-derived growth factor?SDF-1??,CXCR4 plays a key role in leukocyte trafficking,hematopoiesis,organs development and cancer metastases.More importantly,CXCR4 is also one of the principle co-receptors for human immunodeficiency virus type 1?HIV-1?,and thus are important therapeutic target.The diverse role of CXCR4 in physiology and disease underscore the importance of our better understanding of the biochemical or biophysical characteristics and regulatory mechanism of CXCR4 functions in living cells.The role of dimerization and oligomerization of G-protein coupled receptors?GPCRs?on their signal transduction is highly controversial.Delineating this issue can greatly facilitate rational drug design.With single-molecule imaging,we show that chemokine receptor CXCR4exists mainly as monomer in native mammalian living cells and forms dimers and higher-order oligomers at high expression level,such as in cancer cells.Chemotaxis tests demonstrate that the signal transduction activity of CXCR4 does not only depend on its expression level,but also indicating a close relation to the oligomeric status of CXCR4.Moreover,binding ligands can effectively up-regulate or down-regulate the oligomeric level of CXCR4,which suggests that binding ligands may realize their pivotal roles by regulating the oligomeric status of CXCR4rather than simply inducing conformational changes.We presented two methods to express and produce CXCR4 by mammalian expression system and insects system.we found that the CXCR4 expressing in insect cells showed oligomers and was insoluble in aqueous solution,therefore,it is very difficult to purify.We constructed the stably expressing CXCR4-His₁₀,CXCR4-EGFP-His₁₀ and CXCR4-mCherry-His₁₀ T-Rex293 cells.With the large scale culture stably expressing CXCR4 cells,we found that density of CXCR4 decreased with the number of passage cell increasing.We failed to purify CXCR4 because it is extremely hydrophobic and it showed the low efficiency of binding to nickel column.In the purification of CXCR4-EGFP-His₁₀ and CXCR4-mCherry-His₁₀,we found that the efficiency of the binding to nickel column was higher.In vitro,the K_D values of SDF-1?binding to CXCR4-EGFP-His₁₀ and CXCR4-mCherry-His₁₀ were respectively 1.17?M and 1.7?M.In the studies of aggregation of CXCR4 in solution,we found the CXCR4-EGFP and CXCR4-mCherry in aqueous solution can not produce FRET signal.So by detecting oligomeric status of CXCR4-EGFP with single molecule imaging,we found that oligomeric status of CXCR4-EGFP in 10 nM,10 pM,10 fM concentrations no change with different surfactants.Endogenous ligand SDF-1?can induce an increase of CXCR4 oligomeric status with the concentration of 10 nM,while with 10 pM and 10 fM,the ligand SDF-1?has no effect on the aggregation status.AMD3100 and reverse agonist TC14012 have no effect on the aggregation status of CXCR4.SDS decreased the oligomers of CXCR4.These experimental data shows that aggregation of CXCR4 and detergent forms a relatively stable complex.Using PTX to block the signal transduction,the effect of ligand SDF-1?on CXCR4aggregation with and without PTX are different obviously.The above explanation is that CXCR4 is closely related to the aggregation staus and signal transduction.The aggregation status of CXCR4 relate to signal transduction.The stimulation of agonist to CXCR4internalization and the use of CHZ inhibit the internalization found that there are obvious differences in the aggregation status,which indicates the existence of correlation with internalization and dimerization.In this research oligomeric status of CXCR4 correlated to signal transduction and diffusion,the diffusion coefficient of monomers is higher than that of dimers,and that of polymers is lower than that of dimers.When SDF-1?stimulated CXCR4,diffusion coefficient of CXCR4become to slow.The diffusion coefficient of monomer was the slowest,and the dimers and polymers were basically unchanged.When PTX blocks signal transduction,ligand,ligand make diffusion coefficient of CXCR4 unchangeable.It shows that oligomeric status of CXCR4 relate to diffuse and signal transduction.