Study on relationship between conserved motif of sphingosine 1-phosphate receptor type 1 and FTY720-induced internalization / 中国免疫学杂志

ABSTRACT

Objective:To study the relation between conserved motif of sphingosine 1-phosphate receptor type 1(S1P1) and FTY720-induced internalization.Methods:With HA-S1P1(WT)-Myc-EGFP-N1 fusion vector as template,HA-S1P1(R142N)-Myc-EGFP-N1 fusion vector was constructed by overlap PCR.The conserved ERY motif of wild type S1P1 was mutated into ENY.The recombinant vectors were confirmed by sequencing,then they were transfected into HEK293 cells by Polyfect.The transfected HEK293 cells were selected with G418.Cells were incubated for 3,6,12 hours in the absence or presence of 100 nmol/L FTY720,then S1P1 gene expression was analyzed by fluorescence microscopy.Results:Sequencing confirmed HA-S1P1(R142N)-Myc-EGFP-N1 vectors was successfully constructed.S1P1(WT) protein and S1P1(R142N) protein were expressed on the stably transfected-HEK293 cell surface.FTY720 induced S1P1(WT) internalization,but not S1P1(R142N).Conclusion:FTY720-induced S1P1 internalization is related with conserved ERY motif.