S1P1受体ERY保守基序突变对FTY720诱导其内化的影响

1型1-磷酸鞘氨醇受体(S1P1)是淋巴细胞表面的重要受体。为研究S1P1中ERY保守基序与FTY720诱导其内化的关系,以HA-S1P1-Myc-EGFP-N1为模板,应用重叠PCR的方法,将S1P1第142位的Arg(R)突变为Asn(N),连同N端HA标签一起,克隆入pcDNA3.1(+)真核表达载体。同时PCR扩增野生型S1P1,克隆入pcDNA3.1(+)中。限制性酶切消化、PCR和测序鉴定后,经Polyfect转染入HEK293细胞。G418筛选出稳定细胞株。100 nmol/L FTY720处理12 h后,抗HA-PE抗体染色,用流式细胞仪检测S1P1在HEK293细胞上的表达情况。结果显示HA-S1P1(WT)-pcDNA3.1(+)和HA-S1P1(R142N)-pcDNA3.1(+)载体被成功构建,HA-S1P1(WT)和HA-S1P1(R142N)蛋白表达在HEK293稳定细胞株的表面。FTY720能诱导HA-S1P1(WT)内化,但不能诱导HA-S1P1(R142N)内化。提示FTY720诱导S1P1内化与ERY保守基序有关。

Mutation of the conserved ERY motif of sphingosine 1-phosphate receptor type 1 affects FTY720-induced internalization

Sphingosine 1-phosphate receptor type 1(S1P1) is the dominant receptor on lymphocytes.In order to study the relation between the conserved ERY motif of S1P1 and FTY720-induced internalization,the gene encoding R142N mutant S1P1 was amplified from HA-S1P1-Myc-EGFP-N1 by overlap-PCR and the wild type S1P1 gene was amplified by PCR.These genes including N-terminal hemagglutinin(HA)-tag were cloned into pcDNA3.1(+) vector.The recombinant vectors were confirmed by restriction enzyme digestion,PCR and sequencing,then they were transfected into HEK293 cells by Polyfect.The transfected HEK293 cells were selected with G418.Cells were incubated for 12 hours in the absence and presence of 100 nM FTY720,then were stained by anti-HA-PE and the S1P1 gene expression was analyzed by flow cytometry. The results of the restriction enzyme digestion,PCR and sequencing confirmed HA-S1P1(WT)-pcDNA3.1(+) and HA-S1P1(R142N)-pcDNA3.1(+) vectors were successfully constructed.HA-S1P1(WT) protein and HA-S1P1(R142N) protein were expressed on the stably transfected HEK293 cell surface.FTY720 induced HA-S1P1(WT) internalization,but not HA-S1P1(R142N).FTY720-induced S1P1 internalization is related with ERY motif.