重组PAC1-EC1(N)对表达不同PAC1变体的细胞系细胞活性的影响

Biological Effects of Recombinant PAC1-EC1(N) on the Viability of Cell Lines with Different PAC1 Isoforms

垂体腺苷酸环化酶激活多肽(pituitary adenylate cyclase-activating polypeptide,PACAP)特异受体PAC1(normal型)N端胞外域[简称PAC1-EC1(N)]具有调控PAC1活性的作用。为研究PAC1-EC1(N)对表达不同PAC1变体的细胞系活性的影响,利用基因工程技术获得C端带有6个his纯化标签的重组PAC1-EC1(N)。质谱检测、SDS-PAGE电泳和Western blot检测结果均显示成功表达和纯化目的蛋白PAC1-EC1(N)。Western blot检测确定兔眼角膜基质细胞、人神经母细胞瘤细胞SH-SY5Y、人前列腺癌细胞22RV1中分别表达一个或多个分子量不同的PAC1变体。重组PAC1-EC1(N)作用3株细胞,MTT方法检测细胞活性,结果显示:PAC1-EC1(N)有效促进只表达一个小分子PAC1变体的兔眼角膜基质细胞的增殖;但显著抑制只表达一个大分子PAC1变体的人神经母细胞瘤细胞SH-SY5Y的活性。对于表达3种大小不一的PAC1变体的人前列腺癌细胞22RV1,PAC1-EC1(N)对细胞的活性具有复杂的调控。显示PAC1-EC1(N)对表达PAC1变体数量不同,类型不同的细胞具有不同的生物活性的作用,提示了PAC1变体可能具有较复杂的自我调控机制。

PAC1 is a specific receptor for pituitary adenylate cyclase-activating polypeptide （PACAP）. The N terminal first extracellular region of the PAC1 （ PAC1-EC1 ） is involved in the regulation for the activation of PAC1. To study the effects of the EC1 domain of the PAC1 normal （N） isoform [ PAC1-EC1 （N）] on the cell lines expressing different PAC1 isoforms, the recombinant protein PAC1-EC1 （N） with 6-His-tag at the Cterminus was expressed in an engineered Escherichia coli strain and purified by Ni-NTA affinity chromatography. Mass spectrum, SDS-PAGE and Western blot were used to characterize the recombinant PAC1-EC1 （N）. Western blot were used to identify the expression pattern of PAC1 isoforms in three cell lines including rabbit corneal stromal cells, human neuroblastoma cells SH-SY5Y and human prostate cancer cells 22RV1. MTT assays were used to detect different biological effects of the recombinant PAC1-EC1 （N） on the cell lines with different PAC1 isoforms. The results showed that PAC1-EC1 （N） had significantly different biological effects on the cell lines with different PAC1 isoforms. PAC1-EC1 （N） stimulated the viability of rabbit corneal stromal ceils expressing only one PAC1 isoform with a smaller molecular weight, but inhibited the viability of human neuroblastoma cells SH-SY5Y expressing one type of PAC1 isoform with a larger molecular weight. Meanwhile, PAC1-EC1 （N） caused complex biological effects on human prostate cancer 22RV1 ceils with three types of PAC1 isoforms. These results showed that PAC1-EC1 （N） exerts different effects on the viabilities of the cells with different expression patterns of PAC1 isoforms, indicating that the PAC1 owns a complex self-regulation mechanism of activation.