β_1整合素基因缺失对G_i蛋白分布的影响

β_1 integrin deletion altered G_i distribution

目的 了解跨膜蛋白 β1整合素对信号分子G蛋白细胞内构型的影响 ,为进一步研究 β1整合素在信号传导中的作用打下基础 .方法 采用荧光双标记及重叠合显微镜分析技术 ,观察野生型胚胎干细胞系 (D3细胞 ) ,β1整合素基因敲除胚胎干细胞 (G2 0 1细胞 ) ,β1整合素基因敲除后再植入胚胎干细胞 (G2 0 1N)及小鼠胚胎心肌细胞中多种G蛋白 (Gαs,Gαi1-3 ,Gαo,Gαi/o/t/z)细胞内构型特点和差异 .结果 Gαs,Gαo,Gαi/o/t/z在上述几种细胞系中均呈弥漫性分布 ,未能发现明显差异 ,与此不同的是Gαi1-3 在D3细胞源性心肌细胞内呈网状分布 ,在非心肌细胞内呈线条状分布 ,而在G2 0 1细胞中Gi 上述特异性构型被打乱 ,呈近似于弥漫样分布 ,值得注意的是在β1整合素基因重新恢复的G2 0 1N细胞Gi构型又呈网样或线条样分布 ,与野生型胚胎干细胞中Gi 的构型相一致 .结论 β1整合素能够影响信号分子Gi蛋白的细胞内定位 ,推测β1整合素可通过改变Gi 的分布而影响Gi

AIM To investigate the relationship between β 1 integrin and subcellular distribution of signaling molecule G protein. METHODS Immunofluorecence staining and de convolution technique were used to observe the distribution pattern of G pro tein subunits (G αs , G αi1-3 , G αo , G αi/o/t/z ) in wide type embryonic stem (ES) cell line (D3 cells), β 1 integrin deficient ES cells (G201 cells), β 1 integrin rescue ES cell line (G201N) and cardiomyocytes from murine embryos. RESULTS G αs , G αo and G αi/o/t/z proteins were found to be relatively homogeneously distributed in all cell lines. Significant differences became evident in the G αi subunit pattern between D3 and G201 cells. A network like pattern of G αi1-3 was observed in D3 cells derived cardiomyocytes and a linear like pattern in non cardiomyocytes, however these characteristic distributions were lost in β 1 integrin (-/-) cells and G αi1-3 displayed an almost homogeneously distribution very similar to the Gs. Moreover, G i changed to its normal pattern in β 1 integrin rescued G201N cells. CONCLUSION β 1 integrin deletion altered intracellular distribution of G i protein and it implied that signal transduction mediated by G i protein could be also influenced by β 1 integrin deficiency.