ten Dam G B, Poels L G, Wieringa B
Department of Cell Biology and Histology, Faculty of Medical Sciences, University of Nijmegen, The Netherlands.
Mol Biol Rep. 1998 Nov;25(4):197-204. doi: 10.1023/a:1006817322524.
We have designed a new cell surface expression plasmid to study the structural and membrane-topological requirements for functioning of different isoforms of CD45, a leucocyte specific member of the protein tyrosine phosphatase (PTPase) family of proteins. Use of this vector in cell transfection experiments enabled us to produce multiple CD45 isoforms (ABC, B, Null), with their extracellular segment intact, and the entire membrane spanning and intracellular C-terminal domain replaced by a GPI-membrane-anchor and VSV-tag. Our strategy facilitated the identification and analysis of chimeric proteins and selection of cell clones from low transfection efficiency experiments. We demonstrate here that simple expression of GPI-anchored CD45 isoforms on transfected Cos-1 cells does not facilitate binding to CD22+ lymphoid cells. This suggests that not only the mere presence of CD45 extracellular domains but also their assembly into higher order structures at the cell surface, is necessary in order to engage in the recognition and/or signalling processes normally used by B- and T-cells.
我们设计了一种新的细胞表面表达质粒，用于研究蛋白质酪氨酸磷酸酶（PTPase）家族中白细胞特异性成员CD45不同异构体发挥功能所需的结构和膜拓扑学要求。在细胞转染实验中使用该载体，使我们能够产生多种CD45异构体（ABC、B、Null），其细胞外区段完整，整个跨膜和细胞内C末端结构域被糖基磷脂酰肌醇（GPI）膜锚和水疱性口炎病毒（VSV）标签取代。我们的策略有助于嵌合蛋白的鉴定和分析，以及从低转染效率实验中选择细胞克隆。我们在此证明，在转染的Cos-1细胞上简单表达GPI锚定的CD45异构体并不能促进与CD22 + 淋巴细胞的结合。这表明，为了参与B细胞和T细胞通常使用的识别和/或信号传导过程，不仅需要CD45细胞外结构域的存在，还需要它们在细胞表面组装成更高阶结构。
