Chien M L, Foster J L, Douglas J L, Garcia J V
Department of Virology and Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
J Virol. 1997 Jun;71(6):4564-70. doi: 10.1128/JVI.71.6.4564-4570.1997.
The amphotropic murine leukemia virus (MuLV) can infect cells from a number of mammals, including humans, via its specific receptor. Basic knowledge of amphotropic MuLV receptor expression is likely to be useful in the development and improvement of gene therapy protocols based on amphotropic-pseudotyped vectors. To investigate the expression of the human receptor for the amphotropic MuLV (GLVR-2, newly termed Pit2), we determined its mRNA levels in several cell lines and found them to vary significantly. Induction of increased levels of mRNA after removal of phosphate from the media was observed in two osteosarcoma cell lines. The increase in GLVR-2 mRNA resulted in a concomitant rise in the levels of a 71-kDa protein specifically recognized by affinity-purified antibodies against GLVR-2. Using these antibodies, we were able to confirm the intracellular topology of the large hydrophilic domain between the proposed sixth and seventh transmembrane domains of the GLVR-2 protein. This assignment is in agreement with the fourth extracellular loop being outside the cell, consistent with the proposal that the fourth extracellular loop of GLVR-2 contains the envelope binding site.
嗜双性小鼠白血病病毒(MuLV)可通过其特异性受体感染包括人类在内的多种哺乳动物的细胞。嗜双性MuLV受体表达的基础知识可能有助于基于嗜双性假型载体的基因治疗方案的开发和改进。为了研究嗜双性MuLV的人类受体(GLVR-2,新命名为Pit2)的表达,我们测定了几种细胞系中的mRNA水平,发现它们有显著差异。在两种骨肉瘤细胞系中观察到,从培养基中去除磷酸盐后,mRNA水平升高。GLVR-2 mRNA的增加导致一种71 kDa蛋白的水平随之升高,该蛋白可被抗GLVR-2的亲和纯化抗体特异性识别。利用这些抗体,我们能够确定GLVR-2蛋白假定的第六和第七跨膜结构域之间的大亲水结构域的细胞内拓扑结构。这一结果与第四细胞外环位于细胞外一致,这与GLVR-2的第四细胞外环包含包膜结合位点的提议相符。