Sippel C J, Shen T, Perlmutter D H
Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 1996 Dec 20;271(51):33095-104. doi: 10.1074/jbc.271.51.33095.
Recent studies of the rat liver canalicular bile acid transporter/ecto-ATPase/cell CAM 105 (CBATP), a member of the carcinoembryonic antigen (CEA) supergene family, indicate that it is a multifunctional protein possessing bile acid efflux, ecto-ATPase, and intercellular aggregating properties. Cheung et al. (Cheung, P. H., Luo, W., Qiu, Y., Zhang, K. E., Millron, P., Lin, S. H. (1993) J. Biol. Chem. 268, 24303-24310) have shown that the amino-terminal Ig V-like domain of this protein is required for its aggregating properties, much like the homologous amino-terminal domain of CEA is required for its aggregating properties. The amino-terminal domains of both CBATP and CEA include a consensus ATPase sequence. Site-directed mutagenesis within this ATPase consensus sequence completely eliminates the ecto-ATPase activity of CBATP (Sippel, C. J., McCollum, M., Perlmutter, D. H. (1994) J. Biol. Chem. 269, 2820-2826). In this study we examined the possibility that it is this ATPase consensus sequence which is required for the cell aggregating properties of CBATP and CEA and whether there is a relationship between ATPase, aggregating, and bile acid efflux activities. For this we used a baculovirus vector to express in Sf9 cells wild type as well as mutant and chimeric CBATP and CEA molecules. The results indicate that Arg-98 in the ATPase consensus sequence of CBATP and the corresponding residue of CEA are essential for the aggregating properties of these molecules. Moreover Arg-98 is essential for CBATP to interact with itself, CEA to interact with itself, and CBATP to interact with CEA. However, the role of Arg-98 in aggregation is distinct from its role in ecto-ATPase activity and the aggregating properties cannot be attributed to a change in ATP metabolism in the pericellular milieu.
大鼠肝胆小管胆汁酸转运体/胞外ATP酶/细胞黏附分子105（CBATP）是癌胚抗原（CEA）超基因家族的成员，近期对其研究表明，它是一种多功能蛋白，具有胆汁酸外排、胞外ATP酶和细胞间聚集特性。张等人（Cheung, P. H., Luo, W., Qiu, Y., Zhang, K. E., Millron, P., Lin, S. H. (1993) J. Biol. Chem. 268, 24303 - 24310）已表明，该蛋白的氨基末端Ig V样结构域对其聚集特性是必需的，这与CEA的同源氨基末端结构域对其聚集特性的要求非常相似。CBATP和CEA的氨基末端结构域都包含一个共有ATP酶序列。在该ATP酶共有序列内进行的定点诱变完全消除了CBATP的胞外ATP酶活性（Sippel, C. J., McCollum, M., Perlmutter, D. H. (1994) J. Biol. Chem. 269, 2820 - 2826）。在本研究中，我们探讨了是否正是这个ATP酶共有序列是CBATP和CEA细胞聚集特性所必需的，以及ATP酶、聚集和胆汁酸外排活性之间是否存在关联。为此，我们使用杆状病毒载体在Sf9细胞中表达野生型以及突变型和嵌合型CBATP和CEA分子。结果表明，CBATP的ATP酶共有序列中的精氨酸 - 98以及CEA的相应残基对这些分子的聚集特性至关重要。此外，精氨酸 - 98对于CBATP自身相互作用、CEA自身相互作用以及CBATP与CEA相互作用都是必需的。然而，精氨酸 - 98在聚集中的作用与其在胞外ATP酶活性中的作用不同，并且聚集特性不能归因于细胞周围环境中ATP代谢的变化。
