Mapping the membrane topology and extracellular ligand binding domains of the retinol binding protein receptor
Mapping the membrane topology and extracellular ligand binding domains of the retinol binding protein receptor
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DOI：
10.1021/bi8002082
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发表时间：
2008-05-13
期刊：
影响因子：
2.9
通讯作者：
Sun, Hui
中科院分区：
文献类型：
--
作者：
Kawaguchi, Riki;Yu, Jiarnei;Sun, Hui
STRA6是一种多跨膜结构域蛋白，与其他已知功能蛋白不同源。它作为血浆视黄醇结合蛋白（RBP）的高亲和力受体，并介导维生素A-RBP复合物对维生素A的细胞摄取。与维生素A的多种作用和STRA6广泛的组织表达模式相一致，STRA6的突变与人类严重的病理表型相关。STRA6生化功能的结构基础尚不清楚。尽管计算机程序预测了STRA6的I - I跨膜结构域，但其拓扑结构从未进行过实验研究。阐明STRA6的跨膜拓扑结构对于理解其结构和功能至关重要。通过将表位标签插入STRA6的所有可能的细胞外和细胞内结构域，我们系统地分析了每个标签在活细胞表面的可及性，每个标签在渗透细胞中的可及性，以及每个标签对RBP结合和STRA6介导的维生素A从维生素A-RBP复合物中摄取的影响。此外，我们采用一种新的赖氨酸接近技术，结合细胞表面生物素化和串联亲和纯化，研究了表位标记法未显示的蛋白质区域。这些研究不仅揭示了STRA6的胞外、跨膜和胞内结构域，而且还揭示了STRA6在RBP结合中的胞外区域。
STRA6 is a multitransmembrane domain protein not homologous to any other proteins with known function. It functions as the high-affinity receptor for plasma retinol binding protein (RBP) and mediates cellular uptake of vitamin A from the vitamin A-RBP complex. Consistent with the diverse roles of vitamin A and the wide tissue expression pattern of STRA6, mutations in STRA6 are associated with severe pathological phenotypes in humans. The structural basis for STRA6's biochemical function is unknown. Although computer programs predict I I transmembrane domains for STRA6, its topology has never been studied experimentally. Elucidating the transmembrane topology of STRA6 is critical for understanding its structure and function. By inserting an epitope tag into all possible extracellular and intracellular domains of STRA6, we systematically analyzed the accessibility of each tag on the surface of live cells, the accessibility of each tag in permeabilized cells, and the effect of each tag on RBP binding and STRA6-mediated vitamin A uptake from the vitamin A-RBP complex. In addition, we used a new lysine accessibility technique combining cell-surface biotinylation and tandem-affinity purification to study a region of the protein not revealed by the epitope tagging method. These studies not only revealed STRA6's extracellular, transmembrane, and intracellular domains but also implicated extracellular regions of STRA6 in RBP binding.