ANKYRIN BINDS TO 2 DISTINCT CYTOPLASMIC DOMAINS OF NA,K-ATPASE ALPHA-SUBUNIT
ANKYRIN BINDS TO 2 DISTINCT CYTOPLASMIC DOMAINS OF NA,K-ATPASE ALPHA-SUBUNIT
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DOI：
10.1073/pnas.91.8.2965
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发表时间：
1994-04-12
影响因子：
11.1
通讯作者：
MORROW, JS
中科院分区：
文献类型：
--
作者：
DEVARAJAN, P;SCARAMUZZINO, DA;MORROW, JS
锚蛋白已经成为一种普遍存在的蛋白质，将整合的膜转运蛋白如Na，K-ATP酶连接到基础血影蛋白细胞骨架。这种相互作用由Na，K-ATP酶的α亚基介导;然而，Na，K-ATP酶中锚蛋白结合位点的性质尚不清楚。作为确定这种相互作用机制的一个步骤，人红细胞血影蛋白的锚蛋白结合区和Na，K-ATP酶α亚基的五个推定的胞质结构域中的每一个已被制备为细菌中的重组融合蛋白，并在体外分析了它们与红细胞和肾锚蛋白（分别为Ank 1和Ank 3）的相互作用。Spectrin与Ank 1和Ank 3结合，正如预期的那样。固定在生物亲和柱上的两个Na，K-ATP酶结构域也与这两种锚蛋白特异性相互作用。这些ATP酶结构域由密码子140-290（结构域II）和345-784（结构域III）编码，其中结构域II显示最大的表观亲和力。结构域II中的序列在Na，K-ATP酶的物种和同种型之间高度保守，并且与H，K-ATP酶中的胞质结构域和Ca-ATP酶中的有限序列区域同源。相反，结构域II与其他锚蛋白结合蛋白如带3和Na+通道蛋白没有显著的同源性。这些结果确定了一个明确的功能，保守的，但以前不了解的Na，K-ATP酶的α亚基的区域，并建议锚蛋白与膜转运蛋白的相互作用可能涉及复杂的三级结构决定簇不容易推导出的一级序列。
Ankyrin has emerged as a ubiquitous protein linking integral membrane transport proteins such as Na, K-ATPase to an underlying spectrin cytoskeleton. This interaction is mediated by the alpha subunit of Na, K-ATPase; however, the nature of the ankyrin binding site in Na, K-ATPase is unknown. As a step to determine the mechanism of this interaction, the ankyrin binding region of human erythrocyte spectrin and each of five putative cytoplasmic domains of the Na, K-ATPase alpha subunit have been prepared as recombinant fusion proteins in bacteria and analyzed for their interaction with erythrocyte and kidney ankyrin (Ank1 and Ank3, respectively) in vitro. Spectrin binds both Ank1 and Ank3 avidly, as expected. Two of the Na, K-ATPase domains, immobilized on a bioaffinity column, also interact specifically with both of these ankyrins. These ATPase domains are encoded by codons 140-290 (domain II) and 345-784 (domain III), with domain II displaying the greatest apparent affinity. Sequences in domain II are highly conserved between species and isoforms of Na, K-ATPase and are homologous to a cytoplasmic domain in H, K-ATPase and to a limited region of sequence in Ca-ATPase. Conversely, domain II shares no significant homology with other ankyrin binding proteins such as band 3 and Na+-channel proteins. These results identify a clear function for a conserved but previously not understood region of the alpha subunit of Na, K-ATPase and suggest that the interaction of ankyrin with membrane transport proteins may involve complex tertiary structural determinants not easily deduced from the primary sequence.