Devarajan P, Scaramuzzino D A, Morrow J S
Department of Pediatrics, Yale University School of Medicine, New Haven, CT 06510.
Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):2965-9. doi: 10.1073/pnas.91.8.2965.
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.
锚蛋白已成为一种普遍存在的蛋白质，它将诸如钠钾ATP酶等整合膜转运蛋白与底层的血影蛋白细胞骨架相连。这种相互作用由钠钾ATP酶的α亚基介导；然而，钠钾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，人类红细胞血影蛋白的锚蛋白结合区域以及钠钾ATP酶α亚基的五个假定细胞质结构域已在细菌中制备为重组融合蛋白，并在体外分析了它们与红细胞和肾脏锚蛋白（分别为Ank1和Ank3）的相互作用。正如预期的那样，血影蛋白与Ank1和Ank3都能强烈结合。固定在生物亲和柱上的两个钠钾ATP酶结构域也与这两种锚蛋白发生特异性相互作用。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表现出最大的表观亲和力。结构域II中的序列在钠钾ATP酶的不同物种和同工型之间高度保守，并且与氢钾ATP酶中的一个细胞质结构域以及钙ATP酶中的一个有限序列区域同源。相反，结构域II与其他锚蛋白结合蛋白如带3和钠通道蛋白没有显著的同源性。这些结果确定了钠钾ATP酶α亚基中一个保守但以前未被理解的区域的明确功能，并表明锚蛋白与膜转运蛋白的相互作用可能涉及不容易从一级序列推断出来的复杂三级结构决定因素。
