Morgans C W, Kopito R R
Department of Biological Sciences, Stanford University, CA 94305-5020.
J Cell Sci. 1993 Aug;105 ( Pt 4):1137-42. doi: 10.1242/jcs.105.4.1137.
The 89 kDa NH2-terminal domain of erythrocyte ankyrin is composed almost entirely of 22 tandem repeats of a 33 amino acid sequence and constitutes the binding site for the cytoplasmic NH2-terminal domain of the erythrocyte anion exchanger, AE1. We have developed an assay to evaluate the in vivo interaction between a fragment of ankyrin corresponding to this domain (ANK90) and two non-erythroid anion exchangers, AE2 and AE3, that share considerable structural homology with AE1. Association was assessed by co-immunoprecipitation of ANK90-anion exchanger complexes from detergent extracts of cells cotransfected with plasmids encoding the ankyrin fragment and the anion exchanger or mutants thereof. ANK90 was co-immunoprecipitated with AE1 but not with an AE1 deletion mutant lacking the cytoplasmic NH2-terminal domain. Using this assay, we show that the brain anion exchanger AE3, but not the closely related homologue, AE2, is capable of binding to ankyrin.
红细胞锚蛋白89 kDa的氨基末端结构域几乎完全由33个氨基酸序列的22个串联重复序列组成，构成了红细胞阴离子交换蛋白AE1细胞质氨基末端结构域的结合位点。我们开发了一种检测方法，以评估对应于该结构域的锚蛋白片段（ANK90）与两种非红细胞阴离子交换蛋白AE2和AE3之间的体内相互作用，这两种蛋白与AE1具有相当大的结构同源性。通过从共转染了编码锚蛋白片段和阴离子交换蛋白或其突变体的质粒的细胞的去污剂提取物中对ANK90 - 阴离子交换蛋白复合物进行共免疫沉淀来评估结合情况。ANK90与AE1共免疫沉淀，但不与缺乏细胞质氨基末端结构域的AE1缺失突变体共免疫沉淀。使用该检测方法，我们发现脑阴离子交换蛋白AE3能够与锚蛋白结合，而与之密切相关的同源物AE2则不能。
