Peters L L, John K M, Lu F M, Eicher E M, Higgins A, Yialamas M, Turtzo L C, Otsuka A J, Lux S E
Division of Hematology/Oncology, Children's Hospital, Boston, Massachusetts 02115, USA.
J Cell Biol. 1995 Jul;130(2):313-30. doi: 10.1083/jcb.130.2.313.
We cloned a novel ankyrin, Ank3, from mouse kidney cDNA. The full-length transcript is predicted to encode a 214-kD protein containing an 89 kD, NH2 terminal "repeat" domain; a 65 kD, central "spectrin-binding" domain; and a 56 kD, COOH-terminal "regulatory" domain. The Ank3 gene maps to mouse Chromosome 10, approximately 36 cM from the centromere, a locus distinct from Ank1 and Ank2. Ank3 is the major kidney ankyrin. Multiple transcripts of approximately 7.5, 6.9, 6.3, 5.7, 5.1, and 4.6 kb are highly expressed in kidney where Ank1 and Ank2 mRNAs are barely detectable. The smaller mRNAs (< or = 6.3 kb) lack the entire repeat domain. These transcripts have a unique 5'untranslated region and NH2-terminal sequence and encode a predicted protein of 121 kD. Two small sequences of 21 and 18 amino acids are alternatively spliced at the junction of the repeat and spectrin-binding domains in the larger (> or = 6.9 kb) RNAs. Alternative splicing of a 588 bp sequence (corresponding to a 21.5-kD acidic amino acid sequence) within the regulatory domain also occurs. Ank3 is much more widely expressed than previously described ankyrins. By Northern hybridization or immunocytochemistry, it is present in most epithelial cells, in neuronal axons, in muscle cells, and in megakaryocytes/platelets, macrophages, and the interstitial cells of Leydig (testis). On immunoblots, an antibody raised to a unique regions of the regulatory domain detects multiple Ank3 isoforms in the kidney (215, 200, 170, 120, 105 kD) and in other tissues. The 215/200 kD and 120/105-kD kidney proteins are close to the sizes predicted for the 7.5/6.9- and 6.3/5.7-kb RNAs (with/without the 588-bp acidic insert). Interestingly, it appears that Ank3 exhibits a polarized distribution only in tissues that express the approximately 7.0-kb isoforms, the only isoforms in the kidney that contain the repeat domain. In tissues where smaller transcripts (< or = 6.3 kb) are expressed. Ank3 is diffusely distributed in some or all cells and may be associated with cytoplasmic structures. We conclude that Ank3 is a broadly distributed epithelial ankyrin and is the major ankyrin in the kidney and other tissues, where it plays an important role in the polarized distribution of many integral membrane proteins.
我们从小鼠肾脏cDNA中克隆出一种新型锚蛋白Ank3。全长转录本预计编码一个214-kD的蛋白质，该蛋白质包含一个89 kD的NH2末端“重复”结构域、一个65 kD的中央“血影蛋白结合”结构域和一个56 kD的COOH末端“调节”结构域。Ank3基因定位于小鼠第10号染色体，距着丝粒约36 cM，这是一个与Ank1和Ank2不同的位点。Ank3是肾脏中的主要锚蛋白。约7.5、6.9、6.3、5.7、5.1和4.6 kb的多种转录本在肾脏中高表达，而Ank1和Ank2的mRNA几乎检测不到。较小的mRNA（≤6.3 kb）缺乏整个重复结构域。这些转录本有一个独特的5'非翻译区和NH2末端序列，并编码一个预测的121 kD蛋白质。在较大（≥6.9 kb）的RNA中，21个和18个氨基酸的两个小序列在重复结构域和血影蛋白结合结构域的交界处进行可变剪接。调节结构域内一个588 bp序列（对应于一个21.5-kD酸性氨基酸序列）也发生可变剪接。Ank3的表达比先前描述的锚蛋白广泛得多。通过Northern杂交或免疫细胞化学方法发现，它存在于大多数上皮细胞、神经元轴突、肌肉细胞以及巨核细胞/血小板、巨噬细胞和睾丸间质细胞（睾丸）中。在免疫印迹中，针对调节结构域独特区域产生的抗体可检测到肾脏（215、200、170、120、105 kD）和其他组织中的多种Ank3异构体。215/200 kD和120/105-kD的肾脏蛋白接近7.5/6.9-和6.3/5.7-kb RNA预测的大小（有/无588-bp酸性插入片段）。有趣的是，似乎Ank3仅在表达约7.0-kb异构体的组织中呈现极化分布，这是肾脏中仅有的包含重复结构域的异构体。在表达较小转录本（≤6.3 kb）的组织中，Ank3在一些或所有细胞中呈弥散分布，可能与细胞质结构相关。我们得出结论，Ank3是一种广泛分布的上皮锚蛋白，是肾脏和其他组织中的主要锚蛋白，在许多整合膜蛋白的极化分布中起重要作用。
