ALTERNATIVELY SPLICED ISOFORMS OF THE PUTATIVE RENAL NA-K-CL COTRANSPORTER ARE DIFFERENTIALLY DISTRIBUTED WITHIN THE RABBIT KIDNEY
ALTERNATIVELY SPLICED ISOFORMS OF THE PUTATIVE RENAL NA-K-CL COTRANSPORTER ARE DIFFERENTIALLY DISTRIBUTED WITHIN THE RABBIT KIDNEY
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DOI：
10.1073/pnas.91.10.4544
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发表时间：
1994-05-10
影响因子：
11.1
通讯作者：
FORBUSH, B
中科院分区：
文献类型：
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作者：
PAYNE, JA;FORBUSH, B
我们使用源自 Na-K-Cl 协同转运蛋白分泌形式的 cDNA 探针来筛选兔肾皮质和髓质 cDNA 文库。从多个克隆中鉴定出 4750 个碱基的序列。该 DNA 编码含有 1099 个氨基酸的蛋白质，其长度与鲨鱼直肠腺分泌的 Na-K-Cl 协同转运蛋白有 61% 的相同性。根据氨基酸水病分析，我们预测这种假定的肾脏 Na-K-Cl 协同转运蛋白具有 12 个跨膜螺旋和较大的 N 端和 C 端细胞质区域。预测细胞外环上有两个 N 连接糖基化位点。蛋白激酶 A 调节的三个潜在位点位于 C 端胞质结构域。大多数分离的肾 cDNA 克隆在所有重叠区域都是相同的；然而，有一个 96 bp 的区域存在三种不同但同源的变体（A、B 和 F）。该分歧区域被鉴定为选择性剪接盒式外显子，因为鉴定出的克隆含有内含子DNA以及限制该区域的共有剪接受体位点。组织 Northern 印迹分析显示有一条大约 5.1 kb 的宽条带，这是肾脏特有的。使用在三个盒式外显子中的每一个上合成的反义寡核苷酸对皮质和髓质 mRNA 进行高严格 Northern blot 分析，结果表明同种型在肾脏内差异分布 - B 几乎完全在皮质中，F 几乎完全在髓质中，A 几乎均匀分布。
We have used cDNA probes derived from the secretory form of the Na-K-Cl cotransporter to screen both cortical and medullary rabbit kidney cDNA libraries. A sequence of 4750 bases was identified from multiple clones. The DNA encodes a protein containing 1099 amino acids, which is 61% identical over its length to the secretory Na-K-Cl cotransporter from shark rectal gland. From analysis of amino acid hydropathy, we predict that this putative renal Na-K-Cl cotransporter has 12 transmembrane helices and large N- and C-terminal cytoplasmic regions. Two sites for N-linked glycosylation are predicted on an extracellular loop. Three potential sites for modulation by protein kinase A are in the C-terminal cytoplasmic domain. Most of the isolated renal cDNA clones were identical over all regions of overlap; however, there was a 96-bp region for which there were three different but homologous variants (A, B, and F). This region of divergence was identified as an alternatively spliced cassette exon since clones were identified that contained intronic DNA as well as consensus splice acceptor sites that bounded the region. Tissue Northern blot analysis revealed a broad band at approximate to 5.1 kb that was unique to the kidney. High-stringency Northern blot analysis of cortical and medullary mRNA using antisense oligonucleotides synthesized over each of the three cassette exons revealed that the isoforms were differentially distributed within the kidney-B almost exclusively in cortex, F almost exclusively in medulla, and A about equally distributed.