CLONING, EXPRESSION, AND IN-SITU LOCALIZATION OF RAT INTESTINAL CGMP-DEPENDENT PROTEIN-KINASE-II
CLONING, EXPRESSION, AND IN-SITU LOCALIZATION OF RAT INTESTINAL CGMP-DEPENDENT PROTEIN-KINASE-II
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DOI：
10.1073/pnas.91.20.9426
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发表时间：
1994-09-27
影响因子：
11.1
通讯作者：
WALTER, U
中科院分区：
文献类型：
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作者：
JARCHAU, T;HAUSLER, C;WALTER, U
使用逆转录酶PCR和编码已知的cGMP依赖性蛋白激酶的两种保守分子的寡核苷酸引物，随后通过筛选大鼠肠道cDNA文库，从大鼠肠道中克隆了膜相关cGMP依赖性蛋白激酶（cGK II）的cDNA。全长克隆编码761个氨基酸的蛋白质，估计大小为87 kDa。在该克隆的衍生氨基酸序列中发现了来自纯化的猪肠粘膜cGK II的8个肽的序列，鉴定其为大鼠肠cGK II。系统发育分析表明，大鼠肠道CGK II是哺乳动物的CGK I比果蝇DG 1基因产物和最近克隆的小鼠脑CGK II亚型最密切相关。与其他几种cGK序列一样，cGK II的序列含有亮氨酸/异亮氨酸七肽重复序列，该重复序列与cGK I的二聚体形成有关。在HEK 293细胞中表达cGK II cDNA，然后进行亚细胞分级分离，结果显示cGK II定位于细胞颗粒部分，与内源性大鼠cGK II的膜结合一致。在北方印迹上，主要的cGK II，poly（A）RNA形式为4.8 kb，次要形式为6.2和3.1 kb。cGK II RNA在大鼠肠粘膜中高度表达，在大鼠脑和肾脏中的丰度低20倍。原位杂交结果表明，大鼠小肠内源性cGK Ⅱ RNA主要分布于绒毛上皮细胞，并在一定程度上分布于隐窝细胞。
The cDNA for a membrane-associated cGMP-dependent protein kinase (cGK II) was cloned from rat intestine using reverse transcriptase PCR and oligonucleotide primers encoding two conserved moths of known cGMP-dependent protein kinases and subsequently by screening a rat intestine cDNA library. A full-length clone encodes a protein of 761 amino acids with an estimated size of 87 kDa. Sequences of eight peptides from purified pig intestinal mucosa cGK II were found in the derived amino acid sequence of this clone, identifying it as rat intestinal cGK II. Phylogenetic analysis showed that rat intestinal cGK II is less related to mammalian cGK I than to the Drosophila DG1 gene product and most closely related to a recently cloned mouse brain CGKII isoform. Like several other cGK sequences, that of cGK II contained a leucine/isoleucine heptad repeat moth that has been implicated in dimer formation in cGK I. Expression of cGK II cDNA in HEK 293 cells followed by subcellular fractionation revealed cGK II localization in the cell particulate fraction, consistent with the membrane association of endogenous rat cGK II. On Northern blots, the major cGK II, poly(A) RNA form was 4.8 kb, with minor forms of 6.2 and 3.1 kb. The cGK II RNA was highly expressed in rat intestinal mucosa and was 20 times less abundant in rat brain and kidney. The localization of endogenous cGK II RNA in rat small intestine was shown by in situ hybridization to be in villous epithelial cells and to some extent in crypt cells.