Dransfield D T, Bradford A J, Smith J, Martin M, Roy C, Mangeat P H, Goldenring J R
Institute of Molecular Medicine and Genetics, Medical College of Georgia and the Augusta Veterans Affairs Medical Center, 30912, USA.
EMBO J. 1997 Jan 2;16(1):35-43. doi: 10.1093/emboj/16.1.35.
cAMP-dependent protein kinase (A-kinase) anchoring proteins (AKAPs) are responsible for the subcellular sequestration of the type II A-kinase. Previously, we identified a 78 kDa AKAP which was enriched in gastric parietal cells. We have now purified the 78 kDa AKAP to homogeneity from gastric fundic mucosal supernates using type II A-kinase regulatory subunit (RII) affinity chromatography. The purified 78 kDa AKAP was recognized by monoclonal antibodies against ezrin, the canalicular actin-associated protein. Recombinant ezrin produced in either Sf9 cells or bacteria also bound RII. Recombinant radixin and moesin, ezrin-related proteins, also bound RII in blot overlay. Analysis of recombinant truncations of ezrin mapped the RII binding site to a region between amino acids 373 and 439. This region contained a 14-amino-acid amphipathic alpha-helical putative RII binding region. A synthetic peptide containing the amphipathic helical region (ezrin409-438) blocked RII binding to ezrin, but a peptide with a leucine to proline substitution at amino acid 421 failed to inhibit RII binding. In mouse fundic mucosa, RII immunoreactivity redistributed from a predominantly cytosolic location in resting parietal cells, to a canalicular pattern in mucosa from animals stimulated with gastrin. These results demonstrate that ezrin is a major AKAP in gastric parietal cells and may function to tether type II A-kinase to a region near the secretory canaliculus.
环磷酸腺苷（cAMP）依赖性蛋白激酶（A激酶）锚定蛋白（AKAPs）负责II型A激酶的亚细胞隔离。此前，我们鉴定出一种在胃壁细胞中富集的78 kDa AKAP。现在，我们使用II型A激酶调节亚基（RII）亲和色谱法从胃底黏膜上清液中纯化出了均一的78 kDa AKAP。纯化后的78 kDa AKAP可被抗埃兹蛋白（微管肌动蛋白相关蛋白）的单克隆抗体识别。在Sf9细胞或细菌中产生的重组埃兹蛋白也能与RII结合。埃兹蛋白相关蛋白重组根蛋白和埃莫辛在印迹覆盖实验中也能与RII结合。对埃兹蛋白重组截短体的分析将RII结合位点定位到氨基酸373和439之间的区域。该区域包含一个14个氨基酸的两亲性α螺旋假定RII结合区域。含有两亲性螺旋区域的合成肽（埃兹蛋白409 - 438）可阻断RII与埃兹蛋白的结合，但在氨基酸421处将亮氨酸替换为脯氨酸的肽未能抑制RII结合。在小鼠胃底黏膜中，RII免疫反应性从静息壁细胞中主要位于胞质的位置重新分布到胃泌素刺激动物的黏膜中的微管模式。这些结果表明，埃兹蛋白是胃壁细胞中的一种主要AKAP，可能起到将II型A激酶拴系到分泌微管附近区域的作用。
