CYCLIC AMP AS A MEDIATOR OF PARIETAL CELL SECRETION

环磷酸腺苷作为壁细胞分泌的介质

基本信息

批准号：
6177091
负责人：
JAMES Richard GOLDENRING
金额：
$ 22.51万
依托单位：
AUGUSTA UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-04-01 至 2002-03-31
项目状态：
已结题

项目摘要

Polarized epithelial cells, by definition, segregate the functions of their apical and basolateral membranes. This separation of functional membrane domains allows epithelial cells to translate messages received at one membrane surface into responses at the opposite plasma membrane domain. In the gastric parietal cell, activation of second messenger systems at the basolateral membrane results in the fusion of tubulovesicles containing the H/K-ATPase with an apically oriented intracellular canalicular target membrane. In order to carry out the complex integrated processes required, polarized cells have evolved intricate mechanisms to sequester critical links in the signaling pathway within discrete subcellular domains. In the case of the Type II cAMP-dependent protein kinases (A-kinases), the kinase has been localized to both cytosolic and particulate fractions in epithelial cells and neurons. The Type II kinase associates with particulate fractions through the binding of its regulatory subunits (RII) to A- kinase anchoring proteins (AKAPs). We have indentified A-kinase anchoring proteins (AKAPs) in parietal cells which may be involved in localizing A-kinase within particular subcellular domains. In particular, we have recently recognized that the major parietal cell AKAP, AKAP78, is ezrin, the apical F-actin associated protein. These data suggest that Type II A-kinase may be anchored to the secretory canaliculus, the site of membrane fusion during parietal cell secretion. We have also cloned from parietal cells a second novel AKAP, AKAP120, which appears to be present in a number epithelial tissues. We have hypothesized that AKAPs, in particular AKAP78/erin and AKAP120, map function as sites for the assembly of critical kinase/phosphatase signaling complexes anchored to discrete areas of the parietal cell. The present proposal has four specific aims: First, we will establish definitively the association of RII with ezrin by characterizing the RII binding site and the immunocytochemical co-localization of the two proteins. Second, since ezrin is phophorylated in response to histamine, we will determine the effects of phosphorylation on ezrin/RII interactions and characterize ezrin kinase. Third, we will characterize the association of RII with the novel AKAP120 protein. Fourth, we will seek to identify parietal cell proteins which interact with ezrin and AKAP120. These investigations will provide important insights into the mechanisms by which epithelial cells in general, and parietal cells specifically, segregate their second messenger-dependent signaling systems.

根据定义，极化上皮细胞分离以下细胞的功能：它们的顶膜和基底外侧膜。这种分离功能性膜结构域允许上皮细胞翻译在一个膜表面接收到的消息转化为在另一膜表面的响应相反的质膜结构域。在胃壁细胞中，基底外侧第二信使系统的激活膜导致含有以下成分的管泡融合 H/K-ATP酶具有顶端定向的细胞内小管目标膜。为了进行复杂的综合所需的过程，极化细胞已经进化出复杂的隔离信号通路中关键环节的机制在离散的亚细胞域内。对于 II 型的情况 cAMP 依赖性蛋白激酶（A-激酶），该激酶已被定位于上皮细胞中的细胞质和颗粒部分细胞和神经元。 II 型激酶与颗粒相关通过其调节亚基 (RII) 与 A- 的结合来获得分数激酶锚定蛋白（AKAP）。我们已经鉴定出A-激酶壁细胞中的锚定蛋白（AKAP）可能是参与特定亚细胞内 A-激酶的定位域。特别是，我们最近认识到，主要壁细胞 AKAP，AKAP78，是埃兹蛋白 (ezrin)，与顶端 F-肌动蛋白相关蛋白质。这些数据表明 II 型 A 激酶可能是锚定于分泌小管，膜融合部位壁细胞分泌过程中。我们还从顶叶克隆了细胞是第二个新的 AKAP，AKAP120，它似乎是存在于许多上皮组织中。我们假设 AKAP，特别是 AKAP78/erin 和 AKAP120，地图功能作为关键激酶/磷酸酶信号传导的组装位点复合物锚定在壁细胞的离散区域。这本提案有四个具体目标：首先，我们将建立通过表征，明确 RII 与埃兹蛋白的关联 RII 结合位点和免疫细胞化学共定位两种蛋白质。其次，由于埃兹蛋白被磷酸化以响应组胺，我们将确定磷酸化对 ezrin/RII 相互作用并表征 ezrin 激酶。第三，我们将描述 RII 与小说 AKAP120 的关联蛋白质。第四，我们将寻求鉴定壁细胞蛋白与 ezrin 和 AKAP120 相互作用。这些调查将为了解上皮细胞的机制提供了重要的见解一般细胞，特别是壁细胞，将它们的第二信使依赖性信号系统。