EXPRESSION AND FUNCTION OF THE GUANYLIN LIGAND FAMILY

大肠埃希菌耐热肠毒素与其结合的研究 肠道受体在毒素诱导的致病作用中起关键作用 分泌物和腹泻病。但是，sta的操作可以是 分子模拟的结果；sta受体的主要作用可能是 是调解观音林家族中一个或多个成员的行为 哺乳动物的多肽配体。此应用程序的目标是 阐明鸟苷素在体内的作用。此应用程序将解决 三个具体目标：1)我们将检验顺式活跃的假设 鸟苷素基因的元件赋予细胞特异性表达 肠上皮细胞。我们将使用体外转录分析 鸟苷素-荧光素酶报告基因构建及体内转基因 鸟苷素-人生长激素构建小鼠研究元素 它调节鸟苷素基因的表达。鸟苷素的鉴别 引导表达到绒毛、肠细胞和杯状细胞的启动子区域 细胞可能增强肠道鸟苷素的表达策略 粘蛋白、鸟苷素受体与囊性纤维化 跨膜调节剂(CFTR)。2)我们将用转基因小鼠 肠特异性启动子-鸟苷基因构建检测 假设肠道内鸟苷素过度表达将导致 基础肠道氯分泌量增加。我们将确定其效果 鸟苷素过表达及鸟苷素/STA的刺激作用 肠道分泌物与鸟苷二磷酸受体(G-STAR)表达(G-STAR) StAR信使核糖核酸、鸟苷环化酶活性和配体结合)。转基因 线条不仅对描述体内效应很有用 但它们也可能被用作分泌性腹泻的模型 或用于治疗部分可见的胎粪性肠梗阻 更正了CF小鼠模型。3)我们将使用有针对性的技术 破坏鸟苷基因，以检验丢失鸟苷的假设 鸟苷素在肠道中的表达会减少净肠液 体内分泌。我们将在这种损失中描述肠道分泌物的特征。 功能模型类似于目标2中描述的针对 互补性过表达模型。这种“基因敲除”的动物模型可能 也有助于研究其他鸟苷素相关农药的作用， 例如尿鸟苷或异常合成鸟苷，以及用于未来的研究 涉及与G-STAR基因已经在其中的动物杂交的 有针对性的。总而言之，我们将使用分子遗传学方法来定义 鸟苷素的体内作用和细胞定位。我们将发展 动物模型，对解决这一特定目标很有用 建议并阐明腹泻病的发病机制以及 CFTR介导的肠道基础氯离子分泌。

Binding of Escherichia coli heat-stable enterotoxin (STa) to its intestinal receptor is critical to the initiation of toxin-induced secretion and diarrheal disease. However, the action of STa may be a result of molecular mimicry; the primary role for the STa receptor may be to mediate the action of one or more members of the guanylin family of mammalian peptide ligands. The goal of this application is to elucidate the in vivo function of guanylin. This application will address three specific aims: 1) We will test the hypothesis that cis-active elements of the guanylin gene confer cell-specific expression to intestinal epithelial cells. We will use in vitro transcriptional assays with guanylin-luciferase reporter gene constructs and in vivo transgenic mice with guanylin-human growth hormone constructs to study elements which regulate guanylin gene expression. Identification of guanylin promoter regions that direct expression to villus enterocytes and goblet cells may enhance strategies for expression of guanylin in the intestine in proximity to mucin, the guanylin receptor and the cystic fibrosis transmembrane regulator (CFTR). 2) We will make transgenic mice with intestinal specific promoter-guanylin gene constructs to test the hypothesis that intestinal overexpression of guanylin will result in increased basal intestinal Cl secretion. We will determine the effect of overexpression of guanylin on basal and guanylin/STa stimulated intestinal secretion, and guanylin-STa receptor (G-STaR) expression (G- STaR mRNA, guanylate cyclase activity and ligand binding). Transgenic lines will not only be useful for characterization of the in vivo effects of guanylin but they may also be useful as a model of secretory diarrhea or in treating the meconium ileus equivalent seen in the partially corrected CF mouse model. 3) We will use the technique of targeted disruption of the guanylin gene, to test the hypothesis that loss of guanylin expression in the intestine will decrease net intestinal fluid secretion in vivo. We will characterize intestinal secretion in this loss of function model akin to the studies described in Aim 2 for the complementary overexpression model. This "knock-out" animal model may also be useful to study the actions of other guanylin-related pesticides, e.g., uroguanylin or aberrant synthetic guanylins and for future studies involving crossbreeding to animals in which the G-STaR gene has been targeted. In summary, we will use molecular genetic approaches to define the in vivo role and cellular localization of guanylin. We will develop animal models which be useful to address the specific aims of this proposal and to elucidate the mechanisms of diarrheal disease as well as basal intestinal Cl secretion mediated through the CFTR.