CFTR-dependent protein interactions regulate diarrhea

描述(申请人提供)：需要检验的假设是溶血磷脂酸(LPA)通过依赖CFTR的蛋白质相互作用来抑制分泌性腹泻。本实验室与这笔赠款相关的长期目标是：(I)更好地了解调节LPA依赖的CFTR抑制的动态蛋白质-蛋白质相互作用；(Ii)了解这些相互作用与分泌性腹泻的相关性。这项赠款的具体目的是(目的1)检验LPA抑制霍乱毒素诱导的小鼠分泌性腹泻和依赖CFTR的分泌性腹泻的假说；(AIM 2)检验LPA引起的抑制依赖CFTR的氯离子转运需要由LPA2、CFTR和NHERF2组成的大分子复合体的假说。为了推进国家糖尿病、消化和肾脏疾病研究所(NIDDK)的研究任务，拟议的研究将产生对了解、治疗和预防消化系统疾病(如分泌性腹泻)至关重要的重要基础科学信息。在目标1中，我们将验证LPA抑制霍乱毒素诱导的小鼠分泌性腹泻和CFTR依赖的分泌性腹泻的假设。在子目标1a中，我们将测试LPA是否抑制培养的肠道上皮细胞和切除的小鼠肠道组织中的CFTR功能。在子目标1b中，我们将测试LPA是否能抑制霍乱毒素诱导的CFTR依赖性分泌性腹泻。在子目标1c中，我们将测试LPA是否不能抑制LPA2受体基因敲除小鼠的CFTR功能。在目标2中，我们将检验这样的假设，即由LPA2、CFTR和NHERF2组成的大分子复合体是LPA诱导的抑制CFTR依赖的氯离子转运所必需的。在子目标2a中，我们将确定LPA2、CFTR和NHERF2是否在体外组装成大分子复合体。在次级目标2b中，我们将在培养的上皮细胞和小鼠肠道上皮细胞中交联先前存在的大分子复合体(LPA2、CFTR和NHERF2)的成分。在子目标2c中，我们将测试LPA是否由于LPA2与CFTR之间的物理相互作用(由NHERF2介导)而抑制CFTRCl-转运体。目前，LPA抑制分泌性腹泻的分子机制尚不清楚。该项目是了解LPA有益作用的分子机制的关键一步，从而可能改进预防分泌性腹泻的治疗方法。

DESCRIPTION (provided by applicant): The hypothesis to be tested is that lysophosphatidic acid (LPA) inhibits secretory diarrhea through CFTR-dependent protein interactions. The long-term objectives of this laboratory as related to this grant are (i) to gain a better understanding of the dynamic protein-protein interactions that regulate LPA-dependent inhibition of CFTR and (ii) to understand the relevance of these interactions in secretory diarrhea. The specific aims of the grant are (AIM 1) to test the hypothesis that LPA inhibits cholera toxin-induced and CFTR-dependent secretory diarrhea in mice and (AIM 2) to test the hypothesis that a macromolecular complex consisting of LPA2, CFTR, and NHERF2 is required for the LPA-elicited inhibition of CFTR-dependent Cl-transport. To advance the research mission of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), the proposed research will yield important basic science information essential to understanding, treating, and preventing digestive diseases such as secretory diarrhea. In Aim 1, we will test the hypothesis that LPA inhibits cholera toxin-induced and CFTR-dependent secretory diarrhea in mice. In subaim 1a, we will test whether LPA inhibits CFTR function in cultured gut epithelial cells and in excised mouse intestinal tissue. In subaim 1 b, we will test whether LPA inhibits cholera toxin-induced CFTR-dependent secretory diarrhea. In subaim 1c, we will test whether LPA does not inhibit CFTR function in LPA2 receptor knockout mice. In Aim 2, we will test the hypothesis that a macromolecular complex consisting of LPA2, CFTR, and NHERF2 is required for the LPA-elicited inhibition of CFTR-dependent Cl-transport. In subaim 2a, we will determine if LPA2, CFTR, and NHERF2 are assembled in a macromolecular complex in vitro. In subaim 2b, we will cross-link the components of the preexisting macromolecular complex (LPA2, CFTR, and NHERF2) in cultured epithelia and in mouse intestinal epithelial cells. In subaim 2c, we will test whether LPA inhibits the CFTR Cl-transporter due to a physical interaction between LPA2, and CFTR (mediated by NHERF2). At present, the molecular mechanisms responsible for LPA-mediated inhibition of secretory diarrhea are unclear. This project is a critical step in understanding the molecular mechanisms underlying the beneficial effects of LPA, thereby making possible improved treatments in the prevention of secretory diarrhea.