CFTR-dependent protein interactions regulate diarrhea

CFTR 依赖性蛋白质相互作用调节腹泻

• 批准号: 7614197
• 负责人: Anjaparavanda P Naren
• 金额: $ 25.7万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-15 至 2011-05-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7614197
• 关键词: 2-Mercaptoethanol; Antibodies; Basic Science; Binding; Biological Assay; C-terminal; Cells; Chimeric Proteins; Cholera Toxin; Complex; Coupling; Cultured Cells; Cyclic AMP-Dependent Protein Kinases; Cystic Fibrosis Transmembrane Conductance Regulator; Diarrhea; Diet; Digestive System Disorders; Disease; Edg4 Protein; Epithelial Cells; Epithelium; Food; GTP-Binding Proteins; Gastrointestinal tract structure; Goals; Grant; Hand; Health; Human; In Vitro; Individual; Intestines; Knockout Mice; Laboratories; Lead; Length; Lysophospholipids; Macromolecular Complexes; Measurement; Mediating; Mediator of activation protein; Membrane; Methods; Mission; Modeling; Molecular; Monitor; Mus; National Institute of Diabetes and Digestive and Kidney Diseases; PDZ protein; Peptides; Physiological; Prevention; Proteins; Recombinant Proteins; Recombinants; Research; Research Personnel; Signal Transduction; Symptoms; Tail; Testing; Tissues; clinically relevant; crosslink; dithiobis(succinimidylpropionate); improved; lysophosphatidic acid; mouse model; overexpression; polypeptide C; prevent; programs; protein protein interaction; research study

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.

描述（由申请方提供）：待检验的假设是溶血磷脂酸（LPA）通过CFTR依赖性蛋白质相互作用抑制分泌性腹泻。本实验室的长期目标与该资助有关，即（i）更好地了解调节CFTR的LPA依赖性抑制的动态蛋白质-蛋白质相互作用，以及（ii）了解这些相互作用与分泌性腹泻的相关性。该资助的具体目的是（AIM 1）检验LPA抑制小鼠霍乱毒素诱导和CFTR依赖性分泌性腹泻的假设，以及（AIM 2）检验LPA诱导的CFTR依赖性Cl-转运抑制需要由LPA 2、CFTR和NHERF 2组成的大分子复合物的假设。为了推进国家糖尿病、消化和肾脏疾病研究所（NIDDK）的研究使命，拟议的研究将产生重要的基础科学信息，这些信息对理解、治疗和预防分泌性腹泻等消化系统疾病至关重要。在目的1中，我们将检验LPA抑制小鼠中霍乱毒素诱导的和CFTR依赖的分泌性腹泻的假设。在子目标1a中，我们将测试LPA是否抑制培养的肠上皮细胞和切除的小鼠肠组织中的CFTR功能。在子目标1 B中，我们将测试LPA是否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是否不抑制LPA 2受体敲除小鼠中的CFTR功能。在目标2中，我们将检验由LPA 2、CFTR和NHERF 2组成的大分子复合物是LPA引起的CFTR依赖性Cl-转运抑制所需的假设。在子目标2a中，我们将确定LPA 2、CFTR和NHERF 2是否在体外组装成大分子复合物。在子目标2b中，我们将在培养的上皮细胞和小鼠肠上皮细胞中交联预先存在的大分子复合物（LPA 2、CFTR和NHERF 2）的组分。在子目标2c中，我们将测试LPA是否由于LPA 2和CFTR之间的物理相互作用（由NHERF 2介导）而抑制CFTR Cl-转运蛋白。目前，LPA介导的分泌性腹泻抑制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，从而可能改善预防分泌性腹泻的治疗方法。