LPA2 receptor-containing complexes in regulating secretory diarrhea

描述(申请人提供)：本方案的统一假设是2型溶血磷脂酸受体(LPA2受体)和Na/H交换调节因子-2(NHERF2)的大分子复合体在分泌性腹泻的发病过程中发挥重要作用。我们的目标是在分子水平上研究在霍乱引起的腹泻中起关键作用的生理和病理生理条件下大分子复合体的形成和调节。此外，这项研究还将在腹泻动物模型中研究LPA2受体介导的信号事件调节细胞内依赖CFTR的电解质分泌和液体分泌的机制。目的1：验证LPA2受体主要在肠腔表面表达并在某些形式的腹泻疾病中下调的假说，以及LPA2受体的激活抑制质膜上区域化的cAMP生成的假说。目的2：验证LPA通过进一步将其聚集到质膜上的微区来调节含有LPA2的大分子复合体的形成，以及这一过程是由NHERF2依赖的蛋白质-蛋白质相互作用介导的假说。目的3：检验通过沉默NHERF2来破坏大分子复合体改变质膜上区域化的cAMP水平的假说，以及在体内NHERF2基因敲除小鼠对CTX的反应产生改变cAMP的假说。这对于从分子水平上了解致死性分泌性腹泻的发病过程，以及对该病可能的治疗干预具有重要意义。拟议的研究在概念的推进和技术/技术的发展方面都具有很大的创新性。

DESCRIPTION (provided by applicant): The unifying hypothesis of this proposal is that macromolecular complex of type 2 lysophosphatidic acid receptor (LPA2 receptor) and Na+/H+ exchange regulatory factor-2 (NHERF2) plays important roles in the pathogenic process of secretory diarrhea. We aim to study the formation and regulation of the macromolecular complex at the molecular level under physiological and pathophysiological conditions that play a critical role in cholera induced diarrhea. Moreover, the research will study the mechanism through which LPA2 receptor- mediated signaling events regulate CFTR-dependent electrolyte secretion in cells and fluid secretion in an animal model of diarrhea. Three aims will be studied: Aim 1: To test the hypothesis that LPA2 receptor is primarily expressed on the luminal surface of the gut and is down regulated in certain forms of diarrheal diseases, and to test the hypothesis that activation of LPA2 receptor inhibits compartmentalized cAMP generation at the plasma membrane. Aim 2: To test the hypothesis that LPA can regulate the formation of the LPA2-containing macromolecular complex by further clustering it to microdomain on the plasma membrane and that the process is mediated by NHERF2-dependent protein-protein interactions Aim 3: To test the hypothesis that disruption of the macromolecular complex by silencing NHERF2 alters compartmentalized cAMP levels at the plasma membrane and that in vivo NHERF2 knockout mice generate altered cAMP in response to CTX. The proposal is of great importance and significance for understanding the pathogenic process of the deadly secretory diarrheal diseases at the molecular level, and for possible therapeutic intervention of the disease. The proposed study is highly innovative in both conceptual advance and in technology/technique development.