Alcohol and Cell Adhesion

描述（由申请人提供）：胎儿酒精谱系障碍（FASD）是西方世界最常见的可预防的智力迟钝原因。我们的研究涉及FASD，并有三个广泛的目标：更好地了解乙醇如何破坏胎儿发育；确定增加孕期饮酒妇女生育FASD儿童易感性的生物学因素；为预防或减轻FASD的合理药物设计提供科学依据。我们的主要研究重点是L1细胞粘附分子，这是一种与FASD病理生理有关的发育关键蛋白。乙醇能有效抑制L1粘附和L1介导的神经突生长，光标记鉴定出L1细胞外结构域（L1- ecd）上的一个结合袋，在这个结合袋中，点突变改变了乙醇的作用。该研究将测试三个主要假设：1)在L1 - Ig1和Ig4之间的界面上有一个特定的位置，乙醇通过破坏L1的马蹄形构象来改变L1的功能，从而有利于亲同性结合；2) L1胞质结构域（CD）内的磷酸化通过引起L1胞外结构域（ECD）的酒精结合袋的构象变化来调节L1对乙醇的敏感性；3)乙醇抑制L1粘附有助于乙醇抑制L1介导的神经突生长（L1MNO）。我们将确定改变乙醇结合袋的突变对L1的结构和功能的影响。结构变化将通过自旋标记和双电子-电子共振（DEER）协议以及氮醇光标记的可用性的变化来检测。我们将利用瞬时转染L1构建体的完整NIH/3T3细胞，通过检测突变对L1粘附的影响、酒精对L1粘附的抑制以及乙醇对细胞粘附抑制的拮抗剂阻断，将结构与功能联系起来。我们将在L1-CD中使用激酶抑制剂和特定激酶底物的突变来确定这些位点如何调节乙醇敏感性。我们将评估L1-CD磷酸化对L1-ECD结构的影响，通过确定L1-CD突变是否会阻止插入L1-ECD中酒精结合袋内的两个半胱氨酸报告蛋白之间形成二硫键。我们还将研究L1- ECD中抗体表位的掩蔽或揭露，以及酒精抑制L1- cd中激酶底物突变时L1粘附的作用。我们将确定酒精抑制小脑颗粒神经元L1粘附和L1MNO的药理学是否相似。我们还将研究阻断乙醇对L1粘附抑制的突变是否也阻断了乙醇对L1MNO的抑制。这些实验将扩大我们对乙醇如何破坏胎儿发育的认识，为什么个体对乙醇致畸的敏感性不同，以及如何设计药物来减少乙醇毒性。

DESCRIPTION (provided by applicant): Fetal alcohol spectrum disorder (FASD) is the most common preventable cause of mental retardation in the Western World. Our research addresses FASD and has three broad goals: to better understand how ethanol disrupts fetal development; to identify biological factors that increase the susceptibility of women who drink during pregnancy to bear children with FASD; and to provide a scientific foundation for rational drug design to prevent or mitigate FASD. Our major focus is the L1 cell adhesion molecule, a developmentally critical protein that is implicated in the pathophysiology of FASD. Ethanol potently inhibits L1 adhesion and L1-mediated neurite outgrowth, and photolabeling identifies a binding pocket on the extracellular domain of L1 (L1-ECD) in which point mutations alter the effects of ethanol. The proposed research will test three principal hypotheses: 1) there is a specific location at the interface between L1 Ig1 and Ig4 at which ethanol alters L1 function by disrupting the horseshoe conformation of L1 that favors homophilic binding; 2) phosphorylation within the L1 cytoplasmic domain (CD) modulates L1 sensitivity to ethanol by causing conformational changes in the alcohol binding pocket of the L1 extracellular domain (ECD); 3) ethanol inhibition of L1 adhesion contributes to ethanol inhibition of L1-mediated neurite outgrowth (L1MNO). We will determine the effects of mutations that alter the ethanol binding pocket on the structure and function of L1. Structural changes will be detected by spinlabeling and double electron-electron resonance (DEER) protocols and by changes in the availability of sites for photolabeling by azialcohols. We will correlate structure with function by examining the effects of mutations on L1 adhesion, alcohol inhibition of L1 adhesion, and antagonist blockade of ethanol inhibition of cell adhesion using intact NIH/3T3 cells transiently transfected with L1 constructs. We will employ kinase inhibitors and mutations of specific kinase substrates in the L1-CD to determine how these sites regulate ethanol sensitivity. We will evaluate the effects of L1-CD phosphorylation on L1-ECD structure by determining whether L1-CD mutations prevent the formation of disulfide bonds between two cysteine reporters inserted within the alcohol binding pocket in the L1-ECD. We will also study masking or unmasking of antibody epitopes within the L1- ECD and alcohol inhibition of L1 adhesion in response to mutation of kinase substrates in the L1-CD. We will determine whether the pharmacology is similar for alcohol inhibition of L1 adhesion and L1MNO in cerebellar granule neurons. We will also study whether mutations that block ethanol inhibition of L1 adhesion also block ethanol inhibition of L1MNO. These experiments will expand our knowledge of how ethanol disrupts fetal development, why individuals differ in sensitivity to ethanol teratogenesis, and how drugs might be designed to reduce ethanol toxicity.