Alcohol and Cell Adhesion

描述(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精神发育迟滞原因。我们的研究针对FASD，有三个广泛的目标：更好地了解乙醇是如何干扰胎儿发育的；确定增加怀孕期间饮酒的妇女生育FASD孩子的生物因素；以及为合理设计预防或减轻FASD的药物提供科学基础。我们的主要焦点是L1细胞黏附分子，这是一种与FASD病理生理学有关的发育关键蛋白。乙醇有效地抑制L1黏附和L1介导的轴突生长，光标记发现L1胞外区(L1-ECD)上有一个结合口袋，其中的点突变改变了乙醇的作用。这项拟议的研究将检验三个主要假设：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的马蹄形构象改变L1的功能，从而有利于同亲结合；2)L1细胞质结构域(CD)内的磷酸化通过引起L1胞外区(ECD)的酒精结合口袋的构象变化来调节L1对乙醇的敏感性；3)乙醇抑制L1黏附有助于乙醇抑制L1介导的神经突起生长(L1MNO)。我们将确定改变乙醇结合口袋的突变对L1结构和功能的影响。结构的变化将通过自旋标记和双电子-电子共振(DER)方案以及通过叠氮醇光标记位置的变化来检测。我们将通过检测突变对L1黏附的影响，酒精抑制L1黏附，以及拮抗剂阻断酒精抑制细胞黏附，将结构与功能联系起来。我们将使用激酶抑制剂和L1-CD中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。我们将通过确定L1-CD突变是否阻止插入在L1-ECD的酒精结合口袋中的两个半胱氨酸报告分子之间形成二硫键来评估L1-CD磷酸化对L1-ECD结构的影响。我们还将研究L1-ECD中抗体表位的掩蔽或取消掩蔽，以及酒精对L1-CD中的激酶底物突变反应中L1黏附的抑制。我们将确定酒精抑制小脑颗粒神经元L1黏附和L1MnO的药理是否相似。我们还将研究阻断乙醇抑制L1黏附的突变是否也阻断乙醇对L1MNO的抑制。这些实验将扩大我们的知识，了解乙醇如何破坏胎儿发育，为什么个体对乙醇畸形的敏感度不同，以及药物可能是如何设计来降低乙醇毒性的。

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.