Cloning a Membrane Estrogen Receptor in Developing Brain

克隆发育中大脑中的膜雌激素受体

• 批准号: 6615099
• 负责人: DOMINIQUE CLAUDE TORAN-ALLERAND
• 金额: $ 16.35万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6615099
• 关键词: biotechnology; cell membrane; complementary DNA; developmental neurobiology; estrogen receptors; fluorescent in situ hybridization; genetic mapping; laboratory mouse; matrix assisted laser desorption ionization; membrane proteins; messenger RNA; molecular biology information system; molecular cloning; molecular genetics; protein structure; receptor expression; structural biology

DESCRIPTION (provided by applicant): The proposed studies are designed to obtain preliminary data concerning the molecular structure and chromosome localization of a novel and unique plasma membrane-associated, estrogen receptor (ER) that is neither ER-alpha nor ER-beta, which we have designated "ER-X". The NIMH Exploratory / Developmental R21 Grant Program would enable me then to amass a sufficient body of data to permit me to apply for R01 funding to fully characterize the properties of this potentially very important, novel receptor. Expertise in molecular biology, protein chemistry and genetics are currently available to me in my laboratory, the Genome Center (Columbia) and the Keck Foundation Biotechnology Resource Laboratory (Yale). ER-X is a developmentally regulated ~62-63 kDa protein which: (i) is expressed and enriched in caveolar-like microdomains of highly purified P7, but not adult, neocortical and uterine plasma membranes of wild-type and ERKO mice; (ii) binds 3H-estrogen with high affinity but with ligand specificities different from ER-alpha and ER-beta; (iii) exhibits homology with the ligand binding domain of ER-alpha; (iv) mediates estrogen activation of the MAP kinase cascade; and (v) becomes up-regulated in adult neocortical tissue surrounding an ischemic infarct (stroke) and in the pregnant uterus. The question underlying this proposal is whether "ER-X" is an alternative splicing variant of ER-alpha or is encoded by a new gene. The experiments will be carried out on wild-type and ERKO postnatal neocortex. We will focus on characterizing the molecular structure of "ER-X" by two complementary approaches. (1) Using a cDNA-based approach, we will characterize the "ER-X" protein indirectly by first determining the mRNA, using rapid amplification of cDNA 5'ends (5'RACE) and comparing wild-type and ERKO neocortex. (2) Using a protein-based approach, we will determine the protein structure directly, using Maldi-Mass Spectrometry and protein data base searching. Chromosomal assignment will be determined by karyotyping and mapping its subchromosomal position on wild-type, embryonic mouse metaphase chromosomes using fluorescent in situ hybridization (FISH) on DAPI banded chromosomes. The experiments will allow me to build a body of data with a high level of impact in the field by providing new information about some mechanisms underlying the actions of estrogen in the developing and injured brain, leading the way to pharmacologic intervention and drug development.

描述（由申请人提供）：所提议的研究旨在获得有关一种新颖且独特的质膜相关雌激素受体（ER）的分子结构和染色体定位的初步数据，该受体既不是 ER-α 也不是 ER-β，我们指定了“ER-X”。 NIMH 探索性/发展性 R21 资助计划将使我能够积累足够的数据，使我能够申请 R01 资助，以充分表征这种可能非常重要的新型受体的特性。目前，我的实验室、基因组中心（哥伦比亚）和凯克基金会生物技术资源实验室（耶鲁）拥有分子生物学、蛋白质化学和遗传学方面的专业知识。 ER-X 是一种发育调节的约 62-63 kDa 蛋白，其： (i) 在高度纯化的 P7 的小窝样微结构域中表达和富集，但不在野生型和 ERKO 小鼠的成年、新皮质和子宫质膜中表达和富集； (ii) 以高亲和力结合 3H-雌激素，但具有不同于 ER-α 和 ER-β 的配体特异性； (iii) 与 ER-α 的配体结合结构域表现出同源性； (iv) 介导 MAP 激酶级联的雌激素激活； (v) 在缺血性梗塞（中风）周围的成人新皮质组织和怀孕子宫中上调。这一提议背后的问题是“ER-X”是否是 ER-α 的替代剪接变体，或者是由新基因编码的。实验将在野生型和 ERKO 产后新皮质上进行。我们将重点通过两种互补的方法来表征“ER-X”的分子结构。 (1) 使用基于 cDNA 的方法，我们将通过首先确定 mRNA、使用 cDNA 5' 末端 (5'RACE) 的快速扩增并比较野生型和 ERKO 新皮质来间接表征“ER-X”蛋白。 (2)使用基于蛋白质的方法，我们将使用Maldi-质谱法和蛋白质数据库搜索直接确定蛋白质结构。染色体分配将通过核型分析确定，并使用 DAPI 带状染色体上的荧光原位杂交 (FISH) 绘制其在野生型胚胎小鼠中期染色体上的亚染色体位置。这些实验将使我能够通过提供有关雌激素在发育和受损大脑中作用的一些机制的新信息，建立在该领域具有高影响力的数据体，从而为药理干预和药物开发奠定基础。