Cloning a Membrane Estrogen Receptor in Developing Brain

描述（由申请人提供）：拟定研究旨在获得关于一种新型和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，我们将其命名为“ER-X”。NIMH探索/开发R21资助计划将使我能够收集足够的数据，使我能够申请R 01资金，以充分表征这种潜在的非常重要的新型受体的特性。分子生物学、蛋白质化学和遗传学方面的专业知识目前在我的实验室、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）提供给我。ER-X是一种发育调节的~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，但在野生型和ERKO小鼠的成年、新皮质和子宫质膜中不表达和富集;（ii）以高亲和力结合3 H-雌激素，但配体特异性不同于ER-α和ER-β;（iii）与ER-α的配体结合结构域表现出同源性;（iv）介导MAP激酶级联的雌激素活化;和（v）在缺血性梗塞（中风）周围的成人新皮质组织和妊娠子宫中上调。这一提议背后的问题是“ER-X”是否是ER-α的选择性剪接变体，或者是由一个新基因编码的。实验将在野生型和ERKO出生后新皮层上进行。我们将集中在表征的“ER-X”的分子结构的两种互补的方法。(1)使用基于cDNA的方法，我们将通过首先确定mRNA，使用cDNA 5 '末端的快速扩增（5' RACE）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，我们将直接确定蛋白质结构，使用Maldi质谱和蛋白质数据库搜索。将通过核型分析和使用荧光原位杂交（FISH）对DAPI显带染色体定位其在野生型胚胎小鼠中期染色体上的亚染色体位置来确定染色体分配。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，建立一个在该领域具有高度影响力的数据库，从而为药理学干预和药物开发开辟道路。

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.