Cloning a Membrane Estrogen Receptor in Developing Brain

描述(申请人提供)：拟议的研究旨在获得关于一种新的和独特的质膜相关的雌激素受体(ER)的分子结构和染色体定位的初步数据，它既不是ER-α也不是ER-β，我们将其命名为ER-X。NIMH探索性/发展性R21赠款计划将使我能够积累足够的数据，使我能够申请R01资金，以充分表征这种潜在的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)都有分子生物学、蛋白质化学和遗传学方面的专业知识。ER-X是一种受发育调控的~62-63 kDa蛋白质，它：(I)在高纯度P7的空泡样微域中表达和丰富，但不在野生型和Erko小鼠的成体、新皮质和子宫质膜中表达；(Ii)与~3H-雌激素高亲和力结合，但具有不同于ER-α和ER-β的配体特异性；(Iii)与ER-α的配体结合结构域同源；(Iv)介导MAP激酶级联反应的雌激素激活；以及(V)在缺血性脑梗塞(中风)周围的成人新皮质组织和妊娠子宫中上调表达。这一提议背后的问题是，“ER-X”是ER-α的另一种剪接变体，还是由新基因编码。实验将在野生型和Erko出生后的新皮质上进行。我们将通过两种互补的方法来表征“ER-X”的分子结构。(1)采用基于c DNA的方法，我们将通过首先测定m RNA，利用5‘末端快速扩增(5’RACE)和比较野生型和Erko新皮质来间接地表征“ER-X”蛋白。(2)采用基于蛋白质的方法，利用MALDI-MS和蛋白质数据库搜索，直接确定蛋白质的结构。染色体的分配将通过核型分析和利用DAPI显带染色体上的荧光原位杂交(FISH)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。这些实验将使我能够通过提供有关雌激素在发育中和受伤的大脑中的作用机制的新信息，建立在该领域具有高水平影响的数据体系，从而引领药物干预和药物开发。

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.