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.
