Elucidating the mechanisms by which estrogens and other steroids exert their effect upon steroid-sensitive cells is critical for understanding basic reproductive function, the behavior of tumors of reproductive tissue origin, and the actions of estrogenic environmental agents. Although it is generally accepted that estrogens and many other steroid hormones trigger RNA-dependent protein synthesis, steroids are also known to exert a variety of short-term effects (taking seconds to minutes) on their target organs. Compared to the genomic actions of steroids, the mechanism of these fast, non-genomic effects have not been adequately explained. Our hypothesis is that a subpopulation of estrogen receptors (ER) residing in the plasma membrane mediate cellular rabid actions of estrogens. Our proposal defines a model in which to test this hypothesis: Estrogen- induced prolactin release from pituitary tumor cells.
Specific aims designed to test this hypothesis are: 1) Correlate the enrichment of membrane ER (mER) in GH/B6 pituitary tumor cells with the increased rapid (1-5 minute) prolactin release response to estrogen (including characterization of time course, dose response to membrane-impermeable steroid conjugates, and the effect of serum-containing vs. defined growth media) and specifically define the effect as non-genomic (not altering prolactin gene expression at short times); 2) Use ER antibodies (including our own) to different epitopes to study their effect on prolactin release and thus map functions of the epitopes in relationship to the response; 3) Localize mER to the plasma membrane in GH/B6 cells using immunological techniques (such as immunocytochemistry, immunoprecipitation with concomitant radioactive hormone affinity labeling, and trypsin removal of antigen from whole cells) and use epitope mapping with other available antibodies to confirm the identity of mER as an ER and examine exposure of the antigen in the membrane; isolate separate cell populations enriched and depleted for mER by immunoselection; and 4) Characterize mER (in relationship to intracellular ER) with respect to steroid binding, attachment to the membrane, modifications of the protein due to or causing its residence in the membrane, and modulators of mER quantity. Future goals of this study include: 1) Screening of a cDNA library made from mER- enriched cells to ask whether this special form of the receptor is differentially transcribed and/or processed at the nucleic acid level, or results solely from post-translational modifications; 2) Application of these techniques and reagents to demonstrate an mER in other normal and cancerous reproductive tissues; 3) Screening of both natural and synthetic environmental estrogens for non-genomic toxicity effects; and 4) Correlation of the presence of this receptor to membrane physiological mechanisms for eliciting secretion and other rapid responses (such as ion channel function) in a variety of reproductive tissues and cell lines derived from them.
