Rosemary Steinmetz Project Title: Estrogen Receptor-Beta Activation of Prolactin Gene Expression Estrogens modulate the growth and differentiation of many body tissues by influencing the activation or deactivation of estrogen-responsive genes. The first, and most critical, step that is involved in this activation is the binding of estrogen to its endogenous intracellular receptor (ER). ER- alpha (ERa), the first identified and best characterized ER, is composed of several regions or functional domains. Each of these domains carry out a specific function which is absolutely essential for the ER to induce gene activation. Recently, a new ER variant ER-Beta (ER-B) has been discovered. ER-B differs from ERa both in size and the sequence of the amino acids which comprise it. These differences may cause this new receptor to interact with new genes or new ligands or allow it to interact with already identified estrogen-sensitive genes in a manner different from that of ERa. Little is known concerning how this new receptor interacts physically with the genes it governs. The overall objective of this study is to determine the functional domains of ER-B that are required for the activation of the estrogen-responsive prolactin gene. Based on information concerning the structure of ER-B and its homology to ERa, the first specific aim is to construct a series of expression vectors which will express either truncated or mutated ER-B receptors. Using theses mutants, for in vitro transfection assays, we will be able to identify which domain(s) of the ER-B molecule bind various estrogenic compounds and which are required for gene activation. ER-B has been detected in a variety of tissues including the pituitary. Prolactin is a pituitary hormone that is exquisitely estrogen-sensitive. Prolactin is involved in many physiologic functions including reproduction, lactation, sexual development, cellular immunity and behavior. Little is known concerning the regulation of the prolactin gene by ER-B. Estrogens bind to the ER and in synergy with the pituitary-specific factor Pit-1 stimulate prolactin synthesis and release. While the nature of the interaction between Pit-1 and the ERa variant has been well characterized, the interaction between Pit-1 and ER-B has not been determined. The second specific aim of this study is to characterize the molecular interactions between Pit-1 and ER-B that are involved in prolactin gene activation. This will be accomplished by using the prepared mutant receptors in gene transfer studies. Each of the mutant receptors will be introduced into heterologous cell cultures in combination with Pit-1 and a prolactin reporter gene. These cells will then be treated with selected estrogenic compounds and the transcriptional activation of the prolactin reporter gene will be quantified. These studies will determine which regions of the ER-B work synergistically with Pit-1 and which are required for induction of prolactin by estrogenic compounds. Results of these studies will begin to determine the physical interactions between this new receptor isoform and the genes that it controls, as well as, expanding our existing knowledge concerning the mechanisms which govern prolactin homeostasis. These results will advance our knowledge concerning the actions of this newly discovered receptor.
