Physiological Role of Pituitary ER mRNA Variants The estrogen receptor (ER) is a ligand-activated transcriptional regulatory protein with discrete functional domains. Recent studies have demonstrated that its structural integrity is critically important in governing cellular function. Though most variant ER mRNA forms have been found in neoplastic tissues, our laboratory has recently identified an ER message in normal rats which is pituitary-specific. This ER mRNA comprises about 20% of the total ER message in rat pituitary and represents the only variant ER mRNA identified in normal rat tissue to date. Preliminary structural comparison between the pituitary-specific form (5.5kb) and the more ubiquitous form (6.2kb) has demonstrated that approximately 400 bp of the 700 bp size difference between them occurs in the 5' untranslated or coding regions of the message. The pituitary- specific message also appears to be expressed in mature female rats but not in mature males. Studies in this proposal are designed to further define the molecular structure and physiological significance of the pituitary-specific ER mRNA, and to investigate the ER status of human pituitary tissue. In the rat, ribonuclease H experiments will be performed to provide gross structural information about the 5' untranslated and polyadenylate tails of the two mRNAs. A polyadenylate enriched randomly primed rat pituitary cDNA library will be screened in order to clone and sequence the pituitary-specific form. The absolute and relative amounts of each ER mRNA will be determined in ovariectomized and estrogen (E2) replaced mature females, in immature females (basal and E2 stimulated) and in each stage of the estrous cycle. The significance of coding region differences between forms will be evaluated by E2 and DNA binding studies of each message's in vitro translation product. The reverse hemolytic plaque assay and the polymerase chain reaction (PCR) will be combined to determine if particular anterior pituitary cell types contain the 5.5kb or 6.2kb messages. The above experiments, both structural and functional in nature, should provide definitive information on the role of the 5.5kb ER message. Investigation into the ER status of human pituitary will be two-fold. First, autopsy samples will be collected and polyadenylated RNA will be isolated to determine if a pituitary-specific of ER exists in the human. Second, the ER mRNA status of human pituitary tumors will be determined using PCR (several different areas within the coding region will be examined), and these results will be correlated with E2 binding data to determine if the presence or integrity of the ER is an important factor in the regulation of tumor function or growth.
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