A servomechanism operates between progesterone and prolactin and their receptors in the regulation of uterine gene expression. Prolactin receptor is regulated by progesterone and prolactin augments the progesterone-dependent increase in uteroglobin mRNA. The search for new transcription factors that coordinate the convergence of these two signaling pathways culminated in the cloning of RUSH, whose alpha and beta isoforms are the products of a steroid-dependent alternative splicing mechanism. Preferential expression of each isoform correlates with either activation or repression of the target gene. Both isoforms have DNA-binding domains and nuclear localization signals, suggesting they may be functionally antagonistic. This hypothesis will be tested in aim one. RUSH-1alpha protein consists of seven sequential motifs (I, Ia, II-VI) that are characteristic of ATPases/helicases. The truncated RUSH-1beta protein lacks motifs IV-VI. The goal of aim two is to characterize the DNA-dependent ATPase and DNA helicase activities of each isoform, and to evaluate functional differences inferred from their structural dissimilarities. Progesterone-induced transcriptional activation of the RUSH promoter (- 131/-26) is regulated by Sp1/progesterone receptor/NF-Y interactions. This same region mediates repression by estrogen, and autoregulation is achieved via two RUSH sites. The goal of aim three is continued interrogation of the promoter to characterize the mechanisms of estrogen-repression and prolactin-augmented, progesterone-dependent transcription. Classic prolactin-receptor signaling is via the Jak/Stat phosphorylation cascade. RUSH proteins are physically affiliated with Jak2, and tyrosine-phosphorylation is required for RUSH-DNA-binding. The goal of aim four is to determine whether RUSH is phorphorylated by Jak2. Null mouse models have contributed to genetic analyses of prolactin signaling. The goal of aim 5 is to define the importance of RUSH in the regulation of gene transcription in a knockout mouse model. Understanding the mechanism of prolactin and progesterone interaction in the endometrium may be important to good reproductive health.
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