The goals of these studies are to examine the biochemical regulation of endocrine systems, and particularly regulation of steroid hormone receptor function. Steroid hormones play important roles in the regulation of a variety of functions including reproduction and various metabolic processes. Little is known about the mechanism by which steroid hormones, through their receptors, act to specifically induce mRNA of target genes. Even less is known about the regulation of the activity of the steroid hormone receptors. Phosphorylation plays an important role in the regulation of many enzyme systems and phosphorylation of several steroid receptors has been demonstrated both in vivo and in vitro. Moreover, there is evidence that the receptors are phosphorylated at more than one site, suggesting that phosphorylation may play a role in regulating a number of receptor activities. This proposal will examine the effect of these phosphorylations on receptor function. The chicken progesterone receptor will be used as the model system for steroid receptor action to accomplish three specific aims. First, the in vivo phosphorylation site(s) will be identified and the peptides characterized. Second, enzymes which can phosphorylate and dehosphorylate these same sites in vitro will be isolated. There is evidence that phosphorylation alters hormone binding of this receptor. Using receptor phosphorylated in vivo and a hormone binding assay, the phosphatase(s) which dephosphorylates the receptor will be isolated. Kinases which regenerate hormone binding and which phosphorylate the receptor at the same site or sites as are phosphorylated in vivo will be isolated. Third, phosphorylated and dephosphorylated receptor will be prepared in vitro and the effects of these modification on receptor functions, including hormone binding and DNA binding, will be examined. If the mechanism by which receptor activity is regulated can be determined, it may be possible to specifically regulate or alter the activity of receptors as needed for clinical purposes. It has been suggested that tamoxifen, an antiestrogen, acts by preventing the dephosphorylation and recycling of estrogen receptor. It is possible that other receptor antagonists may act through a similar mechanism. Knowledge of these regulatory mechanisms should provide a basis for developing methods to specifically alter receptor function.
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