Many observations with intact cells as well as cell-free systems suggest that receptors of the steroid hormone superfamily, along with other transcription factors, are regulated by phosphorylation. All receptors that have been analyzed carefully so far have turned out to be phosphoproteins. They are basally phosphorylated in the absence of ligand, and in many cases become hyperphosphorylated in the presence of hormone or other agonists, and sometimes of antagonists. Several studies indicate that hyperphosphorylation of receptors follows activation, and may require nuclear binding of the receptor. Serines are the predominant phosphorylated residues detected in receptors, with minor amounts of threonine. Tyrosine phosphorylation of the estrogen receptor is a subject of controversy. With various receptors, evidence has been found for phosphorylation in vivo of the N-terminal, hormone-binding, and DNA-binding domains, as well as of the hinge region. All but one of the phosphorylated sites identified in progesterone and glucocorticoid receptors by phosphopeptide mapping and sequencing are in the N-terminal domain; one is in the hinge region. Even after hormone treatment most of those sites are only partly phosphorylated, which means that several subpopulations of receptors, characterized by different states of phosphorylation and potentially different biological activities, must coexist. The majority of identified phosphorylated sites lie in consensus sequences for the PDPK. Many parallels can be discerned between phosphorylation of receptors and of other transcription factors. For example, several transcription factors become hyperphosphorylated on stimulation, and much indirect evidence points to regulation of both receptors and transcription factors by kinases and phosphatases, with cycling between different phosphorylated states. Functions of receptors that are regulated by phosphorylation are only beginning to be investigated. With transcription factors a substantial body of information is already available, and functions that appear to be thus regulated include dimerization, interactions with other proteins, binding to DNA, nuclear-cytoplasmic localization, and transcriptional activity. These and other functions may be found to be regulated by phosphorylation of receptors.
Binding of (3H)-corticosterone in cytosol of hippocampus and hypothalamus has been measured in adrenalectomized (ADX) rats in the presence or absence of corticosterone replacement therapy (suspended shortly before receptor analysis). Corticosterone pellet implantation into female rats or oral corticosterone administration in salinized drinking water given to males for 3 weeks reduced (3H)-corticosterone binding by half in the hippocampus. This reduction was observed whether corticosterone or dexamethasone was employed as competitor to determine nonspecific binding, thus eliminating transcortin as the cause of the corticosterone effect on binding. Scatchard analysis of binding data revealed that the reduction was mostly due to decreased number of receptors. Animals pretreated with corticosterone had a reduction in thymus weight, indicating further the biological effectiveness of the treatment. Further, serum corticosterone in ADX rats pretreated with corticosterone (but with therapy suspended for 24 h) was very low and similar to that of untreated ADX rats. Uptake studies after injection of (3H)-corticosterone intravenously into ADX rats showed that the injected hormone was absent from blood and brain tissues 1 day later, ruling out (in addition to the measurement of serum corticosterone) that the reduction in binding was due to occupation of receptor sites by exogenous corticosterone remaining after withdrawal from therapy. It is suggested that down-regulation of glucocorticoid receptors in brain follows the chronic corticosterone administration. These data are discussed in relation with evidence for down-regulation of other classes of steroid receptors in several tissues, and the consequence that changes in receptor binding in brain may have on the feedback mechanism of corticoids at the central level.
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