Glucocorticoid receptor (GR) exchanges between an active nuclear form and a complexed inactive, steroid-sensitive cytoplasmic form. Using a semi-quantitative indirect immunofluorescence assay to measure the kinetics of subcellular redistribution of GR in response to challenge during G(o), we have found that the ability to bind DNA is an important determinant for localization and tight binding of GR to the nucleus. The transfer of GR DNA-binding mutants to the nucleus after treatment with hormone agonists and antagonists was markedly reduced. Further, mutant receptors localized to the nucleus were only weakly associated with the nuclear compartment as they were released into cytosol upon hypotonic lysis of the cell membrane. Moreover, after agonist withdrawal, GR redistributed to the cytoplasm more rapidly when unable to bind DNA. By contrast, withdrawal of the hormone antagonist RU486 was found to yield a form of wild type GR that was completely unable to redistribute to the cytoplasm. However, this did not appear to result from a block in nuclear export as selective inactivation of nuclear import with energy inhibitor released RU486-withdrawn GRs from the nucleus at the same rates as agonist-withdrawn receptors. In addition, GR mutants unable to bind DNA, which retained a significant presence in the cytoplasm both during and after antagonist treatment, also failed to redistribute. The effect of RU486 treatment did not appear to be mediated through a block in reassociation of GR into a steroid-responsive form as RU486-withdrawn wild type receptors retained full potential to activate transcription from a glucocorticoid-responsive promoter after a second challenge with hormone. Therefore, reassociation of GR into a steroid-responsive form appears to be independent of signals important for the retention of GR in the cytoplasm.
An immunocolloidal gold electron microscopy method is described allowing the ultrastructural localization and quantitation of the regulatory subunits RI and RII and the catalytic subunit C of cAMP-dependent protein kinase. Using a postembedding indirect immunogold labeling procedure that employs specific antisera, the catalytic and regulatory subunits were localized in electron-dense regions of the nucleus and in cytoplasmic areas with a minimum of nonspecific staining. Antigenic domains were localized in regions of the heterochromatin, nucleolus, interchromatin granules, and in the endoplasmic reticulum of different cell types, such as rat hepatocytes, ovarian granulosa cells, and spermatogonia, as well as cultured H411E hepatoma cells.Morphometric quantitation of the relative staining density of nuclear antigens indicated a marked modulation of the number of subunits per unit area under various physiologic conditions. For instance, following partial hepatectomy in rats, the staining density of the nuclear RI and C subunits was markedly increased 16 h after surgery. Glucagon treatment of rats increased the staining density of only the nuclear catalytic subunit. Dibutyryl cAMP treatment of H411E hepatoma cells led to a marked increase in the nuclear staining density of all three subunits of cAMP-dependent protein kinase. These studies demonstrate that specific antisera against cAMP-dependent protein kinase subunits may be used in combination with immunogold electron microscopy to identify the ultrastructural location of the subunits and to provide a semi-quantitative estimate of their relative cellular density.Cyclic AMP (cAMP) is the intracellular mediator of the regulatory signal of several polypeptide hormones and catecholamines. It plays a key role in the modulation of nuclear events such as the specific regulation of transcription of a number of cAMP-induced enzymes and proteins (1-4), phosphorylation/dephosphorylation of histones and nonhistone chromosomal proteins (5, 6), and nuclear mechanisms that may be involved in cAMP-regulated control of cellular proliferation (7). However, the precise sequence of molecular steps initiated by cAMP in these nuclear events remains to be elucidated. From our present knowledge of cAMP action it appears that cAMP-dependent protein kinase, the only identified mediator of cAMP action in eukaryotic cells, forms an important link in these nuclear events. Two isoenzyme forms Abbreviations used in this paper: PEG-GMA, polyethylene glycolglycol methacrylate mix; TBS, 3% bovine serum albumin in 0.05 M Tris, pH 7.5.0.2 M NaCI. of cAMP-dependent protein kinase (type I and type II) are found in mammalian tissues in various ratios depending on the cell type (8). These tetrameric holoenzymes contain a regulatory subunit dimer (R2) and two catalytic subunits (C) (9, 10). The type I and type II isoenzymes differ only in their regulatory subunits (RI and RI1) which both are the major cAMP-binding proteins in eukaryotic cells identified so far. The potential relevance of the cAM...
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