Abstract
The nuclear matrix is a putative skeletal structure which has been implicated in many nuclear functions. To assess a possible role of the nuclear matrix in glucocorticoid action, purified rat liver nuclei containing glucocorticoid-receptor complexes were treated with DNase I +/- RNase A followed by 1.6 M NaCl, thus yielding salt-extractable and salt-resistant (nuclear matrix) fractions. The subnuclear distribution of hormone-receptor complexes was determined by following the fate of unmetabolized radiolabel after injection of labeled triamcinolone acetonide into adrenalectomized animals and subjecting various subfractions to immunoblotting using a monoclonal antibody which recognizes the glucocorticoid receptor. Both techniques indicated that 50-70% of the total nuclear hormone-receptor complexes were recovered in the nuclear matrix fraction. Previous results (Kaufmann, S. H., and Shaper, J. H. (1984) Exp. Cell Res. 155, 477-495) suggest that a variety of nuclear polypeptides become nuclease- and salt-resistant as a result of the formation of intermolecular disulfide bonds. The following evidence suggests that disulfide bonds mediate the association between the glucocorticoid receptor and the nuclear matrix. When nuclei were isolated in the absence of sulfhydryl-blocking and -cross-linking reagents, sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions revealed that the receptor was present as a high molecular weight disulfide-cross-linked complex. When nuclei were isolated in the presence of the irreversible sulfhydryl-blocking reagent iodoacetamide, the disulfide bonds which cross-linked the receptor into high molecular weight complexes were absent; and 85-100% of the hormone-receptor complexes were salt-extractable. When nuclei (isolated in the absence of iodoacetamide) were treated with the sulfhydryl-cross-linking reagent sodium tetrathionate, greater than 95% of the nuclear hormone-receptor complexes became resistant to extraction with nucleases and 1.6 M NaCl. The implications of these results for other matrix-associated nuclear functions are discussed.
Highlights
The nuclear matrix is a putative skeletal structure According to one current model, the eukaryotic nucleus is which has been implicated in many nuclear functionso. rganized around a proteinaceoustructural framework
- mechanism by which one portion of the glucocorticoid recep- 2 300 tors become nuclease- and salt-resistantwhile another portion E 200 remains salt-extractable? We demonstrate that theglucocorticoid receptor becomes nuclease- and salt-resistant concomitant with the formation of intermolecular disulfide bonds in nuclei
RNA but diminished only slightly the nuclei containing the receptor (Fig. 2, see Fig. 4 A, lane amount of nuclease- andsalt-resistant binding (Fig. 3A). 1)were treated with DNase I and RNase A followed by 1.6 M. These results indicatethat theglucocorticoid receptor can be NaCl, approximately equal amounts of the immunoreactive recovered in the nuclear matrix fraction and suggest further 94-kDa polypeptide were detected in the 1.6 M NaCl extract that RNA does not mediate the association of the glucocorti- (Fig. 4 A, lane 2) and the1.6 M NaC1-insolublepellet
Summary
The subnuclear distributioonf hormone-receptor complexes was determined by (i) folgenerally isolated by treating purified nuclei with nucleases followed by polyanions or buffers of high ionic strength Other investigators have been unable to whichrecognizes the glucocorticoid receptor Both demonstrate inhigher eukaryoticorganismsintranuclear techniques indicated that 50-70% of the total nuclear components which are intrinsically resistant tonucleases and hormone-receptorcomplexeswererecoveredin the buffers of low or high ionic strength It is not clear that the salt-resistant glucocorticoid-binding sites previously described in nuclei from rat liver (39-41), hepatoma tissue culture cells (42), rat thymocytes (43), rat intestine (44), mouse fibroblasts (451, and mouse lymphoma cells (38, 46) are associated with the nuclear matrix. We demonstrate that theglucocorticoid receptor becomes nuclease- and salt-resistant concomitant with the formation of intermolecular disulfide bonds in nuclei. Aliquots of purified nuclei were treated with DNase I k
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