Nuclear import of the glucocorticoid receptor: From plasma membrane glucocorticoid binding sites to nuclear localization signal binding proteins and import models.

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Title: Nuclear import of the glucocorticoid receptor: From plasma membrane glucocorticoid binding sites to nuclear localization signal binding proteins and import models.
Authors: LaCasse, Eric C.
Date: 1994
Abstract: Relatively little is known about the molecular mechanisms involved in the import of proteins into the nucleus. This lack of knowledge extends to he mode of nuclear entry of steroid-receptor complexes and modulation of steroid hormone action during its passage across the nuclear envelope. The main goal of this thesis was to identify the molecular machinery that interacts with the nuclear localization signals located in steroid hormone receptors. Synthetic peptides corresponding to the glucocorticoid and thyroid hormone receptor nuclear localization signals were radioiodinated and incubated with cytosol, high salt- and detergent-extracted rat liver nuclei or nuclear envelopes in the presence of a crosslinking agent. Two specifically labeled polypeptides of 60 and 76 kDa were identified with both synthetic peptides in all fractions by autoradiography after SDS-Page. The two general conclusions drawn from my data are: first, that these two specifically labeled polypeptides may act as shuttling vectors in nuclear transport; and second, the binding sites may be part of a general mechanism for nuclear entry of most nuclear proteins. Nuclear import of proteins is comprised of two steps, the first step being the binding of the protein to he outer nuclear envelope and nuclear pore complex, and the second step being the translocation of the protein through the nuclear pore complex into the nucleus. I established an in vitro system, which consist of isolated rat liver nuclei, capable of the first step but not the second step, to study glucocorticoid receptor nuclear binding. I have also demonstrated that digitonin-permeabilized cells can be used to study both steps (binding and import) of the glucocorticoid receptor translocation into nuclei in an ATP-dependent manner similar to other nuclear proteins. These in vitro models will allow us to further define the mechanisms involved in the nuclear import of the glucocorticoid receptor, particularly the hormone-induced nuclear translocation of the receptor, and to establish the role of the 60- and 76-kDa polypeptides in nuclear import. The ligand-induced nuclear translocation of the glucocorticoid receptor is dependent on the intracellular hormone concentration which is influenced by membrane binding sites for the glucocorticoid ligand. With the affinity label, dexamethasone 21-mesylate, I have identified a 45-kDa microsomal membrane site that displays the characteristics of a low affinity glucocorticoid binder. This protein may limit the amount of free hormone for the receptor and therefore blunt the action of the steroid hormone in the nucleus.
URL: http://hdl.handle.net/10393/6470
http://dx.doi.org/10.20381/ruor-11291
CollectionTh├Ęses, 1910 - 2010 // Theses, 1910 - 2010
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