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PMID: 8035791 Published · ppublish English Comparative Study Journal Article Research Support, U.S. Gov't, P.H.S.

Differential roles of heat shock protein 70 in the in vitro nuclear import of glucocorticoid receptor and simian virus 40 large tumor antigen.

Molecular and cellular biology ·Vol. 14 ·No. 8 ·1994-08-00 ·Pages 5088-98

Yang J, DeFranco DB

Abstract

Nuclear import of glucocorticoid receptors (GRs) was analyzed in vitro with digitonin-permeabilized cells (S. A. Adam, R. Sterne-Marr, and L. Gerace, J. Cell Biol. 111:807-816, 1990). Indirect immunofluorescence methods were used to monitor the transport of GRs from rat hepatoma and fibroblast cell cytosol into HeLa nuclei. In vitro nuclear import of GRs was shown to be hormone dependent and to require ATP and incubation at ambient temperatures (i.e., 30 degrees C). Hormone-dependent dissociation of GR-bound proteins, such as the 90-kDa heat shock protein, hsp90, is part of an activation process that is obligatory for the expression of the receptor's DNA-binding activity. Inhibition of in vitro GR activation by Na2MoO4 blocked hormone-dependent nuclear import, demonstrating that receptor activation is required for nuclear import. The addition to GR-containing cytosol of antiserum directed against the cytosolic 70-kDa heat shock protein, hsp70, while effective in blocking the nuclear import of simian virus 40 large tumor antigen (SV40 TAg), did not affect hormone-dependent nuclear import of endogenous, full-length GRs or an exogenously added truncated GR protein (i.e., XGR556) that lacks a hormone-binding domain but possesses a constitutively active nuclear localization signal sequence (NLS). Depletion of hsp70 from HeLa cell cytosol did not affect the nuclear import of exogenously added XGR556 but led to inhibition of SV40 TAg nuclear import. Thus, two closely related NLSs, one contained within GRs and the other contained within SV40 TAg, are distinguished by their differential requirements for hsp70 in vitro.

MeSH Terms
Amino Acid Sequence Animals Antigens, Polyomavirus Transforming/metabolism Biological Transport Cell Compartmentation Cell Nucleus/metabolism HeLa Cells Heat-Shock Proteins/metabolism Humans In Vitro Techniques Molecular Sequence Data Rats Receptors, Glucocorticoid/metabolism Sequence Alignment Sequence Homology, Amino Acid
Chemicals
Antigens, Polyomavirus Transforming Heat-Shock Proteins Receptors, Glucocorticoid
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Yang J
Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260.
DeFranco D B
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/mcb.14.8.5088-5098.1994
Published
1994-08-00
Pages
5088-98
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC359027
Subset
IM
Grants
NCI NIH HHS · CA-43047 · United States
External Links
PubMed source DOI: 10.1128/mcb.14.8.5088-5098.1994 PMC Full Text View journal details
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