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J. Biol. Chem., Vol. 265, Issue 33, 20123-20130, 11, 1990
ER Sanchez, M Hirst, LC Scherrer, HY Tang, MJ Welsh, JM Harmon, SS Simons Jr, GM Ringold and WB Pratt
In this work, we examine the cellular localization and protein interactions
of mouse glucocorticoid receptors that have been overexpressed in Chinese
hamster ovary (CHO) cells (Hirst, M. A., Northrop, J. P., Danielsen, M.,
and Ringold, G. M. (1990) Mol. Endocrinol. 4, 162-170). We demonstrate that
wild-type unliganded mouse glucocorticoid receptor, which is expressed in
CHO cells to a level approximately 10 times that of L cells, is localized
entirely to the nucleus by indirect immunofluorescence with the BuGR
antireceptor monoclonal antibody. Overexpressed receptors that have either
no hormone binding activity or no DNA binding activity because of point
mutations also localize to the nucleus, providing genetic proof that the
nuclear localization cannot reflect a steroid-mediated shift of the
receptor from the cytoplasm to the nucleus and that DNA binding activity is
not required for nuclear localization. Like unliganded progesterone
receptors, which also associate in a loosely bound "docking" complex with
the nucleus, the mouse glucocorticoid receptor overexpressed in CHO cells
is associated with both hsp90 and hsp70. This is in contrast to the
untransformed mouse glucocorticoid receptor in L cell cytosol, which is
associated with hsp90 but not hsp70. The difference in hsp70 association
between cell types could reflect overexpression of the receptor in CHO
cells. However, like receptors in CHO cells selected for very high levels
of overexpression, receptors in CHO cells selected for an intermediate
level of receptor expression that is comparable to that of L cells are also
bound to hsp70. This observation argues against an explanation of hsp70
association based purely on receptor overexpression, and we speculate that
association of the unliganded glucocorticoid receptor with hsp70 might be a
consequence of its nuclear localization in the CHO cells. Although there
are differences between the mouse receptor in CHO cells and L cells, the
nuclear localization signal of the untransformed mouse receptor reacts
equivalently with the AP64 antibody against NL1 in cytosols prepared from
both cell types.
Hormone-free mouse glucocorticoid receptors overexpressed in Chinese hamster ovary cells are localized to the nucleus and are associated with both hsp70 and hsp90
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109.
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