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Demonstration by Confocal Microscopy that Unliganded Overexpressed Glucocorticoid Receptors are Distributed in a Nonrandom Manner throughout All Planes of the Nucleus

Department of Pharmacology, University of South Carolina School of Medicine Columbia, South Carolina 29208
Department of Anatomy, Cell Biology, and Neurosciences, University of South Carolina School of Medicine Columbia, South Carolina 29208
Department of Pharmacology, University of Michigan Medical School Ann Arbor, Michigan 48109
Institute of Cancer and Developmental Biology, Syntex, Inc. Palo Alto, California 94304

Address requests for reprints to: Paul R. Housley, Department of Pharmacology, University of South Carolina School of Medicine, Room D-6, Building 1, Columbia, South Carolina 29208.

Abstract

Mouse glucocorticoid receptors (GR) that are overexpressed in Chinese hamster ovary (CHO) cells behave like progesterone receptors, in that the unliganded receptor localizes to the nucleus where it resides in a loosely bound docking complex, probably in association with the 90-kDa heat shock protein (hsp90) and hsp70. In this paper we examine the localization of the overexpressed GR within the CHO cell nucleus by confocal microscopy. In hormone- free cells the receptor distributes in a mottled pattern throughout all planes of the nucleus. The receptor is not present in nucleoli and shows no preferential localization in the periphery vs. the center of the nucleus. The mottled distribution in each plane of the nucleus demonstrates clearly that there are regions that do not contain receptor; thus, the distribution of the GR is not random. When triamcinolone acetonide is added to the CHO cells, there is no detectable change in receptor distribution. Overexpressed receptors that have either no ormonebinding activity or no DNA-binding activity because of point mutations localize in the same mottled pattern as the wild-type receptor. These observations are consistent with the proposal that the overexpressed GR can enter the nucleus in its unliganded state and proceed to loci distributed throughout the nucleus, where it is retained in an inactive docking complex until the binding of hormone triggers its progression to high affinity sites where the primary events in transcriptional activation occur. As there is no detectable change in localization with the addition of ligand, we suggest that the docking complex may be located very near or possibly at the site where the primary events in transcriptional activation occur.

FOOTNOTES

This work was supported by a fellowship grant from the Interamerican Development Bank and Universidade de Sao Paulo (to V.R.M.), American Cancer Society Grant IN-107 (to P.R.H.), and NIH Grants DK-36905 (to P.R.H.), CA-28010 (to W.B.P.), HL-40424 (to L.T.), and GM-25821 (to G.M.R.).

^* Present address: Faculdade de Medicina, Disciplina de Oncologia, Universidade de Sao Paulo, Sao Paulo, Brazil.

Received for publication October 16, 1990. Revision received November 26, 1990. Accepted for publication November 29, 1990.

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