Protein-protein interactions are implied in glucocorticoid receptor mutant 465*-mediated cell death.

Hong Chen,E Brad Thompson,Ganesan Srinivasan

doi:10.1074/jbc.272.41.25873

Abstract

Previously we have shown that ICR-27, a clone of glucocorticoid-resistant human leukemic T cells, showed rapid cell loss upon transient transfection with plasmids expressing certain fragments of the human glucocorticoid receptor lacking the ligand binding domain. An extreme example was the frameshift GR mutant 465*, mutated after amino acid 465. This generated a novel 21-amino acid "tail," beginning within the second zinc finger of the human glucocorticoid receptor DNA binding domain, a region required for ICR-27 cell death caused by hologlucocorticoid receptor plus hormone. The cell loss mediated by 465* was faster but quantitatively equivalent to that caused by hologlucocorticoid receptor plus hormone. We are therefore investigating the mechanism of action of 465*. We overexpressed 465* with or without a glutathione S-transferase tag fused to its N terminus and tested its ability to affect glucocorticoid response element (GRE)-driven reactions in vitro. Partially purified 465* showed little binding to a consensus GRE, caused virtually no stimulation of transcription from a GRE, and failed to inhibit GR-driven transcription. However, GST-465* "trapped" several proteins from ICR-27 cell extracts, including c-Jun; recombinant c-Jun also was bound in vitro. In co-transfection assays of CV-1 cells, 465* expression reduced phorbol ester (12-O-tetradecanoylphorbol-13-acetate) transcriptional activation from a promoter containing multiple AP-1 sites. Further studies proved the repressive activity of 465* was c-Jun-specific and not due to squelching artifacts. The data suggest that interaction of 465* with other proteins, such as c-Jun, might be responsible for its cell killing function.

Highlights

Glucocorticoids exert their effects on target cells by binding to an intracellular GR1 [1]
Transfection of 465* caused cell loss only in the FIG. 12. 465* does not activate estrogen response element (ERE)-chloramphenicol acetyltransferase (CAT), nor does it interfere with ER-mediated activation of ERE-CAT
CV-1 cells were cotransfected with 2 ␮g of ERE-CAT reporter plasmid, 1 ␮g of internal control RSV-␤-galactosidase vector, and 2 ␮g of human estrogen receptor (hER) or pRSh465* or the combination of both as indicated

Summary

Introduction

Glucocorticoids exert their effects on target cells by binding to an intracellular GR1 [1]. In the course of transfection experiments aimed at mapping the regions of the hGR required for its lethal effect, we discovered that certain fragments of the receptor could reduce the number of viable test cells independently of added steroid [13]. All of these hGR mutants lacked a large portion of their C termini, such that the entire LBD was missing. In our assay system, 465* was fully as active in reducing cell numbers as other C-terminal truncated hGR mutants that contained the entire DBD along with varying amounts of the hinge region. We show that the 465* fragment interacts with c-Jun and interferes with its site-specific regulation of transcription

Methods

Results

Conclusion