The human glucocorticoid receptor (bGR) gene is transcribed into two alternative splicing products, the classic, ligand-binding hGRa and a nonligand-binding isofonn, hGRB, which have the same first 727 amino acids but differ in the C-terminus. We recently demonstrated that bGRβ mRNA is expressed in virtually all human tissues and that this isofonn is a dominant negative inhibitor of hGRa in a COS-7 co-transfection system. To examine the presence and define the quantity of hGR6 protein in human tissues and to better understand its mechanism of action, we produced and characterized new polyclonal antibodies directed against specific C-terminal region sequences of the a and βisofonns. Using these affinity purified antibodies, we demonstrated the presence of both isoforms in a panel of human tissues and HcLa cells. In the latter, both hGRa and hGRB translocated into the nucleus after addition of dexamethasone. These data suggested that hGRB, like hGRa, was anchored to hsp90 as part of a cytoplasmic neterooligomer, and was probably released from the hsp complex by forming heteroduncrs with ligand-bound hGRa. Indeed, an antibody against hsp90 crossprecipitated both the hGRa and hGRβ isofonns, while both isoform-specific antibodies precipitated "activated" cytosolic and nuclear glucocorticoid receptors and hsp90. By increasing the expression of the βisofonn in HeLa cells through transfection of an expression vector, we observed a decrease in the dexamethasone-iixluced activation of co-transfected MMTV-luc, suggesting that the β isofonn could inhibit the effects of endogenous hGRa. We conclude that the nonligand -binding hGRβ isoform is widely expressed throughout the human body and present in the cytoplasm of human cells, in complex with hsp90 and other proteins. In the presence of glucocorticoid, hGRβ helerodimerizes with ligand-bound hGRa and translocates into the nucleus to act as a dominant negative inhibitor of the classic receptor. We suggest that hGRβ isofonn may be a determinant of target tissue sensitivity to glucocorticoids and may participate in patbophysiologic states of giucocorticoid resistance or hypersensitivity.
|Journal||Journal of Investigative Medicine|
|Publication status||Published - 1 Jan 1996|
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)