Evidence that the beta-isoform of the human glucocorticoid receptor does not act as a physiologically significant repressor.
Alternative splicing of the human glucocorticoid receptor (hGR) primary transcript generates two receptor isoforms, hGRalpha and hGRbeta, with different carboxyl termini diverging at amino acid 727. By reverse transcriptase-polymerase chain reactions it was previously demonstrated that the hGRbeta message had a widespread tissue distribution. To demonstrate the presence of hGRbeta as protein we produced specific rabbit antisera to hGRbeta, as well as a hGRbeta-specific mouse monoclonal IgM antibody, by peptide immunizations. By SDS-polyacrylamide gel electrophoresis and Western immunoblotting we showed that hGRbeta is endogenously expressed at the protein level in HeLa cells and human lymphatic leukemia cells. Using an antibody directed against an epitope shared by both isoforms we showed a relatively lower expression of the hGRbeta form. We also showed that hGRbeta bound to hsp90 by immunoprecipitation of in vitro translated hGRbeta in reticulocyte lysate with hsp90-specific antibodies, a coprecipitation occurring also in the presence of dexamethasone. We could not demonstrate that hGRbeta inhibited the effects of dexamethasone-activated hGRalpha on a glucocorticoid-responsive reporter gene. In conclusion, low hGRbeta expression levels and hGRbeta-hsp90 interaction maintained in the presence of ligand and lack of inhibition of hormone-activated hGRalpha effects challenge the concept of the hGRbeta isoform as a proposed dominant negative inhibitor of hGRalpha activity.
Hecht
K
K
Department of Medical Nutrition, Karolinska Institute, Huddinge University Hospital, Novum, S-141 86 Huddinge, Sweden.
Carlstedt-Duke
J
J
Stierna
P
P
Gustafsson
J
J
Brönnegârd
M
M
Wikström
A C
AC
D017398:Alternative Splicing; D000818:Animals; D019556:COS Cells; D003907:Dexamethasone / Q000494:pharmacology; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D012097:Repressor Proteins / Q000502:physiology*
D018841:HSP90 Heat-Shock Proteins; D012333:RNA, Messenger; D011965:Receptors, Glucocorticoid; D012097:Repressor Proteins; D003907:Dexamethasone