Comparison of the 90-kilodalton heat shock protein interaction with in vitro translated glucocorticoid and estrogen receptors.
The rat glucocorticoid receptor is a 795-amino acid protein with the hormone binding domain located in the C-terminal portion of the molecule. In the absence of hormone, this domain displays a protein inactivation activity that represses the nuclear localization, DNA binding, and transcriptional regulatory activities of the receptor. This inactivation activity, which appears to be mediated by the 90-kilodalton heat shock protein (HSP90), is stronger in the glucocorticoid receptor than the corresponding activity of the estrogen receptor hormone binding domain. In order to analyze these differences, we have directly compared in vitro translated glucocorticoid and estrogen receptors in terms of their interaction with HSP90 by a coimmunoprecipitation assay employing two monoclonal antibodies, AC88 and 8D3, which react with different regions of the HSP90 molecule. Intact forms of both the glucocorticoid receptor and the estrogen receptor coimmunoprecipitated with endogenous HSP90 in reticulocyte lysates, indicating that both receptors were capable of binding to HSP90 when translated in vitro. By assaying a series of receptor deletion mutants, we found that the sequences required for HSP90 binding mapped to a similar region within the hormone binding domain of both receptors. While the hormone binding domain was found to be the only structural requirement for HSP90 binding to the glucocorticoid receptor, additional sequences N-terminal to the hormone binding domain were shown to be required for HSP90 binding to the estrogen receptor. These results are consistent with a postulate that differences in the protein inactivation activities of the glucocorticoid and estrogen receptor hormone binding domains may be secondary to differences in the interactions of these domains with HSP90.
Schlatter
L K
LK
Department of Medicine, Case Western Reserve University School of Medicine, University Hospitals of Cleveland, Ohio 44106.
Howard
K J
KJ
Parker
M G
MG
Distelhorst
C W
CW
D000818:Animals; D006360:Heat-Shock Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D009154:Mutation / Q000235:genetics; D011233:Precipitin Tests; D014176:Protein Biosynthesis / Q000235:genetics / Q000502:physiology; D051381:Rats; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism*; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism*
D006360:Heat-Shock Proteins; D011960:Receptors, Estrogen; D011965:Receptors, Glucocorticoid