Glucocorticoid receptor phosphorylation and interaction with the 90-kDa heat shock protein.

Abstract

In this dissertation, two mechanisms of regulation of glucocorticoid receptor (GR) function were investigated: (1) phosphorylation of the GR and (2) interaction of the GR with the 90 kilodalton heat shock protein (hsp90). In order to study receptor phosphorylation, immunopurified mouse GR from L cells grown in the presence of $\sp{32}$P was digested with proteases and fragments were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis, autoradiography and Western-blotting. The receptor is endogenously phosphorylated only on serine residues. From examination of $\sp{32}$P-labeled receptor fragments, I have deduced that one phosphate is located between amino acid residues 398 and 442, a region containing the BuGR epitope and about one third of the DNA binding domain, and the remaining three phosphates appear to be clustered just to the amino-terminal side of the BuGR epitope in a region defined by residues 313 to 369. In order to study the interaction of the GR with the hsp90, I translated GR, GR derivatives, and thyroid hormone receptor (TR) in both reticulocyte lysate and in wheat germ extract. GR synthesized in reticulocyte lysate is immunoadsorbed by the 8D3 monoclonal antibody directed against the hsp90 and the GR has a normal ability to bind glucocorticoid in a high affinity manner. GR is immunoadsorbed by 8D3 as soon as the translation products are full length. When newly synthesized receptor is bound with steroid and incubated at 25$\sp\circ$C, it is converted to a form that binds to DNA. In contrast to the GR, the reticulocyte lysate translated TR is not associated with hsp90 and it is synthesized in its DNA binding form without any requirement for transformation. When the GR is translated in wheat germ extract, which contains no detectable hsp90, it is translated in its DNA binding form in the same manner as the TR synthesized in reticulocyte lysate. These observations provide direct evidence that binding of GR to hsp90 is an early event and that binding of GR to hsp90 is associated with repression of receptor DNA binding capacity. Translation of GR deletion mutants in reticulocyte lysate followed by immunoadsorption by 8D3 identifies amino acid residues 395-659 of the mouse GR as sufficient and residues 604-659 as possibly required for GR binding to hsp90. In addition synthetic peptides corresponding to residues 587-606 (peptide B) and 637-665 (peptide E) of the mouse GR block association of mouse GR with hsp90 in reticulocyte lysates. These observations support a model in which hsp90 binds to the GR at a minimum of two sites with one site composed of residues corresponding to peptide B and another site composed of residues corresponding to peptide E.