Glucocorticoid receptor phosphorylation in mouse L-cells

This paper summarizes our observations on the phosphorylation state of untransformed and transformed glucocorticoid receptors isolated from 32P-labeled L-cells. The 300-350-kDa 9S untransformed murine glucocorticoid receptor complex is composed of a 100-kDa steroid-binding phosphoprotein and one or possibly two units of the 90-kDa heat shock protein (hsp90), which is also a phosphoprotein. Transformation of this complex to the 4S DNA-binding state is accompanied by dissociation of hsp90. When receptors in cytosol are transformed by heating at 25[deg]C, there is no gross change in the degree of phosphorylation of the steroid-binding protein. Both receptors that are bound to DNA after transformation under cell-free conditions and receptors that are located in the nucleus of cells incubated at 37[deg]C in the presence of glucocorticoid are labeled with 32P. The results of experiments in which the 32P-labeled receptor was submitted to limited proteolysis suggest that the 16-kDa DNA-binding domain is phosphorylated and that the 28-kDa steroid-binding domain is not.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26917/1/0000483.pd

Glucocorticoid Receptor Transformation and Dna Binding.

The overall goal of this thesis is to probe the mechanism whereby glucocorticoid receptors are transformed from a non-DNA-binding form to their active DNA-binding form. In the first portion of the work, I have examined the effect of an endogenous inhibitor purified from rat liver cytosol on receptor binding to DNA. The inhibitor binds to transformed receptors in whole cytosol and prevents their binding to DNA. Although the inhibitor is not a protease, I have shown that the ability of the inhibitor preparation to cause the apparent release of receptors from DNA is due to a contaminating serine protease. In the second portion of this thesis, I have examined the role of sulfhydryl groups in determining the DNA binding activity of the transformed receptor and in determining the transformation process. Treatment of rat liver cytosol containing temperature-transformed, ('3)H dexamethasone-bound receptors at 0(DEGREES)C with the sulfhydryl modifying reagent methyl methanethiosulfonate (MMTS) inhibits the DNA-binding activity of the receptor, and DNA-binding activity is restored after addition of dithiothreitol (DTT). Incubation of rat liver cytosol containing untransformed glucocorticoid-receptor complexes at 25(DEGREES)C with hydrogen peroxide prevents their transformation to the DNA-binding form as shown by their inability to bind to DNA-cellulose after addition of DTT. This inhibition of transformation is however reversible, as the peroxide-treated receptors will bind to DNA if they are warmed a second time in the presence of DTT. These results strongly suggest that sulfhydryl moieties on the receptor must be reduced for temperature-mediated transformation to occur and that sulfhydryl moieties are absolutely required for the receptor to bind to DNA. In the last portion of this thesis, I have examined the relationship between receptor phosphorylation and DNA binding. Untransformed receptor complexes purified from cytosol prepared from mouse L cells grown in medium containing ('32)P orthophosphate contain two components, a 100 k-Da and a 90-kDa subunit, both of which are phosphoproteins. On transformation, the receptor dissociates from the 90-kDa protein. Transformation of the complex under cell free conditions does not result in a dephosphorylation of the 100-kDa steroid-binding protein. Transformed receptor that has been bound to DNA and purified by monoclonal antibody is still in a phosphorylated form. These results suggest that dephosphorylation is not required for receptor binding to DNA.Ph.D.PharmacologyUniversity of Michiganhttp://deepblue.lib.umich.edu/bitstream/2027.42/161333/1/8702846.pd