Three distinct forms of the mouse glucocorticoid receptor (GC-R) have been characterized. The untransformed receptor (9.1S; Rs = 8.3 nm; Mr = 319K) is an oligomeric protein which does not bind to DNA or nuclei. After hormone-binding, two transformed (DNA- and nuclear-binding) receptor forms can be generated, apparently by subunit dissociation. One of these is an oligomeric protein (5.2S; Rs = 6-8.3 nm; Mr = 132-182K), while the other is the GC-R monomer (3.8S; RS = 6 nm; Mr = 96K). Recent studies have shown that receptor transformation (subunit dissociation) apparently involves the dephosphorylation of some cytosolic substance. Additional studies indicated that the 5.2S transformed GC-R may be composed of a receptor monomer (96K) plus a low molecular weight RNA (21-36K). The experiments proposed here are directed at three basic questions. First, what are the actual molecular weights of the oligomeric receptor forms? The oligomeric GC-R species (9.1S, 5.2S) will be covalently affinity-labeled with [3H]dexamethasone mesylate, following by covalent cross-linking of the receptor subunits (either chemically or with UV-irradiation). The molecular weights of the receptor oligomers will then be determined hydrodynamically (S and Rs) and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This should allow an accurate determination of the molecular weights of the GC-R oligomers under denaturing conditions, and allow conclusions to be drawn about their subunit composition. The second question addresses the relevance and structure of the RNA to which the GC-R binds. We will determine if the RNA is present in the 9.1S, oligomeric, untransformed GC-R using a variety of techniques, including a direct analysis for the presence of RNA in purified, untransformed GC-R preparations. These studies may elucidate the possible role of RNA in the structure and function of the receptor. We will also characterize and, if possible, determine the sequence of the RNA. The last question deals with the role of phosphorylation/dephosphorylation in the transformation state of the GC-R. We will attempt to reconstitute the 9.1S untransformed GC-R by incubating the transformed GC-R and determine if dephosphorylating it causes receptor transformation. All of these studies should be helpful in elucidating the structure of the GC-R and the molecular mechanisms of transformation to its DNA-binding (gene regulatory) form.
| Ali, M; Vedeckis, W V (1990) Heat shock-induced loss of the glucocorticoid receptor protein in cultured cells. Receptor 1:121-32 |
| Vedeckis, W V; Ali, M; Allen, H R (1989) Regulation of glucocorticoid receptor protein and mRNA levels. Cancer Res 49:2295s-2302s |
