In cell free lysates steroid receptors may exist as large heterooligomers (nontransformed receptors). A number of treatments may affect transformation, a process which releases the receptor from adhering protein. This process frees the DNA binding domain, allowing the receptor to bind to hormone regulatory elements of sensitive genes. Quite recently it has been noted that receptors stimulate gene action constituitively with the ability of a steroid to induce an effect residing in/or adjacent to the hormone binding domain. Thus to understand the ability of a steroid to effect a response the protein components of the non-transformed 9S receptor must be identified. At present, several associated proteins have been detected and they include 90 kDa, and 70 kDa heat shock proteins, a 59 kDa protein termed p59, smaller proteins of 50, 35, and 26 kDa. However, the assembly of the proteins and the steroid receptors is unknown. The objective of this proposal is to delineate the effect, if any, of growth factors and the interactions of the associated proteins, including heat shock proteins, p59, with mammalian progesterone and estrogen receptors. Furthermore we shall ascertain if the growth factors have any effect on associated protein phosphorylation. We shall accomplish this by purifying steroid free non- transformed receptors from both cytosol and nuclei. In some instances the receptor complexes shall be covalently crosslinked. Both crosslinked and native nontransformed receptors will be subjected to sequential immunoaffinity chromatography and steroid affinity chromatography. Each of the major forms will be isolated and analyzed by sucrose density gradient ultracentrifugation, high performance liquid chromatography, one- and two- dimensional electrophoresis and immunoblotting.
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| Tai, P K; Chang, H; Albers, M W et al. (1993) P59 (FK506 binding protein 59) interaction with heat shock proteins is highly conserved and may involve proteins other than steroid receptors. Biochemistry 32:8842-7 |
| Tai, P K; Albers, M W; Chang, H et al. (1992) Association of a 59-kilodalton immunophilin with the glucocorticoid receptor complex. Science 256:1315-8 |
