We have achieved advances on three fronts in our continuing studies of the initial, intracellular events of glucocorticoid hormone and steroid hormone action in general. The first step in steroid hormone action is ligand binding to the receptor. With glucocorticoid receptors (GRs), this binding requires the prior association of heat shock protein 90 (hsp90), although the precise location of hsp90 contact with GR is unknown. In collaboration with Dr. Bill Pratt (Michigan), we have defined a 7 amino acid sequence at the N-terminal end of the ligand binding domain that is necessary for both steroid and hsp90 binding. The function of this sequence was neither to provide a spacer between protein domains nor to expose a protein surface by introducing a bend in an alpha-helix. Instead, these seven amino acids appear to directly or indirectly affect hsp90 binding to the GR ligand binding domain. These are the first studies to examine both steroid and hsp90 binding and open up a new avenue for defining GR-hsp90 interactions. A subsequent major step in steroid receptor action is the DNA binding of the activated complex of ligand-bound receptors. In pursuing our earlier observations that sub-populations of GR, and progesterone and estrogen receptors, display unequal DNA binding properties, we have found that this behaviour for progesterone receptors reflects unequal rates of DNA binding that can be eliminated by low concentrations of salts such as ammonium sulfate. These different rates of DNA binding by receptor sub- populations offer a new level of control for selective regulation of gene expression in intact cells. Our third development concerns continuing studies of a 21 bp sequence located at -3.6 kb of the rat tyrosine aminotransferase gene, which causes both a left shift in the dose-response curve of GR-agonist complexes and an increase the partial agonist activity of GR-antagonist complexes. We have now succeeded in cloning GMEB-2, one of the two novel proteins (GMEB-1 and -2) that bind to this element. The GMEB-2 from rat has been characterized and found to be indistinguishable from the endogenous counterpart in rat cells. The identification of GMEB- 2, along with the preparation of an antibody to GMEB-2, will facilitate our future investigations of a potentially very important means of modulating GR regulated gene activation. GMEB-2 also shares a unique domain (KDWKR) with proteins derived from diverse organisms: Drosophila (DEAF-I), Rat (Suppressin) and C. elegans (three unknown ORF's). Collectively, these data suggest that the 67 kDa GMEB-2 not only is an important factor for the modulation of GR bound to biologically active DNA sequences but also may belong to a novel family of transcription factors. Collectively, these findings contribute to our long term goal of defining the action of steroid hormones at a molecular level and of understanding their role in human physiology.