GH1 cells are a clonal strain of rat pituitary tumor cells which synthesize GH and PRL. We have previously demonstrated that these cells respond to physiological concentrations of L-T3 and L-T4 when cultured with medium supplemented with thyroidectomized calf serum to achieve a thyroid hormone-depleted state under cell culture conditions. In this study, we describe a method to deplete euthyroid calf serum of L-T3 and L-T4 using an anion exchange resin. We demonstrate that the procedure only minimally alters the low molecular weight anion components of the serum and does not change the total protein content or the electrophoretic pattern of serum proteins. Moreover, we show that euthyroid calf serum depleted of L-T3 and L-T4 by this procedure yields serum which, when used as a medium supplement, results in biological responses identical to those obtained with media supplemented with thyroidectomized calf serum. In addition, resin treatment does not alter the growth-promoting properties of the serum if the thyroid hormone concentration is restored. This procedure should be useful in preparing thyroid hormone-depleted serum for cell culture studies in situations where thyroidectomy is not feasible or would require surgical procedures on a large number of small animals.

The nuclear hormone receptors comprise a superfamily of ligand-modulated transcription factors that regulate homeostasis, reproduction, development, and differentiation. Three amino acids within the zinc finger DNA binding motif determine target gene specificity. Groups of receptors exist with similar DNA binding specificity. A complex carboxy terminal region mediates ligand binding, dimerization, and hormone-relieved transcriptional inactivation. We summarize the current understanding of these phenomena and suggest a novel model that structurally and functionally links these events. This "regulatory zipper model" may explain the mechanism by which ligand activates nuclear hormone receptors.

We describe the cloning and characterization of a new family of nuclear receptor coregulators (NRCs) which modulate the function of nuclear hormone receptors in a ligand-dependent manner. NRCs are expressed as alternatively spliced isoforms which may exhibit different intrinsic activities and receptor specificities. The NRCs are organized into several modular structures and contain a single functional LXXLL motif which associates with members of the steroid hormone and thyroid hormone/retinoid receptor subfamilies with high affinity. Human NRC (hNRC) harbors a potent N-terminal activation domain (AD1), which is as active as the herpesvirus VP16 activation domain, and a second activation domain (AD2) which overlaps with the receptorinteracting LXXLL region. The C-terminal region of hNRC appears to function as an inhibitory domain which influences the overall transcriptional activity of the protein. Our results suggest that NRC binds to liganded receptors as a dimer and this association leads to a structural change in NRC resulting in activation. hNRC binds CREB-binding protein (CBP) with high affinity in vivo, suggesting that hNRC may be an important functional component of a CBP complex involved in mediating the transcriptional effects of nuclear hormone receptors.Nuclear hormone receptors comprise a superfamily of ligand-dependent transcription factors involved in controlling diverse cellular processes, including growth, differentiation, development, and homeostasis (37). The nuclear hormone receptor superfamily includes type I receptors which mediate the effects of glucocorticoids (glucocorticoid receptor , and the PPARs. These receptors share a similar modular structure consisting of an Nterminal A/B domain, a DNA-binding C domain, and a D, E, and F ligand-binding domain (LBD) (4, 37). The DNA-binding C domain is highly conserved among members of type I and type II nuclear hormone receptors. Although the LBDs of nuclear hormone receptors (ϳ300 amino acids) are diverse in sequence, accounting for ligand specificity, they exhibit certain similarities in their overall structure (4, 13). Thus, the LBDs of all nuclear receptors are organized into 12 helical regions which play an important role in determining the conformation of the LBD in the presence and absence of ligand (59) and in mediating heterodimerization of type II receptors with the RXRs (2, 13).[Ligand-dependent conformational changes in the LBD are thought to recruit coactivators or coregulators to the DNAbound receptor, which leads to transcriptional activation (37). The activation function mediated through the LBD has been referred to as activation function 2 (AF2) (38, 54). The majority of the coactivators, identified in a yeast two-hybrid screen, fall into two main groups: the p160/SRC family (SRC-1 (5,20,28). Coactivators which fall outside these groups include PGC-1 (44), ARA70 (62), p/CAF (3, 60), and NRIF3, which exhibits specificity for only the TRs and the RXRs (33). Using a biochemical approach, another class of factors have been identified...