Role of Important Hydrophobic Amino Acids in the Interaction between the Glucocorticoid Receptor τ1-Core Activation Domain and Target Factors ² Tova Almlo ¨f,* Annika E. Wallberg, Jan-A ° ke Gustafsson, and Anthony P. H. Wright Department of Biosciences, Karolinska Institute, NoVum, S-141 57 Huddinge, Sweden ReceiVed December 10, 1997; ReVised Manuscript ReceiVed March 25, 1998 ABSTRACT: In this work, we determined how altered-function mutants affecting hydrophobic residues within the τ1-core activation domain of the human glucocorticoid receptor (GR) influence its physical interaction with different target proteins of the transcriptional machinery. Screening of putative target proteins showed that the τ1-core can interact with the C-terminal part of the CREB-binding protein (CBP). In addition, the previously identified interactions of the τ1-core with the TATA-binding protein (TBP) and the Ada2 adaptor protein were localized to the C- and N-terminal regions of these proteins, respectively. A panel of mutations within the τ1-core that either decrease or increase activation potential was used to probe the interaction of the τ1-core domain with TBP, Ada2, and CBP. We found that the pattern of effects caused by the mutations was similar for each of the interactions and that the effects on binding generally reflected effects on gene activation potential. Thus, the predominant effect of the mutations appears to influence a property of the τ1-core that is common to all three interactions, rather than properties that are differentially required by each of the target factor interactions, individually. Such a property could be the ability of the domain to adopt a folded conformation that is generally necessary for interaction with target factors. We have also shown that TBP, Ada2, and CBP can interact with both the τ1-core and the GR ligand-binding domain, offering a possible mechanism for synergistic interaction between the τ1-core and other receptor activation domains. However, other target proteins (e.g., RIP140, and SRC-1), which interact with the GR C terminus, did not show significant interactions with the τ1-core under our conditions. The GR 1 is a member of a large family of ligand inducible nuclear receptors (1, 2). This receptor mediates the effects of glucocorticoids by regulating transcription of target genes. Binding of glucocorticoids causes dissociation of the recep- tor from an inactive complex containing heat shock pro- teins, allowing it to bind to DNA. The receptor binds as a homodimer to specific glucocorticoid response elements (GREs) present within genes regulated in response to glucocorticoids. Transcriptional activation by GR is medi- ated mainly by two transactivation functions, τ1 (residues 77-262) located in the N-terminal part of the receptor and τ2 (residues 526-556) in the C-terminal part of the receptor (3, 4). An additional, highly conserved region located at the very C terminus of the receptor may also be involved in transcriptional activation (5). Among these transactivation domains, the τ1 domain constitutes the major transactivation function (4). This domain also functions in isolation when fused to the DNA-binding domains of either the GR (6) or the LexA repressor protein (7). The τ1 domain contains a 58 amino acid peptide, the τ1-core, that retains almost all the τ1 activity when assayed in yeast (8). However, struc- tural studies using circular dichroism and nuclear magnetic resonance spectroscopy have shown that the τ1 domain and the τ1-core are largely unstructured in aqueous solution (9). Thus, as seen in a number of other proteins (10-13), a very small segment of the GR protein with poor propensity for structure formation still functions as a powerful mediator of gene activation. It is possible that unstructured activation domains con- tribute to gene activation via relatively nonspecific ionic or hydrophobic interactions with target proteins, but, in the case of the GR τ1-core domain, the phenotypes of amino acid substitution mutants that alter activity are critically dependent on their position within the protein (7). This observation is more easily interpreted in the context of a structured acti- vation domain. Consistent with this view, the τ1-core does adopt an R-helical conformation in the nonpolar solvent tri- fluoroethanol (9). Proline substitution mutants within the predicted R-helical segments reduce the activity of the τ1- core, suggesting that these putative R-helices may play a role in vivo (14). One possibility is that a structured conformation of the τ1-core is induced or stabilized during interaction with target proteins. Three recent reports support this model more directly in the case of the activation domains of the mam- malian activator, c-myc (13), and the herpes simplex virus protein, VP16 (15, 16). The mechanism by which DNA-bound activator proteins stimulate transcription is poorly understood, but it is thought to involve direct or indirect contacts between activation ² This work was supported by the Swedish Natural Science Research Council (Grants K-AA/KU9756-308 and I-AA/LS9756-307) and the Swedish Medical Research Council (Grant 13x-2819). * To whom correspondence should be addressed. Telephone: +46- 8-608 9158. Fax: +46-8-774 5538. E-mail: tova.almlof@cbt.ki.se. 1 Abbreviations: GR, glucocorticoid receptor; CREB, cAMP re- sponse element-binding protein; CBP, CREB-binding protein; TBP, TATA-binding protein; GREs, glucocorticoid responsive elements; GST, glutathione S-transferase; IPTG, isopropyl 1-thio--D-galactose; AF-2, activation function-2. 9586 Biochemistry 1998, 37, 9586-9594 S0006-2960(97)03029-8 CCC: $15.00 © 1998 American Chemical Society Published on Web 06/11/1998