Abstract-The armadillo-related protein ␤-catenin has multiple functions in cardiac tissue homeostasis: stabilization of ␤-catenin has been implicated in adult cardiac hypertrophy, and downregulation initiates heart formation in embryogenesis. The protein is also part of the cadherin/catenin complex at the cell membrane, where depletion might result in disturbed cell-cell interaction similar to N-cadherin knockout models. Here, we analyzed the in vivo role of ␤-catenin in adult cardiac hypertrophy initiated by angiotensin II (Ang II). The cardiac-specific mifepristone-inducible ␣MHC-CrePR1 transgene was used to induce ␤-catenin depletion (loxP-flanked exons 3 to 6, ␤-cat ⌬ex3-6 mice) or stabilization (loxP-flanked exon 3, ␤-cat ⌬ex3 mice). Levels of ␤-catenin were altered both in membrane and nuclear extracts. Analysis of the ␤-catenin target genes Axin2 and Tcf-4 confirmed increased ␤-catenin-dependent transcription in ␤-catenin stabilized mice. In both models, transgenic mice were viable and healthy at age 6 months. ␤-Catenin appeared dispensable for cell membrane function. Ang II infusion induced cardiac hypertrophy both in wild-type mice and in mice with ␤-catenin depletion. In contrast, mice with stabilized ␤-catenin had decreased cross-sectional area at baseline and an abrogated hypertrophic response to Ang II infusion. Stabilizing ␤-catenin led to impaired fractional shortening compared with control littermates after Ang II stimulation. This functional deterioration was associated with altered expression of the T-box proteins Tbx5 and Tbx20 at baseline and after Ang II stimulation. In addition, atrophy-related protein IGFBP5 was upregulated in ␤-catenin-stabilized mice. These data suggest that ␤-catenin downregulation is required for adaptive cardiac hypertrophy. (Circ Res.

In recent years, it has become clear that in many proteins, significant regions are encoded by amino acid sequences that do not automatically fold into their fully condensed, functional structures. Characterization of the conformational propensities and function of the nonglobular protein sequences represents a major challenge. Striking among proteins with unfolded regions are numbers of transcription factors, including steroid receptors. In many cases, the unfolded or partially folded regions of such proteins take shape when the protein interacts with its proper binding partner(s), that is, the molecules to which it must bind to carry out its function. The AF1 domain of the androgen receptor (AR) shows little structure, when expressed as a recombinant peptide. It has been shown previously that AF1 interacts with transcription factor TFIIF in vitro. Using Fourier transform infrared (FTIR), we tested whether this interaction can induce structure in the AR AF1. Our results demonstrate that the recombinant AR AF1 can acquire significantly higher helical content after interacting with RAP74, a subunit of the TFIIF complex. We further show that this induced conformation in the AR AF1 is well-suited for its interaction with SRC-1.

The actions of the male sex hormones testosterone and dihydrotestosterone are mediated by the intracellular androgen receptor (AR) 1 (reviewed in Refs. 1 and 2). In the absence of hormone, the receptor is sequestered in the cytosol with molecular chaperone proteins, which dissociate upon hormone binding. The hormone-bound receptor translocates to the nucleus and is targeted to specific genes through the recognition and binding to the DNA response element, 5Ј-AGA/TACA/TnnnT/ AGTTCT/C-3Ј, which in turn leads to activation of gene transcription (3-10). The activated receptor also represses gene expression through protein-DNA interactions at negative response elements (11,12) or through interactions with other transcription factors (13)(14)(15)(16)(17).In addition to the well characterized DNA-binding domain (DBD) and ligand-binding domain (LBD), regions of the proteins important for transactivation have been mapped to the amino-terminal domain (NTD; 18 -21). These studies have revealed a modular nature for the AR-transactivation domain, with the region between amino acids 142 and 485, containing the TAU-1/AF-1 and TAU-5/AF-5 determinants, being critical for receptor-dependent activation (20, 21). Sequences within the AR-NTD have been shown to mediate protein-protein interactions with the carboxyl-terminal LBD (22-28), the general transcription factors TFIIF (29) and TFIIH (30), members of the p160 family of nuclear receptor coactivator proteins (31-34), and the general coactivator CREB-binding protein (35,36).TFIIF is a tetramer of two subunits, RAP30 and RAP74. TFIIF recruits TFIIE and TFIIH to the preinitiation complex (PIC) and interacts directly with the RNA polymerase II enzyme and prevents pausing of the enzyme during subsequent transcription elongation (37-39). Previously, we have demonstrated that the isolated transactivation function of the human AR, amino acids 142 to 485, interacts with the large subunit of TFIIF, termed RAP74, and that this interaction was capable of reversing AR-dependent squelching of basal transcription under cell-free conditions (29). More recently, we have shown that binding of RAP74 results in the AR-transactivation domain adopting a protease-resistant conformation (40).In the present study we have extended these observations to map the region(s) of RAP74 involved in this interaction with the AR. Using a series of deletion constructs of RAP74 we show that sequences within both the amino-and carboxyl-terminal domains of the protein are sufficient to bind the AR-transactivation function and to reverse receptor-dependent squelching of transcription. In the context of the holo-TFIIF, the carboxylterminal binding site may be the main binding site. Introduction of point mutations into the AR-transactivation domain revealed that sequences near the amino terminus are important for RAP74 binding. These mutations fail to disrupt the interaction of the AR with the p160 coactivator protein SRC-1a. Thus, TFIIF and SRC-1a interact with distinct regions of the AR-transactivation domain. The impl...

In Saccharomyces cerevisiae, the SUP70 gene encodes the CAG-decoding tRNAGlnCUG. A mutant allele, sup70-65, induces pseudohyphal growth on rich medium, an inappropriate nitrogen starvation response. This mutant tRNA is also a UAG nonsense suppressor via first base wobble. To investigate the basis of the pseudohyphal phenotype, 10 novel sup70 UAG suppressor alleles were identified, defining positions in the tRNAGlnCUG anticodon stem that restrict first base wobble. However, none conferred pseudohyphal growth, showing altered CUG anticodon presentation cannot itself induce pseudohyphal growth. Northern blot analysis revealed the sup70-65 tRNAGlnCUG is unstable, inefficiently charged, and 80% reduced in its effective concentration. A stochastic model simulation of translation predicted compromised expression of CAG-rich ORFs in the tRNAGlnCUG-depleted sup70-65 mutant. This prediction was validated by demonstrating that luciferase expression in the mutant was 60% reduced by introducing multiple tandem CAG (but not CAA) codons into this ORF. In addition, the sup70-65 pseudohyphal phenotype was partly complemented by overexpressing CAA-decoding tRNAGlnUUG, an inefficient wobble-decoder of CAG. We thus show that introducing codons decoded by a rare tRNA near the 5′ end of an ORF can reduce eukaryote translational expression, and that the mutant tRNACUGGln constitutive pseudohyphal differentiation phenotype correlates strongly with reduced CAG decoding efficiency.