Abstract
Transcriptional coactivators are involved in gene activation by nuclear hormone receptors. The receptor-associated coactivator 3 (RAC3) was recently identified to be highly related to the steroid receptor coactivator-1 and transcriptional intermediate factor 2, thereby establishing a novel family of nuclear receptor coactivators. In this study, we identified a RAC3 fragment containing three LXXLL motifs conserved among this family, which is sufficient to mediate nuclear receptor interaction in vivo and in vitro. Point mutations that disrupt ligand-dependent activation function of the receptor inhibited the interaction. We found that a 162-amino acid fragment of RAC3 conferred transcriptional activation and recruited the CREB-binding protein and that three distinct LXXLL motifs mediated the transcriptional activation. A trimeric far Western analysis demonstrated the formation of a ternary complex containing CREB-binding protein, RAC3, and the receptor. In addition, we showed that RAC3, transcriptional intermediate factor 2, and steroid receptor coactivator-1 are expressed in specific tissues and cancer cells and that RAC3 transcript is directly up-regulated by retinoid treatment. These results suggest that RAC3 may contribute to amplified transcriptional responses through both recruitment of additional coactivators and autoregulation by the receptor-coactivator complex.
Highlights
Transcriptional regulation by nuclear receptors for steroids, thyroid hormones (TR),1 retinoids (RAR), and vitamin D3 controls key aspects of animal development, reproduction, homeostasis, and adult organ physiology
We showed that receptorassociated coactivator 3 (RAC3) interacts directly with CBP through its activation domain and that RAC3 can form a bridge for the interaction between CBP and nuclear receptors, suggesting that one mechanism of transcriptional activation by these coactivators is recruitment of additional cofactors
Significant interactions were observed with liganded RAR, TR, and estrogen receptor (Fig. 1C), demonstrating that this receptor-interacting domain (RID) domain mediates a ligand-dependent interaction between RAC3 and nuclear receptors
Summary
Transcriptional regulation by nuclear receptors for steroids, thyroid hormones (TR), retinoids (RAR), and vitamin D3 controls key aspects of animal development, reproduction, homeostasis, and adult organ physiology (for reviews, see Refs. 1–5). The steroid/nuclear receptor coactivator gene family contains the steroid receptor coactivator-1 (SRC-1; known as NCoA-1) (34 –38), the transcriptional intermediate factor 2 (TIF2, known as GRIP1) (39 – 41), and the receptor-associated coactivator 3 (RAC3, known as ACTR, p/CIP, and AIB1) [42,43,44]. Sequence comparison of these proteins reveals that they share an overall identity of about 40%, but with striking similarity at the N-terminal basic helix-loop-helix (bHLH) and period-aryl hydrocarbon receptor-single minded (PAS) “A” and “B” domains. The bHLH-PAS domain function in protein-protein interactions, heterodimerization, and target gene selection in many members of this family [45,46,47], but the
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