RAC3, a steroid/nuclear receptor-associated coactivator that is related to SRC-1 and TIF2 - PubMed (original) (raw)
RAC3, a steroid/nuclear receptor-associated coactivator that is related to SRC-1 and TIF2
H Li et al. Proc Natl Acad Sci U S A. 1997.
Abstract
Steroids, thyroid hormones, vitamin D3, and retinoids are lipophilic small molecules that regulate diverse biological effects such as cell differentiation, development, and homeostasis. The actions of these hormones are mediated by steroid/nuclear receptors which function as ligand-dependent transcriptional regulators. Transcriptional activation by ligand-bound receptors is a complex process requiring dissociation and recruitment of several additional cofactors. We report here the cloning and characterization of receptor-associated coactivator 3 (RAC3), a human transcriptional coactivator for steroid/nuclear receptors. RAC3 interacts with several liganded receptors through a mechanism which requires their respective ligand-dependent activation domains. RAC3 can activate transcription when tethered to a heterologous DNA-binding domain. Overexpression of RAC3 enhances the ligand-dependent transcriptional activation by the receptors in mammalian cells. Sequence analysis reveals that RAC3 is related to steroid receptor coactivator 1 (SRC-1) and transcriptional intermediate factor 2 (TIF2), two of the most potent coactivators for steroid/nuclear receptors. Thus, RAC3 is a member of a growing coactivator network that should be useful as a tool for understanding hormone action and as a target for developing new therapeutic agents that can block hormone-dependent neoplasia.
Figures
Figure 1
RAC3 interacts with nuclear receptors. The protein–protein interactions between RAC3 and several steroid/nuclear receptors were analyzed by the yeast two-hybrid system. The original yeast two-hybrid clone, RAC3.1 in pGAD10 vector, was retransformed together with yeast expression vectors for Gal4-DBD fusion of selected hormone receptors into Y190 cells. The receptors used include RAR (full-length hRARα), RXR (full-length hRXRα), VDR (LBD of hVDR), PPAR (LBD of mPPARα), TR (LBD of hTRβ), and COUP (LBD of COUP-TFI) (h-, human; m-, murine). Three independent colonies from each transformation were selected and analyzed for expression of β-galactosidase activity by liquid _o_-nitrophenyl β-
d
-galactoside (ONPG) assay after treatment with 1 μM corresponding ligands (filled bars), or with an equal concentration of vehicle alone (open bars). Under the conditions used in these experiments, ligand treatment did not produce detectable β-galactosidase activity from the Gal4 DBD-receptor fusion alone. atRA, all-trans retinoic acid; VitD3, 1,25-dihydroxyvitamin D3; Wy, Wy 14,642; T3, 3,5,3′-triiodothyronine.
Figure 2
AF2-AD is required for interaction with RAC3. The indicated Gal4-AD fusion (RAC3.1) and Gal4-DBD fusion were cotransformed into yeast Y190 cells, and the β-galactosidase activities from three independent colonies were determined. The yeast cells were treated with 1 μM indicated ligands (filled bars) or with an equal concentration of vehicle alone (open bars). RAR403: full-length hRARα truncated at amino acid 403; RXR443, full-length hRXRα truncated at amino acid 443; VDR364, LBD of hVDR truncated at amino acid 364.
Figure 3
RAC3 contains a transcriptional activation domain. The ability of RAC3 to stimulate transcription was tested in both mammalian and yeast cells. (A) Mammalian cells. The Gal4 DBD alone (−) or its fusion with RAC3.1 or VP16 activation domain was expressed in CV-1 cells together with a luciferase reporter containing five copies of Gal4-binding sites (Gal4-tk-Luc) or the parental vector without Gal4-binding sites (tk-Luc). The relative luciferase activities are averages of three independent transfections normalized to β-galactosidase activity. (B) Yeast cells. Three independent colonies expressing Gal4 DBD fusion of RAC3.1 in Y190 cells were analyzed for their β-galactosidase activities. The average β-galactosidase units with standard deviation are shown. The control experiment (−) is β-galactosidase activity of Y190 cells transformed with Gal4 DBD alone.
Figure 4
RAC3 is related to SRC-1 and TIF2. (A) Deduced amino acid sequences of full-length RAC3. The regions corresponding to the basic helix–loop–helix (bHLH) and Per-AhR-Sim (PAS “A” and “B”) domains are shown in boxes. The C-terminal glutamine-rich (Q-rich) domain is underlined. Six LXXLL or LLXXL motifs (i to vi) at the central region are shown in boxes. (L indicates hydrophobic residues including leucine, isoleucine, and valine). The starting and ending amino acids encoded by the two-hybrid clone RAC3.1 are shown within arrows. (B) Comparison of the bHLH and PAS domains of RAC3 with TIF2, SRC-1, and other bHLH-PAS domain proteins. The white letters are conserved residues determined when more than half of residues in all sequences are similar. (C) Sequence alignment of the LXXLL motifs. The starting and ending amino acids are shown at right in parentheses. The first six motifs are surrounded by highly charged residues, and motifs ii, iv, v, and vi were predicted to form α-helical structures. (D) Schematic diagram of the domain structures of full-length human RAC3, TIF2, and SRC-1. The starting and ending residues of indicated domains are shown. The domains encoded by RAC3.1, TIF2.1, GRIP1, and SRC-1(.8) clones are indicated (arrows). The RAR-binding and p300-binding domains defined in mSRC-1 are also indicated. The numbers at the right are the length of individual proteins and the percentage similarity. The pairwise similarities were calculated to be 65% between RAC3 and TIF2, 64% between TIF2 and SRC1, and 59% between RAC3 and SRC1.
Figure 5
RAC3 enhances transcriptional activation by steroid/nuclear receptors. (A) The pCMX-F.RAC3 construct expressing full-length RAC3 was transfected into A549 cells together with a construct expressing Gal4-DBD fusion of the LBD of hRARα (Gal-RAR) and a Gal4-tk-Luc reporter. The relative luciferase activities are averages from three independent experiments after normalization to β-galactosidase activity used as internal controls for transfection efficiency. Transfected cells were treated with vehicle alone (−) or with 100 nM of all-_trans_-retinoic acid for 24 hr after transfection. Overexpression of RAC3 does not have an effect on the luciferase reporter lacking Gal4-binding sites (not shown). (B) RAC3 enhances transcriptional activation by PR on MMTV-LTR promoter. Transfected cells were treated with or without 100 nM progesterone in the presence or absence of coexpressed RAC3 or SRC-1 in CV-1 cells.
References
- Mangelsdorf D J, Evans R M. Cell. 1995;83:841–850. - PubMed
- Beato M, Herrlich P, Schültz G. Cell. 1995;83:851–857. - PubMed
- Kastner P, Mark M, Chambon P. Cell. 1995;83:859–869. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Molecular Biology Databases
Miscellaneous