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Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists
Published on: November 15, 2013
FLASH interacts with p160 coactivator subtypes and differentially suppresses transcriptional activity of steroid
Tomoshige Kino1, Takamasa Ichijo, George P Chrousos
1Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Development, Building 10, Clinical Research Center, Room 1-3140 , 10 Center Drive, MSC 1109, Bethesda, MD 20892-1109, USA. kinot@mail.nih.gov
Abstract:
We previously reported that tumor necrosis factor alpha receptor- and Fas-associated FLASH interacts with one of the p160 nuclear receptor coactivators, glucocorticoid receptor-interacting protein (GRIP) 1, at its nuclear receptor-binding (NRB) domain, and that inhibits the transcriptional activity of the glucocorticoid receptor (GR) by interfering with association of GR and GRIP1. Here, we further examined the specificity of FLASH suppressive effect and the physical/functional interactions between this protein and two other p160 family subtypes. The suppressive effect of FLASH on GR transactivation was observed in several cell lines and on the chromatin-integrated mouse mammary tumor virus (MMTV) promoter. FLASH strongly interacted with the NRB domain of the thyroid hormone receptor activator molecule (TRAM) 1, a member of the steroid hormone receptor coactivator (SRC) 3/nuclear receptor coactivator (N-CoA) 3 subtypes, as well as with SRC2/N-CoA2 p160 coactivator GRIP1, while its interaction with SRC1a, one of the SRC1/N-CoA1 proteins, was faint in yeast two-hybrid assays. Accordingly, FLASH strongly suppressed TRAM1- and GRIP1-induced enhancement of GR-stimulated transactivation of the MMTV promoter in HCT116 cells, while it did not affect SRC1a-induced potentiation of transcription. Furthermore, FLASH suppressed androgen- and progesterone receptor-induced transcriptional activity, but did not influence estrogen receptor-induced transactivation, possibly due to their preferential use of p160 coactivators in HCT116 and HeLa cells. Thus, FLASH differentially suppresses steroid hormone receptor-induced transcriptional activity by interfering with their association with SRC2/N-CoA2 and SRC3/N-CoA3 but not with SRC1/N-CoA1.
Insights
Tumor necrosis factor receptor-associated FLASH protein specifically inhibits steroid hormone receptor activity. It interferes with coactivators SRC2 and SRC3, but not SRC1, affecting gene transcription.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Nuclear receptors (NRs) regulate gene expression, requiring coactivators for activity.
- p160 family coactivators, including GRIP1 (SRC2), TRAM1 (SRC3), and SRC1, are crucial for NR function.
- FLASH (Fas-associated death domain-like interleukin-1β-converting enzyme-associated protein) is a known inhibitor of glucocorticoid receptor (GR) activity.
Purpose of the Study:
- To investigate the specificity of FLASH's inhibitory effect on nuclear receptor transcriptional activity.
- To elucidate the physical and functional interactions between FLASH and different p160 coactivator subtypes (SRC1, SRC2, SRC3).
Main Methods:
- Yeast two-hybrid assays to assess protein-protein interactions.
- Reporter gene assays using the mouse mammary tumor virus (MMTV) promoter in cell lines (HCT116, HeLa).
- Assessment of FLASH's effect on the transactivation activity of various steroid hormone receptors (GR, androgen receptor, progesterone receptor, estrogen receptor).
Main Results:
- FLASH strongly interacted with the nuclear receptor-binding (NRB) domains of GRIP1 (SRC2) and TRAM1 (SRC3), but weakly with SRC1.
- FLASH significantly suppressed GR, androgen receptor, and progesterone receptor transcriptional activity, mediated by SRC2 and SRC3.
- FLASH did not inhibit estrogen receptor-mediated transactivation or transcription enhanced by SRC1.
Conclusions:
- FLASH exhibits differential suppression of steroid hormone receptor activity based on coactivator usage.
- FLASH specifically interferes with the recruitment of SRC2 and SRC3 coactivators, but not SRC1, to nuclear receptors.
- This differential interaction mechanism highlights the specificity of FLASH as a regulator of nuclear receptor signaling.
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