Activation of a Delayed-Early Gene Encoding MHR3 by the Ecdysone Receptor Heterodimer EcR-B1–USP-1 but Not by EcR-B1–USP-2
Open accessAbstract
MHR3, a homolog of the retinoid orphan receptor (ROR), is a transcription factor in the nuclear hormone receptor family that is induced by 20-hydroxyecdysone (20E) in the epidermis of the tobacco hornworm, Manduca sexta. Its 2.7-kb 5' flanking region was found to contain four putative ecdysone receptor response elements (EcREs) and a monomeric (GGGTCA) nuclear receptor binding site. Activation of this promoter fused to a chloramphenicol acetyltransferase (CAT) reporter by 2 micrograms of 20E per ml in Manduca GV1 cells was similar to that of endogenous MHR3, with detectable CAT by 3 h. When the ecdysone receptor B1 (EcR-B1) and Ultraspiracle 1 (USP-1) were expressed at high levels under the control of a constitutive promoter, CAT levels after a 3-h exposure to 20E increased two- to sixfold. In contrast, high expression of EcR-B1 and USP-2 caused little increase in CAT levels in response to 20E. Moreover, expression of USP-2 prevented activation by EcR-B1-USP-1. Deletion experiments showed that the upstream region, including the three most proximal putative EcREs, was responsible for most of the 20E activation, with the EcRE3 at -671 and the adjacent GGGTCA being most critical. The EcRE1 at -342 was necessary but not sufficient for the activational response but was the only one of the three putative EcREs to bind the EcR-B1-USP-1 complex in gel mobility shift assays and was responsible for the silencing action of EcR-B1-USP-1 in the absence of hormone. EcRE2 and EcRE3 each specifically bound other protein(s) in the cell extract, but not EcR and USP, and so are not EcREs in this cellular context. When cell extracts were used, the EcR-B1-USP-2 heterodimer showed no binding to EcRE1, and the presence of excess USP-2 prevented the binding of EcR-B1-USP-1 to this element. In contrast, in vitro-transcribed-translated USP-1 and USP-2 both formed heterodimeric complexes with EcR-B1 that bound ponasterone A with the same Kd (7 x 10(-10) M) and bound to both EcRE1 and heat shock protein 27 EcRE. Thus, factors present in the cell extract appear to modulate the differential actions of the two USP isoforms.
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20E regulates EcRB1/USP1 heterodimer binding to EcRE to regulate gene transcription.
“20E regulates EcRB1/USP1 heterodimer binding to EcRE to regulate gene transcription [36].”
From G-protein-coupled receptor participates in 20-hydroxyecdysone signaling on the plasma membrane (2014) · cites this paper as [36]
In M. sexta, one ecdysone response element (EcRE1, GGGGTCAATGAACCG) was identified in 20E reporter gene hormone receptor 3 (HR3).
“In M. sexta, one ecdysone response element (EcRE1, GGGGTCAATGAACCG) was identified in 20E reporter gene hormone receptor 3 (HR3).”
From G-protein-coupled receptor participates in 20-hydroxyecdysone signaling on the plasma membrane (2014) · cites this paper as [36]
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- Authors
- Lan, Q., Hiruma, K., Hu, X., Jindra, M., & Riddiford, L. M.
- Affiliations
- University of Washington (US)
- Indiana University Bloomington (US)
- Corresponding author
- Riddiford, Lynn M.
- Venue
- Molecular and Cellular Biology
- Publisher
- Informa UK Limited
- Published
- 1999-07-01
- Volume
- 19
- Issue
- 7
- Pages
- 4897-4906
- ISSN
- 0270-7306 (ISSN-L)
- 1098-5549
- PMC
- PMC84291
- OpenAlex
- W1951209055
- Cited by
- 107
- References
- 60
- Citations by year
- 2025: 2 · 2023: 3 · 2022: 2 · 2020: 1 · 2019: 3 · 2018: 1 · 2017: 2 · 2016: 5 · 2015: 4 · 2014: 10 · 2013: 2 · 2012: 2
- FWCI
- 3.37
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Molecular and Cellular Biology (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- UNI ScholarWorks (University of Northern Iowa) (submittedVersion)
- Funders
- American Cancer Society
- National Institute of Allergy and Infectious Diseases (AI2459)
- Topics
- Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neurobiology and Insect Physiology Research
- Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Nuclear Receptors and Signaling
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Estrogen and related hormone effects
- Keywords
- ecdysone receptor, ultraspiracle, 20-hydroxyecdysone, transcriptional activation, heterodimer, nuclear receptor binding sites, Manduca sexta, epidermis, promoter regulation
- MeSH terms
- Animals, Base Sequence, Cell Line, DNA-Binding Proteins, Ecdysone, Ecdysterone, Gene Expression Regulation, Molecular Sequence Data, Promoter Regions, Genetic, Receptors, Steroid, RNA, Transcription Factors, Genes, Insect, DNA, Complementary, Manduca, Dimerization, Protein Isoforms, Drosophila Proteins
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