Distinct retinoid X receptor activation function-2 residues mediate transactivation in homodimeric and vitamin D receptor heterodimeric contexts

P. D. Thompson, L. S. Remus, Jui-Cheng Hsieh, P. W. Jurutka, G Kerr Whitfield, M. A. Galligan, C. Encinas Dominguez, C. A. Haussler, Mark R Haussler

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Abstract

The vitamin D receptor (VDR) stimulates transcription as a 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-activated heterodimer with retinoid X receptor (RXR). RXR also forms homodimers to mediate 9-cis retinoic acid (9-cis RA)-induced gene expression. Both receptors possess a C-terminal hormone-dependent activation function-2 (AF-2), a highly conserved region that binds coactivators to transduce the transcriptional signal. By replacing single amino acids within the AF-2 of human RXRα (hRXRa) or mouse RXRβ (mRXRβ), the contribution of these residues to transactivation by the RXR-VDR heterodimer and the RXR-RXR homodimer was evaluated. In 9-cis RA-responsive homodimers, the second and fourth positions of the AF-2 (leucine and glutamate respectively) are essential. However, in the context of an RXR-VDR heterodimer activated by 1,25(OH)2D3, alteration of these two RXR residues has little effect. Instead, AF-2 residues located towards the C-terminus, such as the penultimate position (L455 in hRXRα or L441 in mRXRβ), are crucial for RXR-VDR heterodimers. Indeed, L455A mutant RXR exerts a dominant negative effect on RXR-VDR transcriptional responsiveness to 1,25(OH)2D3. Further experiments with a mutant hRXRα (F313A) which elicits 9-cis RA-independent transactivation as a homodimer demonstrate that, when heterodimerized with VDR, this RXR mutant is incapable of activating the RXR-VDR heterocomplex in the absence of the VDR ligand. Taken together, these results indicate that RXR is a subordinate, yet essential transcriptional partner in RXR-VDR-mediated activation of gene expression. Furthermore, a functional switch in RXR AF-2 signaling occurs between RXR residues in the homodimeric versus the heterodimeric states, likely reflecting different interactions between subregions of the AF-2 and coactivator(s).

Original languageEnglish (US)
Pages (from-to)211-227
Number of pages17
JournalJournal of Molecular Endocrinology
Volume27
Issue number2
DOIs
StatePublished - 2001

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Retinoid X Receptors
Calcitriol Receptors
Transcriptional Activation
Aminoacylation
Gene Expression

ASJC Scopus subject areas

  • Endocrinology

Cite this

Distinct retinoid X receptor activation function-2 residues mediate transactivation in homodimeric and vitamin D receptor heterodimeric contexts. / Thompson, P. D.; Remus, L. S.; Hsieh, Jui-Cheng; Jurutka, P. W.; Whitfield, G Kerr; Galligan, M. A.; Encinas Dominguez, C.; Haussler, C. A.; Haussler, Mark R.

In: Journal of Molecular Endocrinology, Vol. 27, No. 2, 2001, p. 211-227.

Research output: Contribution to journalArticle

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AU - Jurutka, P. W.

AU - Whitfield, G Kerr

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AB - The vitamin D receptor (VDR) stimulates transcription as a 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-activated heterodimer with retinoid X receptor (RXR). RXR also forms homodimers to mediate 9-cis retinoic acid (9-cis RA)-induced gene expression. Both receptors possess a C-terminal hormone-dependent activation function-2 (AF-2), a highly conserved region that binds coactivators to transduce the transcriptional signal. By replacing single amino acids within the AF-2 of human RXRα (hRXRa) or mouse RXRβ (mRXRβ), the contribution of these residues to transactivation by the RXR-VDR heterodimer and the RXR-RXR homodimer was evaluated. In 9-cis RA-responsive homodimers, the second and fourth positions of the AF-2 (leucine and glutamate respectively) are essential. However, in the context of an RXR-VDR heterodimer activated by 1,25(OH)2D3, alteration of these two RXR residues has little effect. Instead, AF-2 residues located towards the C-terminus, such as the penultimate position (L455 in hRXRα or L441 in mRXRβ), are crucial for RXR-VDR heterodimers. Indeed, L455A mutant RXR exerts a dominant negative effect on RXR-VDR transcriptional responsiveness to 1,25(OH)2D3. Further experiments with a mutant hRXRα (F313A) which elicits 9-cis RA-independent transactivation as a homodimer demonstrate that, when heterodimerized with VDR, this RXR mutant is incapable of activating the RXR-VDR heterocomplex in the absence of the VDR ligand. Taken together, these results indicate that RXR is a subordinate, yet essential transcriptional partner in RXR-VDR-mediated activation of gene expression. Furthermore, a functional switch in RXR AF-2 signaling occurs between RXR residues in the homodimeric versus the heterodimeric states, likely reflecting different interactions between subregions of the AF-2 and coactivator(s).

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