Ligand-dependent interactions of coactivators steroid receptor coactivator-1 and peroxisome proliferator-activated receptor binding protein with nuclear hormone receptors can be imaged in live cells and are required for transcription

Llopis, Juan; Westin, Stefan; Ricote, Mercedes; Wang, Jiahong; Cho, Charles Y.; Kurokawa, Riki; Mullen, Tina-Marie; Rose, David W.; Rosenfeld, Michael G.; Tsien, Roger Y.; Glass, Christopher K.

doi:10.1073/pnas.97.8.4363

Cited by 135 publications

(105 citation statements)

References 32 publications

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“…By contrast, TRAP220 failed to enhance ER␣-mediated transcription of the 3ERE-TATA-LUC reporter gene (Fig 1B). Nonetheless, chromatin immunoprecipitation experiments have suggested that TRAP/DRIP proteins are recruited to NR-regulated promoters, including ER␣-and AR-responsive promoters (51,55,56), and microinjection of HeLa cell nuclei with anti-TRAP220/PBP antibodies down-regulated ER␣ activation of a reporter gene (57). In addition, a recent report has shown that the TRAP complex can be purified from HeLa cell nuclear extracts using GST-ER␣ LBD in the presence of agonist ligand (50).…”

Section: Trap220 Nuclear Receptor Binding Specificitymentioning

confidence: 99%

An Extended LXXLL Motif Sequence Determines the Nuclear Receptor Binding Specificity of TRAP220

Coulthard¹,

Matsuda

Heery

2003

Journal of Biological Chemistry

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The interaction of coactivators with the ligand-binding domain of nuclear receptors (NRs) is mediated by amphipathic ␣-helices containing the signature motif LXXLL. TRAP220 contains two LXXLL motifs (LXM1 and LXM2) that are required for its interaction with NRs. Here we show that the nuclear receptor interaction domain (NID) of TRAP220 interacts weakly with Class I NRs. In contrast, SRC1 NID binds strongly to both Class I and Class II NRs. Interaction assays using nine amino acid LXXLL core motifs derived from SRC1 and TRAP220 revealed no discriminatory NR binding preferences. However, an extended LXM1 sequence containing amino acids ؊4 to ؉9, (where the first conserved leucine is ؉1) showed selective binding to thyroid hormone receptor and reduced binding to estrogen receptor. Replacement of either TRAP220 LXXLL motif with the corresponding 13 amino acids of SRC1 LXM2 strongly enhanced the interaction of the TRAP220 NID with the estrogen receptor. Mutational analysis revealed combinatorial effects of the LXM1 core and flanking sequences in the determination of the NR binding specificity of the TRAP220 NID. In contrast, a mutation that increased the spacing between TRAP220 LXM1 and LXM2 had little effect on the binding properties of this domain. Thus, a 13-amino acid sequence comprising an extended LXXLL motif acts as the key determinant of the NR binding specificity of TRAP220. Finally, we show that the NR binding specificity of full-length TRAP220 can be altered by swapping extended LXM sequences.

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Section: Trap220 Nuclear Receptor Binding Specificitymentioning

confidence: 99%

An Extended LXXLL Motif Sequence Determines the Nuclear Receptor Binding Specificity of TRAP220

Coulthard¹,

Matsuda

Heery

2003

Journal of Biological Chemistry

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“…interaction with cofactors that allow chromatin remodeling and recruitment of the basic transcription machinery) also possibly influence nuclear receptor mobility. With that respect, FRET has emerged as a technique to characterize the mechanisms of cofactor recruitment following ligand binding in living cells (31,32).…”

mentioning

confidence: 99%

Fluorescence Imaging Reveals the Nuclear Behavior of Peroxisome Proliferator-activated Receptor/Retinoid X Receptor Heterodimers in the Absence and Presence of Ligand*♦

Feige

Gelman

Tudor

et al. 2005

Journal of Biological Chemistry

119

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In a global approach combining fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and fluorescence resonance energy transfer (FRET), we address the behavior in living cells of the peroxisome proliferator-activated receptors (PPARs), a family of nuclear receptors involved in lipid and glucose metabolism, inflammation control, and wound healing. We first demonstrate that unlike several other nuclear receptors, PPARs do not form speckles upon ligand activation. The subnuclear structures that may be observed under some experimental conditions result from overexpression of the protein and our immunolabeling experiments suggest that these structures are subjected to degradation by the proteasome. Interestingly and in contrast to a general assumption, PPARs readily heterodimerize with retinoid X receptor (RXR) in the absence of ligand in living cells. PPAR diffusion coefficients indicate that all the receptors are engaged in complexes of very high molecular masses and/or interact with relatively immobile nuclear components. PPARs are not immobilized by ligand binding. However, they exhibit a ligand-induced reduction of mobility, probably due to enhanced interactions with cofactors and/or chromatin. Our study draws attention to the limitations and pitfalls of fluorescent chimera imaging and demonstrates the usefulness of the combination of FCS, FRAP, and FRET to assess the behavior of nuclear receptors and their mode of action in living cells.

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“…FRET was quantified on the basis of the acceptor photobleaching method, developed for laser-scanning confocal microscopy [28][29][30]. HEK293T cells were co-transfected with different pairs of ECFP and EYFP fusion protein expression vectors.…”

Section: Fret Analysis Of Living Cellsmentioning

confidence: 99%

“…The efficiency of FRET was calculated as E=(D post - [28][29][30][31], where D pre is defined as the average fluorescence intensity of seven pre-images of the ROI, and D post is calculated as the average intensity of seven post-images. It is assumed that FRET did not occur where D post was less than D pre .…”

Section: Fret Analysis Of Living Cellsmentioning

confidence: 99%

Visualization of bHLH transcription factor interactions in living mammalian cell nuclei and developing chicken neural tube by FRET

et al. 2006

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Ligand-dependent interactions of coactivators steroid receptor coactivator-1 and peroxisome proliferator-activated receptor binding protein with nuclear hormone receptors can be imaged in live cells and are required for transcription

Cited by 135 publications

References 32 publications

An Extended LXXLL Motif Sequence Determines the Nuclear Receptor Binding Specificity of TRAP220

An Extended LXXLL Motif Sequence Determines the Nuclear Receptor Binding Specificity of TRAP220

Fluorescence Imaging Reveals the Nuclear Behavior of Peroxisome Proliferator-activated Receptor/Retinoid X Receptor Heterodimers in the Absence and Presence of Ligand*♦

Visualization of bHLH transcription factor interactions in living mammalian cell nuclei and developing chicken neural tube by FRET

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