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A neuronal-specific differentiation protein that directly modulates retinoid receptor transcriptional activation.

Henry II KW, Spencer ML, Theodosiou M, Lou D, Noonan DJ - Nucl. Recept. (2003)

Bottom Line: Expression of NPDC-1 was also observed to repress transcription mediated by retinoid receptors as well as by several other nuclear receptor family members, although not in a universal manner.CONCLUSIONS: These results suggest that NPDC-1, through direct interaction with retinoid receptors, functions to enhance the transcription complex formation and DNA binding function of retinoid receptors, but ultimately repress retinoid receptor-mediated gene expression.As with NPDC-1, retinoids and their receptors have been implicated in brain development and these data provide a point of convergence for NPDC-1 and retinoid mediation of neuronal differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky, 800 Rose Street, Lexington, KY 40536, USA. dnoonan@pop.uky.edu

ABSTRACT
BACKGROUND: The specificity of a nuclear receptor's ability to modulate gene expression resides in its ability to bind a specific lipophilic ligand, associate with specific dimerization partners and bind specific DNA sequences in the promoter regions of genes. This sequence of events appears to be the basis for targeting an additional regulatory complex composed of a variety of protein and RNA components that deliver signals for facilitation or inhibition of the RNA polymerase complex. Characterization of the tissue and cell-specific components of these coregulatory complexes appear to be integral to our understanding of nuclear receptor regulation of transcription. RESULTS: A novel yeast screen sensitive to retinoid-X receptor (RXR) transcriptional activation resulted in the isolation of the rat homologue of the mouse NPDC-1 gene. NPDC-1 has been shown to be involved in the control of neural cell proliferation and differentiation, possibly through interactions with the cell cycle promoting transcription factor E2F-1. Although the amino acid sequence of NPDC-1 is highly conserved between mouse, rat and human homologues, their tissue specific expression was seen to vary. A potential for direct protein:protein interaction between NPDC-1, RXR and retinoic acid receptor beta (RARbeta) was observed in vitro and NPDC-1 facilitated RXR homodimer and RAR-RXR heterodimer DNA binding in vitro. Expression of NPDC-1 was also observed to repress transcription mediated by retinoid receptors as well as by several other nuclear receptor family members, although not in a universal manner. CONCLUSIONS: These results suggest that NPDC-1, through direct interaction with retinoid receptors, functions to enhance the transcription complex formation and DNA binding function of retinoid receptors, but ultimately repress retinoid receptor-mediated gene expression. As with NPDC-1, retinoids and their receptors have been implicated in brain development and these data provide a point of convergence for NPDC-1 and retinoid mediation of neuronal differentiation.

No MeSH data available.


Related in: MedlinePlus

RXR, RAR and E2F-1 proteins form complexes with hNPDC-1 In Vivo PC12 cells were transfected with pCMV-HA-E2F-1, pRS-hRARβ, or pRS-hRXRα and incubated for 48 hours. Subsequently clarified cell lysates were prepared, and incubated for with a 50% slurry of Trx-S-hNPDC beads (bacterially expressed hNPDC fused at its carboxyl terminus with S-peptide and thioredoxin tags and affinity purified by binding to S-protein agarose beads as described in the Methods section). Ligands, atRA and 9cRA were diluted to a concentration of 1 μM into clarified lysate before the addition hNPDC. Beads were collected, washed, bound protein was eluted from beads with 2X SDS electrophoresis sample buffer and proteins were separated on 12.5% SDS-PAGE gels. Proteins were transferred to nitrocellulose membranes and probed for the presence of HA-tagged E2F-1, RARβ, or RXRα••••••••••••••
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Figure 4: RXR, RAR and E2F-1 proteins form complexes with hNPDC-1 In Vivo PC12 cells were transfected with pCMV-HA-E2F-1, pRS-hRARβ, or pRS-hRXRα and incubated for 48 hours. Subsequently clarified cell lysates were prepared, and incubated for with a 50% slurry of Trx-S-hNPDC beads (bacterially expressed hNPDC fused at its carboxyl terminus with S-peptide and thioredoxin tags and affinity purified by binding to S-protein agarose beads as described in the Methods section). Ligands, atRA and 9cRA were diluted to a concentration of 1 μM into clarified lysate before the addition hNPDC. Beads were collected, washed, bound protein was eluted from beads with 2X SDS electrophoresis sample buffer and proteins were separated on 12.5% SDS-PAGE gels. Proteins were transferred to nitrocellulose membranes and probed for the presence of HA-tagged E2F-1, RARβ, or RXRα••••••••••••••

Mentions: To test if human NPDC-1 could interact with in vivo expressed E2F-1, RARβ or RXRα, recombinant Trx-S-NPDC-1 was used to pull-down protein from mammalian cell lysates (Fig. 4). PC12 cells were transfected with pCMV-HA-E2F-1, pRS-hRARβ, or pRS-hRXRα and incubated for 48 hours. Clarified cell lysates were prepared, and incubated for 1 hour at 4°C with a 50% slurry of Trx-S-NPDC beads. Ligands, atRA and 9cRA were diluted to a concentration of 1 micromolar into clarified lysate before the addition of recombinant hNPDC-1. Beads were collected, washed with binding buffer and incubated with S-protein agarose bound to pull out thioredoxin-S-tagged NPDC-1. Bound protein was eluted from beads with electrophoresis sample buffer, run on 12.5% SDS-PAGE gels, Western blotted to nitrocellulose membranes and probed with specific antisera for the presence of HA-tagged E2F-1 (Fig. 4A), RARβ, or RXRα ••••• 4B).


A neuronal-specific differentiation protein that directly modulates retinoid receptor transcriptional activation.

Henry II KW, Spencer ML, Theodosiou M, Lou D, Noonan DJ - Nucl. Recept. (2003)

RXR, RAR and E2F-1 proteins form complexes with hNPDC-1 In Vivo PC12 cells were transfected with pCMV-HA-E2F-1, pRS-hRARβ, or pRS-hRXRα and incubated for 48 hours. Subsequently clarified cell lysates were prepared, and incubated for with a 50% slurry of Trx-S-hNPDC beads (bacterially expressed hNPDC fused at its carboxyl terminus with S-peptide and thioredoxin tags and affinity purified by binding to S-protein agarose beads as described in the Methods section). Ligands, atRA and 9cRA were diluted to a concentration of 1 μM into clarified lysate before the addition hNPDC. Beads were collected, washed, bound protein was eluted from beads with 2X SDS electrophoresis sample buffer and proteins were separated on 12.5% SDS-PAGE gels. Proteins were transferred to nitrocellulose membranes and probed for the presence of HA-tagged E2F-1, RARβ, or RXRα••••••••••••••
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC222963&req=5

Figure 4: RXR, RAR and E2F-1 proteins form complexes with hNPDC-1 In Vivo PC12 cells were transfected with pCMV-HA-E2F-1, pRS-hRARβ, or pRS-hRXRα and incubated for 48 hours. Subsequently clarified cell lysates were prepared, and incubated for with a 50% slurry of Trx-S-hNPDC beads (bacterially expressed hNPDC fused at its carboxyl terminus with S-peptide and thioredoxin tags and affinity purified by binding to S-protein agarose beads as described in the Methods section). Ligands, atRA and 9cRA were diluted to a concentration of 1 μM into clarified lysate before the addition hNPDC. Beads were collected, washed, bound protein was eluted from beads with 2X SDS electrophoresis sample buffer and proteins were separated on 12.5% SDS-PAGE gels. Proteins were transferred to nitrocellulose membranes and probed for the presence of HA-tagged E2F-1, RARβ, or RXRα••••••••••••••
Mentions: To test if human NPDC-1 could interact with in vivo expressed E2F-1, RARβ or RXRα, recombinant Trx-S-NPDC-1 was used to pull-down protein from mammalian cell lysates (Fig. 4). PC12 cells were transfected with pCMV-HA-E2F-1, pRS-hRARβ, or pRS-hRXRα and incubated for 48 hours. Clarified cell lysates were prepared, and incubated for 1 hour at 4°C with a 50% slurry of Trx-S-NPDC beads. Ligands, atRA and 9cRA were diluted to a concentration of 1 micromolar into clarified lysate before the addition of recombinant hNPDC-1. Beads were collected, washed with binding buffer and incubated with S-protein agarose bound to pull out thioredoxin-S-tagged NPDC-1. Bound protein was eluted from beads with electrophoresis sample buffer, run on 12.5% SDS-PAGE gels, Western blotted to nitrocellulose membranes and probed with specific antisera for the presence of HA-tagged E2F-1 (Fig. 4A), RARβ, or RXRα ••••• 4B).

Bottom Line: Expression of NPDC-1 was also observed to repress transcription mediated by retinoid receptors as well as by several other nuclear receptor family members, although not in a universal manner.CONCLUSIONS: These results suggest that NPDC-1, through direct interaction with retinoid receptors, functions to enhance the transcription complex formation and DNA binding function of retinoid receptors, but ultimately repress retinoid receptor-mediated gene expression.As with NPDC-1, retinoids and their receptors have been implicated in brain development and these data provide a point of convergence for NPDC-1 and retinoid mediation of neuronal differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky, 800 Rose Street, Lexington, KY 40536, USA. dnoonan@pop.uky.edu

ABSTRACT
BACKGROUND: The specificity of a nuclear receptor's ability to modulate gene expression resides in its ability to bind a specific lipophilic ligand, associate with specific dimerization partners and bind specific DNA sequences in the promoter regions of genes. This sequence of events appears to be the basis for targeting an additional regulatory complex composed of a variety of protein and RNA components that deliver signals for facilitation or inhibition of the RNA polymerase complex. Characterization of the tissue and cell-specific components of these coregulatory complexes appear to be integral to our understanding of nuclear receptor regulation of transcription. RESULTS: A novel yeast screen sensitive to retinoid-X receptor (RXR) transcriptional activation resulted in the isolation of the rat homologue of the mouse NPDC-1 gene. NPDC-1 has been shown to be involved in the control of neural cell proliferation and differentiation, possibly through interactions with the cell cycle promoting transcription factor E2F-1. Although the amino acid sequence of NPDC-1 is highly conserved between mouse, rat and human homologues, their tissue specific expression was seen to vary. A potential for direct protein:protein interaction between NPDC-1, RXR and retinoic acid receptor beta (RARbeta) was observed in vitro and NPDC-1 facilitated RXR homodimer and RAR-RXR heterodimer DNA binding in vitro. Expression of NPDC-1 was also observed to repress transcription mediated by retinoid receptors as well as by several other nuclear receptor family members, although not in a universal manner. CONCLUSIONS: These results suggest that NPDC-1, through direct interaction with retinoid receptors, functions to enhance the transcription complex formation and DNA binding function of retinoid receptors, but ultimately repress retinoid receptor-mediated gene expression. As with NPDC-1, retinoids and their receptors have been implicated in brain development and these data provide a point of convergence for NPDC-1 and retinoid mediation of neuronal differentiation.

No MeSH data available.


Related in: MedlinePlus