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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.


RXR binds the amino terminus of hNPDC-1. Approximately 10 μg of recombinant S-tagged hNPDC-1 or NPDC-1 deletion mutant (A) was pre-coupled to S-protein to generate a 50% slurry of NPDC-1-beads. Reactions containing 30 μl NPDC-beads, 5 μg of recombinant GST-RXRα and 300 μl of 1X gel-shift buffer were incubated at 4°C for 1 hour with rotation. S-protein pellets were collected by centrifugation and washed 3 times in gel-shift buffer. Proteins were released from the pellet by resuspending S-protein agarose in 2X SDS-PAGE sample buffer. The amount of GST-RXRα bound to beads was analyzed by Western blotting onto nitrocellulose filters and probing blots with anti-RXRα (B, top panel). To normalize for unequal expression of the various deletion mutants, duplicate reactions without GST-RXRα were performed and subjected to coomassie staining instead of immunoblotting, and NIH Image Software was used to normalize the bands in the immunoblot (B, top panel) to the amount of S-tagged NPDC-1 precipitated in the coomassie stained gel. Normalized binding is depicted in B, bottom panel.
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Figure 9: RXR binds the amino terminus of hNPDC-1. Approximately 10 μg of recombinant S-tagged hNPDC-1 or NPDC-1 deletion mutant (A) was pre-coupled to S-protein to generate a 50% slurry of NPDC-1-beads. Reactions containing 30 μl NPDC-beads, 5 μg of recombinant GST-RXRα and 300 μl of 1X gel-shift buffer were incubated at 4°C for 1 hour with rotation. S-protein pellets were collected by centrifugation and washed 3 times in gel-shift buffer. Proteins were released from the pellet by resuspending S-protein agarose in 2X SDS-PAGE sample buffer. The amount of GST-RXRα bound to beads was analyzed by Western blotting onto nitrocellulose filters and probing blots with anti-RXRα (B, top panel). To normalize for unequal expression of the various deletion mutants, duplicate reactions without GST-RXRα were performed and subjected to coomassie staining instead of immunoblotting, and NIH Image Software was used to normalize the bands in the immunoblot (B, top panel) to the amount of S-tagged NPDC-1 precipitated in the coomassie stained gel. Normalized binding is depicted in B, bottom panel.

Mentions: In the above studies we were able to demonstrate binding of NPDC-1 to retinoid receptors, but the transcription studies suggest this may not be through its LXXLL motifs. To localize NDPC-1 binding to RXR, a series of deletion constructs were made sequentially deleting sequence information from the amino and carboxyl termini of hNPDC-1 (Fig. 9A). These deletion constructs were used to generate recombinant hNPDC-1 protein and were analyzed in standard pull-down assays for their ability to interact with recombinant hRXRα protein (Fig. 9B). As seen in Fig. 9B, deletion of a region between amino acids 76 and 112 substantially reduces NPDC-1 interactions, suggesting the putative coil-coil domain residing in this region may play an important role in NPDC-1 interactions with RXRα.


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 binds the amino terminus of hNPDC-1. Approximately 10 μg of recombinant S-tagged hNPDC-1 or NPDC-1 deletion mutant (A) was pre-coupled to S-protein to generate a 50% slurry of NPDC-1-beads. Reactions containing 30 μl NPDC-beads, 5 μg of recombinant GST-RXRα and 300 μl of 1X gel-shift buffer were incubated at 4°C for 1 hour with rotation. S-protein pellets were collected by centrifugation and washed 3 times in gel-shift buffer. Proteins were released from the pellet by resuspending S-protein agarose in 2X SDS-PAGE sample buffer. The amount of GST-RXRα bound to beads was analyzed by Western blotting onto nitrocellulose filters and probing blots with anti-RXRα (B, top panel). To normalize for unequal expression of the various deletion mutants, duplicate reactions without GST-RXRα were performed and subjected to coomassie staining instead of immunoblotting, and NIH Image Software was used to normalize the bands in the immunoblot (B, top panel) to the amount of S-tagged NPDC-1 precipitated in the coomassie stained gel. Normalized binding is depicted in B, bottom panel.
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Related In: Results  -  Collection

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Figure 9: RXR binds the amino terminus of hNPDC-1. Approximately 10 μg of recombinant S-tagged hNPDC-1 or NPDC-1 deletion mutant (A) was pre-coupled to S-protein to generate a 50% slurry of NPDC-1-beads. Reactions containing 30 μl NPDC-beads, 5 μg of recombinant GST-RXRα and 300 μl of 1X gel-shift buffer were incubated at 4°C for 1 hour with rotation. S-protein pellets were collected by centrifugation and washed 3 times in gel-shift buffer. Proteins were released from the pellet by resuspending S-protein agarose in 2X SDS-PAGE sample buffer. The amount of GST-RXRα bound to beads was analyzed by Western blotting onto nitrocellulose filters and probing blots with anti-RXRα (B, top panel). To normalize for unequal expression of the various deletion mutants, duplicate reactions without GST-RXRα were performed and subjected to coomassie staining instead of immunoblotting, and NIH Image Software was used to normalize the bands in the immunoblot (B, top panel) to the amount of S-tagged NPDC-1 precipitated in the coomassie stained gel. Normalized binding is depicted in B, bottom panel.
Mentions: In the above studies we were able to demonstrate binding of NPDC-1 to retinoid receptors, but the transcription studies suggest this may not be through its LXXLL motifs. To localize NDPC-1 binding to RXR, a series of deletion constructs were made sequentially deleting sequence information from the amino and carboxyl termini of hNPDC-1 (Fig. 9A). These deletion constructs were used to generate recombinant hNPDC-1 protein and were analyzed in standard pull-down assays for their ability to interact with recombinant hRXRα protein (Fig. 9B). As seen in Fig. 9B, deletion of a region between amino acids 76 and 112 substantially reduces NPDC-1 interactions, suggesting the putative coil-coil domain residing in this region may play an important role in NPDC-1 interactions with RXRα.

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.