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The table below describes different point mutations at a given position and provides links to other documents. The sentence(s) where the point mutations in VDR_HUMAN at position 236 were found are listed after the table.
Position 236 in 1I1 Vitamin D3 (VDR) family
Position 231 in 1I Vitamin D3-like (VDR,PXR,CAR) family
Position 224 in 1 Thyroid hormone like (TR,RAR,ROR,PPAR,VDR family
In particular , one mutant (VDR (Y236A)) exhibited normal ligand binding and heterodimerization with the retinoid X receptor (RXR) but was transcriptionally inactive
Yeast two-hybrid studies and in vitro protein interaction assays demonstrated that VDR (Y236A) was selectively impaired in interaction with AF-2-interacting coactivator proteins such as SRC-1 and GRIP-1
Specifically , a Y236A helix H3 mutation in the VDR selectively disrupted 1 , 25-(OH)2D3-induced interactions between VDR and SRC-1 or GRIP-1
Ligand-binding Assay-- GST-VDR wild-type and helix H3 point mutants (H229A , D232A , and Y236A) were expressed in the DH5 alpha strain of Escherichia coli and purified by glutathione-agarose affinity as described previously (23 , 24 )
The pAS1-VDR-(116-427) wild-type and Y236A mutant each contained the GAL4 DNA-binding domain (amino acids 1-147) and the carboxyl-terminal region of VDR (amino acids 116-427)
In Vitro Protein Interaction Assay-- GST-VDR-(116-427) wild-type or Y236A mutant and GST-RXR alpha -(235-493) were each individually expressed in the DH5 alpha strain of E
35S-Labeled VDR-(4-427) wild-type or helix H3 point mutants (S225A , H229A , D232A , S235A , Y236A , and K240A) and SRC-1 or GRIP-1 were each generated using the TNT-coupled transcription-translation system according to the manufacturer (Promega , Madison , WI)
Altering these amino acids individually to alanine residues reduced (H229A or Y236A) or abolished (D232A) this autonomous transactivation activity (Fig. 1)
COS-7 cells were transfected with 5 µg of a (GAL4)5-TATA-GH reporter construct and increasing amounts (0.05 , 0.10 , or 0.25 µg) of pSG5 GAL4 , pSG5 GAL4-VDR-(195-238) wild-type , or the H229A , D232A , or Y236A mutants
To investigate the functional importance of amino acid residues within helix H3 for 1 , 25-(OH)2D3-activated transcription by full-length VDR , point mutations were introduced into the full-length pSG5-VDR-(4-427) plasmid , and the ability of these mutants (S225A , H229A , D232A , S235A , Y236A , and K240A) to activate transcription of a (VDRE)4-TATA-GH reporter gene in COS-7 cells was examined
Each transfection included 3 µg of a (VDRE)4-TATA-GH reporter construct and either 10 ng of pSG5-VDR-(4-427) wild-type or a mutant version (S225A , H229A , D232A , S235A , Y236A , or K240A)
Extracts were prepared from duplicate plates of COS-7 cells transfected with the wild-type pSG5-VDR-(4-427) and the helix H3 mutants (S225A , H229A , D232A , S235A , Y236A , and K240A) in pSG5-VDR-(4-427)
Similarly , 35S-labeled full-length VDR mutants (S225A , S235A , Y236A , and K240A) showed ligand-dependent interaction with RXR that was comparable with wild-type (lanes 7-8 , 19-20 , 23-24 , and 27-28)
A , interactions of 35S-labeled wild-type VDR-(4-427) or S225A , H229A , D232A , S235A , Y236A , and K240A mutants with purified GST-RXR alpha were analyzed in a GST pull-down assay
The Y236A mutation appeared to bind ligand with an affinity comparable with wild-type VDR
The Y236A mutant had a K D similar to wild-type VDR (0.2-0.3 nM) , whereas the H229A and D232A mutants had 4.5- and 8-fold lower affinities , respectively
In contrast , the loss of 1 , 25-(OH)2D3-mediated transactivation by the Y236A mutation could not be explained by this defect because it bound ligand with an affinity similar to that of wild-type VDR , and it interacted in a ligand-dependent manner with RXR
Bacterial-expressed and -purified GST-VDR-(116-427) wild-type ((image)) and mutants (open circle , Y236A ; black-square , H229A ; (image) , D232A) , or GST (black-triangle) (0.5 µg of protein) were combined with 4.5 µg of nuclear extract obtained from HeLa cells and incubated with increasing concentrations of 1 , 25-(OH)2-[3H]D3
Interactions between the Wild-Type VDR or Helix H3 Mutant Y236A and Coactivator Proteins-- These data suggested that the Y236A mutant must be defective in some other aspect of receptor action that did not include hormone binding or RXR heterodimerization
Therefore , the yeast two-hybrid system was used to assess the ability of wild-type VDR and mutant Y236A to interact with nuclear receptor coactivator proteins (Fig. 5 )
Interestingly , the helix H3 point mutant Y236A (pAS1-VDR-(116-427) Y236A) showed markedly reduced interaction with RIP-140 and essentially no interaction with SRC-1
Both wild-type and mutant Y236A retained strong interaction with RXR alpha in this system (data not shown)
No interaction was observed with the AD-GAL4 parent plasmid for either wild-type VDR or mutant Y236A
Thus , 1 , 25-(OH)2D3-dependent interactions between VDR (Y236A) and AF-2 coactivator proteins were dramatically impaired compared with wild-type VDR as determined by the yeast two-hybrid system
Interaction of wild-type VDR and VDR (Y236A) with SRC-1 and RIP-140 in a two-hybrid system
Yeast expressing the AS1-VDR-(116-427) wild-type or mutant pAS1-VDR-(116-427) Y236A and pAD-SRC-1 , pAD-RIP-140 , or pAD-GAL4 were grown for 16 h at 30 °C in the absence or presence of 10 -8 M 1 , 25-(OH)2D3
To confirm these in vivo yeast two-hybrid data , interactions between VDR (Y236A) and p160 coactivator proteins were examined in vitro by GST pull-down analysis
Only minimal interaction was detected with the liganded GST-VDR Y236A mutant (compare lanes 4 and 6)
Purified GST-VDR-(116-427) Y236A showed only very weak interaction in the presence of hormone (lane 6) (Fig. 6 B)
These findings indicate that mutant Y236A , despite retaining the ability to bind hormone and to heterodimerize with RXR , is selectively impaired in interaction with coactivator proteins both in yeast and in in vitro binding assays
In vitro interaction of wild-type VDR and VDR (Y236A) with full-length SRC-1 or GRIP-1
A , interactions of 35S-labeled full-length SRC-1 with purified GST-VDR-(116-427) wild-type or GST-VDR-(116-427) mutant Y236A were determined in a GST pull-down assay
In vitro transcribed and translated 35S-labeled SRC-1 was incubated with either 5 µg of GST (lane 2) , with 5 µg of GST-VDR-(116-427) wild-type (lanes 3-4) , or with 5 µg of GST-VDR-(116-427) mutant Y236A (lanes 5-6) in the absence (lanes 3 and 5) or presence (lanes 4 and 6) of 10 -8 M 1 , 25-(OH)2D3
B , interactions of 35S-labeled full-length GRIP-1 with purified GST-VDR-(116-427) wild-type or GST-VDR-(116-427) mutant Y236A was determined in a GST pull-down assay
In vitro transcribed and translated 35S-labeled GRIP-1 was incubated with either 5 µg of GST (lane 2) , with 5 µg of GST-VDR-(116-427) wild-type (lanes 3-4) , or with 5 µg of GST-VDR-(116-427) mutant Y236A (lanes 5-6) in the absence (lanes 3 and 5) or presence (lanes 4 and 6) of 10 -8 M 1 , 25-(OH)2D3
Effect of SRC-1 and GRIP-1 Coactivators on 1 , 25-(OH)2D3-Activated Transcription by Wild-type VDR and VDR (Y236A)-- Transient reporter gene expression assays in COS-7 cells were used to determine the effects of SRC-1 or GRIP-1 on 1 , 25-(OH)2D3-activated transcriptional activity by VDR and the Y236A mutant
As shown in Fig. 7 , cells were cotransfected with the full-length wild-type VDR or mutant Y236A and full-length SRC-1 or the pCR 3.1 parent vector using a (VDRE)4-TATA-GH reporter construct
The transcriptional enhancement observed with wild-type VDR was not apparent with mutant Y236A even in the presence of excess SRC-1
Similar results were obtained for both wild-type VDR and mutant Y236A in cells cotransfected with additional exogenous full-length GRIP-1
Additionally , although the wild-type VDR was maximally activated at 10 -8 M 1 , 25-(OH)2D3 , the Y236A mutant was inactive even when the cells were treated with higher concentrations of ligand (10 -6 M) ruling out the possibility of a partial ligand-binding defect (data not shown)
The overexpression of SRC-1 was previously shown to restore the transcriptional activity of a helix H3 point mutant (T277A in TR beta ) (29 ) ; however , we found that neither the overexpression of full-length SRC-1 or GRIP-1 could promote transactivation by the VDR mutant Y236A
Expression of SRC-1 or GRIP-1 augments ligand-activated transcription by wild-type VDR but not by the VDR (Y236A) mutant
COS-7 cells were cotransfected with 3 µg of a (VDRE)4-TATA-GH reporter construct and 10 ng of pSG5-VDR-(4-427) wild-type or 10 ng of pSG5-VDR-(4-427) mutant Y236A and 1 µg of either pCR 3.1 , pCR 3.1 SRC-1 , pSG5 , or pSG5 GRIP-1
This was most strikingly apparent in the Y236A mutation within the VDR helix H3 activation domain
While the Y236A mutation did not affect the ability of VDR to bind ligand or to heterodimerize with RXR , alteration of this single tyrosine residue selectively impaired both 1 , 25-(OH)2D3-dependent interaction with the AF-2 coactivators SRC-1 and GRIP-1 as well as 1 , 25-(OH)2D3-activated transcription
The essential nature of this helix H3 residue in coactivator function is apparent in Fig. 7 , wherein overexpression of SRC-1 or GRIP-1 dramatically augments 1 , 25-(OH)2D3-activated transcription mediated by wild-type VDR , but each exhibited absolutely no effect on transactivation mediated by the VDR (Y236A) mutant
However , our data also demonstrate that inactivation of the helix H3 domain through the Y236A mutation abolishes 1 , 25-(OH)2D3-mediated transactivation , indicating that the AF-2 domain is also nonfunctional in this context
Ligand-binding Assay--GST-VDR wild-type and helix H3 point mutants (H229A , D232A , and Y236A) were expressed in the DH5 strain of Escherichia coli and purified by glutathione-agarose affinity as described previously (23 , 24)
In Vitro Protein Interaction Assay--GST-VDR-(116 427) wild-type or Y236A mutant and GST-RXR -(235 493) were each individually expressed in the DH5 strain of E
To investigate the functional importance of amino acid residues within helix H3 for 1 , 25-(OH)2D3-activated transcription by full-length VDR , point mutations were introduced into the full-length pSG5-VDR-(4 427) plasmid , and the ability of these mutants (S225A , H229A , D232A , S235A , Y236A , and FIG
Both wildtype and mutant Y236A retained strong interaction with RXR in this system (data not shown)
Each transfection included --> g of a (VDRE)4-TATA-GH reporter construct and either 10 ng of pSG5-VDR-(4 427) wild-type or a mutant version (S225A , H229A , D232A , S235A , Y236A , or K240A)
VDR Helix H3 in Transactivation and Coactivator Interaction 14354 mutant Y236A
A , interactions of 35S-labeled wild-type VDR-(4 427) or S225A , H229A , D232A , S235A , Y236A , and K240A mutants with purified GST-RXR were analyzed in a GST pulldown assay
Bacterial-expressed and -purified GSTVDR-(116 427) wild-type (q) and mutants (E , Y236A ; f , H229A ; , D232A) , or GST (OE) (0.5 g of protein) were combined with 4.5 g of nuclear extract obtained from HeLa cells and incubated with increasing concentrations of 1 , 25-(OH)2[3H]D3
VDR Helix H3 in Transactivation and Coactivator Interaction 14355 was not apparent with mutant Y236A even in the presence of excess SRC-1
The overexpression of SRC-1 was previously shown to restore the transcriptional activity of a helix H3 point mutant (T277A in TR ) (29) ; however , we found that neither the overexpression of full-length SRC-1 or GRIP-1 could promote transactivation by the VDR mutant Y236A
In vitro transcribed and translated 35S-labeled SRC-1 was incubated with either 5 g of GST (lane 2) , with 5 g of GST-VDR-(116 427) wild-type (lanes 3 4) , or with 5 g of GST-VDR-(116 427) mutant Y236A (lanes 5 6) in the absence (lanes -->and 5) or presence (lanes 4 and 6) of 10 8 M 1 , 25-(OH)2D3
In vitro transcribed and translated 35S-labeled GRIP-1 was incubated with either 5 g of GST (lane 2) , with 5 g of GST-VDR-(116 427) wild-type (lanes 3 4) , or with 5 g of GST-VDR-(116 427) mutant Y236A (lanes 5 6) in the absence (lanes -->and 5) or presence (lanes 4 and 6) of 10 8 M 1 , 25-(OH)2D3
COS-7 cells were cotransfected with --> g of a (VDRE)4-TATA-GH reporter construct and 10 ng of pSG5-VDR-(4 427) wild-type or 10 ng of pSG5-VDR-(4 427) mutant Y236A and 1 g of either pCR 3.1 , pCR 3.1 SRC-1 , pSG5 , or pSG5 GRIP-1
Reference #2 (Mohr SC et al.): Y236A
Table 1 Natural* and designed mutations in VDR-LBD and their effects Change Effect (Reference) C190W* Familial VDRR-II [57] S225A Kd for ligand unchanged [44] H229A Kd for ligand increased by 27X [44] D232A Kd for ligand increased by 30X [44] V234A Kd for ligand almost unchanged [44] S235A Kd for ligand almost unchanged [44] Y236A Kd for ligand increased by 3.5X [44] S237A Kd for ligand increased by 27X [44] K240A Kd for ligand increased by 27X [44] I242A Kd for ligand increased by 27X [44] F244G Impaired transactivation [54] K246A Kd for ligand increased by 2X [44] L254G Impaired transactivation ; no RXR heterodimer formed [54] R274L* Ligand binding severely decreased ; VDRR-II [43] S275A Kd for ligand increased by 5.3X [44] M284A Ligand binding significantly reduced (Ray et al. , unpublished) M284S Ligand binding significantly reduced (Ray et al. , unpublished) W286A No ligand binding (Ray et al. , unpublished) W286F No ligand binding (Ray et al. , unpublished) C288G Ligand binding 'attenuated severely' at 37°C ; decreased 3X at 4°C , [25] Y295STOP* No ligand binding [55] D299A No ligand-binding (Ray et al. , unpublished) K302A No change in ligand-binding (Ray et al. , unpublished) H305Q* Ligand binding decreased 5X [55] I314S* Reduced transactivation ; high ligand concentration restores it ; RXR interaction diminished [53] C337G Ligand binding moderately decreased at 37°C [25] C369G Ligand binding only mildly affected at 37°C [25] K386Q Very slight decrease in ligand binding [52] L387STOP No ligand binding [52] R391C* Transactivation decreased together with RXR binding [53] L404STOP Kd for ligand increased by 10X [52] L417A No change in ligand binding ; transcriptional activation eliminated [56] L417S Ligand binding normal ; transactivation abolished (also coactivator binding) [52] V418S Significant reduction in ligand-binding (Ray et al. , unpublished) E420A No change in ligand binding ; transcriptional activation eliminated [56] E420Q Ligand binding normal ; transactivation abolished (also coactivator binding) [52] E425Q No effect on ligand binding or transactivation [52] S.C
2005, NucleaRDB.
cmbi.kun.nl), 21-Apr-2005