This data was extracted from Medline abstracts and full texts (when available) in an automated manner.
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 NR1I3_MOUSE at position 350 were found are listed after the table.
Position 350 in 1I3 Constitutive Androstane alpha (CAR1) family
Position 410 in 1I Vitamin D3-like (VDR,PXR,CAR) family
Position 398 in 1 Thyroid hormone like (TR,RAR,ROR,PPAR,VDR family
Reference #1
Ueda A, Kakizaki S, Negishi M, Sueyoshi T Mol Pharmacol 2002 Jun;61(6):1284-8.
Accordingly , Thr350 and Gly354 were mutated to methionine (mCAR T350M) and glutamine (mCAR G354Q) , respectively
The mutant mCAR T350M lost its ability to show repressed activity by progesterone , whereas mCAR G354Q retained a degree of repression by progesterone (Fig. 2 A)
The double mutation (mCAR T350M / G354Q) showed activity similar to that of the Thr350 mutant (mCAR T350M)
In dose-dependent experiments , a low micromolar dose of progesterone was sufficient to repress the activity of wild-type mCAR , whereas the mutant mCAR T350M was not repressed by 10 µM progesterone (Fig. 3 A)
These residues , Thr350 and Gly354 , in mCAR were singularly and doubly mutated to the corresponding residues in hCAR: T350M , G354Q , and dm , respectively
A , repression of NR1-luciferase reporter gene activity by progesterone was examined for wild-type CAR((image)) and mutant mCAR T350M (open circle ) as described under Materials and Methods
SRC-1 Effect on Wild-Type and T350M mCAR
Coexpression of SRC-1 similarly increased the trans-activation activity of both wild-type mCAR and mutant mCAR T350M 4-fold (Fig. 4 )
KN-62 similarly repressed the activities of both wild-type mCAR and mutant mCAR T350M , indicating that Thr350 plays no role in the repression by KN-62
The wild-type mCAR or the mutant mCAR T350M was cotransfected with or without SRC-1 expression plasmid in HepG2 cells in various concentrations of progesterone (0.1 , 0.3 , 1 , 3 , and 10 µM)
Repression by KN-62 of the mCAR T350M activity
A , repression of NR1-luciferase reporter gene activity by progesterone was examined for wild-type CAR( ) and mutant mCART350M (E) as described under Materials and Methods
Reference #1 (Ueda A et al.): Thr350
Experiments were also performed to understand the role of Thr350 in the coactivation of mCAR by SRC-1
Using appropriate nucleotide primers , methionine 340 in hCAR was mutated to threonine , Thr350 to methionine , and glycine 354 to encode glutamine
Alignment of 13 residues with the corresponding residues of hCAR (from positions 336 to 348) revealed two amino acid differences: Thr350 and Gly354 of mCAR were substituted in hCAR with Met340 and Gln344 , respectively
Accordingly , Thr350 and Gly354 were mutated to methionine (mCAR T350M) and glutamine (mCAR G354Q) , respectively
The double mutation (mCAR T350M / G354Q) showed activity similar to that of the Thr350 mutant (mCAR T350M)
These results indicate that Thr350 of mCAR is a primary determinant conferring hormone responsiveness to mCAR
These residues , Thr350 and Gly354 , in mCAR were singularly and doubly mutated to the corresponding residues in hCAR: T350M , G354Q , and dm , respectively
Thr350 resides within or near a predicted AF2 domain of mCAR
The role of Thr350 in regulating coactivation was examined by cotransfection of an SRC-1 expression plasmid
These results suggest that Thr350 plays no role in the coactivation by SRC-1 in the absence of progesterone under the experimental conditions used but it does regulate the coactivation in the presence of progesterone
The question arose as to whether Thr350 is involved in the repression by KN-62 as well as by steroid hormones
Next , the role of Thr350 in repression of KN-62 was examined (Fig. 5 B)
KN-62 similarly repressed the activities of both wild-type mCAR and mutant mCAR T350M , indicating that Thr350 plays no role in the repression by KN-62
However , the repression was not affected by the mutation of Thr350
Thr350 does not seem to play a role either in the coregulation by SRC-1 or in the repression by KN-62
This is in sharp contrast to the critical role of Thr350 in the repression by steroid hormones
These results suggest that Thr350 plays no role in the coactivation by SRC-1 in the absence of progesterone under the experimental conditions used but it does regulate the Fig. 2
Reference #2 (Jyrkkarinne J et al.): Thr350
The importance of residues 201-263 appears to contrast with the recent report that residue 350 in helix 12 regulates the inhibition of mCAR by steroids.29 Substitution of Thr350 with Met , the corresponding residue in hCAR , abolished the inhibition by progesterone and testosterone
However , the response to the potent inhibitor androstenol was not measured in that study.29 Figure 5A indicates that mutation of Thr350 to Ala did not change the response of mCAR to steroids or TCPOBOP
Substitution of Thr350 with Met did not affect the inhibition by androstenol or testosterone
The response to TCPOBOP was decreased but not completely abolished with Thr350Met mutant
Figure 5C indicates that TCPOBOP enhanced SRC-1 interaction with mCAR and Thr350Ala mutant , to a lesser extent with Thr350Met mutant but not with hCAR
Interestingly , SRC-1 interaction was enhanced by progesterone with Thr350Met mutant only , in agreement with data in Figure 5A
In support of this idea , androstanol was recently shown to enhance interactions of the corepressor SMRT between mCAR with a mammalian two-hybrid assay in CV-1 cells.33 Ueda et al. reported that testosterone and progesterone inhibited mCAR depending on type of residue 350 , and thus this residue would directly regulate steroid-responsiveness of mCAR.29 They also showed that overexpression of SRC-1 prevented the suppression by progesterone of wild-type mCAR but not Thr350Met mutant , which implied that progesterone might inhibit mCAR through dissociation of SRC-1
Our data also indicate that the role of residue 350 in steroid inhibition is limited: the Thr350Ala mutant behaved exactly like the wild-type mCAR , and the responses to androstenol or testosterone are not changed by either Ala or Met mutations
The striking difference was that conversion of Thr350 to Met changed progesterone to an activator in both mammalian and yeast assays
These distinct orientations may explain why progesterone enhanced interactions of the Thr350Met mutant with both NCoR and SRC-1
Moreover , mCAR lacking the entire helix 12 was still able to recruit NCoR in response to androstenol and also testosterone and progesterone , suggesting that amino acids in helix 12 including Thr350 are not essential for steroid recognition
Thus , they would not be directly linked to specific steroid recognition by mCAR , which was the focus of this investigation and the suggested role for the residue Thr350.29 On the basis of a homology model built on the pregnane X receptor , it was hypothesized that androstenol might bind to the mCAR coactivator surface (and not to the ligand-binding pocket) due to its mimicry of hydrophobic residues Leu352 , Leu353 , Ile356 , and Cys357 in helix 12
Reference #2 (Jyrkkarinne J et al.): Thr350Ala
Figure 5C indicates that TCPOBOP enhanced SRC-1 interaction with mCAR and Thr350Ala mutant , to a lesser extent with Thr350Met mutant but not with hCAR
Our data also indicate that the role of residue 350 in steroid inhibition is limited: the Thr350Ala mutant behaved exactly like the wild-type mCAR , and the responses to androstenol or testosterone are not changed by either Ala or Met mutations
Reference #2 (Jyrkkarinne J et al.): Thr350Met
The response to TCPOBOP was decreased but not completely abolished with Thr350Met mutant
Figure 5C indicates that TCPOBOP enhanced SRC-1 interaction with mCAR and Thr350Ala mutant , to a lesser extent with Thr350Met mutant but not with hCAR
Interestingly , SRC-1 interaction was enhanced by progesterone with Thr350Met mutant only , in agreement with data in Figure 5A
In support of this idea , androstanol was recently shown to enhance interactions of the corepressor SMRT between mCAR with a mammalian two-hybrid assay in CV-1 cells.33 Ueda et al. reported that testosterone and progesterone inhibited mCAR depending on type of residue 350 , and thus this residue would directly regulate steroid-responsiveness of mCAR.29 They also showed that overexpression of SRC-1 prevented the suppression by progesterone of wild-type mCAR but not Thr350Met mutant , which implied that progesterone might inhibit mCAR through dissociation of SRC-1
These distinct orientations may explain why progesterone enhanced interactions of the Thr350Met mutant with both NCoR and SRC-1
2005, NucleaRDB.
cmbi.kun.nl), 21-Apr-2005