NucleaRDB: Extraction of mutation data from the literature

This data was extracted from Medline abstracts and full texts (when available) in an automated manner.

The table below describes the selected point mutation and provides links to other documents. The sentence(s) where the point mutation C357S was found are listed after the table.

The mutated residues are also indicated in the family sequence alignments and hyperlinked to the corresponding mutation pages.

Point mutation C357S in NR1I3_MOUSE

Relevant sentences:

C357

• Moreover , these amino acids and C357 were shown to be involved in the response of CAR to androstanol

• We showed that this blockade is not due to an intramolecular disulfide bridge , but is probably caused by an interaction between C357 and Y336

• The mutagenesis of C357 reduced CoA interaction of CAR in the absence of ligand and drastically increased inducibility by TCPOBOP

• Remarkably , C357A became nearly 30-fold inducible by TCPOBOP (compared with 2.2-fold for wild-type CAR)

• The inverse agonistic effect of androstanol was completely lost with L352A , L353A , I356A , C357A , and the AF-2 deletion

• In the case of the mutants L353A , G354A , I356A , and C357A this combined ligand treatment resulted in additive agonistic effect

• The point mutants L352A , L353A , E355A , I356A , and C357A and the AF-2 deletion reduced the basal activity by 90% or more , S358A by 75% , and G354A by only 10%

• These include the reduced TCPOBOP inducibility of C357A (only 10.5-fold) and reduced loss of basal activity of E355A (only 80%) and S358A (only 60%) compared with wild-type CAR

• In summary , the mutagenesis of L352 , L353 , I356 , and C357 resulted in drastically reduced basal activities of CAR in living cells

• In contrast , C357A shows strong agonist responsiveness

• The most interesting mutation is C357A , which showed in the absence of ligand or in the presence of inverse agonist only weak interaction with TIF2 , but strong interaction in the presence of agonist

• This reduction was not as drastic as observed with the mutants L352A , L353A , I356A , and C357A and the AF-2 deletion (Fig. 3(image) )

• Two hypotheses could explain the drastically increased ligand-dependent responsiveness of C357A

• C357 may be able to form a disulfide bridge with other cysteines in the LBD , which would lock wild-type CAR into its agonistic conformation

• Alternatively , the sulfhydryl group of C357 could interact with Y336 , which is homologous to the F422-H397 interaction in VDR (24 ) (see Fig. 7(image) )

• For the second hypothesis C357 was mutated into serine and tyrosine , Y336 was mutated into alanine , cysteine , and phenylalanine , and the point- mutants Y336A / C357A , Y336C / C357Y , and L352A / C357A were created

• This excludes the possibility that C357 is involved in an intramolecular disulfide bridge

• The mutant C357S showed the same functional profile as C357A , suggesting that both the hydroxyl group of a serine and the short hydrophobic side chain of an alanine at position 357 drastically decrease CoA interaction and increase agonist inducibility

• This indicates that the sulfhydryl group of C357 is critical for keeping the basal activity of CAR high and its responsiveness to ligand low

• The reduction in basal activity and increase in agonist response of Y336A was not as drastic as with C357A , but showed the same tendency

• All three double-point mutants , Y336A / C357A , Y336C / C357Y , and L352A / C357A , showed nearly abolished response to ligand , which was expected because of the profile of the individual mutants Y336C , C357Y , and L352

• Interestingly , C357A / Y336A and L352A / C357A reduced the basal activity of CAR by 80-90% , whereas Y336C / C357Y did not influence it significantly

• Taken together , the functional profiles of all three double-point mutants suggest that C357 , Y336 , and the CoA interacting amino acid L352 are functionally linked , such as F422 , H397 , and L417 in VDR (24 , 25 , 26 )

• Finally , a dose response to TCPOBOP was performed with wild-type CAR in comparison to C357A using the PBREM reporter gene system in MCF-7 cells (Fig. 6C(image) )

• The half-maximal activation (EC50) value of wild-type CAR (110 nM) was found to be only less than two times lower than that of C357A (200 nM)

• This demonstrates that a mutagenesis of C357 into alanine has only minor effects on the affinity of CAR for TCPOBOP but does not clarify whether or not this amino acid is involved in a direct contact with the agonist

• Model for Stabilization of Helix 12 in CAR Compared with VDR The structural relation of helices 11 and 12 in mouse CAR (left) and human VDR (right) and their link via an interaction between Y336 and C357 (CAR) or H397 and F422 (VDR) is indicated by a dashed line

• The Importance of the Role of C357 in CAR’s Constitutive Activity Reporter gene assays were performed with extracts from MCF-7 cells that were transiently transfected with a luciferase reporter construct containing one copy of the PBREM of the mouse CYP2B10 gene and expression vectors for wild-type and point- mutated mouse CAR (as indicated)

• The most surprising observation of this study is the strong agonist responsiveness of the mutant C357A , which represents a rare case of a gain of function mutant

• The amino acid C357 is exposed toward the hydrophobic core of mouse CAR and therefore unlikely to be involved in any direct CoA contact , but it seems to block full agonist responsiveness of the receptor

• C357 appears to be very specific in its interaction with Y336 and is not involved in the formation of disulfide bridges

• A modeling of the structure of helices 11 and 12 on the basis of the VDR structure (Fig. 7(image) ) predicts that C357 interacts with Y336

• In fact , mutagenesis of Y336 alone and in combination with C357 suggests that both amino acids are functionally linked , such as F422 and H397 in VDR

• The specificity of the interaction between these two amino acids is supported by the observation that the double mutant Y336C / C357Y showed no effect on the basal activity of CAR , i.e. that these two residues stabilize helix 12 independent of their orientation

• In contrast to VDR , in CAR the stabilization of helix 12 by the C357-Y336 interaction occurs even in the absence of ligand

• A mutation of C357 or Y336 allows a free movement of helix 12 in the absence of ligand and provides CAR with a functional profile comparable to classical endocrine NRs , i.e. low basal activity and high ligand inducibility

• The mutation C357A increases ligand inducibility from a very low basal activity , but it lowers the ligand affinity by a factor of nearly 2

• The C357-Y336 interaction seems to be reinforced and optimized by the agonist TCPOBOP , whereas the Y336C / C357Y double mutant of CAR has lost its responsiveness to ligand

• The principal role of C357 seems to be the stabilization of helix 12 via an interaction with Y336 , but it is possible that Y336 directly contacts the ligand in the way that H397 does in VDR

• We agree with Dussault et al. (32 ) concerning the impact of K205 , since the mutant K205A has a similar functional profile as C357A (data not shown)

• Within the adopted orphan receptor , movement of helix 12 seems to be reduced via an interaction between C357 and Y336 , which is only optimized by contact with a ligand

• The amino acid sequences of VDR and CAR were aligned and the amino acid residues V418 , L419 , V421 , F422 , G423 , and H397 of VDR were mutated in silico to the respective amino acid residues of mouse CAR (L353 , G354 , I356 , C357 , S358 , and Y336 , respectively) by using the mutation command of Swiss PDB Viewer (v.3.7b2)

C357S

• The mutant C357S showed the same functional profile as C357A , suggesting that both the hydroxyl group of a serine and the short hydrophobic side chain of an alanine at position 357 drastically decrease CoA interaction and increase agonist inducibility

F.Horn (nucleardbcmbi.kun.nl), 21-Apr-2005