Abstract
The constitutive androstane receptor (CAR) is an interesting member of the nuclear receptor superfamily because of its exceptionally high constitutive activity due to ligand-independent interaction of the ligand-binding domain with co-activator proteins. This study compares the agonist-dependent and agonist-independent activities of human CAR with those of mouse CAR and the vitamin D receptor and demonstrates that the constitutive activity of CAR is mediated by at least three contacts between the amino acids of helix 12, partner amino acids in helices 4 and 11, and a charge clamp between helices 12 and 3. The stabilization of helix 12 by a contact between its C terminus and the lysine of helix 4 has the same impact in human and mouse CARs. In addition, the charge clamp between the glutamate in helix 12 and the lysine in helix 3 is also important for the constitutive activity of both receptor orthologs but less critical for the agonist-dependent stabilization of their respective helices 12. Interestingly, Cys-357 in mouse CAR has significantly more impact on the stabilization of helix 12 than does the orthologous position Cys-347 in human CAR. This deficit appears to be compensated by a more dominant role of Ile-330 in human CAR over Leu-340 in mouse CAR because it is more efficient than Cys-347 in controlling the flexibility of helix 12 in the presence of an agonist. The constitutive activity of other members of the nuclear receptor superfamily could be explained by a homologous hydrophobic interaction between large, non-polar amino acids of helices 11 and 12.
Highlights
Nuclear receptors (NRs)1 form a large family of transcription factors (48 human members) and have critical roles in most aspects of vertebrate development and adult physiology by transducing the effects of small lipophilic compounds into a transcriptional response [1]
The crystal structures of two adopted NRs, the pregnane X receptor (PXR; NR1I2) [10] and the peroxisome proliferator-activated receptor (NR1C1-3) [11], indicated that helix 12 takes a continuum of positions that span the extremes of complete repression and complete activation, such that fairly minor changes in the position of the helix can have a large impact on the transcriptional activity of NRs
The phenobarbitalresponsive enhancer modules (PBREMs), two copies of DR4(T/T), or twice the response elements (REs) NR1 were fused with the tk promoter driving the luciferase gene
Summary
Nuclear receptors (NRs) form a large family of transcription factors (48 human members) and have critical roles in most aspects of vertebrate development and adult physiology by transducing the effects of small lipophilic compounds into a transcriptional response [1]. CoAs and co-repressors make direct contacts with a hydrophobic cleft on the surface of the NR LBD, and the ability to discriminate between both classes of co-regulators is determined by the position of helix 12 [7] This biphasic situation allows NR ligands to activate or repress the transcription of specific target genes. CAR and PXR play key roles in the response to chemical stress and regulate an overlapping set of genes, some of which encode proteins such as cytochrome P450 monooxygenases (CYPs) that are involved in the detoxification of potentially harmful xenobiotics and endobiotics [13]. The relative impact of four different possibilities for the stabilization of helix 12 via amino acid contacts was assessed in comparison with human VDR and mouse CAR
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