Functional characterization of the pregnane X receptor

Author

He Zhang, University of Rhode Island

Date of Award

2002

Degree Type

Dissertation

First Advisor

Bingfang Yan

Abstract

Cytochrome P450 3A enzymes (CYP3A) are involved in the metabolism of two thirds of drugs and other xenobiotics, and highly induced by structurally diverse xenobiotics. Induction of CYP3A causes many drug-drug interactions, and an orphan nuclear receptor, the pregnane X receptor (PXR) is established to mediate the induction of CYP3A in mouse and human. Structurally, the PXR has a N-terminal uncharacterized domain, a conserved DNA binding domain (DBD) and a diverse C-terminal ligand binding domain (LBD). The purpose of the studies described in this dissertation is to characterize the role of the N-terminal domain and the LBD in PXR-mediated signaling pathway. The full-length PXR, rPXR-1, is cloned from rat liver cDNA library. rPXR is highly expressed in the liver and intestine. In the presence of CYP3A inducers, rPXR-1 activates a synthetic reporter gene containing the PXR response element in the promoter of Cyp3a23. Replacing the rPXR-LBD with the hPXR-LBD changes the ligand specificity of rPXR-1 to that of hPXR. Studies on the specific amino acids in the rPXR-LBD indicate that the ligand specificity is determined by specific amino acids in the LBD. For example, mutation of Q407 of rPXR-LBD to arginine diminishes its affinity for pregnenolone-16α-carbonitrile (PCN) and thus decreases the activity of PCN to 40% of the wild type. One isoform of rPXR-1, rPXR-2 has a stop codon in the DBD resulted from an insert, so it has two open reading frames with an alternative start codon. rPXR-2 has no transactivation activity but it dramatically potentiates the activity of rPXR1 through the protein containing the N-terminal fragment of PXR. The N-terminal fragment is further found to weaken the interaction between PXR and corepressor, SUN-CoR, which could be the mechanism of the potentiation effect. In general, these studies establish the determinant role of PXR in mediating the induction of CYP3A, and the availability of nuclear receptor cofactors regulates the activity of PXR.

Recommended Citation

Zhang, He, "Functional characterization of the pregnane X receptor" (2002). Open Access Dissertations. Paper 1847.
https://digitalcommons.uri.edu/oa_diss/1847