An Examination of the Car-rxr Heterodimer and Response Element Transactivation

Open Access
Author:
Agrawal, Rishi Raj
Area of Honors:
Toxicology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Curtis John Omiecinski, Thesis Supervisor
  • Gary H Perdew, Honors Advisor
Keywords:
  • Proteins
  • Nuclear Receptors
  • Xenobiotics
  • Metabolism
  • Molecular Biology
  • Toxicology
Abstract:
In most organisms, nuclear receptors (NRs) comprise a superfamily of critical regulatory proteins that sense a variety of endogenous and xenobiotic compounds within animal cells. These include steroids, hormones, drugs and toxins. In response to chemical signals delivered by these substances, NRs generally act as transcriptional modulators that regulate the expression of important genes encoding enzymes that control critical metabolic and homeostatic pathways. One “xenosensing” NR is the constitutive androstane receptor (CAR), initially identified for its role as a regulator of cytochrome P450 (CYP) gene expression. CAR binds response elements of gene promoter regions as a heterodimer with RXR, another NR protein. This heterodimerization interaction comprises the focus of our studies. In humans, alternative splice variants of CAR exist that feature polymorphic heterodimerization interfaces. Also, the expression of RXR itself is programmed by three separate genes. We hypothesize that these variant receptors may differentially interact, leading to altered functional responses. Specifically, we tested CAR’s ability to interact with different isoforms of RXR by assessing differential binding of the respective dimers to an endogenous CYP2B6 gene promoter. Additionally, we tested whether different “versions” of the CAR-RXR heterodimer may display variable activation when targeting different CYP response elements. The results demonstrated a significantly lesser interaction of hCAR with hRXRβ as compared with hRXRα or hRXRγ, a finding that was consistent among all structural isoforms of hCAR. Additionally, we identified a greater level of reporter transactivation by hCAR-hRXR heterodimers as compared with hCAR2/3-hRXR heterodimers, likely due to the structural modifications interposed in the hCAR splice variants’ respective ligand-binding domains. Finally, a preference for association with the DR-4X3 response element was identified in the CAR activation profile, corroborating previous findings from our laboratory. In conclusion, these investigations characterized significant differences among the interactions of receptor heterodimer pairs, implying that gene regulation programs may be subject to “tuning” in target cells as a function of NR diversity.