Superfund Research Program

A Proteomic Analysis of the AHR signaling Network

Project Leader: John J. LaPres
Grant Number: P42ES004911
Funding Period: 2006-2013

Project-Specific Links

Final Progress Reports

Year:   2012 

The aryl hydrocarbon receptor (AHR) mediates virtually all of the toxic effects of the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), as well as, related compounds. AHR-mediated toxic endpoints include immune suppression, chloracne, tissue hyperplasia, and metabolic dysfunction. The AHR is a member of the Per-ARNT-Sim (PAS) superfamily of transcription factors. The AHR, like all PAS proteins, functions as an environmental sensor. To fulfill its sensor function, the AHR is maintained in the cell in a high affinity state bound to two other proteins, the heat shock protein of 90 kDa (hsp90) and the immunophilin-like chaperone, ARA9. These two chaperones play a crucial role in cellular localization, ligand affinity, and transcriptional activity of the AHR. Published reports and preliminary data demonstrate that other proteins are capable of influencing the activity of the AHR through protein:protein interaction. Until the start of this project, a comprehensively analysis of the protein interaction network (PIN) of the AHR in a mammalian system had not been published. Using cutting edge technology (i.e. tandem affinity purification (TAP) and mass spectrometry (MS)) the researchers have characterized the AHR-PIN in the mouse hepatoma cell line, Hepa1c1c7.

The most reproducible protein found to bind the TAP-AHR was a component of the ATP synthase complex, ATP5∝1. This protein is found in the mitochondria and acts as part of the F1 portion of the complex. This raised the possibility that the AHR could be found in the mitochondria, similar to other ligand activated receptors (e.g. gluccocorticoid receptor). Immunhistochemistry and cell fractionation experiments demonstrate that the AHR can localize to the mitochondria. To explore the possibility that the receptor can impact mitochondrial function, assessments have been made in Hepa1c1c7 and Hepac1c12 (lacking functional AHR) cells. In the presence of TCDD, the hepa1c1c7 exhibit a dose-dependent increase in mitochondrial inner membrane polarization. Recently, the researchers have shown that these cells also display an increase oxygen consumption rate (OCR) under similar conditions. In contrast, the Hepac1c12 cells display an endogenous hyperpolarization compared to the Hepa1c1c7 cells that is refractory to TCDD exposure. In addition, these cells do not alter their OCR upon ligand exposure. Finally, the research team has begun creating a mitochondrial targeted version of the AHR and GFP in the Hepac1c12 cells to further delineate the role of mitochondrial AHR in cellular energetic and metabolism. Taken together, the data suggest that the traditional signaling pathway for the AHR is not adequate to cover its growing list of "moonlighting" roles. The identification and characterization as a mitochondrial protein just adds to this list. It also opens the door to novel routes of exploration to help understand the relationship between exposure to environmental pollutants, such as TCDD, and metabolic disorders, such as wasting syndrome and diabetes.