A Proteomic Analysis of the AHR signaling Network

Michigan State University, East Lansing MI

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Abstract

Aryl hydrocarbon receptor (AHR) agonists, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are some of[unreadable] the most toxic chemicals known to man. They also hold 4 of the top 10 positions within the EPA-ATSDR[unreadable] registry of priority substances that contaminate National Priority List. The toxicity of these compounds is[unreadable] primarily dependent upon the presence of a functional AHR signaling complex. This complex, in the[unreadable] absence of ligand consists of the AHR bound to a dimer of the heat shock protein of 90 kDa (Hsp90), the[unreadable] immunophilin-like protein, ARA9 (also known as XAP2 and AIP) and possibly several other factors (eg[unreadable] pp60src, p21). The role these chaperones play and their mechanism of action remains largely unknown.[unreadable] Our recent preliminary experiments suggest that ARA9 may function by recruiting other cellular factors to the[unreadable] AHR cytosolic complex. The role these cellular factors and other signaling systems play in the formation and[unreadable] integrity of the AHR cytosolic complex (upstream events) and how these other complex proteins influence[unreadable] AHR mediated toxicity (downstream events) has not been thoroughly explored. These signals may play[unreadable] important roles in the tissue specific biology and toxicity of AHR agonists. Our preliminary data and recent[unreadable] literature have led us to hypothesize: Secondary signaling, both upstream and downstream, plays an[unreadable] important role in AHR mediated signaling and toxicity through direct influence of the activity of the AHR[unreadable] cytosolic complex and perturbations of downstream signaling cascades. To address the hypothesis this[unreadable] project will look at the effects of secondary signaling on AHR biology in four specific aims (SA).[unreadable] SA1) Identify and characterize the proteins capable of interacting with the AHR in liver and immune cells in[unreadable] the absence and presence of ligand using tandem affinity purification, mass spectrometry and retroviral[unreadable] mediated gene transfer.[unreadable] SA2) Determine the fate of AHR complex members following ligand exposure using mass spectrometry and[unreadable] retroviral mediated gene transfer.[unreadable] SA3) Characterize the role of AHR-interacting proteins in ligand-induced signaling using RNAi, transient[unreadable] transfections and functional assays.[unreadable] SA4) Create a functional interaction network map for the AHR using proteins identified in the first aims and[unreadable] published reports and determine its overlap with regulatory networks.[unreadable] The completion of these aims will create a detailed picture of the AHR protein interaction network (AHR-PIN)[unreadable] and directly relate the proteins in this PIN to functional consequences for AHR mediated toxicity. Finally, the[unreadable] computational model that will be developed will generate new mechanistic directions for understanding the[unreadable] toxicity of AHR ligands and allow more accurate risk assessment for Superfund sites.