Redefining the role of the endogenous XAP2 and C-terminal hsp70-interacting protein on the endogenous Ah receptors expressed in mouse and rat cell lines

Abstract

Studies using transient expression systems have implicated the XAP2 protein in the control of aryl hydrocarbon receptor (AHR) stability and subcellular location. Thus, studies were performed in cell lines that expressed endogenous rat or mouse Ahb-1 (C57BL/6) or Ahb-2 (C3H) AHRs with similar levels of endogenous XAP2. Unliganded rat and mouse Ahb-2 receptor complexes associated with reduced levels of XAP2 and exhibited dynamic nucleocytoplasmic shuttling in comparison with Ahb-1 receptors. Rat and mouse Ahb-2 receptors also exhibited a greater magnitude of ligand-induced degradation than Ahb-1 receptors. Small interfering RNA reduction of endogenous XAP2 by >80% had minimal impact on the level of Ahb-2 receptors but resulted in a 25-30% reduction of Ah b-1 receptors. XAP2 reduction resulted in increased susceptibility of the Ahb-1 receptor to ligand-induced degradation yet produced higher levels of endogenous CYP1A1 induction. Stable expression of the Ah b-2 receptor in the C57BL/6 background resulted in a protein with reduced association with XAP2, dynamic nucleocytoplasmic shuttling, and increased levels of ligand-induced degradation. Small interfering RNA reduction of endogenous XAP2 in a C-terminal hsp70-interacting protein knockout mouse cell line, exhibited a 25-30% reduction in the level of endogenous Ahb-1 AHR and showed high levels of ligand-induced degradation. Thus, endogenous XAP2 exerts a negative function on a small fraction of the endogenous Ah b-1 receptor complex but appears to have a minimal impact on endogenous rat or Ahb-2 receptors. This implies that the analysis of the AHR-mediated signaling via rat and mouse Ahb-2 receptors may better represent the physiology of this signal transduction pathway. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.