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

Richard S Pollenz,Edward J Dougherty

doi:10.1074/jbc.m506619200

Richard S Pollenz, Edward J Dougherty

Open Access

https://doi.org/10.1074/jbc.m506619200

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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 Ah(b-1) (C57BL/6) or Ah(b-2) (C3H) AHRs with similar levels of endogenous XAP2. Unliganded rat and mouse Ah(b-2) receptor complexes associated with reduced levels of XAP2 and exhibited dynamic nucleocytoplasmic shuttling in comparison with Ah(b-1) receptors. Rat and mouse Ah(b-2) receptors also exhibited a greater magnitude of ligand-induced degradation than Ah(b-1) receptors. Small interfering RNA reduction of endogenous XAP2 by >80% had minimal impact on the level of Ah(b-2) receptors but resulted in a 25-30% reduction of Ah(b-1) receptors. XAP2 reduction resulted in increased susceptibility of the Ah(b-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 Ah(b-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 Ah(b-2) receptors. This implies that the analysis of the AHR-mediated signaling via rat and mouse Ah(b-2) receptors may better represent the physiology of this signal transduction pathway.

Highlights

Another common finding among the various studies is that XAP2 expression can impact the subcellular localization of the aryl hydrocarbon receptor (AHR) and appears to prevent dynamic nucleocytoplasmic shuttling (13, 16 –18)
A general assumption has been that the functions ascribed to XAP2 on the Ahb-1 receptor complex are universal for all other AHRs
These results imply that XAP2 may only impact AHR-mediated signaling in the context of the Ahb-1 receptor and suggest 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

Summary

Introduction

Another common finding among the various studies is that XAP2 expression can impact the subcellular localization of the AHR and appears to prevent dynamic nucleocytoplasmic shuttling (13, 16 –18). Reduction of Endogenous XAP2 with siRNA Results in Increased Levels of Ligand-induced Degradation of the AHR and Enhanced Levels of CYP1A1 Induction in Hepa-1 Cells—The reduction of endogenous Ahb-1 receptor by 25–30% is intriguing, since it has been hypothesized that there are distinct pools of AHR1⁄7hsp90 complexes in Hepa-1 cells [12], and previous studies have consistently shown that ϳ20 –25% of the endogenous AHR pool remains in Hepa-1 cells after ligand exposure (Fig. 3) [24, 26, 27].

Results

Conclusion