A Direct Interaction between the Aryl Hydrocarbon Receptor and Retinoblastoma Protein: LINKING DIOXIN SIGNALING TO THE CELL CYCLE

Nie-Lin Ge,Cornelis J Elferink

doi:10.1074/jbc.273.35.22708

Abstract

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor in eukaryotic cells that alters gene expression in response to the environmental contaminant 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD). In 5L hepatoma cells, TCDD induces a G1 cell cycle arrest through a mechanism that involves the AhR. The retinoblastoma tumor suppressor protein (pRb) controls cell cycle progression through G1 in addition to promoting differentiation. We examined whether the human AhR or its dimerization partner, the AhR nuclear translocator, interacts with pRb as a basis of the TCDD-induced cell cycle arrest. In vivo and in vitro assays reveal a direct interaction between pRb and the AhR but not the AhR nuclear translocator protein. Binding between the AhR and pRb occurs through two distinct regions in the AhR. A high affinity site lies within the N-terminal 364 amino acids of the AhR, whereas a lower affinity binding region colocalizes with the glutamine-rich transactivation domain of the receptor. AhR ligand binding is not required for the pRb interaction per se, although immunoprecipitation experiments in 5L cells reveal that pRb associates preferentially with the liganded AhR, consistent with a requirement for ligand-induced nuclear translocation. These observations provide a mechanistic insight into AhR-mediated cell cycle arrest and a new perspective on TCDD-induced toxicity.

Highlights

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor in eukaryotic cells that alters gene expression in response to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
The TCDD-inducible G1 arrest in 5L cells may bespeak an interaction between pRb and the AhR-AhR nuclear translocator (Arnt) complex and is supported by immunocoprecipitation experiments (Fig. 1)
Immunoprecipitates from control or TCDD-treated 5L cell lysates using an antibody against human pRb were analyzed by Western blotting for the presence of AhR, Arnt protein, and pRb

Summary

LINKING DIOXIN SIGNALING TO THE CELL CYCLE*

We examined whether the human AhR or its dimerization partner, the AhR nuclear translocator, interacts with pRb as a basis of the TCDD-induced cell cycle arrest. AhR ligand binding is not required for the pRb interaction per se, immunoprecipitation experiments in 5L cells reveal that pRb associates preferentially with the liganded AhR, consistent with a requirement for ligand-induced nuclear translocation These observations provide a mechanistic insight into AhR-mediated cell cycle arrest and a new perspective on TCDD-induced toxicity. The C terminus of each protein contains a transactivation domain (TAD) Both TADs are functionally competent (e.g. in yeast expression systems), in vivo studies indicate that the TAD of AhR predominates, at least in TCDD-induced CYP1A1 gene expression [14, 15]. The TCDD-inducible G1 cell cycle arrest in 5L cells may involve an interaction between pRb and the AhR-Arnt complex

These observations prompted us to examine whether the

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION