Role of arginine residues 14 and 15 in dictating DNA binding stability and transactivation of the aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator heterodimer.
The aryl hydrocarbon receptor (AHR) and its DNA binding partner, the aryl hydrocarbon receptor nuclear translocator (ARNT) are basic helix-loop-helix/PAS proteins. The goal of the current study was to determine the extent to which residues R14 and R15 contained within the basic region of the AHR contribute to the DNA binding affinity and stability of the AHR/ARNT heterodimer. Towards this end, we first performed equilibrium binding and dissociation rate analyses using a single dioxin response element (DRE-1). While the K(D) and Bmax values obtained from the equilibrium binding analysis were similar for the wild-type AHR (wt AHR) and that containing the substitutions of R14 and R15 with Q residues (Q14Q15 AHR), dissociation rate analyses revealed that the stability of the Q14Q15 AHR DNA binding complex was approximately 10-fold less. Using a two-site DNA binding model, we also found that AHR/ARNT heterodimer does not participate in cooperative binding, as binding of the second dimer appears to be prohibited by occupation of the first. This property was similar regardless of the composition of the amino acids at positions 14 and 15. Finally, reporter assays revealed that the Q14Q15 substitutions severely compromised the ability of the AHR to activate gene expression despite appropriate nuclear localization. The present results revealed that DNA binding stability of the AHR/ARNT heterodimer is an important requirement for its transactivation capabilities and that this stability is governed, in part, by residues R14 and R15 that lie within the basic region of the AHR.
Wache
Susanne C
SC
Department of Molecular and Biomedical Pharmacology, University of Kentucky Medical Center, Lexington, KY 40536, USA.
Hoagland
Erica M
EM
Zeigler
Georgia
G
Swanson
Hollie I
HI
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine / Q000502:physiology*; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator / Q000378:metabolism*; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D015870:Gene Expression; D007700:Kinetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000378:metabolism; D018336:Receptors, Aryl Hydrocarbon / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation
D021122:Protein Subunits; D018336:Receptors, Aryl Hydrocarbon; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D004247:DNA; D001120:Arginine