Sl. Levine et al., A tetratricopeptide repeat half-site in the aryl hydrocarbon receptor is important for DNA binding and trans-activation potential, MOLEC PHARM, 58(6), 2000, pp. 1517-1524
Similar to certain unliganded steroid hormone receptor complexes, the unlig
anded aryl hydrocarbon receptor has been shown to consist of a multimeric c
ore complex that includes the 90-kDa heat shock protein (hsp90) and the imm
unophilin-like hepatitis B X-associated protein 2 (XAP2). Immunophilins and
XAP2 associated with these complexes bind to the carboxyl-terminal end of
hsp90 through an interaction with their tetratricopeptide repeat (TPR) doma
ins. The consensus TPR binding motif contains two domains, A and B. Recentl
y, the carboxyl terminus of XAP2 has been shown to contain a highly conserv
ed TPR domain that is required for the assembly of XAP2 with both hsp90 and
AhR. A search of the murine AhR sequence identified domain B (A-F-A-P) of
the consensus TPR sequence directly adjacent to the carboxyl-terminal side
of the helix-loop-helix region of the murine and human AhR. We hypothesized
that this conserved domain B region may be involved with mediating interac
tions between either AhR-hsp90, AhR-XAP2, and/or AhR-AhR nuclear translocat
or protein. Site-directed mutagenesis of the amino-terminal alanine residue
of this region to an aspartic acid (A78D) completely inhibited 2,3,7,8-tet
rachloro-p-dioxin (TCDD)-dependent activation of a xenobiotic response elem
ent (XRE) driven gene expression construct in transfected COS-1 and BP8 cel
ls. The A82F mutation caused a 40 to 50% decrease in TCDD-dependent activat
ion. The inability of A78D and the reduction of A82F to trans-activate XRE-
driven reporter activity did not result from impaired AhR-XAP2-hsp90 intera
ctions, TCDD-dependent AhR translocation to the nucleus, or AhR-AhR nuclear
translocator protein interactions. In vitro DNA binding analysis demonstra
ted that loss of trans-activation potential by the A78D mutation resulted f
rom impaired XRE binding. This study underscores the potential importance o
f AhR mutations that occur naturally outside of known functional domains.