B. Lefebvre et al., STRUCTURAL DETERMINANTS OF THE LIGAND-BINDING SITE OF THE HUMAN RETINOIC ACID RECEPTOR-ALPHA, Biochemistry, 34(16), 1995, pp. 5477-5485
The ligand-dependent transactivating properties of retinoic acid recep
tors are controlled through a complex structure at the C-terminus of t
hese proteins, commonly referred to as the hormone binding domain. Thi
s domain is involved not only in ligand recognition but also in protei
n-protein interactions such as homo- and heterodimerization processes.
To identify more precisely regions of the human all-trans-retinoic ac
id receptor alpha (hRAR alpha) that are involved in Ligand binding, we
constructed a series of deletion mutants of this molecule and overexp
ressed them in bacteria. We found that the C-terminal part of the D do
main (amino acids 186-198) was necessary for ligand binding. The F dom
ain and the 10 C-terminal amino acids of the E domain were dispensable
for high-affinity binding of various natural and synthetic retinoids.
A further deletion to position 403 resulted in a moderate decrease in
affinity for all-trans-(ATRA) and 9-cis-retinoic acids, whereas the b
inding of two RAR alpha-specific ligands (Am80 and Am580) was abolishe
d. In addition, hRAR alpha and the minimal hormone binding domain (ami
no acids 186-410) bound ATRA with a positive, cooperative mechanism. T
his behavior was not observed with CD367, a conformationally restricte
d synthetic retinoid. The positive cooperativity could be correlated w
ith stable ATRA binding to RAR homodimers, whose formation was trigger
ed by ligand. In the same conditions, only monomeric CD367-RAR alpha c
omplexes were detected. These data indicate that ligand binding to hRA
R alpha requires the presence of part of the D domain, whereas the C-t
erminal end of the E domain is involved in more subtle ligand recognit
ion processes, They also clearly suggest that structurally distinct re
tinoids interact differently with the Ligand-binding site of this rece
ptor.