Je. Driscoll et al., LIGAND-INDUCED CONFORMATIONAL-CHANGES IN THE HUMAN RETINOIC ACID RECEPTOR-GAMMA DETECTED USING MONOCLONAL-ANTIBODIES, The Journal of biological chemistry, 271(38), 1996, pp. 22969-22975
The mechanism by which the naturally occurring ligand for a nuclear ho
rmone receptor regulates transcription remains largely unknown, One ap
proach combines the specificity of monoclonal antibodies, which recogn
ize a three-dimensional epitope, with ligand binding. Using purified r
etinoic acid receptor gamma D and E domains, a panel of six unique mon
oclonal antibodies were isolated and characterized using solid-state r
eceptor binding and retinoic acid receptor (RAR)-RXR heterodimer super
shift formation. Three antibodies are specific for RAR gamma (mAbI, mA
bII, and mAbV) and four recognize a three dimensional epitope (mAbI, m
AbIV, mAbV, and mAbVI). Three antibodies (mAbIII, mAbV, and mAbVI) dis
sociate from the receptor in electrophoretic mobility shift assays upo
n the addition of retinoic acid. In particular, the binding characteri
stics of mAbIII, whose epitope was mapped to a region identified as an
Omega-loop (amino acids 207-222), suggest a model for ligand binding
to the receptor, In this model, ligand binding causes a positioning of
helix 12 into a favorable conformation for interaction with the trans
criptional machinery. The Omega-loop then closes in order to stabilize
this ''active'' position. The results reported here also suggest that
a region of the hinge or D domain of the receptor (amino acids 156-18
8), an area that can play a role in protein-protein interactions, may
also be important in ligand-induced functional changes.