LIGAND-INDUCED CONFORMATIONAL-CHANGES IN THE HUMAN RETINOIC ACID RECEPTOR-GAMMA DETECTED USING MONOCLONAL-ANTIBODIES

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.