In vitro binding of retinoids to the nuclear retinoic acid receptor alpha

We describe a rapid method for measuring in vitro binding properties of new synthetic retinoids to the recently identified nuclear receptor RAR alpha. Transfection of cos-7 cells with the expression vector RAR alpha O produces a 100-fold increase in intracellular RAR alpha concentration which allows us to perform accurate determination of binding parameters of various retinoids. Cytosol and nuclear extracts obtained after freeze drying of the transfected cells are incubated with a new stable tritiated analog of retinoic acid, 3H]CD367. Complete separation between RAR alpha and endogenous cellular retinoic acid binding protein is achieved by high-performance size-exclusion chromatography. These improved techniques provide a useful method for determining binding affinities of analogs to RAR alpha.