SENSITIVE AND SPECIFIC DETECTION OF RETINOID RECEPTOR SUBTYPE PROTEINS IN CULTURED-CELL AND TUMOR EXTRACTS

Subtype-specific antipeptide antibodies have been developed against ea ch of the retinoic acid receptors (RARs alpha, beta, and gamma) and ea ch of the retinoid X receptors (RXRs alpha, beta, and gamma). Each ant ibody reacts specifically with its respective recombinantly expressed protein but not with any of the other retinoid receptor subtypes, by b oth immunoblot and immunoprecipitation technology. We describe a sensi tive and specific assay that combines the binding of cultured cell and tumor extracts to [H-3]all-trans-retinoic acid or [H-3]S-cis-retinoic acid with immunoprecipitation of the hormone-receptor complexes by th e subtype-specific antibodies to determine the levels of functional re tinoid receptor subtype proteins that are present. We also report the use of a hormone-binding assay that uses RAR- and RXR-selective compou nds as competitors of the tritiated retinoids to ascertain the RAR and RXR subfamily profiles of these cells. HeLa cells contain all six ret inoid receptor proteins ranging in concentration from 9 fmol/mg total protein for RAR beta and RXR gamma to 50 fmol/mg for RXR alpha. Hep G2 and HL60 cells express RAR alpha and RXR alpha proteins at approximat ely 20-60 fmol receptor/mg protein, and RAR beta is expressed at lower levels (similar to 5 fmol/mg) in Rep G2 cells. MCF-7 cells in culture express RAR alpha (similar to 32 fmol/mg), RAR gamma (similar to 35 f mol/mg), and RXR alpha (similar to 60 fmol/mg). Estrogen treatment of the MCF-7 cells and MCF-7 cell-derived tumors results in the up-regula tion of RAR alpha protein (similar to 80 and similar to 57 fmol/mg, re spectively) and the downregulation of RXR alpha protein (similar to 12 and similar to 24 fmol/mg, respectively). ME-180 cell-derived tumors express RAR alpha (similar to 7 fmol/mg), RAR gamma (similar to 28 fmo l/mg), and RXR alpha (similar to 30 fmol/mg). These assays have clear ramifications in the development of drug treatment strategies that wou ld make use of retinoid receptor subtype-selective drugs.