Sensitive and specific detection of retinoid receptor subtype proteins in cultured cell and tumor extracts.
Subtype-specific antipeptide antibodies have been developed against each of the retinoic acid receptors (RARs alpha, beta, and gamma) and each of the retinoid X receptors (RXRs alpha, beta, and gamma). Each antibody reacts specifically with its respective recombinantly expressed protein but not with any of the other retinoid receptor subtypes, by both immunoblot and immunoprecipitation technology. We describe a sensitive and specific assay that combines the binding of cultured cell and tumor extracts to [3H]all-trans-retinoic acid or [3H]9-cis-retinoic acid with immunoprecipitation of the hormone-receptor complexes by the subtype-specific antibodies to determine the levels of functional retinoid receptor subtype proteins that are present. We also report the use of a hormone-binding assay that uses RAR- and RXR-selective compounds as competitors of the tritiated retinoids to ascertain the RAR and RXR subfamily profiles of these cells. HeLa cells contain all six retinoid 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 approximately 20-60 fmol receptor/mg protein, and RAR beta is expressed at lower levels (approximately 5 fmol/mg) in Hep G2 cells. MCF-7 cells in culture express RAR alpha (approximately 32 fmol/mg), RAR gamma (approximately 35 fmol/mg), and RXR alpha (approximately 60 fmol/mg).(ABSTRACT TRUNCATED AT 250 WORDS)
Titcomb
M W
MW
Ligand Pharmaceuticals, Inc., Department of Biochemistry, La Jolla, California 92037.
Gottardis
M M
MM
Pike
J W
JW
Allegretto
E A
EA
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D015153:Blotting, Western; D003429:Cross Reactions; D018076:DNA, Complementary / Q000235:genetics; D004354:Drug Screening Assays, Antitumor; D006801:Humans; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000032:analysis* / Q000145:classification; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology; D011233:Precipitin Tests; D018168:Receptors, Retinoic Acid / Q000032:analysis* / Q000145:classification; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D047488:Retinoid X Receptors; D012176:Retinoids / Q000378:metabolism; D014020:Tissue Extracts / Q000737:chemistry*; D014157:Transcription Factors / Q000032:analysis* / Q000145:classification; D014316:Tritium; D014407:Tumor Cells, Cultured / Q000737:chemistry*
D018076:DNA, Complementary; D008024:Ligands; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D018168:Receptors, Retinoic Acid; D011993:Recombinant Fusion Proteins; D047488:Retinoid X Receptors; D012176:Retinoids; D014020:Tissue Extracts; D014157:Transcription Factors; D014316:Tritium