The retinoic acid receptors alpha and beta are expressed in the human promyelocytic leukemia cell line HL-60.
The human promyelocytic leukemia cell line HL-60 can be induced to differentiate into granulocytes upon exposure to retinoids. Previously we have shown that extracts of undifferentiated HL-60 cells possess a specific retinoid-binding activity (RSBP-1) corresponding to an approximate 95 kilodalton (kDa) protein as determined by size-exclusion chromatography. We now extend these observations to reveal a second approximate 95 kDa retinoic acid-binding component (RSBP-2), which is separable from RSBP-1 using anion exchange chromatography. We further show that the chromatographic properties of RSBP-1 and RSBP-2 are identical to those found for the retinoid-binding activities present in extracts of HeLa cells transfected with the human retinoic acid receptor (RAR) expression vectors RAR-beta phi and RAR-alpha phi, respectively. Moreover, an antiserum preparation directed against RAR-beta selectively immunoprecipitated both the retinoid-binding activity in extracts of HeLa cells transfected with RAR-beta phi and that corresponding to RSBP-1 in HL-60 cell extracts. Similarly, an antiserum preparation directed against RAR-alpha immunoprecipitated the retinoid-binding activity in extracts from RAR-alpha phi transfected HeLa cell as well as that corresponding to RSBP-2 in HL-60 cell extracts. Using these antisera, Western blot analyses of extracts from HL-60 cells, and from HeLa cells transfected with either RAR-alpha phi or RAR-beta phi, confirmed that RSBP-2 and RSBP-1 are identical to RAR-alpha and RAR-beta, respectively. However, RAR-alpha, RAR-beta, RSBP-1, and RSBP-2 appeared as an approximate 51 kDa species in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis in contrast with an apparent approximate 95 k mol wt as estimated from size-exclusion chromatography in the presence of 0.6 M KCl.(ABSTRACT TRUNCATED AT 250 WORDS)
Hashimoto
Y
Y
Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Petkovich
M
M
Gaub
M P
MP
Kagechika
H
H
Shudo
K
K
Chambon
P
P
D000906:Antibodies; D001565:Benzoates; D015153:Blotting, Western; D002352:Carrier Proteins / Q000032:analysis*; D002599:Chalcone; D047188:Chalcones; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000378:metabolism*; D008970:Molecular Weight; D018168:Receptors, Retinoic Acid; D013764:Tetrahydronaphthalenes; D014407:Tumor Cells, Cultured / Q000378:metabolism
D000906:Antibodies; D001565:Benzoates; D002352:Carrier Proteins; D047188:Chalcones; D018168:Receptors, Retinoic Acid; D013764:Tetrahydronaphthalenes; C061133:tamibarotene; D002599:Chalcone; C054189:3,5-di-tert-butylchalcone 4'-carboxylic acid