The Retinoic Acid Receptors α and β are Expressed in the Human Promyelocytic Leukemia Cell Line HL-60

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Abstract
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..vphi. and RAR-.alpha..vphi., respectively. Moreover, an antiserum preparation directed againt RAR-.beta. selectively immunoprecipitated both the retinoid-binding activity in extracts of HeLa cells transfected with RAR-.beta..vphi. 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..vphi. 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-a.vphi. or RAR-.beta..vphi., 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. In addition size exclusion analyses in the presence of 0.6 M KCl but in the absence of retinoids indicated that all of these retinoid binding-components eluted as an approximately 42 kDa species. These findings are consistent with the possibility that the approximate 95 kDa species correspond to RAR homodimers formed under high salt conditions in the presence of retinoids.

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