Hydrodynamic characterization and DNA-binding properties of the liganded and unliganded forms of the retinoic-acid receptor alpha from human HL-60 cells.
The hydrodynamic parameters of the retinoic-acid receptor from human myeloblastic leukemia HL-60 cells were accurately investigated. The ligand-bound retinoic-acid receptor (RAR) has a Stokes radius of 3.5 nm when analyzed by size-exclusion chromatography. A 53-kDa protein was detected by Western blot analysis using a polyclonal antibody directed against the F domain of hRAR alpha, in the fractions containing the 3.5-nm complex. Fractionation of a crude nuclear extract from HL-60 cells, untreated with retinoic acid, yielded antibody-revealed material with Stokes radii ranging from 3.5 nm to 6 nm. From the hydrodynamic data, a molecular mass of 52 kDa was calculated for the liganded receptor, whereas no precise value could be deduced for the unliganded receptor form, since it dissociates rapidly into the 3.5-nm form. Gel-retardation experiments showed that the 3.5-nm form of hRAR alpha bound specifically to DNA, whereas binding of the unliganded receptor form was sharply reduced. These findings suggest that the unliganded inactive receptor form dissociates upon ligand binding and acquires a ligand-dependent DNA-binding activity.
Sablonniere
B
B
Laboratoire de Biochimie Structurale, Faculté de Médecine, Lille, France.
Dallery
N
N
Danze
P M
PM
Hublau
P
P
Formstecher
P
P
Dautrevaux
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D018168:Receptors, Retinoic Acid / Q000378:metabolism*; D000072482:Retinoic Acid Receptor alpha; D014212:Tretinoin / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014867:Water / Q000378:metabolism
D008024:Ligands; C000607071:RARA protein, human; D018168:Receptors, Retinoic Acid; D000072482:Retinoic Acid Receptor alpha; D014867:Water; D014212:Tretinoin; D004247:DNA