The AF-1 and AF-2 activating domains of retinoic acid receptor-alpha (RARalpha) and their phosphorylation are differentially involved in parietal endodermal differentiation of F9 cells and retinoid-induced expression of target genes

Mol Endocrinol. 2000 Sep;14(9):1398-410. doi: 10.1210/mend.14.9.0527.


Retinoic acid (RA) induces the differentiation of F9 cells cultured as monolayers into primitive endodermal-like cells, whereas a combination of RA and cAMP leads to parietal endodermal differentiation. In RA receptor alpha-null F9 cells (RARalpha-/- cells), RA still efficiently triggers RARgamma-mediated primitive endodermal differentiation, but parietal endodermal differentiation is markedly delayed. To investigate the role of RARalpha1 activation functions AF-1 and AF-2 and of their phosphorylation sites during RA- and cAMP-induced parietal differentiation, cell lines reexpressing WT or mutated RARalpha1 were established in RARalpha-/- cells. We have found that the protein kinase A (PKA) phosphorylation site and the AF-2AD core (helix 12) of RARalpha1 are required for efficient parietal endodermal differentiation, whereas the AF-1 proline-directed kinase phosphorylation site is dispensible. Interestingly, deletion of the AF-1 activating domain (the A/B region), but not of the AF-2AD core, generates a dominant negative mutant that abrogates primitive endodermal differentiation when expressed in RARalpha-/- cells. We also show that the RARalpha AF-1 and AF-2 activation functions, but not their phosphorylation sites, are involved in the induction of RA-responsive genes in a differential promoter context-dependent manner.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites
  • Cell Differentiation
  • Cell Line
  • Cyclic AMP / physiology
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • Endoderm / cytology
  • Endoderm / physiology
  • Mice
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism*
  • Phosphorylation
  • Receptors, Glucocorticoid / metabolism
  • Receptors, Interferon / genetics
  • Receptors, Interferon / metabolism*
  • Receptors, Retinoic Acid / chemistry
  • Receptors, Retinoic Acid / genetics*
  • Receptors, Retinoic Acid / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Retinoic Acid Receptor alpha
  • Reverse Transcriptase Polymerase Chain Reaction
  • Transfection
  • Tretinoin / pharmacology


  • Ifngr2 protein, mouse
  • Nuclear Proteins
  • Rara protein, mouse
  • Receptors, Glucocorticoid
  • Receptors, Interferon
  • Receptors, Retinoic Acid
  • Recombinant Proteins
  • Retinoic Acid Receptor alpha
  • Tretinoin
  • Cyclic AMP
  • Cyclic AMP-Dependent Protein Kinases