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

Retinoic acid (RA) induces the differentiation of F9 cells cultured as mono layers into primitive endodermal-like cells, whereas a combination of RA an d cAMP leads to parietal endodermal differentiation. In RA receptor alpha-n ull F9 cells (RAR alpha(-/-)cells), RA still efficiently triggers RAR gamma -mediated primitive endodermal differentiation, but parietal endodermal dif ferentiation is markedly delayed. To investigate the role of RAR alpha 1 ac tivation functions AF-1 and AF-2 and of their phosphorylation sites during RA- and cAMP-induced parietal differentiation, cell lines reexpressing WT o r mutated RAR alpha 1 were established in RAR alpha(-/-) cells. We have fou nd that the protein kinase A (PKA) phosphorylation site and the AF-2AD core (helix 12) of RAR alpha 1 are required for efficient parietal endodermal d ifferentiation, whereas the AF-1 proline-directed kinase phosphorylation si te is dispensible. Interestingly, deletion of the AF-1 activating domain (t he A/B region), but not of the AF-2AD core, generates a dominant negative m utant that abrogates primitive endodermal differentiation when expressed in RAR alpha(-/-) cells. We also show that the RAR alpha AF-1 and AF-2 activa tion functions, but not their phosphorylation sites, are involved in the in duction of RA-responsive genes in a differential promoter context-dependent manner.