No abstract

Transcriptional activation by nuclear receptors is achieved through autonomous activation functions (AFs), a constitutive N‐terminal AF‐1 and a C‐terminal, ligand‐dependent AF‐2 that comprises a motif conserved between nuclear receptors. We have performed an extensive mutational analysis of the putative AF‐2 domain of chicken thyroid hormone receptor alpha (cT3R alpha). We show that the AF‐2 region mediates transactivation as well as transcriptional interference (squelching), not only between the thyroid hormone and vitamin (type II) receptors, but also between type II and steroid hormone (type I) receptors. Transcriptional activation and interference require equivalent doses of the cognate ligand, and mutations in the conserved motif that reduce ligand‐induced transactivation also impair transcriptional interference. When fused to the Gal4 DNA binding domain, a 35 amino acid long fragment containing the conserved motif is able to transactivate and squelch, albeit in a ligand‐independent manner. Our results define the AF‐2 of cT3R alpha as an autonomous transactivation domain that, in its natural context, is governed by ligand. We propose that AF‐2 is probably part of a surface for interaction with either a general transcription factor or a putative bridging factor, that might be utilized by type I and II receptors.

Retinoic acid (RA) treatment of SH-SY5Y neuroblastoma cells results in activation of phosphatidylinositol-3-kinase (PI3K) signaling pathway, and this activation is required for RA-induced differentiation. Here we show that RA activates PI3K and ERK1/2 MAPK signaling pathways through a rapid, nongenomic mechanism that does not require new gene transcription or newly synthesized proteins. Activation of PI3K by RA appears to involve the classical nuclear receptor, retinoic acid receptor (RAR), on the basis of the pharmacological profile of the activation, loss, and gain of function experiments with mouse embryo fibroblast-RAR(alpha beta gamma)(L-/L-) null cells, and the physical association between liganded RAR and PI3K activity. The association of RAR with the two subunits of PI3K was differentially regulated by the ligand. Immunoprecipitation experiments performed in SH-SY5Y cells showed stable association between RARalpha and p85, the regulatory subunit of PI3K, independently of the presence of RA. In contrast, ligand administration increased the association of p110, the catalytic subunit of PI3K, to this complex. The intracellular localization of RAR proved to be relevant for PI3K activation. A chimerical RAR fusing c-Src myristylation domain to the N terminus of RARalpha (Myr-RARalpha) was targeted to plasma membrane. Transfection of Myr-RARalpha to mouse embryo fibroblast-RAR(alpha beta gamma)(L-/L-) null cells and COS-7 cells results in strong activation of the PI3K signaling pathway, although both in the absence as well in the presence of RA. Our results support a mechanism in which ligand binding to RAR would play a major role in the assembly and intracellular location of a signaling complex involving RAR and the subunits of PI3K.