17 páginas, 9 figuras.Retinoic Acid (RA) treatment of SH-SY5Y neuroblastoma cells results in activation of phosphatidyl-inositol-3-kinase (PI3K) signaling pathway, and this activation is required for RA-induced differentiation. Here we show that RA activates PI3K and ERK1/2 MAP Kinase signaling pathways through a rapid, non-genomic mechanism that does not require new gene transcription or newly synthesized proteins. Activation of PI3K by RA appears to involve the classical nuclear receptor RAR, on the basis of the pharmacological profile of the activation, loss and gain of function experiments with MEF-RAR(αβγ)L-/L- null cells, and the physical association between liganded RAR and PI3K kinase 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 RARα 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 resulted to be relevant for PI3K activation. A chimerical RAR receptor fusing c-Src myristylation domain to the N-terminal of RARα (Myr-RARα) was targeted to plasma membrane. Transfection of Myr-RARα to MEF-RAR(αβγ)L-/Lnull cells and COS-7 cells results in strong activation of 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.This work was supported by grants of the Spanish former Ministry of Science and Technology and Ministry of Education and Science [SAF2003-00311 and SAF2006-00647 (to D.B.)]; SAF2004-07131 (to A.R.d.L.), European Commission [QLK3-2002-02029 “Anticancer Retinoids” (to A.R.d.L.)], Generalitat Valenciana [GRUPOS 03/15 and ACOMP 06/212 (to D.B.)] and Fondo Europeo de Desarrollo Regional.Peer reviewed