Reelin, a large secreted extracellular matrix glycoprotein, plays a key role in neuronal migration during cortical development and promotes neuronal maturation. The signaling pathway regulating neuronal maturation in the postnatal period are relatively less well understood. In this study, we demonstrated that a heterotrimeric G protein, Go, is a novel target of Reelin-induced signaling to promote neurite outgrowth. In primary hippocampal neurons of Reelin-deficient reeler mice, neurite outgrowth was significantly reduced and rescued upon addition of Reelin. Pertussis toxin (PTX) treatment or transfection with G alpha(o)-siRNA suppressed Reelin-mediated neurite outgrowth in wild-type neurons. Additionally, Reelin treatment led to increased phosphorylation of AKT, GSK3 beta, and JNK, which were all effectively blocked by the PI3K inhibitor, LY294002. By comparison, PTX specifically blocked JNK activation, but not AKT and GSK3 beta. Immunoprecipitation assays disclosed that Reelin increases the active forms of both Src and G alpha(o) and promotes their direct association. Notably, Dab1, a cytoplasmic adaptor molecule that mediates Reelin signaling, did not interact with G alpha(o). Neurite outgrowth by Reelin was induced via activating Src kinase, which directly stimulated G alpha(o), activity, leading to JNK activation. Based on the collective findings, we suggest that Reelin-dependent signaling mechanisms may be split into Src-AKT-dependent and Src-Go-dependent pathways. Our results additionally provide evidence that Reelin receptors cross-communicate with heterologous G protein-coupled receptors (GPCR) independently of the cognate ligands of GPCR. (C) 2015 Elsevier Inc. All rights reserved.