The Reelin signaling pathway plays a critical role in the correct positioning of neurons within the developing brain. Within this pathway, Disabled I (Dab l) serves as an intracellular adaptor that is tyrosine phosphorylated when Reelin, a secreted glycoprotein, binds to the lipoprotein receptors VLDLR and ApoER2 on the surface of neurons. The phosphotyrosine-binding (PTB) domain within its amino terminus enables Dab1 to recognize and bind to a conserved sequence motif within the cytoplasmic tails of the receptors. In addition, the PTB contains a Pleckstrin Homology-like subdomain that binds to phosphoinositides. Here, we show that the phosphoinositide-binding region within Dab1 PTB domain is required for membrane localization and basal tyrosine phosphorylation of Dab1 independently of VLDLR and ApoER2. Furthermore, receptor-independent membrane targeting of Dab1 is required for its interaction with Src and Crk, and disruption of phosphoinositide binding also blocks subsequent Reelin-induced tyrosine phosphorylation of Dab1. (c) 2005 Elsevier Inc. All rights reserved.