The Disabled 1 Phosphotyrosine-Binding Domain Binds to the Internalization Signals of Transmembrane Glycoproteins and to Phospholipids

Abstract

Disabled gene products are important for nervous system development in drosophila and mammals. In mice, the Dab1 protein is thought to function downstream of the extracellular protein Reln during neuronal positioning. The structures of Dab proteins suggest that they mediate protein-protein or protein-membrane docking functions. Here we show that the amino-terminal phosphotyrosine-binding (PTB) domain of Dab1 binds to the transmembrane glycoproteins of the amyloid precursor protein (APP) and low-density lipoprotein receptor families and the cytoplasmic signaling protein Ship. Dab1 associates with the APP cytoplasmic domain in transfected cells and is coexpressed with APP in hippocampal neurons. Screening of a set of altered peptide sequences showed that the sequence GYXNPXY present in APP family members is an optimal binding sequence, with approximately 0.5 μM affinity. Unlike other PTB domains, the Dab1 PTB does not bind to tyrosine-phosphorylated peptide ligands. The PTB domain also binds specifically to phospholipid bilayers containing phosphatidylinositol 4P (PtdIns4P) or PtdIns4,5P₂ in a manner that does not interfere with protein binding. We propose that the PTB domain permits Dab1 to bind specifically to transmembrane proteins containing an NPXY internalization signal.

ACKNOWLEDGMENTS

We thank Steve Hahn for his encouragement in the use of the fluorescence polarization device, Wendy Thomas and John Glomsett for use of the LUV extrusion bomb, John Rasko for plasmid, Jürgen Wehland for helpful suggestions on the peptide arrays, Len Stevens and Lew Cantley for advice, Ching-Shih Chen for a kind gift of di-C₁₆-PtdIns3,4,5P₃, and Andrea Tiepold and Brigitte Kornak for help with the peptide synthesis.

This work was supported by R01-CA-41072 (J.A.C.) and NIH GM58801-01 (F.B.G.).