The seven members of the Dok adapter protein family share a highly conserved phosphotyrosine-binding (PTB) domain. In the case of Dok-1,2 and 3, the PTB domain binds to the lipid phosphatase Ship1, a key component of their inhibitory signaling mechanisms in immune cells. In contrast to most other Dok family members, Dok-4 is expressed widely but is poorly understood, largely because of limited knowledge of its partner molecules. We previously showed that, in contrast to the Dok-1 PTB domain (defined as aa 107-260), the homologous sequence in Dok-4 (aa 100-233) bound very poorly to Ret, a known Dok-4 partner. In the current study, we show that binding of Dok-4 to Ret requires residues C-terminal to the previously defined PTB domain boundaries (up to aa 246). These residues are predicted to form an extension in a critical C-terminal a-helix. We show that the Dok-4 PTB domain also binds the phosphorylated NPXY motifs in Ship1 but not Ship2. Finally, we found that a rare human single nucleotide polymorphism causing a R186H substitution in the PTB domain abolishes tyrosine phosphorylation of Dok-4 by Ret. In addition to providing a clearer understanding of Dok-4 PTB domain structure and function, our findings point to a potential mechanism for Dok-4 inhibitory signaling in T-cells and to the possibility of a rare Dok-4-related phenotype in humans. (C) 2012 Elsevier Inc. All rights reserved.