A Conserved Sequence Motif in the Integrin β3 Cytoplasmic Domain Is Required for Its Specific Interaction with β3-Endonexin

Abstract

Integrin signaling is mediated by interaction of integrin cytoplasmic domains with intracellular signaling molecules. Recently, we identified a novel 111-amino acid polypeptide, termed β3-endonexin, which interacts selectively with the integrin β3 cytoplasmic domain. In the present study we conducted a systematic mutational analysis of both the integrin β3 cytoplasmic domain and β3-endonexin to map sites required for interaction. The interaction of the full-length β3 integrin subunit with β3-endonexin in vitro required the β3 cytoplasmic domain. In a yeast two-hybrid system, both membrane-proximal and membrane-distal residues of the β3 cytoplasmic domain were necessary for interaction with β3-endonexin. In particular, the membrane-distal NITY motif at β3 756-759 was critical for the interaction. Exchange of β3 residues 756-759 (NITY) for the corresponding residues in β1 (NPKY) endowed the β1 cytoplasmic domain with the ability to interact with β3-endonexin. Conversely, exchange of the NPKY motif at β1 772-775 for the NITY motif in β3 abolished interaction of this chimeric cytoplasmic domain with β3-endonexin. Because the NITY motif is present in the β3 but not the β1 cytoplasmic domain, these results explain the selective interaction of this cytoplasmic domain with β3-endonexin. In addition, deletional analysis suggested that a core 91-residue sequence of β3-endonexin is sufficient for specific binding to the β3 cytoplasmic domain. These studies have identified a cytoplasmic domain sequence motif that specifies an integrin-specific protein-protein interaction. Integrin signaling is mediated by interaction of integrin cytoplasmic domains with intracellular signaling molecules. Recently, we identified a novel 111-amino acid polypeptide, termed β3-endonexin, which interacts selectively with the integrin β3 cytoplasmic domain. In the present study we conducted a systematic mutational analysis of both the integrin β3 cytoplasmic domain and β3-endonexin to map sites required for interaction. The interaction of the full-length β3 integrin subunit with β3-endonexin in vitro required the β3 cytoplasmic domain. In a yeast two-hybrid system, both membrane-proximal and membrane-distal residues of the β3 cytoplasmic domain were necessary for interaction with β3-endonexin. In particular, the membrane-distal NITY motif at β3 756-759 was critical for the interaction. Exchange of β3 residues 756-759 (NITY) for the corresponding residues in β1 (NPKY) endowed the β1 cytoplasmic domain with the ability to interact with β3-endonexin. Conversely, exchange of the NPKY motif at β1 772-775 for the NITY motif in β3 abolished interaction of this chimeric cytoplasmic domain with β3-endonexin. Because the NITY motif is present in the β3 but not the β1 cytoplasmic domain, these results explain the selective interaction of this cytoplasmic domain with β3-endonexin. In addition, deletional analysis suggested that a core 91-residue sequence of β3-endonexin is sufficient for specific binding to the β3 cytoplasmic domain. These studies have identified a cytoplasmic domain sequence motif that specifies an integrin-specific protein-protein interaction.