A membrane proximal region of the integrin alpha5 subunit is important for its interaction with nischarin

Abstract

In a previous study [Alahari, Lee and Juliano (2000) J. Cell Biol. 151, 1141–1154], we have identified a novel protein, nischarin, that specifically interacts with the cytoplasmic tail of the α5 integrin subunit. Overexpression of this protein profoundly affects cell migration. To examine the nischarin–α5 interaction in detail, and to find the minimal region required for the interaction, several mutants of nischarin and α5 were created. The results obtained for the yeast two-hybrid system indicate that a 99-aminoacid region of nischarin (from residues 464 to 562) is indispensable for the interaction. Also, we demonstrate that the membrane proximal region (from residues 1017 to 1030) of the α5 cytoplasmic tail is essential for the interaction. To characterize more directly the properties of the interaction between nischarin and α5, we performed surface-plasmon resonance studies in which peptides were immobilized on the surface of a sensor chip, and the recombinant nischarin protein fragments were injected. Consistent with the two-hybrid results, recombinant nischarin binds well to immobilized α5 peptides. In addition, mutational analysis revealed that residues Tyr1018 and Lys1022 are crucial for α5–nischarin interactions. These results provide evidence that nischarin is capable of directly and selectively binding to a portion of the α5 cytoplasmic domain. Further studies demonstrated that the minimal α5 binding region of nischarin does not affect cell migration.