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

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 alpha5 integrin subunit. Overexpression of this protein profoundly affects cell migration. To examine the nischarin-alpha5 interaction in detail, and to find the minimal region required for the interaction, several mutants of nischarin and alpha5 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 alpha5 cytoplasmic tail is essential for the interaction. To characterize more directly the properties of the interaction between nischarin and alpha5, 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 alpha5 peptides. In addition, mutational analysis revealed that residues Tyr(1018) and Lys(1022) are crucial for alpha5-nischarin interactions. These results provide evidence that nischarin is capable of directly and selectively binding to a portion of the alpha5 cytoplasmic domain. Further studies demonstrated that the minimal alpha5 binding region of nischarin does not affect cell migration.