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.
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January 2004
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Research Article|
January 15 2004
A membrane proximal region of the integrin alpha5 subunit is important for its interaction with nischarin
Suresh K. ALAHARI;
Suresh K. ALAHARI
1
Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7365, U.S.A.
1To whom correspondence should be addressed (e-mail alahari@med.unc.edu).
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Hani NASRALLAH
Hani NASRALLAH
Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7365, U.S.A.
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Publisher: Portland Press Ltd
Received:
March 14 2003
Revision Received:
September 17 2003
Accepted:
October 09 2003
Accepted Manuscript online:
October 09 2003
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2004
2004
Biochem J (2004) 377 (2): 449–457.
Article history
Received:
March 14 2003
Revision Received:
September 17 2003
Accepted:
October 09 2003
Accepted Manuscript online:
October 09 2003
Citation
Suresh K. ALAHARI, Hani NASRALLAH; A membrane proximal region of the integrin alpha5 subunit is important for its interaction with nischarin. Biochem J 15 January 2004; 377 (2): 449–457. doi: https://doi.org/10.1042/bj20030411
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