Identification of proteins that associate with integrin α2 by proteomic analysis in human fibrosarcoma HT-1080 cells.

Takayuki Uematsu, Chieko Konishi, Daisuke Hoshino, Xiao Han, Taizo Tomari, Nagayasu Egawa, Yoshikazu Takada, Toshiaki Isobe, Motoharu Seiki, Naohiko Koshikawa

Division of Cancer Cell Research, Institute of Medical Science, The University of Tokyo, Shirokanedai, Minato-ku, Japan.

Journal of cellular physiology 2012 Aug

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Integrins are adhesion receptors for components of the extracellular matrix (ECMs) that regulate multiple cellular functions, such as migration, invasion, proliferation, and survival by mediating bidirectional signal transmission. Even though many proteins have been reported to associate with integrins both on and in cells, systemic analyses of the adhesome have not been carried out. In previous studies, we identified proteins associating with a membrane-type protease, MT1-MMP, using nano-flow liquid chromatography/tandem mass spectrometry (nano-LC/MS/MS) of associated proteins prepared by optimized conditions for cell lysis and purification. Since integrins were identified as MT1-MMP-associated proteins, we next applied this method to analyze integrin-associated proteins. In this study, we expressed integrin α2 fused at the C terminus to a FLAG peptide in HT1080 cells. Cells stably expressing the chimeric protein were lysed with 1% Brij-98 and affinity purified using anti-FLAG antibody. Integrin β1 co-purified with integrin α2 confirming the specificity of the purification procedure. Analysis of the purified mixture by nano-LC/MS/MS identified 70 proteins. Nineteen of these were membrane proteins, including adhesion proteins, receptors, transporters, proteinases, and ion-channel receptors, and the balance were cytoplasmic. Interestingly, eight of the proteins had previously been shown to associate with MT1-MMP. We believe the present study provides a platform to facilitate the study of the mechanisms of cell adhesion, migration, and invasion. Copyright © 2011 Wiley Periodicals, Inc.

Citation

Takayuki Uematsu, Chieko Konishi, Daisuke Hoshino, Xiao Han, Taizo Tomari, Nagayasu Egawa, Yoshikazu Takada, Toshiaki Isobe, Motoharu Seiki, Naohiko Koshikawa. Identification of proteins that associate with integrin α2 by proteomic analysis in human fibrosarcoma HT-1080 cells. Journal of cellular physiology. 2012 Aug;227(8):3072-9