title: Identification of proteins that associate with integrin α2 by proteomic analysis in human fibrosarcoma HT-1080 cells.
abstract: 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.
authors: Division of Cancer Cell Research, Institute of Medical Science, The University of Tokyo, Shirokanedai, Minato-ku, Japan.
authors: Takayuki
authors: T
authors: Uematsu
authors: Chieko
authors: C
authors: Konishi
authors: Daisuke
authors: D
authors: Hoshino
authors: Xiao
authors: X
authors: Han
authors: Taizo
authors: T
authors: Tomari
authors: Nagayasu
authors: N
authors: Egawa
authors: Yoshikazu
authors: Y
authors: Takada
authors: Toshiaki
authors: T
authors: Isobe
authors: Motoharu
authors: M
authors: Seiki
authors: Naohiko
authors: N
authors: Koshikawa
mesh_terms: D045744:Cell Line, Tumor; D005354:Fibrosarcoma / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D039421:Integrin alpha2 / Q000235:genetics* / Q000378:metabolism*; D039641:Integrin beta Chains / Q000302:isolation & purification; D053511:Matrix Metalloproteinase 14 / Q000378:metabolism; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics*
chemical_list: D039421:Integrin alpha2; D039641:Integrin beta Chains; D008565:Membrane Proteins; D009842:Oligopeptides; D010455:Peptides; D011994:Recombinant Proteins; C085706:FLAG peptide; C507086:MMP14 protein, human; D053511:Matrix Metalloproteinase 14