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Proteomic analysis of α4β1 integrin adhesion complexes reveals α-subunit-dependent protein recruitment.

Byron A, Humphries JD, Craig SE, Knight D, Humphries MJ - Proteomics (2012)

Bottom Line: Here, we report the isolation of ligand-induced adhesion complexes associated with wild-type α4β1 integrin, an activated α4β1 variant in the absence of the α cytoplasmic domain (X4C0), and a chimeric α4β1 variant with α5 leg and cytoplasmic domains (α4Pα5L), and the cataloguing of their proteomes by MS.Furthermore, we demonstrate colocalization of MYO18A with active integrin in migrating cells.These datasets provide a resource for future studies of integrin receptor-specific signaling events.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, UK.

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Hierarchical clustering analysis of proteins identified by MS ana-lyses of VCAM-1 and control affinity purifications. (A) Integrin-associated complexes were isolated from K562 cells expressing WT α4 integrin (α4), α4 integrin with a cytoplasmic domain deletion (X4C0), and an α4α5 chimera comprising the N-terminal β-propeller domain of α4 integrin linked to the leg, transmembrane, and cytoplasmic domains of α5 integrin (α4Pα5L). (B) Complete output of hierarchical clustering analysis of identified proteins (see Supporting Information for details). (C–H) Selected clusters are displayed, as indicated by blue dendrogram nodes and bars in (B). Correlations at selected dendrogram nodes are indicated. PAI, phosphoribosylaminoimidazole; TNFR, tumor necrosis factor receptor.
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fig01: Hierarchical clustering analysis of proteins identified by MS ana-lyses of VCAM-1 and control affinity purifications. (A) Integrin-associated complexes were isolated from K562 cells expressing WT α4 integrin (α4), α4 integrin with a cytoplasmic domain deletion (X4C0), and an α4α5 chimera comprising the N-terminal β-propeller domain of α4 integrin linked to the leg, transmembrane, and cytoplasmic domains of α5 integrin (α4Pα5L). (B) Complete output of hierarchical clustering analysis of identified proteins (see Supporting Information for details). (C–H) Selected clusters are displayed, as indicated by blue dendrogram nodes and bars in (B). Correlations at selected dendrogram nodes are indicated. PAI, phosphoribosylaminoimidazole; TNFR, tumor necrosis factor receptor.

Mentions: Integrin activation is coupled to spatial separation of the cytoplasmic domains of the integrin heterodimer [7]. To examine the recruitment of proteins to adhesion complexes in the absence of β integrin tail interactions with an α integrin tail, we used an α4 integrin C-terminal cytoplasmic domain deletion mutant (X4C0) (Fig. 1A) [19]. This mutant may mimic the cytoplasmic domain separation linked to integrin activation because of the loss of the α–β clasp, the loss of lateral association with other cell-surface receptors, the exposure of β1 integrin-binding sites or the reduced steric hindrance around the β1 integrin tail in the absence of the α4 integrin tail. The truncation of the α4 cytoplasmic domain has been used previously to indicate a role for α4 integrin in a cell motility signaling pathway that is distinct from α5 integrin-stimulated migration [9, 12]. To examine differences in recruitment of proteins to α4β1 and α5β1 integrin cytoplasmic domains, we used an α4α5 integrin chimera comprising the N-terminal β-propeller domain of α4 integrin – which determines ligand specificity – linked to the leg, transmembrane and cytoplasmic domains of α5 integrin (α4Pα5L) (Fig. 1A) [20]. K562 human chronic myelogenous leukemia cells were used for our work because this cell line has served as a prototype for many analyses of integrin signaling and adhesion. The integrin complement of K562 cells is dominated by α5β1 integrin, which enabled us to use cells into which wild-type (WT) α4 integrin, X4C0, or α4Pα5L transgenes had been introduced. These cell lines are referred to here as K562-α4, K562-X4C0 (gifts from M. E. Hemler, Dana-Farber Cancer Institute, Boston, MA, USA) [19], and K562-α4Pα5L [20], respectively. All cell lines were cultured in RPMI 1640 medium supplemented with 10% (v/v) FCS (Lonza Bioscience, Wokingham, UK), 2 mM l-glutamine, and Geneticin (1 mg/mL; Invitrogen, Paisley, UK) at 37°C.


Proteomic analysis of α4β1 integrin adhesion complexes reveals α-subunit-dependent protein recruitment.

Byron A, Humphries JD, Craig SE, Knight D, Humphries MJ - Proteomics (2012)

Hierarchical clustering analysis of proteins identified by MS ana-lyses of VCAM-1 and control affinity purifications. (A) Integrin-associated complexes were isolated from K562 cells expressing WT α4 integrin (α4), α4 integrin with a cytoplasmic domain deletion (X4C0), and an α4α5 chimera comprising the N-terminal β-propeller domain of α4 integrin linked to the leg, transmembrane, and cytoplasmic domains of α5 integrin (α4Pα5L). (B) Complete output of hierarchical clustering analysis of identified proteins (see Supporting Information for details). (C–H) Selected clusters are displayed, as indicated by blue dendrogram nodes and bars in (B). Correlations at selected dendrogram nodes are indicated. PAI, phosphoribosylaminoimidazole; TNFR, tumor necrosis factor receptor.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3472074&req=5

fig01: Hierarchical clustering analysis of proteins identified by MS ana-lyses of VCAM-1 and control affinity purifications. (A) Integrin-associated complexes were isolated from K562 cells expressing WT α4 integrin (α4), α4 integrin with a cytoplasmic domain deletion (X4C0), and an α4α5 chimera comprising the N-terminal β-propeller domain of α4 integrin linked to the leg, transmembrane, and cytoplasmic domains of α5 integrin (α4Pα5L). (B) Complete output of hierarchical clustering analysis of identified proteins (see Supporting Information for details). (C–H) Selected clusters are displayed, as indicated by blue dendrogram nodes and bars in (B). Correlations at selected dendrogram nodes are indicated. PAI, phosphoribosylaminoimidazole; TNFR, tumor necrosis factor receptor.
Mentions: Integrin activation is coupled to spatial separation of the cytoplasmic domains of the integrin heterodimer [7]. To examine the recruitment of proteins to adhesion complexes in the absence of β integrin tail interactions with an α integrin tail, we used an α4 integrin C-terminal cytoplasmic domain deletion mutant (X4C0) (Fig. 1A) [19]. This mutant may mimic the cytoplasmic domain separation linked to integrin activation because of the loss of the α–β clasp, the loss of lateral association with other cell-surface receptors, the exposure of β1 integrin-binding sites or the reduced steric hindrance around the β1 integrin tail in the absence of the α4 integrin tail. The truncation of the α4 cytoplasmic domain has been used previously to indicate a role for α4 integrin in a cell motility signaling pathway that is distinct from α5 integrin-stimulated migration [9, 12]. To examine differences in recruitment of proteins to α4β1 and α5β1 integrin cytoplasmic domains, we used an α4α5 integrin chimera comprising the N-terminal β-propeller domain of α4 integrin – which determines ligand specificity – linked to the leg, transmembrane and cytoplasmic domains of α5 integrin (α4Pα5L) (Fig. 1A) [20]. K562 human chronic myelogenous leukemia cells were used for our work because this cell line has served as a prototype for many analyses of integrin signaling and adhesion. The integrin complement of K562 cells is dominated by α5β1 integrin, which enabled us to use cells into which wild-type (WT) α4 integrin, X4C0, or α4Pα5L transgenes had been introduced. These cell lines are referred to here as K562-α4, K562-X4C0 (gifts from M. E. Hemler, Dana-Farber Cancer Institute, Boston, MA, USA) [19], and K562-α4Pα5L [20], respectively. All cell lines were cultured in RPMI 1640 medium supplemented with 10% (v/v) FCS (Lonza Bioscience, Wokingham, UK), 2 mM l-glutamine, and Geneticin (1 mg/mL; Invitrogen, Paisley, UK) at 37°C.

Bottom Line: Here, we report the isolation of ligand-induced adhesion complexes associated with wild-type α4β1 integrin, an activated α4β1 variant in the absence of the α cytoplasmic domain (X4C0), and a chimeric α4β1 variant with α5 leg and cytoplasmic domains (α4Pα5L), and the cataloguing of their proteomes by MS.Furthermore, we demonstrate colocalization of MYO18A with active integrin in migrating cells.These datasets provide a resource for future studies of integrin receptor-specific signaling events.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, UK.

Show MeSH
Related in: MedlinePlus