Proteomic analysis of integrin-associated complexes
Ontology highlight
ABSTRACT:
INSTRUMENT(S): 4000 Q TRAP, instrument model
ORGANISM(S): Homo Sapiens (human)
SUBMITTER: 
Adam Byron
PROVIDER: PRD000148 | Pride | 2009-12-01
REPOSITORIES: Pride
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Project description:The composition and stiffness of the extracellular matrix (ECM) environment that surrounds Multipotent mesenchymal stem cells (MSCs) stem cells dictates transcriptional programming, thereby affecting stem cell lineage decision-making. Cells sense force between themselves and their microenvironment controlling the capability of MSCs to differentiate into adipocytes, osteocytes and chondrocytes. Force sensing is transmitted by integrin receptors and their associated adhesion signalling complexes. To identify regulators of MSC force sensing we sought to catalogue MSC adhesion complex composition. Therefore we isolated integrin-associated adhesion complexes formed in MSCs plated on the ECM ligand fibronectin. We identifed proteins using mass spectrometry that define a MSC specific subset of adhesion complex proteins consisting of key linkages to the actin cytoskeleton together with integrin signalling and force sensing components.
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Project description:Human foreskin fibroblast cells were spread on fibronectin (FN) for 1 h and treated with DMSO or FAK inhibitor (3 uM, AZ13256675 (termed AZ675 for short)) for 1 h. Integrin-associated adhesion complexes isolated from these cells were analysed by mass spectrometry to determine changes in adhesion complex composition upon FAK inhibition. As a negative control, complexes were also isolated from cells attached to transferrin (Tf), which allows integrin-independent adhesion via the transferrin receptor.
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Project description:Integrins are a major class of heterodimeric adhesion receptors composed of an a and b subunit that link the extracellular matrix (ECM) and the cytoskeleton across the cell membrane. When activated by either the intracellular (inside-out signaling) or extracellular (outside-in signaling) environment, integrins undergo a conformational change that increases the ligand binding affinity. As the primary receptor for ECM protein fibronectin, Integrin a5 plays a critical role in zebrafish somitogenesis. To better understand integrin activation in this context, we performed co-immunoprecipitation and Mass Spectrometry (MS) based proteomics using FLAG-tagged Integrin a5 alleles that alter it activation state: constitutive active mutant a5GAAKR and inactive ligand binding deficient mutant a5FYLDD, expressed in maternal zygotic a5 mutant (MZa5-/-) embryos. Our data provide an overview of Integrin associated proteins according to the activation state of the Integrin.
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Project description:Proteomic analysis of integrin activation-state-dependent adhesion complexes. Adhesion complexes were isolated from K562 cells using activation-state-specific monoclonal antibodies coupled to magnetic beads.Tandem mass spectra were extracted using extract_msn (Thermo Fisher Scientific) executed in Mascot Daemon (version 2.2.2; Matrix Science). Peak list files were searched against the IPI Human database (version 3.70) modified to contain ten additional contaminants and reagent sequences of non-human origin. Searches were submitted to an in-house Mascot server (version 2.2.03; Matrix Science). Carbamidomethylation of cysteine was set as a fixed modification, and oxidation of methionine was allowed as a variable modification. Only tryptic peptides were considered, with up to one missed cleavage permitted. Monoisotopic precursor mass values were used, and only doubly and triply charged precursor ions were considered. Mass tolerances for precursor and fragment ions were 0.4 Da and 0.5 Da, respectively.
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Project description:To explore the dynamics of integrin adhesion complex disassembly, isolated adhesion complexes from U2OS cells were subjected to mass spectrometry based proteomic analysis. U2OS cells were allowed to spread on FN, treated with nocodazole for 4 hours to disrupt microtubules. To coordinate microtubule-induced integrin adhesion complex disassembly nocodazole was subsequently washed out and adhesion complexes isolated at 5, 10 and 15 minutes during this process.
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Project description:Soluble VEGFR-1 (sVEGFR-1) acts both as a decoy receptor for VEGFs and as an extracellular matrix protein for α5β1 integrin. A sVEGFR-1-derived peptide that interacts with α5β1 integrin promotes angiogenesis. However, canonical signal downstream integrin activation is not induced, resulting into lack of focal adhesion maturation. We performed a gene expression profile of endothelial cells adhering on sVEGFR-1 compared to that of cells adhering on fibronectin, the principal α5β1 integrin ligand. Three protein kinase-C substrates, adducin, MARCKS, and radixin were differently modulated. Adducin and MARCKS were less phosphorylated whereas radixin was higher phosphorylated in sVEGFR-1 adhering cells, the latter leading to prolonged small GTPase Rac1 activation and induction of a pathway involving the heterotrimeric G protein α13. Altogether, our data indicated endothelial cell acquisition of an highly motile phenotype when adherent on sVEGFR-1. Finally, we indicated radixin as accountable for the angiogenic effect of α5β1 integrin interaction with sVEGFR-1 that in turn depends on an active VEGF-A/VEGFR-2 signaling.
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