Proteomic analysis of α4β1 integrin adhesion complexes reveals α-subunit-dependent protein recruitment.
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.
Affiliation: Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, UK.Show MeSH
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Mentions: To examine the molecular landscape of the isolated integrin adhesion complexes in the context of currently known protein–protein interactions, and as a complementary approach to hierarchical clustering analysis, interaction network analysis was used. We generated a human interactome consisting of physical protein–protein interactions reported in multiple databases (see Supporting Information for details). We mapped onto this interactome 338 (97%) of the 350 proteins enriched in the VCAM-1 affinity purifications. A threshold of one standard deviation greater than the mean enrichment ratio (VCAM-1 over control) was used as a cut-off for specific enrichment in VCAM-1 affinity purifications, which equated to a 1.82-fold change (Supporting Information Fig. S2). To generate a focused dataset, we extracted all proteins reported in a literature-curated database of adhesion-related components (the “adhesome”)  and displayed them as an interaction network (Fig. 2). The proteins (nodes) were annotated to indicate their subcellular localization according to the Gene Ontology (GO) database (release date 28th January 2012) . Almost all proteins in the network were annotated as cytoplasmic, as expected for proteins in a complex associated with the cytoplasmic tails of integrins, and 22 (73%) out of 30 proteins were reported by GO annotation as transmembrane or cell periphery components (e.g. plasma membrane-associated or focal adhesion components). The interaction network was arranged as sets of proteins that were identified using one or multiple cell lines. The overlaps of these sets highlight distinct heterodimer-dependent protein recruitment. As discussed above, α4 and β1 integrins (ITGA4 and ITGB1, respectively) were enriched in all cell lines, and are displayed in the central intersection of all three sets in the network (Fig. 2). Also present in this intersection set are the following proteins: talin-1 (TLN1), a key activator of integrin function; moesin (MSN), a member of a family of plasma membrane-actin cytoskeleton linker proteins; subunit 4 of the Arp2/3 complex (ARPC4), which controls actin polymerization; and adenosine 5′-diphosphate ribosylation factor (Arf) 1 (ARF1), a small guanosine triphosphatase (GTPase) that plays a role in vesicular trafficking. In the context of the adhesome database, these proteins represent a “core” set that is associated with all three types of integrin tested in this study, which implicates several major functions of cell adhesion: integrin activation, cytoskeletal linkage, regulation of the cytoskeleton, and protein trafficking.
Affiliation: Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, UK.