ProLIF: a quantitative assay for investigating integrin cytoplasmic protein interactions and synergistic membrane effects on proteoliposomes

De Franceschi, Nicola and Miihkinen, Mitro and Hamidi, Hellyeh and Alanko, Jonna and Mai, Aanja and Picas, Laura and Levy, Daniel and Mattjus, Peter and Goult, Benjamin T and Goud, Bruno and Ivaska, Johanna (2017) ProLIF: a quantitative assay for investigating integrin cytoplasmic protein interactions and synergistic membrane effects on proteoliposomes. BioRxiv, . (Submitted) (doi:https://doi.org/10.1101/209262) (Full text available)

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Abstract

Integrin transmembrane heterodimeric receptors control a wide range of biological interactions by triggering the assembly of large multiprotein complexes at their cytoplasmic interface. A diverse set of methods have been used to investigate cytoplasmic interactions between integrins and intracellular proteins. These predominantly consist of peptide-based pull-downs and biochemical immuno- isolations from detergent-solubilized cell lysates. However, quantitative methods to probe integrin- protein interactions in a more biologically relevant context where the integrin is embedded within a lipid bilayer have been lacking. Here we describe a technique called ProLIF (Protein-Liposome Interactions by Flow cytometry) to reconstitute recombinant integrin transmembrane domain (TMD) and cytoplasmic tail (CT) fragments on liposomes as individual α or β subunits or as αβ heterodimers and, using flow cytometry, to rapidly and quantitatively measure protein interactions with these membrane-embedded integrins. Importantly, the assay can analyse binding of fluorescent proteins directly from cell lysates without further purification steps. By combining integrins with membrane lipids to generate proteoliposomes, the effects of membrane composition such as PI(4,5)P2 presence on protein recruitment to the integrin CTs can be analyzed. ProLIF requires no specific instrumentation, apart from a standard flow cytometer and can be applied to measure a broad range of membrane-dependent protein-protein interactions with the potential for high-throughput/multiplex analyses.

Item Type: Article
Uncontrolled keywords: Integrin, talin, ProLIF
Subjects: Q Science > QH Natural history > QH581.2 Cell Biology
Divisions: Faculties > Sciences > School of Biosciences
Depositing User: Ben Goult
Date Deposited: 29 Oct 2017 11:01 UTC
Last Modified: 25 May 2018 09:04 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/64160 (The current URI for this page, for reference purposes)
Goult, Benjamin T: https://orcid.org/0000-0002-3438-2807
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