ProLIF - quantitative integrin protein-protein interactions and synergistic membrane effects on proteoliposomes.
J Cell Sci
; 132(4)2018 08 20.
Article
em En
| MEDLINE
| ID: mdl-30072441
ABSTRACT
Integrin transmembrane receptors control a wide range of biological interactions by triggering the assembly of large multiprotein complexes at their cytoplasmic interface. Diverse methods have been used to investigate interactions between integrins and intracellular proteins, and predominantly include peptide-based pulldowns and biochemical immuno-isolations from detergent-solubilised 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 'protein-liposome interactions by flow cytometry' (denoted ProLIF), a technique to reconstitute recombinant integrin transmembrane domains (TMDs) and cytoplasmic tail (CT) fragments in liposomes as individual subunits or as αß heterodimers and, via flow cytometry, allow rapid and quantitative measurement of protein interactions with these membrane-embedded integrins. Importantly, the assay can analyse binding of fluorescent proteins directly from cell lysates without further purification steps. Moreover, the effect of membrane composition, such as PI(4,5)P2 incorporation, on protein recruitment to the integrin CTs can be analysed. ProLIF requires no specific instrumentation and can be applied to measure a broad range of membrane-dependent protein-protein interactions with the potential for high-throughput/multiplex analyses.This article has associated First Person interviews with the first authors of the paper (see doi 10.1242/jcs.223644 and doi 10.1242/jcs.223719).
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteolipídeos
/
Integrinas
/
Membrana Celular
/
Lipossomos
Limite:
Humans
Idioma:
En
Revista:
J Cell Sci
Ano de publicação:
2018
Tipo de documento:
Article
País de afiliação:
Finlândia