ProLIF: quantitative integrin protein-protein interactions and synergistic membrane effects on proteoliposomes

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 pull-downs and biochemical immuno-isolations from detergent-solubilized cell lysates. However, quantitative methods to probe integrin-protein-protein interactions in a more biologically relevant context where the integrin is embedded within a lipid bilayer have been lacking. Here we describe ProLIF (Protein-Liposome Interactions by Flow cytometry), a technique to reconstitute recombinant integrin transmembrane domain (TMD) and cytoplasmic tail (CT) fragments in liposomes as individual 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. 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.