EFR3A: a new raft domain organizing protein?

Abstract

Abstract Membrane rafts play a crucial role in the regulation of many important biological processes. Our data suggest that specific interactions of flotillins with MPP1 are responsible for membrane raft domain organization and regulation in erythroid cells. Interaction of the flotillin-based protein network with specific membrane components underlies the mechanism of raft-domain formation and regulation, including in cells with low expression of MPP1. We sought to identify other flotillin partners via the immobilized recombinant flotillin-2-based affinity approach and MS technique. Thereby EFR3A was identified as a candidate protein which interacts with flotillin-2. This was further confirmed via immunoblotting using anti-EFR3A antibody and via co-immunoprecipitation (Co-IP). Moreover, this newly discovered interaction was demonstrated via overlay assay using recombinant EFR3A and flotillin-2. EFR3A is a stable component of the detergentresistant membrane (DRM) fraction of HeLa cells, and its presence was sensitive to removal of cholesterol. While silencing the EFR3A gene, we observed decreased order of the plasma membrane of living cells or giant plasma membrane vesicles (GPMVs) derived from KnD cells and altered mobility of the raft probe, as indicated via fluorescence lifetime imaging microscopy and spot-variation fluorescence correlation spectroscopy. Moreover, silencing of EFR3A expression was found to disturb epidermal growth factor receptor (EGFR) and phospholipase C gamma (PLCγ) phosphorylation and affect EGF-dependent cytosolic Ca2+ concentration. Altogether, our results suggest hitherto unreported flotillin-2-EFR3A interaction, which appears to be responsible for membrane raft organization and regulation. This implies participation of this interaction in the regulation of multiple cellular processes, including those connected with cell signaling.