Abstract
Annexin A2 (AnxA2) is a cytosolic Ca2+ regulated membrane binding protein that can induce lipid domain formation and plays a role in exocytosis and endocytosis. To better understand the mode of annexin-membrane interaction, we analyzed membrane-bound AnxA2 assemblies by employing a novel 3-armed chemical crosslinker and specific AnxA2 mutant proteins. Our data show that AnxA2 forms crosslinkable oligomers upon binding to membranes containing negatively charged phospholipids. AnxA2 mutants with amino acid substitutions in residues predicted to be involved in lateral protein–protein interaction show compromised oligomer formation, albeit still being capable of binding to negatively charged membranes in the presence of Ca2+. These results suggest that lateral protein–protein interactions are involved in the formation of AnxA2 clusters on a biological membrane.
Highlights
Biological membranes can segregate into microdomains of defined lipid and protein composition that serve diverse but yet very specific tasks
Quartz Crystal Microbalance with Dissipation (QCM-D) analysis was performed as described before [21,23] using a Q-Sense E4 quartz crystal microbalance with dissipation (QCM-D) (Q-Sense, Gothenburg, Sweden) equipped with four temperature controlled flow cells in a parallel configuration connected to a peristaltic pump
Annexin A2 (AnxA2) has been shown by atomic force microscopy to form two-dimensional assemblies on model membranes containing negatively charged phospholipids [13]
Summary
Biological membranes can segregate into microdomains of defined lipid and protein composition that serve diverse but yet very specific tasks. Rafts are well studied in the plasma membrane of eukaryotic cells, where they serve as assembly and transmission platforms in outside-in as well as inside-out signaling and regulate membrane trafficking events to and from the plasma membrane (for review see [1,2,3]) To function in these processes, rafts have to be highly dynamic, both with respect to lipid and protein composition as well as size, they assemble into larger structures and disassemble again on a rapid time scale. It directly binds to headgroups of negatively charged phospholipids and requires the presence of these lipids in membranes (and raft domains) for high-affinity association One such lipid is PI(4,5)P2, and AnxA2 has been shown to interact with this phosphoinositide in a specific manner [9,10]. Mutating residues present at the dimer interface identified positions that reside in close proximity in membrane-bound AnxA2 oligomers and could participate in oligomer formation
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