Phosphoinositide Specificity of and Mechanism of Lipid Domain Formation by Annexin A2-p11 Heterotetramer

Abstract

Annexin A2 is a phospholipid-binding protein that forms a heterotetramer (annexin II-p11 heterotetramer; A2t) with p11 (S100A10). It has been reported that annexin A2 is involved in binding to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) and in inducing membrane microdomain formation. To understand the mechanisms underlying these findings, we determined the membrane binding properties of annexin A2 wild type and mutants both as monomer and as A2t. Our results from surface plasmon resonance analysis showed that A2t and annexin A2 has modest selectivity for PtdIns(4,5)P 2 over other phosphoinositides, which is conferred by conserved basic residues, including Lys 279 and Lys 281 , on the convex surface of annexin A2. Fluorescence microscopy measurements using giant unilamellar vesicles showed that A2t of wild type, but not (K279A) 2 -(p11) 2 or (K281A) 2 -(p11) 2 , specifically induced the formation of 1-m-sized PtdIns(4,5)P 2 clusters, which were stabilized by cholesterol. Collectively, these studies elucidate the structural determinant of the PtdIns(4,5)P 2 selectivity of A2t and suggest that A2t may be involved in the regulation of PtdIns(4,5)P 2 clustering in the cell.Annexins are a family of peripheral proteins that bind anionic phospholipids in a Ca 2ϩ -dependent manner (1-5). Structures and in vitro functions of annexins have been well characterized. Annexins have a variable N-terminal region and a conserved C-terminal core that is composed of four (eight in case of annexin A6) ␣-helical annexin folds (2). The C-terminal core is the Ca 2ϩ -dependent membrane-binding module that contains multiple Ca 2ϩ -binding sites on its convex membrane-binding surface (2). The N-terminal region of annexins is attached to the concave side of the C-terminal core and thought to be involved in interactions with other proteins and post-translational modifications (3-5). In addition to their membrane-binding activities, annexins have been reported to have other in vitro activities, including membrane aggregation and lateral aggregation on the membrane surface (6). Despite the wealth of structural and functional information on annexins, their physiological functions are only beginning to emerge with recent genetic and cell studies (3-5).Annexin A2 is an abundant cellular protein that has been implicated in numerous physiological processes (3-5, 7). Annexin A2 interacts with an EF-hand protein p11 (also known as S100A11) with high affinity via its N-terminal region, forming a symmetric heterotetramer, (annexin A2) 2 -(p11) 2 (A2t) 2 (8, 9). Annexin A2 has been reported to exist either as a monomer or A2t in mammalian cells (3, 10 -12). Annexin A2 and A2t have been shown to have high vesicle aggregating activity (13) and form a monolayer of protein clusters when bound to the lipid bilayer with anionic phospholipids accumulating underneath the protein clusters (14). Mounting evidence indicates that annexin A2 and A2t are involved in organizing cholesterol-rich lipid rafts (15, 16) and linking them to cytoskeletal protein...

Phosphatidylinositol 4,5-Bisphosphate (PtdIns(4,5)P2) Specifically Induces Membrane Penetration and Deformation by Bin/Amphiphysin/Rvs (BAR) Domains

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Deletion of Annexin 2 Light Chain p11 in Nociceptors Causes Deficits in Somatosensory Coding and Pain Behavior

“…p11 has been proposed to organize microdomains in the cell membrane through the formation of cholesterol-stabilized phosphatidylinositol 4,5-bisphosphate clusters. It is possible that these lipid domains within the neuronal membrane organize channel distribution to affect neuronal excitability and that not only trafficking to the membrane but subsequent membrane localizations are determined by interactions with p11 (Gokhale et al, 2005).…”

Molecular Basis of the Potent Membrane-remodeling Activity of the Epsin 1 N-terminal Homology Domain

“…Giant Unilamellar Vesicle (GUV) Tubulation Assay-GUVs were prepared by the electroformation technique as described previously (29,30). The lipid mixtures (POPC/POPE/POPS/ PtdIns(4,5)P 2 /Rh-PE ϭ 46.5:30:20:3:05) were prepared in chloroform/methanol (3:1) at a total concentration of 0.5 mg/ml, and then the lipid solution was spread onto the indium tin oxide electrode surface, and the lipid was dried under vacuum to form a uniform lipid film.…”