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...

The mechanisms by which secretory phospholipase A 2 (PLA 2 ) exerts cellular effects are not fully understood. To elucidate these mechanisms, we systematically and quantitatively assessed the activities of human group IIA, V, and X PLA 2 s on originating and neighboring cells using orthogonal fluorogenic substrates in various mixed cell systems. When HEK293 cells stably expressing each of these PLA 2 s were mixed with non-transfected HEK293 cells, group V and X PLA 2 s showed strong transcellular lipolytic activity, whereas group IIA PLA 2 exhibited much lower transcellular activity. The transcellular activity of group V PLA 2 was highly dependent on the presence of cell surface heparan sulfate proteoglycans of acceptor cells. Activation of RBL-2H3 and DLD-1 cells that express endogenous group V PLA 2 led to the secretion of group V PLA 2 and its transcellular action on neighboring human neutrophils and eosinophils, respectively. Similarly, activation of human bronchial epithelial cells, BEAS-2B, caused large increases in arachidonic acid and leukotriene C 4 release from neighboring human eosinophils. Collectively, these studies show that group V and X PLA 2 s can act transcellularly on mammalian cells and suggest that group V PLA 2 released from neighboring cells may function in triggering the activation of inflammatory cells under physiological conditions.

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