Cholesterol regulates membrane binding and aggregation by annexin 2 at submicromolar Ca 2+ concentration

104

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...

supporting

confidence: 86%

Section: Resultsmentioning

confidence: 99%

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

Gokhale

Abraham

Digman

et al. 2005

104

“…Ax II has also been localized to adhesions between confluent Madin-Darby canine kidney cells (25). Ax II has been found to bind directly to anionic liposomes (11,12), and cholesterol has been reported to enhance this binding (26), suggesting that an Ax II/SHP-2 complex could bind directly to membrane lipid domains enriched in cholesterol. Membrane anchoring could also occur through an intrinsic membrane protein such as CD44, which was recently found to anchor Ax II to detergentresistant, cholesterol-rich membranes (rafts) in mammary epithelial EpH4 cells (27).…”

Section: Localization Of Shp-2 and Ax II To Adhesion Bands And Stressmentioning

confidence: 99%

“…Membrane anchoring could also occur through an intrinsic membrane protein such as CD44, which was recently found to anchor Ax II to detergentresistant, cholesterol-rich membranes (rafts) in mammary epithelial EpH4 cells (27). Both Ax II (27)(28)(29)(30) and SHP-2 (31) have been identified in rafts in cultured cells, and cholesterolsequestering reagents have been found to dissociate Ax II from both chromaffin granules (26) and endosomes (32). Alternatively, it is conceivable that the localization of either Ax II or SHP-2 to sites of cell-cell attachment could occur through direct binding to actin, since both Ax II (33,34) and SHP-2 (35) have been reported to bind to actin filaments in vitro, and both proteins were found along stress fibers in subconfluent endothelial cells (Fig.…”

Section: Localization Of Shp-2 and Ax II To Adhesion Bands And Stressmentioning

confidence: 99%

Regulation of the SHP-2 Tyrosine Phosphatase by a Novel Cholesterol- and Cell Confluence-dependent Mechanism

Burkart

Samii

Corvera

et al. 2003

Endothelial cells approaching confluence exhibit marked decreases in tyrosine phosphorylation of receptor tyrosine kinases and adherens junctions proteins, required for cell cycle arrest and adherens junctions stability. Recently, we demonstrated a close correlation in endothelial cells between membrane cholesterol and tyrosine phosphorylation of adherens junctions proteins. Here, we probe the mechanistic basis for this correlation. We find that as endothelial cells reach confluence, the tyrosine phosphatase SHP-2 is recruited to a low-density membrane fraction in a cholesterol-dependent manner. Binding of SHP-2 to this fraction was not abolished by phenyl phosphate, strongly suggesting that this binding was mediated by other regions of SHP-2 beside its SH2 domains. Annexin II, previously implicated in cholesterol trafficking, was associated in a complex with SHP-2, and both proteins localized to adhesion bands in confluent endothelial monolayers. These studies reveal a novel, cholesterol-dependent mechanism for the recruitment of signaling proteins to specific plasma membrane domains via their interactions with annexin II.Endothelial cells approaching confluence in culture undergo profound morphological and functional alterations, including the formation of intercellular junctions and the cessation of cell growth. These changes are associated with reductions in tyrosine phosphorylation levels of both receptor tyrosine kinases (1, 2) and cadherins and catenins (3), the major components of adherens junctions. The dephosphorylation of adherens junctions proteins appears to be necessary for the formation of stable, confluent endothelial monolayers, since increased tyrosine phosphorylation of adherens junctions proteins leads to the disruption of adherens junctions (4 -6). The mechanisms by which cell density decreases tyrosine phosphorylation of endothelial membrane proteins are unknown.One possible mechanism is that increased cell confluence modulates the local environment of signaling proteins in the plasma membrane, thereby altering their response to extracellular ligands. Recently (7), we identified membrane cholesterol as a potential determinant of tyrosine phosphorylation levels in growing endothelial monolayers. Membrane cholesterol increased markedly with cell density in cultures of growing endothelial cells, exhibiting levels 3-4-fold higher upon reaching confluence. Depletion of cholesterol from confluent monolayers of CPAE 1 cells induced the tyrosine phosphorylation of multiple membrane proteins, including the adherens junctions proteins ␥-catenin and pp120, and disrupted adherens junctions.Cholesterol might regulate tyrosine phosphorylation levels through the localization of tyrosine kinases or phosphatases to specific plasma membrane loci. To begin to identify such kinases or phosphatases, we have used very low (0.01%) levels of the sterol-containing detergent digitonin to identify proteins bound to membranes in a cholesterol-dependent manner (8). At these concentrations, digitonin complexes specifica...

“…Addition of cholesterol to liposome enhances the ability of AIIt to bind with and aggregate phosphatidylserine vesicles at submicromolar Ca 2ϩ levels. Cholesterol also alters the subcellular localization of AIIt at low Ca 2ϩ concentrations (25,26). Arachidonic acid enhances AIIt-mediated fusion in model membranes (13).…”

mentioning

confidence: 99%

Fusion of Lamellar Body with Plasma Membrane Is Driven by the Dual Action of Annexin II Tetramer and Arachidonic Acid

Chattopadhyay

Sun

Wang

et al. 2003

Annexin II has been implicated in membrane fusion during the exocytosis of lamellar bodies from alveolar epithelial type II cells. Most previous studies were based on the fusion assays by using model membranes. In the present study, we investigated annexin II-mediated membrane fusion by using isolated lamellar bodies and plasma membrane as determined by the relief of octadecyl rhodamine B (R18) self-quenching. Immunodepletion of annexin II from type II cell cytosol reduced its fusion activity. Purified annexin II tetramer (AIIt) induced the fusion of lamellar bodies with the plasma membrane in a dose-dependent manner. This fusion is Ca 2؉ -dependent and is highly specific to AIIt because other annexins (I and II monomer, III, IV, V, and VI) were unable to induce the fusion. Modification of the different functional residues of AIIt by N-ethylmaleimide, nitric oxide, or peroxynitrite abolished AIIt-mediated fusion. Arachidonic acid enhanced AIIt-mediated fusion and reduced its Ca 2؉ requirement to an intracellularly achievable level. This effect is due to membranebound arachidonic acid, not free arachidonic acid. Other fatty acids including linolenic acid, palmitoleic acid, myristoleic acid, stearic acid, palmitic acid, and myristic acid had little effect. AIIt-mediated fusion was suppressed by the removal of arachidonic acid from lamellar body and plasma membrane using bovine serum albumin. The addition of arachidonic acid back to the arachidonic acid-depleted membranes restored its fusion activity. Our results suggest that the fusion between lamellar bodies with the plasma membrane is driven by the synergistic action of AIIt and arachidonic acid.Lung surfactant is a surface active material synthesized and secreted by cuboidal alveolar type II cells (1-3). It is composed of phospholipids, mainly dipalmitoylphosphatidylcholine, and surfactant proteins A-D. It minimizes the surface tension at the air-liquid interface by inserting phospholipids between water molecules. As a result, surfactant disrupts the cohesive hydrogen bonds and prevents them from exerting high molecular forces at the alveolar surface. Deficiency of dipalmitoylphosphatidylcholine in the alveolar surface has been associated with infant respiratory distress syndrome.The secretion of surfactant by type II cells involves the translocation, docking, and fusion of lamellar bodies to the plasma membrane. For the lamellar body content to reach its extracellular destination, a continuity of the vesicular lumen and extracellular fluid must be established. This continuity is maintained through the formation of a narrow pore similar to membrane channels (4 -6). The formation of this pore is a complex event involving physical and chemical factors and is regulated by intracellular Ca 2ϩ and proteins (7). Studies on mast cells and chromaffin cells revealed that the opening of fusion pore and extrusion of granule content is influenced by an osmotic force depending upon the osmolarity of the surrounding solution (8).Annexin II plays multidimensional roles in dif...