Title: 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)P2) 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)P2 over other phosphoinositides, which is conferred by conserved basic residues, including Lys279 and Lys281, 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-μm-sized PtdIns(4,5)P2 clusters, which were stabilized by cholesterol. Collectively, these studies elucidate the structural determinant of the PtdIns(4,5)P2 selectivity of A2t and suggest that A2t may be involved in the regulation of PtdIns(4,5)P2 clustering in the cell. 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)P2) 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)P2 over other phosphoinositides, which is conferred by conserved basic residues, including Lys279 and Lys281, 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-μm-sized PtdIns(4,5)P2 clusters, which were stabilized by cholesterol. Collectively, these studies elucidate the structural determinant of the PtdIns(4,5)P2 selectivity of A2t and suggest that A2t may be involved in the regulation of PtdIns(4,5)P2 clustering in the cell. Annexins are a family of peripheral proteins that bind anionic phospholipids in a Ca2+-dependent manner (1Creutz C.E. Science. 1992; 258: 924-931Crossref PubMed Scopus (495) Google Scholar, 2Swairjo M.A. Seaton B.A. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 193-213Crossref PubMed Scopus (190) Google Scholar, 3Gerke V. Moss S.E. Physiol. Rev. 2002; 82: 331-371Crossref PubMed Scopus (1667) Google Scholar, 4Rescher U. Gerke V. J. Cell Sci. 2004; 117: 2631-2639Crossref PubMed Scopus (510) Google Scholar, 5Gerke V. Creutz C.E. Moss S.E. Nat. Rev. Mol. Cell Biol. 2005; 6: 449-461Crossref PubMed Scopus (1165) Google Scholar). 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) α-helical annexin folds (2Swairjo M.A. Seaton B.A. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 193-213Crossref PubMed Scopus (190) Google Scholar). The C-terminal core is the Ca2+-dependent membrane-binding module that contains multiple Ca2+-binding sites on its convex membrane-binding surface (2Swairjo M.A. Seaton B.A. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 193-213Crossref PubMed Scopus (190) Google Scholar). 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 (3Gerke V. Moss S.E. Physiol. Rev. 2002; 82: 331-371Crossref PubMed Scopus (1667) Google Scholar, 4Rescher U. Gerke V. J. Cell Sci. 2004; 117: 2631-2639Crossref PubMed Scopus (510) Google Scholar, 5Gerke V. Creutz C.E. Moss S.E. Nat. Rev. Mol. Cell Biol. 2005; 6: 449-461Crossref PubMed Scopus (1165) Google Scholar). 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 (6Gerke V. Moss S.E. Biochim. Biophys. Acta. 1997; 1357: 129-154Crossref PubMed Scopus (320) Google Scholar). Despite the wealth of structural and functional information on annexins, their physiological functions are only beginning to emerge with recent genetic and cell studies (3Gerke V. Moss S.E. Physiol. Rev. 2002; 82: 331-371Crossref PubMed Scopus (1667) Google Scholar, 4Rescher U. Gerke V. J. Cell Sci. 2004; 117: 2631-2639Crossref PubMed Scopus (510) Google Scholar, 5Gerke V. Creutz C.E. Moss S.E. Nat. Rev. Mol. Cell Biol. 2005; 6: 449-461Crossref PubMed Scopus (1165) Google Scholar). Annexin A2 is an abundant cellular protein that has been implicated in numerous physiological processes (3Gerke V. Moss S.E. Physiol. Rev. 2002; 82: 331-371Crossref PubMed Scopus (1667) Google Scholar, 4Rescher U. Gerke V. J. Cell Sci. 2004; 117: 2631-2639Crossref PubMed Scopus (510) Google Scholar, 5Gerke V. Creutz C.E. Moss S.E. Nat. Rev. Mol. Cell Biol. 2005; 6: 449-461Crossref PubMed Scopus (1165) Google Scholar, 7Waisman D.M. Mol. Cell Biochem. 1995; 149: 301-322Crossref PubMed Scopus (263) Google Scholar). 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) 2The abbreviations used are: A2tannexin II-p11 heterotetramerCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidGUVgiant unilamellar vesicle(s)LUVlarge unilamellar vesicle(s)PHpleckstrin homologyPLCphospholipase CPOPC1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholinePOPE1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolaminePOPI1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoinositolPOPS1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserinePtdIns-(3)Pphosphatidylinositol 3-phosphatePtdIns(4)Pphosphatidylinositol 4-phosphatePtdIns(5)Pphosphatidylinositol 5-phosphatePtdIns(3,4)P2phosphatidylinositol 3,4-bisphosphatePtdIns(3,5)P2phosphatidylinositol 3,5-bisphosphatePtdIns(3,4,5)P3phosphatidylinositol 3,4,5-triphosphatePtdIns(4,5)P2phosphatidylinositol 4,5-bisphosphateSPRsurface plasmon resonanceMARCKSmyristoylated alanine-rich C kinase substrate. (8Johnsson N. Marriott G. Weber K. EMBO J. 1988; 7: 2435-2442Crossref PubMed Scopus (163) Google Scholar, 9Rety S. Sopkova J. Renouard M. Osterloh D. Gerke V. Tabaries S. Russo-Marie F. Lewit-Bentley A. Nat. Struct. Biol. 1999; 6: 89-95Crossref PubMed Scopus (264) Google Scholar). Annexin A2 has been reported to exist either as a monomer or A2t in mammalian cells (3Gerke V. Moss S.E. Physiol. Rev. 2002; 82: 331-371Crossref PubMed Scopus (1667) Google Scholar, 10Harder T. Gerke V. Biochim. Biophys. Acta. 1994; 1223: 375-382Crossref PubMed Scopus (71) Google Scholar, 11Nilius B. Gerke V. Prenen J. Szucs G. Heinke S. Weber K. Droogmans G. J. Biol. Chem. 1996; 271: 30631-30636Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 12Konig J. Prenen J. Nilius B. Gerke V. J. Biol. Chem. 1998; 273: 19679-19684Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Annexin A2 and A2t have been shown to have high vesicle aggregating activity (13Raynal P. Pollard H.B. Biochim. Biophys. Acta. 1994; 1197: 63-93Crossref PubMed Scopus (1034) Google Scholar) and form a monolayer of protein clusters when bound to the lipid bilayer with anionic phospholipids accumulating underneath the protein clusters (14Menke M. Ross M. Gerke V. Steinem C. Chembiochem. 2004; 5: 1003-1006Crossref PubMed Scopus (34) Google Scholar). Mounting evidence indicates that annexin A2 and A2t are involved in organizing cholesterol-rich lipid rafts (15Oliferenko S. Paiha K. Harder T. Gerke V. Schwarzler C. Schwarz H. Beug H. Gunthert U. Huber L.A. J. Cell Biol. 1999; 146: 843-854Crossref PubMed Scopus (357) Google Scholar, 16Babiychuk E.B. Draeger A. J. Cell Biol. 2000; 150: 1113-1124Crossref PubMed Scopus (232) Google Scholar) and linking them to cytoskeletal proteins (17Harder T. Kellner R. Parton R.G. Gruenberg J. Mol. Biol. Cell. 1997; 8: 533-545Crossref PubMed Scopus (188) Google Scholar, 18Merrifield C.J. Rescher U. Almers W. Proust J. Gerke V. Sechi A.S. Moss S.E. Curr. Biol. 2001; 11: 1136-1141Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 19Zobiack N. Rescher U. Laarmann S. Michgehl S. Schmidt M.A. Gerke V. J. Cell Sci. 2002; 115: 91-98Crossref PubMed Google Scholar, 20Benaud C. Gentil B.J. Assard N. Court M. Garin J. Delphin C. Baudier J. J. Cell Biol. 2004; 164: 133-144Crossref PubMed Scopus (152) Google Scholar). It has been also reported that annexin A2 (21Hayes M.J. Merrifield C.J. Shao D. Ayala-Sanmartin J. Schorey C.D. Levine T.P. Proust J. Curran J. Bailly M. Moss S.E. J. Biol. Chem. 2004; 279: 14157-14164Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 22Rescher U. Ruhe D. Ludwig C. Zobiack N. Gerke V. J. Cell Sci. 2004; 117: 3473-3480Crossref PubMed Scopus (146) Google Scholar) and A2t (22Rescher U. Ruhe D. Ludwig C. Zobiack N. Gerke V. J. Cell Sci. 2004; 117: 3473-3480Crossref PubMed Scopus (146) Google Scholar) bind to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) with high specificity and affinity and that this activity is linked to the organization of actin at membrane sites that are enriched in PtdIns(4,5)P2. Together with previous reports showing that annexin A2 binds cholesterol-containing membranes (23Ayala-Sanmartin J. Henry J.P. Pradel L.A. Biochim. Biophys. Acta. 2001; 1510: 18-28Crossref PubMed Scopus (62) Google Scholar, 24Mayran N. Parton R.G. Gruenberg J. EMBO J. 2003; 22: 3242-3253Crossref PubMed Scopus (172) Google Scholar) and PtdIns(4,5)P2 is localized in cholesterol-rich lipid rafts in the plasma membrane (25Pike L.J. Casey L. J. Biol. Chem. 1996; 271: 26453-26456Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, 26Pike L.J. Miller J.M. J. Biol. Chem. 1998; 273: 22298-22304Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 27Waugh M.G. Lawson D. Tan S.K. Hsuan J.J. J. Biol. Chem. 1998; 273: 17115-17121Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 28Waugh M.G. Minogue S. Anderson J.S. dos Santos M. Hsuan J.J. Biochem. Soc. Trans. 2001; 29: 509-511Crossref PubMed Scopus (53) Google Scholar), these results suggest that annexin A2 plays a role in regulating the formation of PtdIns(4,5)P2-rich lipid rafts or lipid raft-like structures. However, it is not known whether the annexin A2 dynamically controls the organization of these structures or it passively binds to the PtdIns(4,5)P2-rich regions. Furthermore, the affinity and specificity of annexin A2 and A2t for PtdIns(4,5)P2 have not been quantitatively determined, which makes it difficult to assess their capability to compete with other PtdIns(4,5)P2-binding proteins under physiological conditions. annexin II-p11 heterotetramer 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid giant unilamellar vesicle(s) large unilamellar vesicle(s) pleckstrin homology phospholipase C 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoinositol 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine phosphatidylinositol 3-phosphate phosphatidylinositol 4-phosphate phosphatidylinositol 5-phosphate phosphatidylinositol 3,4-bisphosphate phosphatidylinositol 3,5-bisphosphate phosphatidylinositol 3,4,5-triphosphate phosphatidylinositol 4,5-bisphosphate surface plasmon resonance myristoylated alanine-rich C kinase substrate. In this study, we systematically and quantitatively determined the phosphoinositide binding specificity and affinity of annexin A2 and A2t by surface plasmon resonance (SPR) analysis and identified the structural determinant of its phosphoinositide specificity. We also investigated the formation of PtdIns(4,5)P2-rich membrane domains on giant unilamellar vesicles (GUV) induced by A2t and mutants under various conditions. Our study provides new insight into the mechanism by which A2t mediates the organization of PtdIns(4,5)P2-rich membrane domains. Materials—1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoinositol (POPI), and cholesterol were all purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). 1,2-Dipalmitoyl derivatives of phosphatidylinositol 3-phosphate (PtdIns(3)P), phosphatidylinositol 4-phosphate (PtdIns(4)P), phosphatidylinositol 5-phosphate (PtdIns(5)P), phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2), phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2), PtdIns(4,5)P2, and phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3) were generous gifts from Dr. Karol Bruzik. The concentrations of the phospholipids were determined by a modified Bartlett analysis (29Kates M. Techniques of Lipidology. 2nd Ed. Elsevier, Amsterdam, The Netherlands1986Google Scholar). 6-Dodecanoyl-2-dimethylaminonaphthalene (LAURDAN), BODIPY® FL C5,C6-phosphatidylinositol 4,5-diphosphate (BODIPY-PtdIns(4,5)P2), fluorescein 5-isothiocyanate (“Isomer I”), and Texas Red™ C2-maleimide were all purchased from Invitrogen. Restriction endonucleases were purchased from New England Biolabs (Beverly, MA). CHAPS and octyl glucoside were purchased from Sigma and Fisher, respectively. The protease inhibitors, pepstatin, leupeptin, and aprotinin and the protease inhibitor mixture tablets were from Roche Applied Science. The Pioneer L1 sensor chip was purchased from Biacore AB (Piscataway, NJ). Vector Construction and Mutagenesis—The cDNA of full-length human annexin A2, which was a generous gift from Dr. Volker Gerke, was subcloned into the vector pET-21a(+) (Novagen, Madison, WI), between the restriction sites BamHI and XhoI. A stop codon was introduced just before the restriction site XhoI in order to exclude the C-terminal hexahistidine tag from the sequence during protein expression. The cDNA of p11 (a generous gift from Dr. James Seilhamer) was also subcloned into pET-21a(+) in a similar fashion. K279A and K281A mutants of annexin A2 were generated by the overlap extension polymerase chain reaction mutagenesis. The vector pGEX-4T-1 (Novagen, Madison, WI), which has an N-terminal glutathione S-transferase tag and a thrombin cleavage site was used for subcloning the cDNA of the phospholipase Cδ1 (PLCδ1) pleckstrin homology (PH) domain between the restriction sites BamHI and XhoI. All of the above constructs were transformed into DH5α cells for plasmid isolation, and their DNA sequences were verified. Subsequently, these plasmids were transformed into BL21(DE3) cells for protein expression. Protein Expression and Purification—One liter of sterile Luria broth medium containing 100 μg/ml ampicillin was inoculated with BL21(DE3) cells harboring each construct and grown at 37 °C until the optical density at 600 nm reached 0.6. Protein expression was then induced with 1 mm isopropyl-1-thio-β-d-galactopyranoside (Research Products, Mount Prospect, IL). After 8 h of incubation at 37 °C, cells were harvested by centrifugation (5,000 × g for 10 min at 4 °C). The resulting pellet was resuspended in 20 ml of precooled lysis buffer, containing 100 mm Tris-HCl (pH 7.5), 200 mm NaCl, 2 mm dithiothreitol, 1 mm EGTA, 0.5 mm phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, 5 μg/ml pepstatin, 5 μg/ml aprotinin, a protease inhibitor tablet, and 0.1% Triton X-100. This suspension was sonicated for 6 min (30 s of sonication followed by 30 s of incubation on ice) and then centrifuged for 1 h (40,000 × g at 4 °C). The supernatant was treated with 50% (NH4)2SO4 (final concentration) for 45 min and centrifuged at 40,000 × g for 20 min to remove insoluble proteins. This supernatant was then applied to an 80-ml butyl-Sepharose column (Amersham Biosciences) pre-equilibrated with 50% (NH4)2SO4 in the lysis buffer. Annexin A2 (or a mutant) was eluted with a linear gradient of (NH4)2SO4 from 50 to 0% in the lysis buffer. The eluted samples were dialyzed against 10 mm HEPES buffer, pH 7.5, containing 50 mm NaCl, 1 mm EGTA, and 1 mm dithiothreitol and applied to a DEAE-Sepharose column (Amersham Biosciences) equilibrated with the same buffer. Elution was carried out with 200 ml of 10 mm HEPES buffer, pH 7.5, containing 1 m NaCl, 1 mm EGTA, and 1 mm dithiothreitol. The recombinant p11 was purified by the same protocol. The purified recombinant annexin A2 was dialyzed against 10 mm HEPES buffer, pH 7.5, containing 50 mm NaCl, 1 mm EGTA, 1 mm dithiothreitol, 6 mm CaCl2, and a 2–5-fold molar excess of p11. These samples were then loaded onto a 5-ml Heparin HP HiTrap™ column (Amersham Biosciences) equilibrated with the same buffer without p11. The column was washed with 100 ml of the same buffer to get rid of excess p11, and the heterotetramer was eluted with 50 ml of elution buffer containing 30 mm HEPES, pH 7.5, 160 mm NaCl, 10 mm EGTA, and 1 mm dithiothreitol. The heterotetrameric nature of A2t was confirmed by the polyacrylamide gel electrophoresis performed under nondenaturing conditions (i.e. in the absence of SDS and dithiothreitol) using a 16% polyacrylamide gel (see Fig. 1). For the expression of the PLCδ1 PH domain, 1 liter of Luria broth containing 100 μg/ml ampicillin was inoculated with BL21(DE3) cells harboring the PH domain construct and grown at 37 °C until the optical density at 600 nm reached 0.4. Protein expression was induced by the addition of 50 mg of isopropyl-1-thio-β-d-galactopyranoside, and cells were harvested by centrifugation (5,000 × g for 10 min at 4 °C) after a 12-h incubation at 25 °C. The resulting pellet was resuspended in 10 ml of 30 mm HEPES buffer, pH 7.5, containing 160 mm NaCl, 50 μm phenylmethylsulfonyl fluoride, and 0.1% Triton X-100. This solution was sonicated for 6 min (30 s of sonication followed by 30 s of cooling on ice) and then centrifuged for 1 h (40,000 × g at 4 °C). After filtering the supernatant into a 50-ml Falcon tube, 500 μl of the glutathione S-transferase-Tag™ resin (Novagen, Madison, WI) were added. After incubating this mixture on ice for 45 min with mild shaking at 80 rpm, it was poured onto a column prerinsed with 50 ml of 30 mm HEPES buffer, pH 7.5, containing 160 mm NaCl. After washing the nonspecifically bound protein with 50 ml of 30 mm HEPES buffer, pH 7.5, containing 160 mm NaCl, 1 ml of the same buffer containing 4 units of thrombin was added in order to cleave the glutathione S-transferase tag, and the column was then sealed for a 6-h incubation at 25 °C. The protein was then eluted in five fractions using 500 μl of 30 mm HEPES buffer, pH 7.5, containing 160 mm NaCl. The protein purity was checked on a 16% polyacrylamide gel, and all of the protein samples were subsequently concentrated. The protein concentrations were determined by the bicinchoninic acid method (Pierce). Chemical Labeling of Proteins—The purified p11 (2 mg/ml) was treated with a 10-fold molar excess of Texas Red™ C2-maleimide for 2 h at room temperature in 30 mm HEPES buffer, pH 7.5, which was purged with a stream of nitrogen gas before use to remove oxygen. The labeling reaction was subsequently quenched by adding an excess amount of 2-mercaptoethanol, and the labeled protein was separated from the reagents using a Sephadex G25 column (Sigma) eluted with 30 mm HEPES buffer, pH 7.5, containing 50 mm NaCl. The fractions corresponding to the protein peak were pooled and dialyzed against 30 mm HEPES buffer, pH 7.5, containing 50 mm NaCl and 6 mm CaCl2 for 24 h at 4 °C. The labeling efficiency of p11 was estimated using the equation, mol of dye/mol of protein = (absorbance of the labeled protein at 582 nm)/((molar absorptivity of Texas Red at 582 nm (ϵ = 112,000 m–1 cm–1) × (protein concentration)). Under our labeling conditions, ∼0.7 mol of Texas Red was incorporated per mol of p11. The labeled p11 was then incubated on ice with purified annexin A2 (wild type or mutants) for 2 h, and the labeled heterotetramer was purified using a HiTrap™ Heparin HP column as described above. The PLCδ1 PH domain was labeled with a 10-fold molar excess of fluorescein-5-isothiocyanate for 2 h at room temperature, and the labeled protein was separated using a Sephadex G25 column eluted with 30 mm HEPES buffer, pH 7.5, containing 160 mm NaCl. SPR Measurements—All SPR equilibrium binding measurements were carried out at 23 °C as described (30Ananthanarayanan B. Stahelin R.V. Digman M.A. Cho W. J. Biol. Chem. 2003; 278: 46886-46894Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 31Stahelin R.V. Long F. Diraviyam K. Bruzik K.S. Murray D. Cho W. J. Biol. Chem. 2002; 277: 26379-26388Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). The sensor chip Pioneer L1 (Biacore) was coated with vesicles according to a protocol described previously (32Stahelin R.V. Cho W. Biochemistry. 2001; 40: 4672-4678Crossref PubMed Scopus (146) Google Scholar). Typically, after washing the sensor chip surface, 90 μl of phospholipid vesicles of different lipid composition were injected at a flow rate of 5 μl/min to give a response of 5000 resonance units. The control surface was then coated with POPC/POPE (80:20) vesicles to give the same resonance unit response as that of the active surface. All of the equilibrium binding measurements were carried out at a steady flow rate of 5 μl/min in order to give sufficient time for the R values of the association phase to attain saturating response units (Req). Req values were then plotted against protein concentrations (C), and the Kd value was determined by a nonlinear least squares analysis of the binding isotherm using the equation Req = Rmax/(1 + Kd/C), where Rmax is the maximal Req value. Spectrofluorometric Measurements on Large Unilamellar Vesicles (LUV)—LUV were prepared by an extrusion method using a Liposofast microextruder and a 100-nm polycarbonate filter (Avestin, Ottawa, Canada). Spectrofluorometric measurements using LAURDAN-containing LUV were carried out with a Hitachi F4500 fluorescence spectrophotometer at an excitation wavelength of 364 nm. Microscopy Measurements on GUV—GUV were prepared by the electroformation method using a home-built device as described previously (33Bagatolli L.A. Gratton E. Biophys. J. 1999; 77: 2090-2101Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). Briefly, GUV were grown in deionized water at 60 °C for 30 min by spreading ∼3 μl of the lipid stock with various compositions on platinum wires. During GUV growth, the platinum wires were connected to a function generator (Hewlett-Packard, Santa Clara, CA) for 30 min, and a low frequency AC field (sinusoidal wave function with a frequency of 10 Hz and an amplitude of 3 V) was applied. After 45 min, the temperature was lowered to 40 °C, and the frequency generator was switched off after the system attained this temperature. All subsequent measurements were carried out at 40 °C in 10 mm HEPES buffer, pH 7.5, with 0.16 m NaCl and different concentrations of Ca2+. All microscopy measurements were carried out using a custom-built combination laser-scanning and multiphoton microscope that was described previously (34Stahelin R.V. Digman M.A. Medkova M. Ananthanarayanan B. Rafter J.D. Melowic H.R. Cho W. J. Biol. Chem. 2004; 279: 29501-29512Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Briefly, a 920-nm ultrafast pulsed beam from a tunable Tsunami laser, set up for femtosecond operation (Spectra Physics, Mountain View, CA), was spatially filtered and launched into the scan head. The beam was directed toward the primary dichroic mirror (Chroma Technology, Brattleboro, VT) and then toward the XY scan mirrors (model 6350, Cambridge Technologies, Cambridge, MA). A Prairie Technologies scan lens (Middleton, WI) was used to focus the laser light, collimated by the 1× Zeiss tube lens, and directed toward a 40× water-corrected 1.2 numerical aperture Zeiss objective, mounted on a Zeiss 200 M platform (Carl Zeiss Inc.). Light excited by a 920-nm ultrafast pulse was collected on a nondescanned pathway by the Peltiercooled 1477P style Hamamatsu photomultiplier tubes. The light was reflected and filtered using appropriate optics. Instrument control was accomplished with the help of ISS amplifiers, an ISS three-axis scanning card (Champaign, IL), and two ISS 200-KHz analog lifetime cards. All the microscopic experiments were controlled by a data acquisition program, SimFCS (Laboratory for Fluorescence Dynamics, University of Illinois, Urbana-Champaign, IL). Phosphoinositide Specificity of Annexin A2 and A2t—It has been recently reported that annexin A2 and A2t have high specificity for PtdIns(4,5)P2 (21Hayes M.J. Merrifield C.J. Shao D. Ayala-Sanmartin J. Schorey C.D. Levine T.P. Proust J. Curran J. Bailly M. Moss S.E. J. Biol. Chem. 2004; 279: 14157-14164Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 22Rescher U. Ruhe D. Ludwig C. Zobiack N. Gerke V. J. Cell Sci. 2004; 117: 3473-3480Crossref PubMed Scopus (146) Google Scholar); however, this putative PtdIns(4,5)P2 specificity has not been quantitatively measured. We therefore prepared mixed vesicles of POPC/POPE containing 3 mol % of each of seven phosphoinositides and quantitatively determined by SPR analysis the affinity of annexin A2 and A2t for these vesicles coated onto the sensor chip. Annexin A2 and p11 have been shown to form a heterotetramer with high affinity (8Johnsson N. Marriott G. Weber K. EMBO J. 1988; 7: 2435-2442Crossref PubMed Scopus (163) Google Scholar, 9Rety S. Sopkova J. Renouard M. Osterloh D. Gerke V. Tabaries S. Russo-Marie F. Lewit-Bentley A. Nat. Struct. Biol. 1999; 6: 89-95Crossref PubMed Scopus (264) Google Scholar). Fig. 1 indicates that A2t exists as a heterotetramer under our experimental conditions. Representative sensorgrams for A2t and vesicles and a binding isotherm generated from the sensorgrams are shown in Fig. 2. The SPR method not only allows sensitive and quantitative determination of Kd values (32Stahelin R.V. Cho W. Biochemistry. 2001; 40: 4672-4678Crossref PubMed Scopus (146) Google Scholar, 35Cho W. Bittova L. Stahelin R.V. Anal. Biochem. 2001; 296: 153-161Crossref PubMed Scopus (114) Google Scholar) but also circumvents the vesicle aggregation during binding measurements, because it uses vesicles immobilized on the sensor chip. This is important, because in the vesicle pelleting assay, the charge and size of vesicles can significantly affect the pelleting efficiency, which in turn complicates the interpretation of binding data (35Cho W. Bittova L. Stahelin R.V. Anal. Biochem. 2001; 296: 153-161Crossref PubMed Scopus (114) Google Scholar). For the first set of measurements, a relatively high Ca2+ concentration (0.1 mm) was employed to ensure that the proteins show detectable affinities for all phosphoinositide-containing vesicles under the same conditions. The control surface was coated with POPC/POPE (80:20) vesicles, because neither annexin A2 nor A2t showed detectable affinity for these zwitterionic vesicles. As summarized in TABLE ONE, A2t showed relatively high affinity (i.e. Kd = 33 nm) for POPC/POPE/PtdIns(4,5)P2 (77:20:3) vesicles, which was >10-fold higher than that for POPI- or POPS-containing vesicles. Also, this affinity is comparable with those of epsin 1 ENTH domain (36Stahelin R.V. Long F. Peter B.J. Murray D. De Camilli P. McMahon H.T. Cho W. J. Biol. Chem. 2003; 278: 28993-28999Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar) and PLCδ1 PH domain (see TABLE ONE) for the same vesicles. However, annexin A2 monomer had >10-fold lower affinity for POPC/POPE/PtdIns(4,5)P2 (77:20:3) vesicles than A2t under the same conditions. The difference was even bigger at lower Ca2+ concentrations. For instance, at 50 μm Ca2+, A2t had a Kd value of ∼100 nm, whereas annexin A2 monomer showed no detectable affinity, suggesting that PtdIns(4,5)P2-dependent membrane binding of annexin A2 monomer is physiologically insignificant. We therefore focused our measurements on A2t hereafter.TABLE ONEPhosphoinositide specificity for A2t, annexin A2, and p11Lipid composition (77:20:3)ProteinKdIncrease in Kda-Fold increase in Kd relative to the binding of A2t to POPC/POPE/PtdIns(4,5)P2 (77:20:3) vesicles.nm-foldPOPC/POPE/PtdIns(4,5)P2A2t32.6 ± 2.81.0POPC/POPE/PtdIns(3,4)P2A2t54.6 ± 2.61.7POPC/POPE/PtdIns(3,5)P2A2t61.4 ± 2.11.9POPC/POPE/PtdIns(3,4,5)P3A2t57.1 ± 1.41.8POPC/POPE/PtdIns(3)PA2t96.0 ± 3.33.0POPC/POPE/PtdIns(4)PA2t126.1 ± 6.63.9POPC/POPE/PtdIns(5)PA2t106.8 ± 4.63.3POPC/POPE/POPIA2t360.5 ± 1811.1POPC/POPE/POPSA2t418.6 ± 2612.8POPC/POPE/PtdIns(4,5)P2A2 alone391.6 ± 7.912.0POPC/POPE/PtdIns(3)PA2 alone713.3 ± 9.021.9POPC/POPE/PtdIns(4,5)P2p11 alone758.3 ± 46.423.3POPC/POPE/PtdIns(3)Pp11 alone730.5 ± 55.322.4POPC/POPE/PtdIns(4,5)P2PLCδ1-PH52.0 ± 9.01.6a -Fold increase in Kd relative to the binding of A2t to POPC/POPE/PtdIns(4,5)P2 (77:20:3) vesicles. Open table in a new tab When compared among phosphoinositides, A2t showed only modest selectivity for PtdIns(4,5)P2 over other phosphoinositides (i.e. its affinity for PtdIns(4,5)P2-containing vesicles was less than 2-fold higher than that for vesicles containing PtdIns(3,4)P2, PtdIns(3,5)P2, and PtdIns(3,4,5)P3, respectively, and less than 4-fold higher than that for vesicles containing each monophosphorylated phosphoinositide). Under the same conditions, the annexin A2 monomer also showed modest selectivity for PtdIns(4,5)P2 over PtdIns(3)P, whereas p11 exhibited no appre