Abstract
Annexin II is a member of a multigene family of Ca2+-regulated, membrane-binding proteins implicated through biochemical and perforated cell experiments in Ca2+-triggered secretion. Within most cells annexin II resides in a tight heterotetrameric complex with a cellular protein ligand, p11, and complex formation is mediated via the N-terminal 14 residues of annexin II including the N-terminal acetyl group. To analyze at the single cell level whether the annexin II-p11 complex is involved in regulated secretion, we used membrane capacitance measurements to follow exocytotic fusion events in bovine aortic endothelial cells manipulated with respect to their annexin II-p11 complex formation. Upon guanosine 5'-O-(thiotriphosphate) (GTPgammaS) stimulation, the endothelial cells show a significant increase in membrane capacitance which is generally preceded by a transient rise in intracellular Ca2+ and thus indicative of the occurrence of Ca2+-regulated secretion. The GTPgammaS-induced capacitance increase is markedly reduced in cells loaded with a synthetic peptide, Ac1-14, which corresponds in sequence to the N-terminal 14 residues of annexin II in their correctly acetylated form and which is capable of disrupting preformed annexin II-p11 complexes. The effect of the peptide is highly specific as the nonacetylated variant, N1-14, which is incapable of disrupting annexin II-p11, does not interfere with the GTPgammaS-induced increase in membrane capacitance. These data show that intact annexin II-p11 complexes are indispensable for regulated exocytosis to occur in an efficient manner in endothelial cells.
Highlights
Changes in the intracellular Ca2ϩ levels are known to regulate a variety of cellular processes ranging from the control of cell architecture to intracellular communication, cellular differentiation, and cell death
guanosine 5-O-(thiotriphosphate) (GTP␥S) Induced Secretion in Cultivated Endothelial Cells— Calf pulmonary artery endothelial (CPAE) cells were chosen to analyze the participation of annexin II-p11 in regulated exocytosis for two major reasons
We show that a substantial increase in cell surface area indicative of secretory vesicle fusion with the plasma membrane occurs in GTP␥S-stimulated endothelial cells and that in the majority of the cells this increase is preceded by a transient rise in intracellular Ca2ϩ
Summary
Antibodies and Proteins—Mouse monoclonal antibodies, H28 and H21, were used for the detection of annexin II and p11, respectively [16]. All secondary antibodies were tested in control experiments by omitting the primary antibody and showed neglectable fluorescence signals on CPAE cells. Immunoblotting experiments employed horseradish peroxidase-coupled pig anti-mouse antibodies (Dako) as secondary antibodies. For N-terminal acetylation, peptides were left on the resin, and the Fmoc (N-(9-fluorenyl)methoxycarbonyl) group was released by treatment with dimethylformamide. Peptides were purified by reverse-phase high performance liquid chromatography on a preparative column (Vydac 218TP 1022) using a linear acetonitrile gradient for elution. All products were characterized by mass spectrometry (MALDI compact III; Kratos Analytical) and by N-terminal sequence analysis of a nonacetylated aliquot on an automated gas-phase sequenator (Knaur model 810). Cells were grown in Dulbecco’s modified Eagle’s medium containing 20% fetal calf serum, 2 mM glutamine, 100 g/ml streptomycin, 100 units/ml penicillin, detached by exposure to 0.05% trypsin in a Ca2ϩ- and Mg2ϩ-free solution, reseeded on gelatin-coated coverslips, and kept in culture 2– 4 days before use
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