The Annexin II-p11 Complex Is Involved in Regulated Exocytosis in Bovine Pulmonary Artery Endothelial Cells*

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) (GTPγS) 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 GTPγS-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 GTPγS-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.