Annexin II in exocytosis: catecholamine secretion requires the translocation of p36 to the subplasmalemmal region in chromaffin cells.

Abstract

Abstract. Annexin II is a Ca2+-dependent membranebinding protein present in a wide variety of cells and tissues. Within cells, annexin II is found either as a 36-kD monomer (p36) or as a heterotetrameric complex (p90) coupled with the S-100-related protein, pll. Annexin II has been suggested to be involved in exocytosis as it can restore the secretory responsiveness of permeabilized chromaffin cells. By quantitative confocal immunofluorescence, immunoreplica analysis and immunoprecipitation, we show here the translocation of p36 from the cytosol to a subplasmalemmal Triton X-100 insoluble fraction in chromaffin cells following nicotinic stimulation. A synthetic peptide corresponding to the NH2-terminal domain of p36 which contains the phosphorylation sites was microinjected into individual chromaffin cells and catecholamine secretion was monitored by amperometry. This peptide blocked cornpletely the nicotine-induced recruitment of p36 to the cell periphery and strongly inhibited exocytosis evoked by either nicotine or high K ÷. The light chain of annexin II, pll, was selectively expressed by adrenergic chromaffin cells, and was only present in the subplasmalemmal Triton X-100 insoluble protein fraction of both resting and stimulated cells, pll can modify the Ca 2÷-and/or the phospholipid-binding properties of p36. We found that less Ca 2+ was required to stimulate the translocation of p36 and to trigger exocytosis in adrenergic chromaffin cells. Our findings suggest that the translocation of p36 to the subplasmalemmal region is an essential event in regulated exocytosis and support the idea that the presence of pll in adrenergic cells may confer a higher Ca 2+ affinity to the exocytotic pathway in these cells.

Phospholipase D1 Production of Phosphatidic Acid at the Plasma Membrane Promotes Exocytosis of Large Dense-core Granules at a Late Stage

“…Immunocytochemistry-Transfected PC12 and chromaffin cells were washed twice with Locke's solution and then incubated for 10 min either in calcium-free Locke's solution (resting conditions) or in Locke's solution containing a depolarizing concentration of potassium (stimulation) before the fixation step and further processed for immunofluorescence as described previously (12,19). The primary antibodies were used at the following dilutions: anti-GH antibody (1/150) and anti-SNAP25 antibody (1/200).…”

Phosphorylation by Protein Kinase C of Annexin 2 in Chromaffin Cells Stimulated by Nicotine

“…and in the subcortical region. Recently, we suggested that the translocation of p36 may be an early event in the process of regulated exocytosis and that translocation of p36 to the cell periphery may precede the phosphorylation process (Chasserot-Golaz et a!., 1996). It will be important to analyze the relationship between the phosphorylation and the translocation steps, especially in view of the postulated role of annexin 2 in catecholamine secretion.…”

Specific release of membrane-bound annexin II and cortical cytoskeletal elements by sequestration of membrane cholesterol.

“…This protein can partially restore Ca2+-dependent secretory activity in digitonin/SLOpermeabilized chromaffin cells, and annexin II can aggregate chromaffin granules at Ca2+ concentrations in the ,tM range (Ali et al, 1989;Drust and Creutz, 1988;Sarafian et al, 1991;Chasserot-Golaz et al, 1996). In the endocytic pathway, immunoelectron microscopic studies have shown that annexin II is associated with plasma membrane and early endosomes of BHK cells and with the apical and basolateral plasma membranes and early endosomes of polarized MDCK cells (Harder and Gerke, 1993).…”